text:
Dr.V.Ravi, Dr.Anita Desai and Dr.S.N.Madhusudana
Department of Neurovirology
Cell cultures
Virus growth and titration
Methods for viral antigen detection
Methods for viral antibody detection
Subculture of a cell line
1. Aedes albopictus C6/36 ( mosquito gut epithelium ) cell line
2. Discard supernatant fluid from a confluent monolayer of C6/36 (Aedes albopictus ) bottle cultures.
3. Add 3 to 5 ml trypsin (Pre-warmed at 37 C ) to each culture bottle.
4. Leave the bottle at room temperature for 3-5 mins.
5. Discard the trypsin.
6. Add 5ml of growth medium to the bottle and gently detach the cells from the surface with the help of a pipette.
7. Add the required amount of medium (split ratio for this cell line is 1: 5) and distribute the diluted cell suspension to fresh flasks/bottles.
8. Incubate at room temperature for 3-7 days.
Primary cell culture technique
Chick embryo fibroblastic culture :
The development of routine cell culture methods has reduced the importance of eggs but they are still valuable for the isolation of many important viruses and for the production of vaccines. Fertile eggs must be obtained ideally from a specific pathogen free flock, should be clean, preferably unwashed and pale shelled to simplify candling. After laying they have to be incubated for 10 days at 37° C With 40-70% humidity and good aeration and turned twice daily. After 6 days they are candled, infertile and dead eggs are discarded. On the day of culture those with satisfactory development of chorioallantioic blood vessels and showing embryonic movement are marked with pencil to indicate the limits of the air sac.
Materials required
1. Embryonated eggs preferably 10-12 days old.
2. Phosphate buffer saline (pH 7.2) .
3. 0.25% trypsin in PBS.
4. Growth medium (MEM, supplemented with 10% bovine serum)
5. Forceps, scissors, egg cup, petri dishes, filtration unit, silicone/teflon
6. Coated magnet, solution bottles and culture flasks.
1. Candle and select 10-11 day old eggs.
2. Place the egg in an egg cup, air sac upwards and wipe clean with spirit.
3. Break the shell with the sharp end of a sterile forceps, and lift the membrane. With a bent forceps pick up the embryo and place it in a petri dish containing PBS.
4. Dissect the embryo in the dish, remove and discard the head, limbs and viscera. Pick up the fibroblastic tissue and transfer into a wide neck bottle containing PBS.
5. Mince the tissue finely with scissors, and wash minced tissue several times in PBS to remove blood cells and debris.
6. Transfer tissue to transfusion bottle containing sterile silicone covered magnet and 50ml of 0.25 % trypsin solution and stopper securely.
7. Trypsinize on magnetic stirrer unit 37° C for 30 mins : avoid frothing of contents.
8. The tissue will disintegrate, forming a turbid suspension of cells. Filter the suspension through sterile gauze and centrifuge filtrate for 10min at 1000 rev/min.
9. Discard supernatant and resuspend cells in 100 ml growth medium.
10. Centrifuge once again and resuspend the cells in fresh medium.
11. Dilute 0.9ml suspension with 0.1ml trypan blue solution and count cells in haemocytometer.
12. Adjust concentration to 1 10 cells / ml growth medium and dispense into tubes or bottles.
13. Incubate at 37° C until monolayer is formed (2-3 days).
14. When cells have formed a monolayer, remove growth medium, inoculate virus, add maintenance medium (MEM, Eagles base with 1-2% bovine serum) and incubate.
Media preparation for cell culture
Dehydrated Tissue Culture Media
1. Take 900ml of triple distilled water.
2. Add the contents of one unit vial of dehydrated media to the water at room temperature with stirring until dissolved.
3. Rinse the vial with a small amount of triple distilled water to remove traces of powder and add to the above solution.
4. Add 2.2 grams of sodium bicarbonate or HEPES Buffer.
5. Adjust the pH if required between 7.1 to 7.4 using 1N HC1 or 1N NaOH or by bubbling carbon dioxide. Note that pH tends to rise during filtration and hence adjust it 0.2 to 0.3 units below the final desired pH.
6. Make up final volume to 1000ml with triple distilled water.
Sterilization of tissue culture Media
1. Sterilize the media by filtering through sterile membrane filter(sterilized by autoclaving at 15 lbs for 15 min & 121° C ) of 0.22 micron or less porosity using positive pressure to minimise loss of carbon dioxide.
2. The following antibiotics can be aseptically added to litre of media :
3. Amphotericin 2.5 mg
4. Gentamycin (50mg/ml. Solution) 1.0 ml
5. Benzyl Penicillin 10000 units
6. Streptomycin 100 mg
Sterility check
Add 0.5 - 1.0 ml of filtered media to a tube containing sterile thioglycolate broth and incubate at 37° C for 48 hours. If the broth is clear after 48 hours the media is sterile.
TVG (Trypsin Versene Glucose)
1. TVG consists of the following components in 1X PBS:
2. Trypsin 0.1%
3. Versene 0.2%
4. Glucose 0.05%
Stock solutions for TVG
1). 10x PBS
NaCl 80.00 gms
KCl 2.00 gms
Na2H P04 14.42 gms
KH2 PO4 2.00 gms
Dist.H2O upto 1 litre
To prepare 1X PBS - add 100 ml of 1X PBS to 900 ml of D/W.
Trypsin 2.00 gms
D/W upto 100ml
Stir the above solution on a magnetic stirrer for 4h or O/N at 4°C. Sterilize by filtering through sterile membrane filter of 0.22u pore size. A sterility check can bedone before using the solution.
EDTA 200 mg
D/W 100 ml
Sterilize by autoclaving at 15 lbs & 121°C for 15 minutes.
Glucose 10.00 gms
D/W upto 100 ml
Sterilize by autoclaving at 15 lbs & 121° C for 15 minutes.
Phenol Red 1.00 gm
D/W upto 100ml
Preparation of working solution of TVG
Prepare 840ml of 1X PBS and to this add 1.0 ml of 1% phenol red (indicator). Sterilize by autoclaving at 15lbs and 121°C for 15 minutes. Cool this sterile solution and then add the following sterilized solutions to it :
Trypsin 2.0% 50 ml
Versene 0.2% 100 ml
Glucose 10.0% 5 ml
Do a sterility test as mentioned above before using this TVG.
1) Autoclave all glassware before washing, at 120°C, 15psi for 20min.
2) The outside surface of glassware can be cleaned with Vim and inside with only Teepol and scrubbed with a clean brush , which is only meant for brushing glassware. Wash in tap water and leave in 5% Teepol solution overnight (can be boiled)
3) Next morning wash in tap water at least 20 times and leave in 10% HC1 overnight.
4) Next day wash in tap water and rinse in 2 changes of demineralised distilled water at least 10 times in each, leave in third bucket of demineralised distilled water overnight.
5) Take out next day, dry it in hot air oven, pack and sterilize.
Packing and sterilization
Pipettes: Wrap the pipette with brown paper and sterilize in a hot air oven at 160°C for two hours.
Petridishes : Wrap the Petri dishes in brown paper and tie with twine and sterilize by hot air oven at 160°C for two hours.
Culture & media storage bottles, measuring cylinders and beakers :
Cover mouth with aluminum foil, over which brown paper is tied with twine at the neck and sterilize in a hotoven at 160°C. Plastic measuring cylinders and beakers are autoclaved at 120°C, psi for 30min.
Plastic centrifuge tubes, Eppendorf tubes, screw cap vials & tubes, :
Arrange neatly the following items in a plastic orglass beaker and cover the mouth with aluminum foil andover that wrap brown paper tied with twine at the neck.
Sterilize by autoclaving at 120°C, 15 psi for 30min.
Coverslips: Coverslips are put into a Petri dish , which is covered with brown paper and tied with twine. Sterilize in a hot air oven at 160°C for two hours.
Filter Apparatus: Wrap filter apparatus first with aluminum foil and then with brown paper, tie it with twine tightly. Sterilize by autoclaving at 120° C,
15 psi for 30min.
Solutions: Plug the mouth of the container with cotton, wrap it with brown paper and tie it with twine. Sterilize by autoclaving at 121° C , 15psi for 20 min.
1) HSV - Demonstration of cytopathic effect (CPE) in Vero cell line.
2) Grow Vero cells to confluence in Nunc flasks / 24 well dishes.
3) Discard the growth medium.
4) Infect the cell line with HSV-1 suspension at an MOI (multiplicity of infection) of 1.
5) Adsorb the virus for 30 minutes at 37° C .
6) Add the maintenance medium - 5ml for Nunc flask/10-15ml for Milk dilution bottle/100ml for a Roux bottle.
7) Incubate the bottles at 37°C in CO incubator.
8) Observe daily for CPE both in the morning and in the evening.
9) HSV produces CPE by 24-48 h. Two forms of CPE are observed. The most common begins with cytoplasmic granulation after which the cells become enlarged or ballooned. These macrocytes then become rounded, take on a refractile appearance, and undergo lytic degeneration. The second type of CPE is formation of multinucleated giant cells.
Measles virus : Demonstration of CPE in Vero cell line
1) Grow Vero cells to form a monolayer in Nunc flasks/ Milk dilution bottles/Roux bottle.
2) Discard the growth medium.
3) Infect the cell line with measles virus suspension at an MOI(multiplicity of infection) of 1.
4) Adsorb the virus for 30 minutes at 37° C.
5) Add the maintenance medium - 5ml for Nunc flask/10-15ml for Milk dilution bottle/100ml for Roux bottle.
6) Incubate bottles at 37°C in CO incubator.
7) Observe daily for CPE both in the morning and in the evening.
8) Measles virus produces CPE after 4-5 days. Two types of CPE are described. One type seen after infection with wild virus and dilute inoccula is giant cell transformation. Multinucleated giant cells containing 10-100 nuclei form as a result of cell fusion induced by virus. The second type of CPE, spindle cell formation, is associated with measles virus , which has been passed repeatedly in tissue culture, or with repeated passage of undiluted inoccula.
Japanese Encephalitis virus : Demonstration of CPE in porcine stable kidney cell line (PS)
1) Grow PS cells to form a monolayer in Nunc flasks/Milk dilution bottles/Roux bottle.
2) Discard the growth medium.
3) Infect the cell line with measles virus suspension at an MOI (multiplicity of infection) of 1.
4) Adsorb the virus for 30 minutes at 37° C.
5) Add the maintenance medium - 5ml for Nunc flask/10-15ml for Milk dilution bottle/100ml for a Roux bottle.
6) Incubate the bottles at 37° C in CO incubator.
7) Observe daily for CPE both in the morning and in the evening.
8) JEV produces CPE after 18-20 hrs. CPE is seen in the form of rounding of cells followed by cell lysis.
Titration of Rabies virus in vivo by intracerebral inoculation
After a virus is propogated in either cell culture or in a suitable animal, we need to know the infectivity titre of the virus material obtained. This can be determined in vivo by inoculating increasing dilutions of the virus material to a susceptible host animal such as laboratory mice and based on mortality seen in different dilutions, the infectivity titre which is the reciprocal of highest dilution showing 50% mortality in the inoculated mice and expressed as LD50 /ml and can calculated by suing either Reed-Muench or Karber formula. As an example, titration of rabies virus is illustrated in this section.
Materials :
1. Microbiological safety cabinet.
2. Sterile Pipettes, 10ml and 1ml
3. A tray with ice flasks. Rabies virus suspension (cell culture supernatant of infected brain emulsion)
4. Mice (Swiss albino or Laka) 4-6 weeks old
5. One ml syringe and needle for I/c inoculation
6. Test tubes.
7. Sterile PBS pH 7.4
8. Sterile bovine or equine serum
9. Cages for housing mice.
1. Working in microbiological safety cabinet, prepare the diluting fluid which is PBS containing 2% serum and dispense 9ml in test tubes labelled 10-1 to 10-7 and keep the test tubes in rack immersed in plenty of ice.
2. To make 10 fold (log) dilutions of the virus material, dilute 1ml of virus in 9ml of diluent to get the initial dilution i.e. 10-1. Subsequently transfer 1ml of previous virus dilution to next dilution by using at each step a fresh pipette, to achieve serial tenfold dilutions.
3.Inocultate 0.03 ml of each virus dilution intracerebrally into mice, starting from the highest dilution (in this case 10 -7). Use at least 6 mice per dilution and transfer these into cages appropriately labelled.
4.Observe the mice for 14 days. Any death occurring within first 5 days should be considered non-specific. Observe for specific signs and symptoms of rabies i.e., ruffling of hair, incoordination, tremors and paralysis of hind and fore limbs and finally death. Note down total number of specific deaths in each dilution and calculate the virus titre by using Reed-Muench or Karber formula.
Microtitration of Poliovirus using Vero cell line
Most of the commonly encountered human viruses produce characteristic cytopathic effect in one or the other cell lines routinely used in virology laboratories. The infectivity titres of these viruses can conveniently be determined by infecting a particular cell line with increasing dilutions of the virus material and determining the highest dilution producing cytopathic effect in 50% of the inoculated cells. The 50% end point dilution which in this case is expressed as TCID 50/ml can be calculated by using either Reed-Muench or Karber formulae. As an example, titration of polio virus I is illustrated in this section.
Materials :
1) Microbiological safety cabinet
2) Sterile test tubes or Pencillin vials
3) Micro pippettes, 200 & 1000 microns
4) Co2 incubator.
5) 96 well flat bottom tissue culture plates(Nunc)
6) Minimum Essential Medium(MEM,Sigma,HI) supplemented with antibiotics
7) Fetal calf serum(FCS)
8) Trypsin versene glucose coln.(TVG)
9) VERO cells.
10) Fluid containing PI.
11) Inverted microscope
Procedure :
1.Make 10 fold serial dilutions, say from 10-3 to 10-7 of the virus material in MEM containing 2% FCS, dispensed in either test tubes or penicillin vials, changing pipettes or tips with each dilution. Keep the tubes in rack immersed in ice.
2. Transfer 100 ul of each dilution to 4 wells of the micro-titre plate, starting from highest dilution to the lowest.
3. Trypsinise one MD bottle containing a confluent monolayer of Vero cells and count the cells and dilute to 4x 105 cells per ml. In MEM containing 10% FCS. Dispense 100 ul of cell suspension in to each of the well containing virus dilution and also include 4 wells as cell control in which 100Ul of cell suspension is mixed with 100 UL of MEM with 2% FCS. While dispensing the virus dilutions and cell suspension it is necessary to keep the plate on ice tray.
4. Cover the plate and keep it in CO2 incubator and adjust the temperature to 37 C.
5. Read the plate under an inverted microscope after 3 to 4 days when a confluent monolayer of Vero cells can be seen in control wells. Look for the cytopathic effect in the wells inoculated with virus dilutions. This consists of rounding of cells, i.e. if 2 of 4 wells inoculated with 10-8 dilution shows cytopathic effect then the titre is 10-8 per 0.1 ml. The titre can also be calculated by the method of Reed and Muench.
Calculation of 50% endpoints
In any biological quantitation, the most desirable endpoint is one representing a situation in which half of the inoculated animals or cells show the reaction (death in the case of animals and in CPE case of cells) and the other half do not. In other words, the endpoint is taken as the highest dilution of the biological material , which produces desired reaction in 50% of the animals or cells. The 50% endpoint can be based on several types of reactions. The most widely used endpoint, based on mortality, is the LD50 (50% lethal dose). This terminology can also be applied to other host systems-for example, tissue cultures - in which the TCID50 represents the dose that gives rise to cytopathic effect in 50% of inoculated cultures. When computing, if closely-placed dilutions are used and in each dilution large number of animals or cells are used, it may be possible to interpolate a correct 50% end point dilution, but it is neither practical nor economical. Reed and Muench devised a simple method for estimation of 50% endpoints based on the large total number of animals, which gives the effect of using at the 2 critical dilutions between which the endpoint lies, larger groups of animals than were actually included in these dilutions.
Calculation of the LD 50 titre by the Reed-Muench method
Let us presume that we have titrated a virus suspension by inoculating mice and death is the reaction. The number of deaths and survivals in each dilution is tabulated as given in table I below:
Accumulated values for the total number of animals that dies or survival are obtained by adding in the direction of lowest to the highest values. The accumulated mortality ratios and the percentage mortality for each dilution is calculated.
In the example depicted in the table it can be seen that mortality in the 10-5, is higher than 50% and in the next higher dilution, 10-6 it is only 13%. So, we need to find the 50% endpoint dilution , which obviously lies between these dilutions. First, we have to calculate the proportionate distance (PD) of the 50% endpoint from these dilutions by using a simple formula.
The proportionate distance obtained thus has to be corrected by the dilution factor, which is the logarithm of the dilution step employed , which in this case is 1 (log of 10). Then the 50% endpoint dilution can be calculated thus:
Negative logarithm of LD50 titre = (negative logarithm of the next dilution above 50% mortality + PD) x dilution factor i.e. in this example, (-5 + 0.4) x 1 =
-5.4 or log LD50 titre = 10-5.4 per 0.03 ml which is the amount of material inoculated I/C. The same methodology can also be applied to compute TCID50 when cell culture system is used for virus titration. Each dilution is inoculated into a minimum of 4 to 6Wells in a 96 well tissue culture plate and observed for CPE , which in this case replaces mortality.
Virus titration by using the plaque assay
1. Prepare confluent monolayers of cells (Porcine kidney for JEV and Hep-2 for Polio virus) in 24 well plates (Nunc).
2. Prepare serial 10-fold dilutions (101 to 107 ) of virus in chilled maintenance medium (MEM, with 1% serum).
3. Remove culture medium and add 0.2ml of virus inoculum, starting from the highest dilution. Ensure that a film of medium completely covers the cell sheet.
4. Incubate the plate at 37° C for 1 hour with intermittent rocking of the plate.
5. Remove the inoculum, preferably with a pipette and then add 1.5 ml of agarose overlay medium (growth medium with 0.3% agarose and 2.5% FCS).
6. Ensure that the overlay medium has spread evenly over the monolayer, leave at room temperature for 10 mins and then incubate at 37° C .
7. Examine the monolayers daily, starting from second day of incubation.
8. Once the plaques have developed, usually by the fourth day post inoculation, count the number of plaques at each dilution, remove the agarose overlay and gently wash the monolayer with PBS.
9. Stain the plate with 0.1% crystal violet solution and count the plaques again.
10. Estimate the virus titre as a plaque forming units per ml (pfu /ml) as follows by counting the number of plaques at an appropriate dilution.
For example :
Number of plaques produced 9
Dilution of virus 1 x 105
Volume of inoculum 0.2 ml
Virus titre = 9 x 1x105 x 1/5 pfu per ml
= 4.5 x 106
Enteroviruses including polio viruses cause a variety of CNS manifestations including aseptic maningitis, encephalitis and paralysis. These viruses are natural inhabitants of human intestinal tract and they can be easily isolated from samples of feces or from rectal swabs. But mere isolation of a enterovirus from stool sample may not prove the diagnosis as these may be present in normal stool specimens also. Therefore isolation should also follow a demonstration of a serological response to corroborate the diagnosis. However isolation of virus from CSF, or from brain postmortem cases is diagnostic.
Isolation of Enteroviruses from fecal specimens:
1. 4-8 gms of feces,
2. 2. Centrifuge tubes.
3. 3.Cryo centrifuge
4. 4. Test tubes
5. 5.Rubber corks size 2,3 5.Micro pipettes
6. 6.CO2 incubator
7. 7. Water bath
8. 8.MEM with antibiotics and FCS 10% and 2%
9. 9.Hep 2 cells (ATCC, CCL 23)
10. 10. RD cells ( ATCC< CCL 136)
11. TVG soln.
12. 96 well flat bottom tissue culture plates.
13. 13.LBM pools of Antisera.
Processing of fecal specimens :
About 4-8 gms of feces is required. The specimens are kept frozen or in cold till processed. A 10% or 20% fecal suspension is made in cold sterile PBS in a tightly stoppered bottle. After vigorous shaking to emulsify the feces, the specimen is allowed to settle in the cold for 10min. The supernatant fluid is then poured in to a centrifuge tube and clarified at 3000 rpm for 10min. The supernatant fluid is removed and to this is added antibiotics to get a final concentration of 1000 units of penicillin and 1000 ug of streptomycin per ml and the sample is held at room temperature for 30min. It is then centrifuged in cold at speed 10,000 rpm for 20 min. The clear supernatant is separated and used immediately or stored frozen.
Inoculation of specimens into cell culture:
For isolation of most enteroviruses a combination of 2 cell lines like Hep 2 and RD will suffice. These cells are grown separately in test tubes and monolayers are obtained by employing usual cell culture techniques, described elsewhere. The sample processed is inoculated in 200 Ul quantities to each of 4 tubes of both Hep 2 and RD cell lines containing 1 ml maintenance medium and incubated in an almost horizontal position at 37 ° C . Cultures showing early degeneration, less than 24hrs are subpassaged. The tubes are observed daily for evidence of typical enterovirus CPE and discarded at the end of 7-10 days if found negative.
Identification of Enterovirus by Neutralisation : Ex: Poliovirus
If typical CPE is seen with either Hep 2 or RD or both cell lines, it is almost certain that an enterovirus has been isolated. The isolate may be identified as one of the polio antibodies. The isolate should be titrated and a dilution containing 100 TCID 50 per 0.1 ml is prepared. Each of the polio antisera P1, P2 and P3 is diluted to contain 50 antibody units, i.e.50 times that necessary to neutralise 100 TCID 50 of virus. Equal quantities of virus and serum are mixed and incubated in water bath at 37 C for 2hrs. The mixture is then inoculated on to monolayers of HEP 2 or RD cell lines in test tubes or microtitre plates. The reading is taken on day 3 and 7 and if the isolate is one of the poliovirus it will be neutralised by the corresponding antisera and hence there will be no CPE in the inoculated cultures. Virus controls not treated with antisera should show typical CPE at the end of 3rd day. Enteroviruses, which produce CPE, can be conveniently typed by neutralization using LBM pool (Lim-Benyesh-Melnick) of antisera. For further details about this technique as well as interpretation of results please refer to Melnick et al (1979). Enteroviruses. In : Diagnostic procedures for viral, Ricketsial and Chlamydial infections (E.H.Lennette, N.J.Schmitt, Eds)
5th edition, pp 471 - 524. American Public Health Association, Washington,USA.
Isolation of virus from CSF :
Enteroviruses can sometimes be isolated from CSF during early stages of the illness. For isolation from CSF 200 Ul of CSF is inoculated on to the monolayers of both Hep 2 and RD cells as described previously. No pre-treatment or addition of antibiotics is required. The tubes are incubated and observed for CPE as in case of fecal specimens. If an isolate is obtained it is identified as described earlier.
Haemagglutination inhibition (HI) test
A wide variety of animal viruses have been shown to possess the capacity of adsorbing to red blood cells and in many cases causing agglutination of these cells. This haemagglutination (HA) test furnishes a relatively simple, quick, convenient and fairly quantitative way of detecting , identifying and titrating viruses.
Virus haemagglutination is readily inhibited by specific antibody and the haemagglutination inhibition test furnishes a delicate way of detecting viral antibody. It may be strain-specific as in the case of the influenza viruses or group-specific as with most of the arboviruses where the test is mainly used for antigenic group screening.
Materials required :
1. 25% kaolin borate saline pH 9.0.
2. 0.4% bovine albumin in borate saline
pH 9.0 (0.4% BABS pH 9.0).
3. Virus adjusting diluents of varying pH (VADs)
4. Goose erythrocytes (RBC) in DGV
5. Antisera
6. Antigens
7. Normal serum
8. Acid citrate dextrose (ACD)
9. Dextrose gelatin veronal (DGV)
10. Borate saline pH 9.0
11. 0.9% NaCl
12. Acetone
13. 2-Mercaptoethanol
Preparation of Reagents
1. Borate saline pH 9.0
1.5 M NaCl 160 ml
0.5 M H3BO4 (Boric acid) 200 ml
1.0 M NaOH 47 ml
Make it to 2 litres with D/W, pH adjusted to 9 with 1M NaOH or 0.5M H3BO3.
2. 0.4% Bovine albumin in 1000ml borate saline pH 9.0 kept O/N in the cold.
Adjust pH 9.0 with 1M NaOH.
3. 25% kaolin in 100 ml borate saline pH 9.0 (Use acid-washed kaolin)
Borate saline is stirred on a magnetic stirrer and kaolin powder is added slowly to make a uniform suspension.
4. Dextrose-gelatin-veronal (DGV) for preservation of goose cells:
A. Gelatin 0.600 g
Diethyl barbituric acid (Veronal) 0.580 g
B. Sodium barbitone 0.380 g
CaCl2 (Anhydrous) 0.020 g
NaCl 8.500 g
Dextrose 10.000 g
Merthiolate (Thiomerosal powder) 0.100 g
MgSO4.7H2O 0.120 g
Add 250 ml of hot D/W to A and 750 ml of D/W to B. Mix A and B.
Autoclave at 10 lbs for 10 minutes.
5. 0.9% NaCl
9 g NaCl in 100 ml D/W. Autoclave at 15 lbs for 15 minutes.
6. Preparation of stock solutions
A. 1.5M sodium chloride (NaCl) - 87.675 g NaCl made to 1 litre with distilled water.
B. 0.5M boric acid (H3BO3) - 30.92 g H3BO3 are dissolved in about 700 ml of hot D/W. The solution is allowed to cool to room temperature and then made to 1 litre with D/W.
C. Concentrated sodium hydroxide (NaOH) - approximately 500 ml of distilled water is added to approximately 500g of NaOH pellets. The concentrated NaOH is about 18M and is very stable.
D. 0.5M dibasic sodium phosphate (Na2HPO4) - 70.99 g Na2HPO4 (anhydrous) made to 1 litre with distilled water.
E. 1M monobasic sodium phosphate (NaH2PO4) - 138.01 g NaH2PO4.H2O made to 1 litre with distilled water.
Collection/washing of goose RBC
a. Collection of RBC
1. Goose RBC are collected under sterile condition.
Take 125 ml of DGV in a 250 ml flask.
2. Take 10 ml ACD in a sterile tube.
3. Withdraw 7.5 ml ACD into 50 ml syringe fitted with a 20 gauge needle.
4. Collect 50 ml goose blood from the wing of jugular vein.
5. Remove needle from syringe and transfer blood from syringe to flask with DGV.
6. Mix well.
7. Store at 4°C (can be kept for up to 2 weeks)
b. Washing of RBC
1. Centrifuge required quantity of RBC suspension in a graduated centrifuge tube for six
Minutes at 2000 rpm.
2. Aspirate off the pipette attached to a rubber bulb without disturbing the RBC.
3. Add 2 ml of normal saline (NS).
4. Mix gently with Pasteur pipette to resuspend cells and add an extra quantity of NS to bring to the original volume.
5. Centrifuge at room temperature for 3 minutes at 200 rpm.
6. Repeat steps 2,3,4 & 5.
7. Centrifuge at 2000 rpm for 6 minutes.
8. Read volume of packed RBC.
9. Remove supernatant fluid.
10. Add 0.9% NaCl to make a 10% suspension of RBC and mix. The cells are ready for use in IV (use washed and packed cells for adsorption of sera).
Titration of Antigen
1. With a wax pencil mark a "U" microtitre plate as shown in figure:
20 40 80 160 320 640 1280 2560 5120 10240 cc** cc
· cc - cell control.
2. With a 0.05 ml dropper, add one drop of 0.4% BABS pH 9.0 to each well.
3. Keep plate in refrigerator for chilling.
4. Keep seven 0.05 ml micro diluters with tips immersed in 0.9% NaCl in a refrigerator for chilling.
5. Dispense 0.9 ml of 0.4% BABS in a tube in an ice bath.
6. Add 0.1 ml of antigen and mix thoroughly (1:10 dilution of antigen).
7. Remove plate and micro dilutor from refrigerator.
8. Blot 0.05 ml micro dilutors on a blotting paper, dip the tips in tube with 1:10 antigen and place in each of the wells marked 1:20 in the plate.
9. With the micro dilutors, prepare serial 2 - fold dilution of the antigen upto well 10 (1:10,240) by swirling the micro dilutors 10 to 15 times.
10. Add 2.4 ml of VADs 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, & 7.2 to labelled tubes.
11. Add 0.1 ml of 10% RBC to each of the tubes and mix well (this gives a dilution of 0.4% RBC).
12. With a 0.05 ml dropper pipette add one drop of RBC to the corresponding row.
13. Tap plate gently to mix cells and the antigen.
14. Cover the plate and incubate at room temperature until cells in the wells of the cell control row settle down and form a button (usually this takes 20 - 30 minutes)
15. Read and record the titration.
16. Complete agglutination consists of a uniform layer on the lower surface of the well. A negative consists of a compact, sharply demarcated button of sedimented cells in the centre of the well, the pattern being identical to that seen in the control wells. The results are coded as follows:
20 40 80 160 320 640 1280 2560
+ + + + + + 0 0
then the antigen titre is the average of the antigen dilutions 640 and 1280 i.e.
(640 + 1280) /2 = 960.
17. The VAD with which the antigen shows the highest titre is the optimum one and should be used for that particular antigen.
18. Determine the dilution, which will contain 8 units per 0.025 ml by dividing the endpoint by 16. For e.g., end point 1280 = (1280/16) = 80. Therefore a 1:80 dilution of the antigen will contain 8 HA units per 0.025 ml.
19. Calculate the quantity of antigen required for the test and dilute the antigen accordingly.
20. Store antigen at 4°C.
Treatment of sera for HI test
The sera may be either extracted with acetone or treated with kaolin. These sera are then adsorbed
With goose erythrocytes to remove heamagglutinins for goose erythrocytes.
a Acetone extraction of sera
1. Label 16x120 mm tubes with specimen number of sera (this should include negative and
positive sera).
2. Add 0.9 ml normal saline to each tube.
3. Add 0.1 ml serum to each correspondingly labelled tube.
4. Keep sera in an ice bath.
5. Add 12 ml chilled acetone to each tube.
6. Stopper each tube and mix by inverting tubes several times.
7. Incubate for 5 minutes mixing occasionally.
8. Centrifuge at 3000 rpm for 5 minutes at 4°C.
9. Decant supernatant fluid.
10. Add 12 ml fresh acetone to each tube.
11. Mix thoroughly.
12. Incubate in cold for 1 hour. Mix the material occasionally.
13. Centrifuge at 4°C for 5 minutes at 2000 rpm.
14. Aspirate off the supernatant.
15. Tap each tube against palm of hand so that sediment forms a uniformly thin layer.
16. Desiccate in a desiccator attached to a vacuum pump for 1-2 hours.
17. Add 0.5 ml borate saline pH 9.0 to each tube and mix until completely resuspended (keep for minimum of 1 hour in cold).
18. Adsorb with goose cells.
a. Kaolin treatment of sera
1. Label 12x75 mm tube with specimen numbers of sera.
2. Add 0.1 ml serum to each correspondingly labelled tube.
3. Add 0.4 ml of kaolin solution (25% in borate saline, pH 9.0), to each tube and mix by shaking tube rack.
4. Incubate for 20 minutes at room temp. shaking every 5 minutes.
5. Centrifuge at 2500 rpm for 30 minutes at room temp.
6. Adsorb with goose cells.
b. Adsorption of sera with goose cells
1. Keep sera (acetone extracted or kaolin treated) an ice bath.
2. Add 0.1 ml of washed and packed goose RBC to each tube (It may be necessary to add more
cells for pig and rabbit sera)
3. Mix well and incubate for 20 minutes in an ice bath, mixing every 5 minutes.
4. Centrifuge at 2000 rpm for 15 minutes at 4°C.
5. Transfer supernatant fluid to fresh tube by decanting (transfer labels at the same time).
The sera are ready to be tested in HI.
Preliminary titration of antigen
The diluted antigens are titrated again before using them in the test.
1. Mark a "U" type microtitre plate as shown below.
Antigen Dilution Cell
Number ------------------------------------------------------------ control
1 2 3 4 5 6 7
2. Add a drop of 0.4% BABS pH 9.0 to each well from dilutions 1-7 and cell controls in rows 1-5
with a 0.05 ml dropper.
3. Keep plate in the refrigerator for chilling.
4. Keep 5 microdiluters (0.05 ml) with tips immersed in 0.9% NaCl for chilling.
5.Blot out the tip of each microdiluter. Dip the tip in each of the antigens and place in each of
the corresponding well dilution no.1.
6. Prepare serial two-fold dilutions of each antigen from well No.1 to well No…
7. Add one drop of 0.4% cells in required VAD with a 0.05 ml dropper pipette to corresponding
rows of antigens including cell control wells.
8. Shake the plate gently and incubate at room temperature till cell controls settle down
(about 30 minutes)
9. Read and record the titration. Adjust if necessary and re-titrate.
Performance of HI test
1. Select antigens to which sera are to be tested.
2. Treat all test sera, antisera homologous to antigens selected and a negative control serum, as shown in treatment of sera.
3. Mark the "U" type microtitre plates to accommodate the number of sera to be tested for each antigen. Also mark the control plate (see below)
4. With a 0.025 ml dropper add a drop of 0.4% BABS pH 9.0 to each well.
5. Dip pretested 0.025 ml micro diluters into each treated serum and place in well No.1
(1:10 dilution).
6. Prepare serial two-fold dilutions with the microdiluters for each antigen and for serum
7. With a 0.025 ml dropper add a drop of test virus antigens containing 8 HI units, to all Wells of the serum dilutions in each test plate.
8. With a 0.025 ml dropper add a drop of 0.4% BABS pH 9.0 to all wells of the serum dilution series in the controls plate. Shake plates to mix, cover and keep in the refrigerator.
9. Mark the plate for post incubation titration.
10. Add 0.05 ml of 0.4% BABS pH 9.0 to each well with dropper.
11. Add 0.05 ml of each antigen to the first well and mix.
12. Incubate all plates at 4° C overnight (about 18 hours)
13. Place a 0.05 ml microdiluter in each of the sells containing antigen diluent mixture in
The post incubating titration plate (well No.1).
14. Prepare serial two-fold dilutions through well No.7.
15. Add one drop of 0.4% goose RBC in respective VAD to each well of titration plate with 0.05ml dropper.
16. Shake and incubate at room temperature.
17. Add one drop 0.4% goose RBC (in respective VAD) to each well of test plates and control plates with a 0.05 ml dropper.
18. Shake plates and incubate at room temperature till the cells in controls settle down.
19. Read and record the cell patterns.
a. Check RBC controls. These should settle down into a button.
b. Check the titration of antigens. Each antigen should contain 4-8 HA units.
c. Check the serum controls. If no agglutination occurs in any dilution of a serum the HI results in test plates are considered valid.
d. Check the negative control serum. It should show complete agglutination in all dilutions. If not, the test is invalid.
e. Check the positive reference serum. The serum must show complete inhibition of
f. Check the test sera. The end point used to designate the titre of HI antibody
present is the highest dilution of serum that inhibits heamagglutination.
Early convalescent sera are treated with 2-ME to detect the presence of IgM antibodies. The acetone extracted sera are treated with 2-ME. Prepare 1M 2-ME in borate saline pH 9.0 : (a) 1.47 ml 2-ME +18.53ml or (b) 1.56 ml + 18.44 ml, depending upon the molecular weight of stock solution. Add one part of 2-ME (1M) to four parts of serum and incubate the mixture for one hour at 37° C and make serum dilutions. For post titration of antigen with 2-ME, 0.2M 2-ME is mixed with equal volume of antigen and incubate overnight at 4° . Antigen dilution is carried out and HA titre is determined. The 2-ME treated and untreated sera are tested in HI as per routine test.
A virus will lose its infectivity when it is mixed with specific antiserum raised against that virus. This is called neutralisation. The neutralised virus is unable to infect any cell culture or experimental animal. This property of the virus can be conveniently employed for its identification and also for detection and quantification of specific antiviral antibodies. In the virus neutralisation tests for antibody assay, a fixed amount of a specific virus is made to react with increasing dilutions of a specific antiserum under standard conditions and the degree of virus neutralisation is determined either in vivo by animal inoculation or in vitro by infectivity in cell culture. On the other hand, for identification of a particular viral isolate, a fixed quantity of virus is reacted with a standard amount of specific antiserum and also with normal serum. The identity of the virus gets established if it is neutralised by specific antiserum and remains infectious with normal serum.
In this section we will illustrate assay of anti-rabies serum by in vivo neutralisation in mice and detection and quantification of polio virus antibodies by in vitro neutralisation in cell culture.
This test is the standard test recommended for estimation of anti-rabies antibodies in human or animal sera, to know the level of protection after vaccination of for diagnosis of the disease. In this test different dilutions of the serum under test is incubated with a standard amount of a fixed rabies virus strain known as CVS (challenge the virus strain) and serum virus mixture is inoculated I/C in to weanling mice and depending on the survival rate in mice the titre can be expressed in international units (IU) in comparison to a standard anti-rabies serum whose unitage is known.
1.Sterile test tubes or penicillin vials.
2.Pipettes, 10 ml, 5 ml and 1 ml
3.Water bath
4.1 ml syringes
5. Weanling mice, 4 weeks old
6. Sterile PBS pH 7.4
7. Bovine or horse serum
8. CVS of known titre. 9.Reference ARS of known unitage.
1. Inactivate sera under test at 56 C for 30 min in water bath.
2. Prepare dilutions of test and reference sera in PBS containing 2% bovine of horse serum. Dilutions should be so adjusted as to get 50% end points preferably in the middle dilutions. To get a dilution of say 1:8 add 0.1 ml of serum to 0.7 ml of diluent and then continue with doubling dilutions i.e 16,32,64 etc.
3. Prepare a dilution of CVS containing 100 LD50/0.03 ml so that when mixed with an equal quantity of serum dilution the challenge is reduced to 50 LD50. To calculate the titre of CVS giving 100 LD 50 we should know the original titre of CVS. For E.g. if the titre is 10-7 /0.03 ml then 100 LD50 will be present in dilution 10-7 / 0.03 ml then 100 LD50 will be present in dilution 10-5.
4. Mix 0.5 ml of serum dilution with 0.5 ml of CVS dilution, 10-5 and incubate in a water
bath at 37° C for 60 min. Simultaneously prepare 3 more dilutions of CVS i.e, 10-6 to 10-8and mix 0.5 ml of this dilution with 0.5 ml of diluent. This is also incubated along with serum-virus dilutions.
5. After 60 min remove the tubes from water bath and chill the tubes by keeping in
Refrigerator at 4° C or in a ice tray.
6. Inoculate I/C 6-8 mice with each serum dilution and to determine the Exact LD50 used for challenge inoculate virus dilutions also in similar manner. Observe the mice for 14 days. Ignore all deaths before 5 days post inoculation. Note down the specific deaths occurring from 6 to 14 days.
7. The 50% end point titre of serum called effective dose 50 (ED50) or neutralising dose 50
(ND50) is calculated by Reed and Muench method. At the same time note down the mortality in mice inoculated with CVS dilutions and calculate the exact LD50 used in the test. This should be approximately 50 LD50.
8. The titre of the test serum can be expressed in IU/ml in comparison to reference serum. E.g. If reference serum has an IU of 5 IU and its ND50 is 1:128 and the test serum shows ND50 of 1:64 then the unitage of test serum is
reciprocal of
test serum ND50
_____________________________ X unitage of reference serum
reciprocal of
reference serum ND50
______ X 5 = 2.5 IU/ml
Antibodies to polio viruses develop after vaccination with polio vaccine or after infection with wild virus. Antibodies to polio and other enteroviruses may be present in normal population and hence demonstration of four fold rise in the titre of antibody is absolutely necessary for the sero- diagnosis of enteroviral infections. For the test, two serum samples, one taken just after the onset of the disease and the second, after a gap of 2 weeks or more is required.
In the test proper, increasing dilutions of patient's serum is mixed with 100 LD50. Of the 3 types of polio viruses and incubated in water bath for 2 hrs. The mixture is then inoculated to preformed monolayers of either Vero or Hep 2 cells in flat bottom 96 well tissue culture plates and incubated for 7 days. Readings are taken at 3rd and 7th day and depending on the CPE seen in different dilutions, titre of the serum sample is calculated by using Reed and Muench method.
1. 96 well flat bottom tissue culture plates
2. Micro pipettes, 200 and 1000 Ul
3. Microbiological safety cabinet (Laminar flow)
4. CO2 incubator.
5. Test tubes or penicillin vials
6. MEM with added antibiotics and containing 2% FCS
7. Water bath 56° C and 37° C
Each serum sample has to be tested separately for antibodies against 3 polio viruses. Before starting the procedure a plan should be drawn for the distribution of samples and controls in the microtitre plates.
1. Inactivate the serum samples at 56 C for min.
2. Dilute the serum samples from 1:16 to 1:2048 in test tubes or penicillin vials using MEM with 2% FCS.
3. Dilute the 3 types of polio viruses, P1, P2 and P3 in MEM to contain 100 TCID 50 per
0.1 ml using MEM with 2% FCS.
4. Mix equal serum quantities of serum dilution and virus dilution in test tubes and incubate in
water bath at 37 C for 2 hrs.
5. Simultaneously prepare three 10 fold dilutions of challenge virus dose starting from the
dilution containing 100 TCID 50. Mix equal quantities of these dilutions and MEM with 2% FCS and incubate in a similar manner.
6. At the end of incubation, dispense 100 ul of serum virus mixture and virus MEM mixture to
wells of the micro titre plate, starting from lower dilution of the serum to the higher. Include 2 wells for cell control and to this add 100 UL of MEM. Each serum dilution and virus dilution should be inoculated to at least 4 wells.
7. Incubate the plates in a CO2 incubator and take the reading at the end of 3rd and 7th day. Look
for the CPE in wells in which virus dilutions were added and calculate the actual TCID50
employed in the test. This should be around 100 TCID50 per 0.1 ml. Note down the highest serum dilution showing complete neutralisation of CPE. Calculate the 50% endpoint dilution by using Reed -Muench formula. If there is initial neutralisation at day 3 followed by breakthrough at day 7, it indicates partial neutralisation due to antigenic relation with other enteroviruses.
Preparation of solutions and reagents for ELISA
1. 0.05M carbonate buffer pH 9.6.
- Dissolve 0.53 g Na2CO3 (anhydrous) in distilled water and make final volume to 100 ml.
- Dissolve 0.42 g NaHCO3 in distilled water and make final volume to 100 ml.
To 32 ml of solution A, slowly B till pH is 9.6 (approximately 68 ml).
2. Phosphate citrate buffer, pH 5.0.
a. Dissolve 11.9 g of Na2HPO4, 2H2O in distilled water and make up to 1 litre (Solution A).
b. Dissolve 7.0 g of citric acid in distilled water and make up to 1 litre (Solution B).
Mix 55 ml of Solution A with 45 ml Solution B.
Adjust pH to 5.0 with either a or b if necessary.
3. 0.01 M Phosphate buffer saline (PBS), pH 7.2.
Dissolve 8.0 g of NaCl, 0.2g of KC1, o.2g of KH2PO4 and 1.5 g of Na2HPO4. 2H2O in distilled water and bring the final volume to 1000ml. Adjust pH to 7.2 with 0.1 N NaOH or 0.1N HC1 if necessary. Add 0.5 ml/L Tween 20 (Polyoxyethylene sorbitan monolaurate) to 1X PBS to make PBST.
1) 0.5ml of a stock of Herpes simplex virus (HSV-1) is added to a Roux bottle with a monolayer of Vero cell line after discarding the growth medium.
2) Adsorb the virus for 30 mins at 37° C and add 100 ml of maintenance medium. Incubate in a CO2 incubator at 37° C.
3) Observe for evidence of CPE (24-48 h).
4) Remove the supernatant medium and harvest the cells with 10-20 ml of sterile PBS, when >90% of the cells show CPE.
5) Centrifuge for 5 mins at 1000 rpm and wash the cells thrice with sterile PBS.
6) Resuspend the cells in a minimal amount of PBS (ex:1ml).
7) Sonicate the suspension at 60 cycles/second continuously for 5 mins and pulsed sonication for 3 mins.
8) Centrifuge the sonicate for 10 mins at 1000 rpm.
9) The supernatant is used as the antigen after estimating the protein concentration.
10) Control Vero cell antigen is prepared in a similar manner with mock infected Vero cells. (Sterile medium to be used instead of the virus suspension).
NOTE : The protein values of the HSV and Vero cell antigens should be matched before it is used in the test.
1) 1 vial of commercial Measles vaccine reconstituted in 0.5 ml of cell culture medium and
made up to 5 ml with same medium and inoculated into 1 Roux bottle with a monolayer of Vero cell line.
2) Adsorption is carried at 37° C for 1 h
3) Maintenance medium is added.
4) Cytopathic effect is first seen on third day and it gradually increases over next four days. On the seventh day extensive CPE can be seen
5) The fluid is then frozen and thawed twice and kept as seed stock.
1. Virus is grown in Roux bottle and fluid harvested aseptically.
2. Spin at 1000 rpm for 30 min.
3. To supernatant add sodium chloride (4gms/100ml) and PEG precipitate at a concentration of 10%. (10gms/100ml); (add Nacl first allow it to dissolve and then add PEG slowly)
4. Stir for half an hour in ice bath and add 0.1% sodium azide while stirring.
5. Keep for two hours or overnight in refrigerator.
6. Spin at 10000 x g for 30 min.
7. Discard supernatant.
8. Resuspend pellet in 1/100th of the original volume with normal saline.
9. Dialyse against sterile normal saline containing 1% sodium azide for 24h with three changes. (Dialysis bags are pretreated by boiling in 1% sodium carbonate solution in distilled water containing lmM EDTA).
10. Collect the dialysed antigen centrifuge and collect supernatant 5MM EDTA + 200MM NaHCO3 in 100 ml D.W. x 20 min. and estimate the protein before using it in the test.
11. Control Vero antigen is prepared in a similar manner with fluid collected from mock infected Vero cells.
1. The infected suckling mouse brain is homogenised with four volumes of chilled 8.5%
aqueous solution of sucrose in homogeniser (Waring blender) (ex. If the total weight of the mouse brain is 2g then 8ml of sucrose solution should be added)
2. The homogenate is added drop wise with brisk mechanical stirring to 20 volumes of
chilled acetone. The mixture is shaken vigorously and allowed to stand for 15 min.
The milky supernatant acetone is decanted, the sediment at this stage being a pink gummy mass which is tightly adherent to the bottom of the centrifuge bottle.
3. A volume of fresh acetone equal to that originally used is added to each bottle and the
preparation are allowed to stand in an ice bath for at least one hour to dehydrate the
sediment; after sufficient time the sediment is readily reduced to a fine suspension by use of a thick glass rod. The bottles are then centrifuged as before. The supernatant fluid is
aspirated out and the sediments from all bottles are pooled into one bottle with the help of small quantity of fresh acetone.
4. After most of the final supernatant acetone has been removed by aspiration the bottle is
closed with a rubber stopper fitted with glass tubing, which is connected to a vacuum pump for drying. The drying is continued for one hour with the bottle kept immersed in an ice bath. The pump is protected from contamination by a trap containing a large quantity of non-absorbent cotton.
5.To the dry powder a volume of 0.9% NaCl is added which is equal to 2/5th of the total
Volume of homogenate originally used (Ex. If the homogenate is 10ml, 4ml of 0.9% NaCl is added for rehydration). The bottle with antigen is left overnight in ice water in the cold room. The next morning it is centrifuged at 10000 rpm for one hour. The supernatant fluid is antigen. For gel diffusion test uncentrifuged antigen is used.
Note : Tris buffer (0.1M) pH 8 to 9 can be used rehydration of sucrose acetone antigen instead of 0.9% NaCl.
Procedure for biotinylation of monoclonal antibody
Reagents :
1) Monoclonal antibodies : Ascitic fluids from BALB/c mice inoculated with desired clones have to be precipitated using the sodium sulphate technique. After dialysis the protein concentration is measured (absorbance at 280nm) and adjusted to 1mg in 0.1M sodium bicarbonate solution (stock solution).
2) Biotin-N-hydroxy succinamide (BRL Bethesda): 1mg of Biotin-n-hydroxy succinamide is dissolved in 1ml of dimethylsulfoxide (Sigma or Merck). Prepare it fresh on the day of use. DO NOT STORE SOLUTION PREPARED EARLIER.
3) Good quality dialysis bags (25000 mol.wt cutoff)
To 1000 ug of monoclonal antibody in 1ml of sodium bicarbonate solution, add 200 ul of stock biotin solution drop-wise (1mg/ml DMSO, it can be 100, 200 & 400 accordingly check before use). After mixing well incubate at room temp. for 4 hours with periodic mixing. At the end of 4 hours dialyse the mixture against sterile normal saline (500 ml x 3 changes) for 18 hours at 4° C to remove unbound biotin. After dialysis measure the exact volume. If the volume is 1 ml then the protein concentration is 500 ug/ml. Distribute 50 ul volumes into 20 Eppendorf tubes (bullets). Add 50 ul of sterile glycerol to each bullet (cryopreservative). Each bullet now contains 50 ug of biotinylated monoclonal antibody. Store at -70° C. Estimate optimal concentration of this diluent for this antibody. DO NOT USE PLAIN PBS SINCE HIGH BACKGROUND IS OBTAINED. The conjugate used is STREPTAVIDIN PEROXIDASE (BRC Bethesda) at a dilution of 1:1000 (50 ul/well in MAC-ELISA). Incubation with biotinylated monoclonal antibody is for 2 hours at 37° C in a water bath (float the plates).
DETECTION OF IgG ANTIBODIES TO HSV-1 BY ELISA
1. Coat ELISA plates with HSV and Vero antigens at a predetermined concentration in carbonate
buffer pH 9.6, 100 ul/well.
2. Incubate overnight at 4° C.
3. Discard and wash the plates thrice with PBST.
4. Quench the plates with 1% skimmed milk powder in PBST, 150 ul/well.
5. Incubate for 2hrs at 37° C.
6. Discard, wash plates thrice with PBST.
7. Add 100 ul/well sera, CSF appropriately diluted in PBST. Include positive, negative
controls and blanks.
8. Incubate for 2 hrs at 37° C.
9. Discard, wash thrice with PBST.
10. 100 ul/well of goat/rabbit anti-human IgG peroxidase conjugate (Bangalore Genei)
(dilution to be used: 1:2000 in PBST with 1% normal goat serum)
11. Incubate for 2 hrs at 37° C.
12. Discard, wash thrice with PBST.
13. Add 100 ul/well of a solution containing OPD (4mg/10ml of PCB, pH 5) with 10 ul
of H2O2.
14. Incubate in the DARK at room temperature for 20 minutes
15. Stop the reaction with 50 ul/well of 4N H2SO4.
16. Read the plate at 492 nm on an ELISA reader.
17. A sample is considered positive if the difference in OD values is >0.1 between the
HSV antigen and Vero antigen at that particular dilution. Antigen is considered
significant at that dilution.
DETECTION OF IgG ANTIBODIES TO MEASLES VIRUS BY ELISA
1. Coat ELISA plates (100 ul /well) with measles virus antigen and Vero antigen at a
predetermined concentration in carbonate buffer pH 9.6
2. Incubate overnight at 4°C.
3. Discard contents of the wells and wash thrice with PBST.
4. Quench with 1% skimmed milk powder in PBST (150 ul/well). Incubate at 37°C for 2 hrs.
5. Discard and wash thrice with PBST.
6. Add the required dilutions of CSF/Serum samples (100 ul/well) and incubate at 37° C for 2 hrs. Include positive , negative controls and blanks.
7. Discard and wash thrice with PBST.
8. Add 100 ul/well of rabbit anti-human IgG peroxidase conjugate (Bangalore Genei) diluted 1:2000 in PBST with 1% normal goat serum and incubate for 2 hrs. at 37° C.
9. Discard and wash thrice with PBST.
10. Add 100 ul/well of a solution containing OPD (4mg/10ml of PCB,pH5) and 10 ul of H2O2. Incubate at RT for 20 mins in the dark.
11. Stop the reaction with 50 ul/well of 4N H2SO4.
12. Read the results at 492 nm on an ELISA reader.
13. A sample is considered positive if the difference in OD values is > 0.1 between the measles virus antigen and Vero antigen at any given dilution.
DETECTION OF IgM ANTIBODIES TO JEV BY AN IGM ANTIBODY CAPTURE ELISA (MACELISA)
1. Coat ELISA plates with 100 ul/well of a predetermined concentration of rabbit anti-human IgM antibodies (DAKOPATTS) in carbonate buffer pH 9.6.
2. Incubate at 4° C overnight.
3. Discard and wash with PBST thrice.
4. Quench with 1% skimmed milk powder in PBST (150 ul/well). Incubate at 37°C for 2 hrs
5. Discard and wash thrice with PBST.
6. Add 100 ul/well of CSF (diluted 1:100 in PBST) in duplicates. Incubate for 4hr at 37° C.
7. Discard and wash with PBST thrice.
8. Add 100 ul/well of JEV mouse brain antigen (50HA units/100 ul diluted in PBS). Incubate at 4° C overnight.
9. Discard and wash with PBST thrice.
10. Add 100ul/well of biotinylated NIV cross-reactive monoclonal antibody to JEV (25ul/10 ml of PBST containing 1% skimmed milk powder and 1% normal goat serum). Incubate at 37° C for 2 hr.
11. Discard and wash with PBST thrice.
12. Add 100 ul/well of streptavidin peroxidase conjugate (DAKOPATTS) diluted 1:5000 in PBST containing 1% normal growth serum. Incubate for 15 mins at RT.
13. Add 100 ul/well of a solution containing OPD (4mg/10ml of PCB,pH5) and 10 ul of
H2O2. Incubate at RT for 20 mins in the dark.
14. Stop the reaction with 50 ul/well of 4N H2SO4.
15. Read the results at 492 nm on an ELISA reader.
16. Appropriate high positive control, low positive control, negative control for CSF and serum as well as blanks should be included in every assay.
17. The results are expressed as MACELISA units which is calculated using the following
formula :
ELISA units = (OD of test sample - OD of negative control)
--------------------------------------------------------- x 100
(OD of weak positive - OD of negative control)
Interpretation of results is as follows :
a. Any test sample with > 100 units is considered positive for IgM antibodies to JEV.
b. A sample with ELISA units between 30-99 is considered probably positive for IgM antibodies to JEV but requires further testing with Dengue and West Nile virus antigens as well as normal mouse brain antigen to exclude false positive reactions.
c. A sample with ELISA units < 30 is considered negative IgM antibodies to JEV.
Western blot analysis for HTLV-1 proteins
a) Preparation of Western blot strips : Use solubilized virus obtained from MT2 or HUT102 cells which has been purified and concentrated 1000 fold by sucrose gradient centrifugation .
b) Polyacrylamide Gel Electrophoresis :
1) Layer the 10% resolving gel and incubate for 15min. at RT with distilled water layered over it.
2) After this removing the water layer 4% stacking gel on to the resolving gel and insert combs into the stacking gel. Incubate at RT for 15 min.
3) Remove the comb and flush the wells with distilled water.
4) Carefully remove the clamps and mount the gel plates on to the Electrophoresis unit and add tank buffer into both lower and upper tanks. Ensure that the upper tank has sufficient buffer to cover the wells in the gel.
5) Denature the protein sample (HTLV-1 antigen) in sample buffer by boiling for 3 min. in a water bath. Load 25 to 50 ul of this denatured sample into each of the wells carefully avoiding spills and air bubbles using a micro pipette.
6) Run the gel in the constant voltage mode at a setting of 60 volts until the dye front reaches the bottom of the gel plate.
7) Remove the gels carefully and use for transferring of proteins to nitrocellulose membranes by Western Blotting.
c) Western Blotting :
1) Transfer Electrophoresed gel to tray containing Western Blot buffer (20% methanol in 1x tris-glycine buffer which has been de-aerated for 15 min.)
2) Place a pre-wetted nitrocellulose sheet, which has been cut to the same dimensions as the gel on the gel ensuring that there are no air bubbles trapped in between.
3) Keep filter paper pads below the gel and above the nitrocellulose paper to make a sandwich.
4) Place this sandwich in the appropriate clip and insert into the Electro blotting tank containing buffer.
5) Ensure that the anode is towards the nitrocellulose membrane side while the cathode is towards the gel.
6) The electroblotting is carried out using constant current mode at 150 MA for 2 hrs.
7) Remove the nitrocellulose membrane (always handle gently using a forceps) and place in a tray containing 1% milk powder in PBS. Incubate at 4° C overnight.
8) The nitrocellulose membrane is removed from the tray and carefully cut into thin strips. Store in a zip lock bag at 4° C for upto 1 month.
9) Place thin strips in a Western Blot immuno assay tray and wash with PBS using a rocking platform.
10) Add 2.5 ml of 1:50 dilution of CSF or 1:100 dilution of serum prepared in PBS containing 1% skimmed milk powder.
11) Incubate at room temperature on the rocking platform for 2 hrs. Wash thrice with PBST on the rocking platform.
12) Add 2.5 ml of appropriate dilution of anti-human IgG peroxidase conjugate prepared in PBST containing 1% milk powder and incubate on the rocking platform at RT for 2 hrs.
13) Wash thrice with PBST on the rocking platform.
14) Add 2ml of substrate solution (Diaminobenzidine 4 mg /10 ml of PBS and 10 ul of H2O2) and rock on the platform for 1 min. and keep the tray in the dark for 10 min. at room temperature.
15) Stop the reaction by adding distilled water to each of the wells.
ELISA procedure for detection of anti HTLV-1 antibodies
1. Use cell culture purified and concentrated virus solubilized virus for ELISA. The optimal
concentration should be pre-determined in a checkerboard titration. Usually 5-10 ug/ml
of viral antigen gives best results.
2. Coat plates with 100 ul of antigen diluted in 50 mM NaHCO3 buffer, pH 9.6 and incubate
at 4° C overnight.
3. Discard contents and rinse thrice with PBS. Add 200 ul of 2.5% BSA solution in PBS
containing 1.25 % milk powder and 5% sucrose to each of the wells and incubate at 37°
C for 2 hrs. Wash plates thrice with PBST.
5. Add 100 ul of CSF/serum samples diluted five fold (1:5 to 1:625) in PBST with 2% milk powder to the wells in the ELISA plate.
6. Incubate at 37° C for 2 hrs. Wash plates thrice with PBST.
7. Add 100 ul of goat anti-human IgG peroxidase conjugate (Bangalore Genei) diluted 1:1000 in PBST containing 1% normal goat serum. Incubate for 2hrs. at RT. Wash thrice in PBST.
8. Add 100 ul of substrate (4 mg % of OPD in PCB with 0.005% H2O2) and incubate in the dark for 20 mins. at RT.
9. Stop the reaction with 50 ul of 4N H2SO4.
10. Measure the OD values at 492nm in an ELISA Reader.


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NHK,MENWAN automatic sliding door,Access control,entrance
Zhejiang Gangdu
Electronic CO.,LTD is professional manufacturer of NHK,MENWAN automatic sliding door,Access control,entrance . NHK and MENWAN automatic sliding door is a famous brand in the field: Access control,include:,micorwave sensor,card and password access plate,RFID card,card reader for ATM door access, magnetic card reader for access control, noncontact palm sensor switches, wireless hand encumbered switch, wireless receiver, remote-controller for auto-door, microwave probe, infrared sensors probe, universal remote control, actendance access machine, fingerprint access controller, auto-door switch for disabled, access for power, access dedicated backup power, electric lick catch, electric interpolaton lock, magnetic locks, exit switch(the plastic), glass electric lock clip without frame, card reading head, sensor beam, pedal sensor switch, remote controller
1.The sliding door use Brushless DC motor :60W
2. moment of force is strong and noise is low. when started.
3. with survey function of optics ruler,high efficiency close-route control and with stable revolvin g torsion and speed.
4. overload protective device prevent from being wear and obstarcle .
1. with long serve life, controller Uses the advanced CPU chip processing control system. Operating parameter be demonstrated in digital.
2. Parameter is adjusted conveniently and nimberly.
3. can link to entrance manage system
The hoisting mechanisms
1. advanced structural design , hoop always move on orbital .
2. convenient altitude control
3. The guards equipment prevented the door from derailed .
4. according to your need, we make hoop of different material .
5. with high intensity, even load, steady move , the hoisting mechanisms can bear 180 kilogram .
The timing belt
The tooth timing belt is of high Intensity and Wearability.
Rear wheel device
Easy to adjust between loose and tight, it prevents the belt vibration practically
Zhejiang Gangdu Electronic CO.,LTD
Address: No.125 JianXin Road, QuZhou city, ZheJiang province, P.R.China
Tel: 86-570-8019790
Fax: 86-570-3853106
Mob: 86- 13616707412
Sales manager: Water. Chen.(Mr.)
Company Contact:
Contact Name: Water.chen
Company Name: Zhejiang Gangdu Electronic Co., Ltd.
Tel: 86-570-8019790 or 86-570-8586578
Fax: 86-570-3853106
Street Address: No.125 Jianxin Road,
Quzhou City, Zhejiang Province,
P.R. China
Member name: Sanwoodcxh
Member Since: 30 August 2008
Total Leads:
14 Sanwoodcxh Import Export Business Leads
Business focus: Automatic Sliding Door, Coin Acceptor, Coin Validator, Access Control, Entrance, Garage Door
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Product Category:
Security or access control systems
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text:
For a brief history of Valentine paint, please see the earlier post about the Valentine ghost sign in Saintes.
The sign writer cleverly positioned the painter to the right of the window, making it look as if he were painting the only area that was not white yet.
Les belles
The painter was originally created by Charles Loupot in the 1920s. Even if his design was later modified, he remained the symbol of Valentine until the 1980s.
Underneath the ghost sign are the names of the two companies that managed successively this advertising space. The original one was the COFRAP. The second company, , which could be reached by calling TRO 15 57, was the AGPP (for Agence Générale de Presse et de Publicité). TRO stood for Trocadéro, a telephone exchange in Paris.
Location: Avenue de la République, Montlieu-la-Garde, Charente-Maritime


text:
I am seeing that forecasts continue to enhance my much-anticipated suspicion that we will see a full collapse of the polar vortex, and this could be happening in the next two to three weeks.
We'll go image by image. This first image above is the Day 10 forecast of heights and temperatures. Areas of lower pressure are marked with the letter L, and are also shown with decreasing numbers closer to the center. Warmer temperatures are shown in warmer colors. We can clearly see that the polar vortex is nowhere near organization, a result of several sudden stratospheric warmings (SSW), an event that forces warm air into the stratosphere and increases chances for cold air about 2-4 weeks later. Typically, if we were to find a sturdy polar vortex, there would be a single low pressure vortice centered over the Arctic. However, in the above forecast image, we see three separate vortices of the polar vortex, none of which are close enough to the Arctic for me to determine that they are in control of said area. What I can determine is that the polar vortex is beginning its dramatic collapse that could bring a brutally cold February IF, and ONLY IF the atmospheric pattern cooperates.
If you saw the title of this post mentioning North America and Europe, you may be intrigued. However, if you look towards Greenland and then look east, you will see several arching formations. This signifies high pressure in the area, possibly leading to a negative NAO, which would then enhance cold weather over those areas.
yesterday's article here. The third vortice over Asia will provoke storm systems to begin making a rebound in the US after a recent dry spell over much of the nation, not helped by an ongoing drought in much of the Plains and Rockies.
I'm seeing good indications that the polar vortex is on its way down, and February should end up nice and cool to round out winter. Just how cold is TBD, but things are looking up.


text:
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When Football Was Football - Everton
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When Everton became founder members of the Football League they were already 10 years old!

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Kasperek & Co. Accountants provides you the attention to your financial life that you deserve.
We have been offering for nearly 25 years a unique blend of personal service, technical proficiency and affordable rates.
Our team takes pride in giving you the assurance that only years of advanced training, technical experience, and investment in continuing professional education, computer technology, and personal relationships can provide!
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Tax Planning and Return Preparation Services
Kasperek & Co. Accountants practices the art of tax planning and return preparation to minimize your taxes.
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text: MAE KATIE HURST, 80, 2221 Sandy Lane Drive, Apopka, died Wednesday. Born in Webster, she moved to Apopka from Lockhart in 1948. She was a homemaker and a member of Lockhart Baptist Church. Survivors: daughters, Margie Louise Bonneville, Christine Riggall, Bobbie Jo Hickman, Betty J. Swearingen, all of Apopka, Dorothy Mae Beach, Fennville, Mich., Edith Lorrine Struve, Orlando; brothers, Leo Harrell, Apopka, Ralph, Daytona Beach, Vernon, Jacksonville; sisters, Verlie, Jacksonville, Evelyn Harrell, Daytona Beach, Inez Hamm, Lakeland; 23 grandchildren; 34 great-grandchildren; three great-great- grandchildren. Baldwin-Fairchild Funeral Home, Apopka.


text:
Thu., Sept. 22, 2011, 8 a.m.
Repeat offender accused of escape plot
A 15-time felon considered by law enforcement to be one of the area's most active criminals is accused of planning an escape at Geiger Corrections Center, and jail staff are investigating why he was placed at the minimum-security facility.
Jeffery Edward Krell, 44, was caught with marijuana at the Spokane County Jail last month, but he had recently been reclassified as a lower security inmate when Geiger staff discovered 15 screws missing from a broken window in his unit at Geiger in Airway Heights, which serves as an extension of the jail for minimum security inmates, said Sgt. Dave Reagan, spokesman for the Spokane County Sheriff's Office.
"I don't know why" Krell was reclassified, Reagan said, adding that staff at Geiger "are kind of scratching their heads, too."
Read the rest of my story here.
You must be logged in to post comments. Please log in here or click the comment box below for options.comments powered by Disqus


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How to Get (and keep) Customers
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text:
Of all the historical things in Boston ... they chose this giant macaroniSchedules are about to get nutty. September means school orientation for the kids, school orientation for the parents, no more summer Fridays, wardrobe changes, bus schedules, and just piles of stuff that I am going to have to deal with! Which leaves me to savor our recent family trip (and for that matter, the whole damn summer) as long as I can. Next weekend can't really be Labor Day Weekend, can it?
Vacations are all about making memories. In just four days in Boston we played in the Boston Common playground, watched 80 different penguins have lunch, explored the Museum of Science, and ate peanuts at Fenway ... and that's not counting the first leg of the trip in Canada. If I could have added another night to our stay at the Omni Parker House hotel, I would have (damn you mortgage) because it felt like home. The historic hotel (and location of the very first Boston Cream Pie) was upscale enough for my husband and me to feel like adults but kid-friendly enough to give little backpacks filled with toys upon arrival.
But trip aside, we make memories every day and often it is the little things that make us smile the most. So as the days get shorter and the weather starts to cool, here is my list of memories from the summer, 2010. And when you are finished reading mine ... make a list of your own and put it somewhere safe so you can look back and smile when there is snow on the ground!
Things that left an impression ...
Going on the Superdooperlooper with my daughter at Hershey Park.
My kids playing Mini Golf for the first time (and oh, I beat my husband's score).
My kids both losing their shit while watching Open Season because the stupid bear didn't want to go back to live with the Park Ranger.
My daughter's camp performance (even if it was to Justin Bieber's song "Baby").
My son swimming in the water -- fully submerged -- for the first time.
My nephew and my kids sitting and laughing while on a glass floor 113 stories above ground, in the CN Tower in Toronto.
My daughter jumping off a diving board for the first time.
Finding out one of my oldest friends got engaged (and knowing it means a trip to L.A. next summer).
Both my kids and my husband passed out on the bed, with a baseball game on in the background. Clearly, true exhaustion had won.
Bowling on a rainy day and getting a Turkey! So what if it was recorded as one roll for each one of the kids I was with ... it was still three strikes in a row.
Going to Yankee Stadium so my kids could eat cotton candy, chocolate chip cookies, and hot dogs. Right, and watch a ballgame.
Finding out that my sister-in-law and my cousin (who is like my little sister) will both have babies in March.
My husband and kids, in the car, singing "Dynamite" by Taio Cruz.
My brother dressing up like a full-fledged pirate for my son's birthday.
Sneaking away on a day-date with my husband to a Broadway show.
Beating my dad in a water-gun fight because my kids helped me gang up on him.
Our basement flood ... and the construction that followed (no one said they all needed to be good memories!).
So much happens in one person's life over the course of two months ... what are some of the things you will remember from this summer?


text:
These American Red, White and Blue Star Cupcake Toppers and this stunning American Flag Star, will make your family Proud.
These fully edible toppers will make your cakes look sensational.
Very patriotic party idea for Independence Day Celebrations or any American Party.
Follow these 3 easy Steps
As Easy as 1 2 3.
Also check out the American Flag Cupcake Toppers.


text:
I don't know about you, but when I got married, deciding how to break up the holidays among our many family members wasn't an easy task. You'd think Kate Middleton and Prince Will might have the same problem, given how different their families are -- how different every family on Earth is compared with the Royals, to be fair. But the amazing duo is yet again proving they have this marriage thing DOWN and that they're going to do things their way. According to reports, Prince George's first Christmas is bound to be extraordinary -- and not at all what you'd expect.
In the past, the Royals hosted pretty stiff Xmas parties complete with actual timetables and instructions handed out to guests. Yikes. As if remembering which fork to use at the royal table isn't bad enough. But Will was apparently inspired by the time he spent with the Middletons last year. They played board games, Pippa probably told an improper joke or two -- it sounds like an absolute orgy in comparison.
To celebrate their baby's first Christmas, Kate and Will are getting both the Middletons and 30 of the Queen's relatives together at the family's Sandringham House to toast the holiday with tea, scones, presents, mulled wine, and lots of games. Nope, not polo -- Pictionary! Can you imagine? How much money would you give to take a peek at the Queen's drawing of a surfboard?
Rumor has it that Prince Harry will be returning from the South Pole and has promised to bring a Wii console (no word on whether Cressida will join the family). Oh, and that Pippa is planning a Wii Olympics tournament. Has anyone reminded them that they're supposed to be royalty and are not allowed to have this much fun?!
Kate and Will have not only managed to bring their two families together -- something many of us struggle to do during the holidays -- but are also taking huge steps to make Christmas a more down-to-Earth occasion filled with laughter and fun. Is there anything these two can't do?
What do you think of Kate and Will's casual Christmas plans? Do you celebrate with both families or do you split up your holidays?
Image via WPA Pool/Getty


text:
Your bridesmaids offer nods of encouragement as you brave the bridal salon for wedding dresses and bridesmaid dresses. They share in the paper cuts that come from stuffing wedding invitation envelopes. They even endure high-heeled pain (and smeared mascara!) standing by your side as you recite those handwritten wedding vows.
This holiday season, it's time to show them some love.
You could panic and get them all a nail polish a week before the wedding, or you could take advantage of the season's best steals and snag your nearest and dearest totally unexpected—and totally adorable—tokens of appreciation they'll go nuts over and, better yet, will actually use. Here, nine items our editors ohhed and ahhed over this year.
Time to go shopping, brides-to-be!
1. For the Bridesmaid You Put on Mother-in-Law Duty: Dramatic Earrings
So glam, so elegant, so affordable—these stunners make any LBD black-tie ready.
Chandelier earrings, $17, Cost Plus World Market
2. For the Bridesmaid Who Has Been Reading Your Horoscope Since Before Your Met Your Fiancé : A Constellation Necklace
Admit it: You've got more than one initial necklace from your own bridesmaid gigs buried in your jewelry box. Break out of the alphabet with a simple gold-plated disc painted with the birth-sign constellation of each of your 'maids.
Gold-plated Taurus locket necklace, $75, Julie Nolan Jewelry available on Etsy
3. For the Bridesmaid Who Checked "Vegan" on the RSVP Card: An Organic Skin-Care Set
Weeks of pre-wedding stress and a weekend of champagne top-offs will leave your girls needing some TLC, stat. Help them give their complexion some love with this set of (all-natural and organic!) travel-size essentials, including a cleanser, moisturizer, blotting sheet, and microdermabrasion scrub mask. They'll be ready for work on Monday in no time!
One Love Organics Essentials to Go, $50, One Love Organics
4. For the Bridesmaid Who Has an Excel Spreadsheet Dedicated to Your Bachelorette Party: A Stash Box
No, not that kind of stash. It's the perfect size for her earrings, hair ties, fave lipsticks, and anything else you need organized—and customizable with each bridesmaid's name and favorite design.
Personalized Lucite box, $78, Minnie & Emma
5. For the Bridesmaid Who Always Offers to Do Your Makeup: A Wedding-Day Makeup Set
This has everything they'll need for touch ups at the reception (or any night out ever): gorge neutral lipstick, mini mascara, and a corrective pen to brighten up tired eyes after a night of dancing. They'll thank you when the photos come out!
Givenchy Red Carpet Look, $60, Sephora
6. For the Bridesmaid Who Introduced You to the Perfect Stationer: Monogrammed Stationery
She'll never be more excited to write you a thank-you note for her gift.
Monogrammed note set, $18, Fig. 2 Design Studio
7. For the Bridesmaid Who Helped You Stuff Envelopes: Ultrarich Hand Creams
Buy the set of 10, jam-packed with moisturizing shea butter, dole 'em out to your bridesmaids, and keep the rest for yourself!
L'Occitane Hand Cream Soiree, $92; Sephora
8. For the Bridesmaid Who Has Her Own Pinterest Board Dedicated to Your Wedding Dress: A Statement Necklace
Gift them a piece they can wear with their bridesmaids' dresses—and then out on Saturday night a week later.
Necklace, $98, C. Wonder
9. For the Bridesmaid Who Is Traveling All the Way Across the Country Just to Be There: Jewelry Roll
So very Liz Taylor on a yacht off the coast of Italy, no? They'll want to stash their jewelry in it year-round.
Leopard jewelry roll, $24, Pottery Barn
You Might Also Like:
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Thoughtful Bridesmaid Gifts Under $100


text:
A pastoral response to the identity confusion of young children confronted with a family secret after the death of a parent
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A pastoral response to the identity confusion of young children confronted with a family secret after the death of a parent
Mariri, L.M.T. (Lepai Meshack Trevor)
Secrecy, by its very definition, remains a difficult topic to study. The presence of secrecy in all social organizations is universal and families happen to be one of the earliest forms of social organization that individuals encounter. Family relationships are shaped, in part, by what is shared and what is held secret by family members. Family secrets may accordingly serve as a stimulus for family functioning and the experience of an individual within the family. Secret is kept as a result of intimidation or shame. Secrecy compounds the trauma by ensuring isolation. Often, the victim comes to doubt his or her own experience of reality, which is at odds with the family's version of the truth. The present study focuses on the disclosure of familial secrets and the impact thereof on adult children who are part of and the secret itself, and the dissertation will be teasing out pastoral care of such adult children. This research interrogates the haunting effects of family secrets on those who happen to be subject of the secret in question. The idea that an individual's behaviour might be traced to a secret kept by a family member in another generation is impressed upon in this study. From a methodological perspective it implies a shift in the analytic process, which does not aim to link co-researchers' indicators with his or her unconscious but to trace these indicators to a muted episode in the family's history. Co-researchers found themselves haunted by someone else's secret, by the silence erected around a deed that took place decades ago. Thus they are held, individually and respectively, within a group dynamic constituted by a specific familial topology that prevents the individual from living life as her or his own. The unspeakable secret suspended within the adult is transmitted upon disclosure, to the child (now young adult) in "undigested" form and lodges within his or her mental landscape as an unmarked tomb of in accessible knowledge. Concealing the true paternal and/or maternal identity from a child is a monumental setback in the history of childhood. There are a couple of issues that emerge in light of disclosure of such a secret and, therefore, a paroxysm of emotions that are experienced. Thus pastoral care seeks toThe study also looks at how the concealment of a family secret generates an obstacle to an individual who happens to be the secret and how this obstacle prevents him/her from becoming a holistic individual. The contention, especially among Africans, is that an unspoken family secret causes the affected individual's puzzling behaviour. Co-researchers relate their own stories that may have been made enigmatic by secrets that have been muted over decades. Following disclosure of such secrets, co-researchers are also dominated by an emotion which is inexpressible, because it is in excess of the facts as they appear. Hence the haunting influence that exists on the co-researchers of element(s) missing. In their engagements following the pastoral and therapeutic alliance established, co-researchers and the researcher endeavour to reconstruct a hidden story. And, by reinterpreting the co-researcher's story and together with the research, they journey pastorally and therapeutically to find a way to get beyond the enigma-laden silence that had dominated until disclosure. The main focus of this research would be to pastorally empower the young adults to deal with the issues that come to the fore in light disclosing a secret that had been muted for decades. The key question that the thesis deals with is: What would it take to remove the obstacle to a co-researcher's being and to reinstate the possibility of his/her existence? bring about healing for the affected
Thesis (PhD)--University of Pretoria, 2014.
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text:
99 Any Street • New York • NY 99999 • Home: 111-111-1111 • Cell: 111-111-1111 • firstname.lastname@example.com
Belinda Reyes
October 2008 – Present
AAA Company New York, NY
- Skilled with the basics to business math and record keeping
- Experienced with vocabulary skills, writing skills, as well as the use of computers and various computer programs: 10-Key, Word, Excel, PowerPoint, Outlook
- Filed, typed, and performed basic office duties
- Performed skills Mistake #1 click herewhich inquired using a calculator, typewriter, and telephone
- Proficient with PC's, fax and copy machines, and printers
- Used postal services to mail letters and parcels
- Inserted, edited, and formatted tables in a document
- Demonstrated the ability to complete forms and prepare envelopes and labels
- Type 50+ WPM
February 2007 – October 2008
BBB Inc New York, NY
- Successfully Mistake #2 click herecompleted a 22-week comprehensive building maintenance training program
- Learned the proper and safe use of a variety of tools including hand tools pneumatic tools, power saws, etc.
- Learned and performed carpentry work such as building fences (privacy and cyclone), wood decks, removing and building walls, building work benches, basic plumbing, electrical, interior and exterior painting and residential roofing
- Responsible for transporting employees and equipment to and from job sites in company vehicle
- Safely towed trailers and equipment behind truck
- Responsible for addressing the maintenance needs of the agency facilities
November 2006 – February 2007
CCC Corporation New York, NY
- Performed overall campus maintenance, under supervision and non supervision
- Roughed in campus office building
- Renovated Mistake #3 click hereschool restrooms
- Repair all types of fixtures and water lines
New York County High School
June 1998 High School New York, NY
Summary Of Qualifications
- Experience with financial administration of retail sales and service operations.
- Management of high volume retail operations. Includes supervision of merchandising, asset management, customer service and maintenance. Effective creative marketing programs and maintain compliance with corporate procedure.
- Skill in direction and development of individual and team personnel. Responsible for training, schedule and motivation of staff personnel.


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Orderlinessprevious next
Also: Organization
Orderliness is having a sense of where things belong and how they relate to each other, and keeping them organized accordingly. When there is a place for everything and everything in its place, then we are able to think and work efficiently. We are also more able to work collaboratively –to share tools and space with others.
To be orderly, we also must have a sense of priorities, of what is most important and what we must let go. We can only put our lives in order if our priorities are clear.
In this sense, orderliness is not so much an external habit as a quality of our mental life. It means that we seek to understand how things relate to each other--their similarities and differences, the cause and effect relationships that connect them, and how these relate to our own values and aspirations.
- What does orderliness look like in a person? (0 responses)
- What does orderliness look like in a society? (0 responses)


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At least 90% of women will suffer from cellulite at one point of their lives or the other. Cellulite is that appearance on the skin that looks more or less like the orange peel. It is actually a dimple appearance on the skin. It is commonly found on women's thighs and is caused by the uneven deposit of fat under the skin's top layer. It is so unfortunate that there are no quick methods of removing the cellulite. But lucky for you since we have come up with some steps that could help you to grammatically decrease the appearance of the cellulite.
What You'll Need…
Anti-cellulite cream
Body brush
You should eat more healthy meals. As cellulite is a result of fat deposits right under the skin, the easiest way of removing this fat would be to adjust your meals and consequently reduce weight. Keep junk foods far away from you. They are full of saturated fats which are harmful to your body as they tend to clog up the body. Eat vegetables and fruits in plenty, drink water in plenty and only eat lean meat.
Do regular exercise
By doing exercise you will be reducing overall fat levels in the body. This helps in the removal of fat deposits which cause cellulite. Try getting 30 minutes of cardiovascular exercises 3 times a week while combining this with regular squats so as to really workout the top of your thighs. You can stand with the feet in shoulder-width. Then bend the knees while pushing your bottom in an outward manner. For two seconds, keep your knees bent. After 2 seconds slowly straighten the legs again while you clench your bottom when getting back up. Do 15 repetitions per set for three sets.
Brush your body
By regularly brushing your body you will be assisting the body in boosting the circulation, breaking down deposits of fat and helps in eliminating body toxins. Before heading on to have a shower in the morning, use a body brush to dry brush the body. Use sweeping circular motions which are in the heart's direction. If you are able to do this each and every day for a few weeks you will realize that your cellulite becomes less noticeable.
Apply anti-cellulite cream each and every day after you shower
Find creams containing Ginko Biloba extract, Retinol and caffeine extract. Using circular sweep motions, really massage the body while using the cream.


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Landing approximately 2 inches above your ankle, this is the mid-point between our low and crew heights.
A good choice for a close fit, these lightweight socks offer a breathable, less-bulky barrier between your foot and the shoe.
Twice as strong as regular cotton, Supima's longer fiber resists pulling, breaking and tearing for superior comfort and resiliency.
Additional Information
Throw up your U! The company that changed the game forever is doing it again with Stance's Utah Anklet from the College Sports Collection, the official socks of school spirit. For the best teams in the league (and their loyal fans) we've created the best looking and best feeling socks in the game. Built with premium materials, active arch support, reinforced heels and toes for added durability, and collegiate colors, these socks are your admission ticket to game day style. Whether you're on the field, part of the courtside team, stomping the hell out of the bleachers or keeping the spirit alive outside of the stadium, we made serious socks for those who take Saturdays seriously. Go Utes!
- Reinforced Heel & Toe
- Seamless Toe Closure
- Machine Wash in 40 °C / 104 °F.
- Avoid Bleaching or Ironing the socks.
- Socks will last longer if you keep them out of the dryer.