text:
|By PR Newswire
Article Rating:
|November 23, 2012 07:55 AM EST
LONDON, November 23, 2012 /PRNewswire/ --
RippleRock, a leader in Agile Project Management training and consulting, in partnership with agil8, experts in Agile, Lean and Scrum, are pleased to have conducted the first public Kanban course, in London, delivered by a UK-based, Lean-Kanban University accredited organisation (RippleRock). Students from the UK and Europe attending the 2-day "Real Kanban" course learnt the core Kanban principles, shared insights around Kanban's relevance and value to their businesses and discussed the practical application to their projects. The highly interactive course incorporated the Get Kanban game, which provided a real-world experience of Kanban in action. Many of the participants were looking to use Kanban in addition to other Agile methods and were able to see how to blend Kanban into their existing ways of working.
Aleem Cummins, from Tesco, gave his views on the course: "I didn't think that strong metrics and Agile ever met until I was blown away by this game changing Kanban training. I can now focus on getting things done in a repeatable, sustainable and quality focused fashion."
"The course was challenging and well received," explained Dan Brown, RippleRock's accredited Kanban Trainer. "It allowed participants to learn how to go back and make a real difference in their work. I'm looking forward to hearing how they apply the practical leanings in their workplace. "
"I was very pleased with the course. We had an interesting mix of delegates, some with many years experience of Agile, all with an eager appetite to learn about Kanban and how they might benefit from its use," said David Hicks, agil8 CEO and accredited Kanban trainer. "The engagement and feedback we got from the participants have proven to us that our Real Kanban class provides exactly the kind of learning that people want on the topic."
David Anderson, CEO, Lean-Kanban University, also provided his approval: "RippleRock is bringing the next level of process improvement with Lean-Kanban University Accredited Kanban Training. They are showing the way for organisations to improve their quality, collaboration, and speed of delivery, and they are doing it in a gradual, evolutionary way. They can improve predictability and reduce risk -- and do it without a disruptive and painful change initiative."
About RippleRock
RippleRock helps companies to adopt both the principles and the practices of enterprise-wide Agile. We combine whichever tools, in the growing Agile toolkit, will enable clients to deliver more value to their users.
Kanban is a valuable way to increase the flow of value and we encourage all our clients adopting Agile to have a working knowledge of Kanban. As well as explore how it can help improve their end-to-end value stream - from ideas to continuous delivery.
About agil8
Agil8 helps clients with the most challenging and complex of Agile implementations to deliver real value, in the real world. Headed-up by Agile Alliance Founder, David Hicks, agil8 provides fully Accredited Training and Consulting services in Agile including Scrum, Kanban and AgilePM/DSDM.
Agil8 sees Kanban as a genuinely unique approach which can be used in any environment, either as an excellent complement to other Agile practices and methods, or on its own where other Agile approaches may be less appropriate.
About LKU Accredited Kanban Training
The Lean-Kanban University Accredited Kanban Training Program exists to provide confidence to consumers about the quality of the Kanban training that they will receive. The program establishes the quality of Kanban training based on the status of 1) the organization, 2) the individual trainer, and 3) the training curriculum. Member organizations, their trainers and their training curriculums must meet the established criteria in all three areas in order to call their Kanban training "accredited" and issue certificates under the terms of the program.
The Kanban Method pioneered by David. J. Anderson is at the core of the Lean-Kanban University Accredited Kanban Training curriculum. This curriculum lays out basic principles and practices of effective implementation, including limiting the amount of work in progress and visualizing the work. The Kanban Method takes an organization's current processes and provides greater visibility into the dynamics of the workflow and state of the work-in-progress. With this new clarity, organizations can start on a path of continual improvement.
For further information:
RippleRock Ltd
Contact: Bazil Arden, [email protected]
Telephone: +44(0)7775-913-932
Agil8 Ltd
Contact: Donna Hicks, [email protected]
Telephone: +44(0)207-681-6078
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Get rid of long grass for Pokemon SapphireHome / GameBoy Advance /
This page contains Pokemon Sapphire Hints for GameBoy Advance called "Get rid of long grass" and has been posted or updated on Mar 17, 2008 by MePokemonMaster.
- Developer: Game Freak
- Publisher: Nintendo
- Genre: Role-Playing Console-style RPG
- Release: Mar 19, 2003
- ESRB: Everyone
Get rid of long grass
You need the HM cut for this. This is a trick of how to get rid of all that annoying long grass that pokemon jump out at you in(also works for the grass you cant ride your bike in.). All you have to do is stand in the grass area you want gone. And then and then use the move cut and the surrounding grass is gone.cool ay?:P
Please submit a problem for any incomplete, non-working or fake cheat listed above.
Stuck in this game? Set a specific, clearly defined question and let other gamers answer your question or view answers to previously asked questions. If you think you are an expert then please try to help others with their questions.
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Nov 14, 1995
|Filing date
|Sep 22, 1993
|Priority date
|Sep 22, 1993
|Fee status
|Also published as
|Publication number
|Mark L. White, Stephen F. Carroll, Jeremy K. Ma
|Original Assignee
|Xoma Corporation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (2), Non-Patent Citations (34), Referenced by (27), Classifications (15), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to methods for determination of the presence of Bactericidal/permeability-increasing protein in a body fluid sample including a blood sample. Bactericidal/permeability-increasing protein (BPI) is a cationic anti-microbial protein which has been purified from the azurophilic granules of human and animal neutrophils (Weiss et al., J. Biol. Chem., 253:2664 (1978), Elsbach et al., J. Biol. Chem., 254:11000 (1979). BPI binds to the lipopolysaccharide (LPS) component of the outer membranes of gram-negative bacteria (Gazzano-Santoro et al., Infect. Immun., 60:4754 (1992)). Recently, a recombinant form of human BPI (rBPI23) has been characterized and compared to that of native BPI. The rBPI23 fragment consists of the amino-terminal 23 kDa portion of holo-BPI and retains the LPS binding properties, as well as the anti-microbial activity, of native BPI (Gazzano-Santoro et al., J. Clin. Invest., 90:1122 (1992), Weiss, et al., J. Clin. Invest., 90:1122 (1992)).
BPI levels have not previously been accurately assayed in any body fluids. Because of the potential therapeutic use of rBPI23 and other BPI proteins and protein products, a sensitive and reproducible assay is needed to measure the presence and amount of BPI in body fluids. In particular, measurements of BPI in body fluids may be useful for diagnostic purposes. Pereira et al, J. Immunol. Methods, 117:115 (1989) discloses a competitive ELISA assay for the determination of BPI in crude granule extracts of human neutrophils. Pereira et al. also disclose that non-specific interactions of cationic proteins in an ELISA assay can be minimized by treatment with polyanions such as heparin or dextran sulfate. See also Pesce et al., J. Immunol. Methods, 87:21 (1986). However, the competitive assay of Pereira et al. is characterized by limited sensitivity. Accordingly, them remains a desire in the art for a more sensitive BPI assay capable of measuring endogenous BPI levels in mammalian body fluids. Also of interest to the present application is the disclosure of von der Mohlen et al., Abstract, 13th International Symposium on Intensive Care and Emergency Medicine, Brussels (March 1993) disclosing the results of assays for serum levels of BPI in patients with gram-negative sepsis and healthy subjects. The abstract disclosed that no BPI was detectable under the conditions of the assay in the serum of healthy subjects while circulating BPI was detected in all septic patients.
The present invention provides methods for quantifying BPI levels in body fluid samples including blood samples according to the method of conducting a BPI immunoassay on a blood sample wherein the blood sample is plasma. Plasma is the blood fluid which remains after the white and red cells are separated frown fresh uncoagulated blood. Serum is the blood fluid which remains when coagulated blood is separated by centrifugation (i.e., plasma without the blood clotting factors. ) As one aspect of the invention it is taught that levels of BPI present in serum are not representative of endogenous levels of BPI in circulating blood while levels of BPI in plasma are. As a further aspect of the invention methods are provided for determining the presence of gram negative sepsis in a subject comprising determining the concentration of endogenous extracellular BPI in a plasma sample obtained from that subject and comparing that concentration with a standard value indicative of gram negative sepsis. Preferred methods according of the invention determine the concentration of extracellular BPI in body fluids such as blood using a sandwich immunoassay and further utilize a cationic nonspecific blocking agent selected from the group consisting of heparin and dextran sulfate in BPI immunoassays. The BPI immunoassays of the invention may also be used to determine the concentration in other body fluids including, but not limited to, serum, urine, lung lavages, vitreous fluid and crevicular fluid.
FIG. 1a depicts the effects of heparin, 8 kDa dextran sulfate, 500 kDa dextran sulfate, 1M NaCl and buffer control on the detection of rBPI in BPI sandwich ELISA assays;
FIG. 1b depicts the effects of heparin, 8 kDa dextran sulfate, 500 kDa dextran sulfate, 1M NaCl and buffer control on the detection of rBPI23 in BPI sandwich ELISA assays;
FIG. 2a depicts the reproducibility of rBPI standard curve for three separate assays in BPI sandwich ELISA assays;
FIG. 2b depicts the reproducibility of rBPI23 standard curve for four separate assays in BPI sandwich ELISA assays;
FIG. 3 depicts the dose-response curves for rLBP, rBPI and rBPI23 in BPI sandwich ELISA assays;
FIG. 4 depicts endogenous BPI levels in matched plasma and serum samples for 20 different healthy human donors;
FIG. 5 depicts endogenous BPI levels in matched plasma and serum samples for 20 different human donors suffering frown sepsis;
FIG. 6 depicts the effect of processing time and centrifugal force of 1300 g or 400 g on endogenous BPI levels measured by BPI sandwich ELISA in human plasma;
FIG. 7 depicts Western Blot immunoreactivity of material captured by affinity-purified anti-BPI23 antibody coated microtiter wells;
FIG. 8 depicts the relationship of Western blot integrated peak area for two BPI bands (Mr 62 and 64 kDa) with BPI sandwich ELISA immunoreactivity for 20 human serum samples;
FIG. 9a depicts a scattergram of BPI levels in the serum of healthy and septic human donors; and
FIG. 9b depicts a scattergram of BPI levels in the plasma of healthy and septic human donors.
The present invention relates to methods for quantifying the presence of extracellular BPI in body fluids including blood comprising conducting a BPI immunoassay on plasma obtained frown said subject. While the assay is useful for determining the presence and quantity of therapeutically administered BPI such an assay is particularly suitable for quantifying the presence of endogenous extracellular BPI in circulating blood as an indication of the presence of sepsis including gram-negative sepsis in a subject. Moreover, quantifying the presence of endogenous extracellular BPI in circulating blood is further contemplated to be useful in prognostic methods for evaluating sepsis patients.
The present invention provides a sandwich ELISA assay for human BPI which exhibits high assay sensitivity, high specificity, and excellent reproducibility. As used herein "BPI" quantitated according to assay methods includes native BPI, recombinant BPI, as well as a recombinant N-terminal fragment of BPI (rBPI23) and other BPI proteins and protein products. Such BPI protein products may be readily quantified in the subnanogram per mL range. The immunological assays are preferably carried out by enzyme linked immunosorbant (ELISA) sandwich assays but competitive assays and immunological assays utilizing other labelling formats may also be used. Preferred assays of the invention utilize anti-BPI antibodies, including monoclonal antibodies and affinity-purified rabbit polyclonal antibodies. Rabbit polyclonal anti-BPI antibodies may be prepared according to conventional methods using BPI as an immunogen. A particularly preferred monoclonal antibody is designated Xoma 6C2 which was selected on the basis of its ability to bind BPI in solution according to conventional methodologies. According to one preferred embodiment of the invention heparin is utilized in dilution buffers. Heparin appears to improve assay performance by both reducing backgrounds and by enhancing assay signals. Similar effects are noted with low molecular weight (8 kDa) dextran sulfate. In contrast, high molecular weight dextran sulfate (500 kDa) reduces assay sensitivity, and actually reverses the beneficial effects of the low molecular weight polyanions. Initial studies revealed non-specific interactions of rBPI23 with the microtiter plate which resulted in high background signals. Administration of heparin at 10 units/mL (approx. 55μg/mL) reduced background signals and also improved assay sensitivity compared to buffer controls. Higher concentrations of heparin (100 units/mL) produced results similar to those observed with 10 units/mL. The inhibition caused by high molecular weight dextran sulfate may result from sterically hindering access of the antibodies to epitopes on the surface of BPI.
The addition of high salt concentrations (1M NaCl) is useful in the immunoassays of the invention for reducing background signals. The use of high salt concentrations produces greater assay sensitivities than heparin, but salt is not as effective as heparin in reducing background signals for some samples. It is believed that the enhanced sensitivity noted when samples are diluted in solutions containing heparin or high salt is due to the disruption of ionic interactions, whereas other possibly hydrophobic forces may also contribute to background signals.
The specificity of the BPI ELISA has been demonstrated in two ways. First, when immunoreactive proteins were "captured" from serum onto ELISA plates and subsequently eluted, electrophoretically separated, blotted and probed with anti-rBPI23 antibodies, the only material detected was a doublet at ca. 60 kDa which comigrated with native BPI extracted from human neutrophils. Similarly, when identical blotted samples were probed with anti-rBPI antibodies, a significant relationship (R2 =0.807, p=0.0001) was found between the ELISA signal measured previously and the intensity of the BPI bands. Second, human LBP, which also binds to LPS and shows considerable sequence homology (44%) with BPI, generated a signal 30,000-fold to 100,000-fold lower than those generated by rBPI or rBPI23, respectively. Even at 100 μg/ml, rLBP generated signals equivalent to less than 3 ng/ml of BPI in the ELISA. Since LBP levels in normal human serum samples have been reported (Leturcq et al, J. Cell. Biochem., 16C:161 (1992)) to be between 1 and 24 μg/ml (mean 7 μg/ml), these data indicate that LBP causes minimal interference in the BPI ELISA.
As one aspect of the invention it has been found that endogenous BPI levels differ significantly depending upon whether they are assayed in human plasma or serum. Whereas normal plasma contains only low levels of BPI (<0.2 to 2.1 ng/ml), the levels in serum samples collected at the same time from the same individuals were on average 37-fold higher (4.9 to 72.1 ng/ml). Moreover, BPI levels varied depending upon the elapsed time for collection and processing. These data are important in the evaluation and interpretation of BPI levels in normal and pathologic individuals, since they suggest (i) that the plasma levels of BPI in normal individuals is quite low, and (ii) that BPI may be released from neutrophils into serum during the process of coagulation. Thus, the endogenous levels of BPI should be measured in plasma, and not in serum, thereby avoiding artifacts caused by release and/or neutrophil destruction in vitro. Similarly, the analysis of clinical samples containing recombinant forms of BPI are best performed in plasma.
Weiss and Olsson, Blood 69:652 (1987) have reported that neutrophils contain an average of 65 μg BPI per 108 cells. Assuming that whole blood contains 5�106 neutrophils per ml, there would be approximately 3.2μg/ml of BPI in 1 mL of blood. Since the concentration of BPI in serum is low (<100 ng/mL), the amount of BPI that is released during coagulation is only a small percentage (ca. 1%) of the total available material. This release of BPI may be of no physiological significance, and merely represent leakage of BPI from damaged neutrophils. Alternatively, the possibility exists that in vivo coagulation may be a general signal for the localized release of anti-microbial agents (including BPI) from neutrophils in response to injury or trauma. Under these conditions, BPI may then act as an anti-bacterial defense mechanism localized at the site of injury.
According to this example affinity purified rabbit anti-rBPI23 antibody was prepared. Specifically, rBPI23 (20 mg) produced according to the methods of Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992) was coupled to 10 mL of cyanogen bromide-activated Sepharose 4B (Sigma Chemical Co., St. Louis, Mo.) in 0.2M bicarbonate, pH 8.6, containing 0.5 NaCl. Approximately 97% of the rBPI23 was coupled to the resin. Pooled antisera (150 mL) from two rabbits hyper-immunized with rBPI23 were diluted with an equal volume of phosphate buffered saline, pH 7.2 (PBS). A portion (50 mL) of the diluted antisera was passed through the 10 mL rBPI23 -Sepharose column; the column was then washed with PBS and bound antibodies were eluted with 0.1M glycine, pH 2.5. Collected fractions were immediately neutralized with 1M phosphate buffer, pH 8.0. Peak fractions were identified by measuring absorbance at 280 nm according to the method of Harlow et al., Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, New York, p. 312 (1988). Recovery was 45 mg of affinity purified anti-rBPI23 antibody, or 300 micrograms of antibody per milliliter of rabbit antisera.
In this example twenty milligrams of affinity purified rabbit anti-BPI23 antibody produced according to the method of Example 1 was incubated with 2 mg of biotinamidocaproate N-hydroxysuccinimide ester (Sigma Chemical Co., St. Louis, Mo.) in 11 mL of 0.1M sodium bicarbonate pH 8.3 for two hours at room temperature. Unconjugated biotin was removed and the alkaline buffer exchanged by fractionating the reaction mixture on a PD-10 column (Pharmacia Biotech Inc., Piscataway, NJ) equilibrated with PBS containing 0.1% sodium azide. The final yield of biotin-labeled antibody was 17.9 mg.
Fifty microliters of affinity purified rabbit anti-BPI23 antibody (1 μg/mL in PBS) were incubated overnight at 2�-8� C. (or alternatively, 1 hour at 37� C.) in the wells of Immulon 2 (Dynatech Laboratories Inc., Chantilly, Va.) microtiter plates. The antibody solution was removed and 200 μL of 1% nonfat milk in PBS (blocking agent) was added to all wells. After blocking the plates for 1 hour at room temperature, the wells were washed 3 times with 300 μL of wash buffer (PBS 0.05% Tween-20).
Blood from individual human donors was collected into two Vacutainer (Becton Dickinson, Rutherford, NJ) tubes; one containing acid citrate dextrose and the second containing a clot activator and serum separator. Within 30 minutes and one hour of collection, both plasma and serum samples from an individual donor were processed simultaneously by centrifugation for 5 minutes at 1300 g. The appropriate fractions were collected and stored at -70� C. in 0.5 mL aliquots. Pooled normal human serum and pooled citrated plasma were obtained from Sigma Chemical Co. (St. Louis, Mo.).
Standards, samples and controls were diluted in triplicate with PBS containing 1% bovine serum albumin, 0.05% Tween 20 (PBS-BSA/Tween) and 10 units/mL of sodium heparin (Sigma Chemical Co., St. Louis, Mo.) in separate 96-well plates. rBPI or rBPI23 standard solutions were prepared as serial two-fold dilutions frown 100 to 0.012 ng/mL. Each replicate and dilution of the standards, samples and controls (50 μL) was transferred to the blocked microtiter plates and incubated for 1 hour at 37� C. After the primary incubation, the wells were washed 3 times with wash buffer. Biotin-labeled rabbit anti-BPI23 antibody was diluted 1/4000in PBS-BSA/Tween and 50 μL was added to all wells. The plates were then incubated for 1 hour at 37� C. Subsequently, all wells were washed 3 times with wash buffer. Alkaline phosphatase-labeled streptavidin (Zymed Laboratories Inc., San Francisco, Calif.) was diluted 1/2000 in PBS-BSA/Tween and 50 μL was added to all wells. After incubation for 15 minutes at 37� C., all wells were washed 3 times with wash buffer and 3 times with deionized water and the substrate p-nitrophenylphosphate (1 mg/mL in 10% diethanolamine buffer) was added in a volume of 50 μL to all wells. Color development was allowed to proceed for 1 hour at room temperature, after which 50 μL of 1 N NaOH was added to stop the reaction. The absorbance at 405 nm was determined for all wells using a Vmax Plate Reader (Molecular Devices Corp., Menlo Park, CA).
The mean absorbance at 405 nm (A405) for all samples and standards (in triplicate) were corrected for background by subtracting the mean A405 of wells receiving only sample dilution buffer (no BPI) in the primary incubation step. A standard curve was then plotted as A405 versus ng/mL of rBPI or rBPI23. The linear range was selected, a linear regression analysis was performed and concentrations were determined for samples and controls by interpolation from the standard curve.
EFFECTS OF HEPARIN, DEXTRAN SULFATE AND NaCl ON SENSITIVITY
In this example, a comparison of the effects of heparin, dextran sulfate (Mr 8 kDa and 500 kDa) or 1M sodium chloride BPI sandwich on assay sensitivity was conducted. The results shown in FIG. 1 indicate that for both rBPI (FIG. 1a) and rBPI23 (FIG. 1b), heparin and low molecular weight dextran sulfate (Mr 8 kDa) were equally effective on a mass basis in reducing backgrounds and increasing assay sensitivity. In contrast, high molecular weight dextran sulfate (Mr 500 kDa) reduced assay sensitivity compared to the buffer control. Although high salt caused the greatest improvement in assay sensitivity, background signals were not reduced for rBPI23 to the same degree as with heparin. Heparin at 10 units/mL was thus utilized for all subsequent assays.
In this example, rBPI and rBPI23 standard curves were found to be reproducible among several separate assays as shown in FIGS. 2a and 2b. Linear regression analysis of concentration with observed A405 demonstrated a linear concentration response for both rBPI and rBPI23 (R2 =0.997 and 0.999 respectively). The linear range was 100 to 6000 pg/mL for the rBPI standard curve and 25 to 800 pg/mL for the rBPI23 standard curve.
MEASUREMENT OF rBPI AND rBPI23 SPIKED INTO POOLED HUMAN PLASMA
In this example, pooled human plasma was spiked with different concentrations of rBPI or rBPI23 and then frozen and thawed prior to measurement in the sandwich ELISA. Recovery of spiked BPI was defined as the amount of BPI measured in spiked human plasma samples minus the concentration in the unspiked control, divided by the actual amount spiked in the sample. The fraction recovered was multiplied by 100 and the results were expressed as a percentage of the input concentration. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/mL to 97% at 3 ng/mL. Recovery of rBPI23 averaged 56% and ranged from 30% at 0.5 ng/mL to 90% at 50,000 ng/mL. Tables I and II summarize the recovery data for each BPI spiked plasma sample.
TABLE I______________________________________Recovery of rBPI23 Spiked into Pooled Citrated Human PlasmaAmountSpiked Amount Measured Amount Recovered Percent(ng/mL) (ng/mL) (ng/mL) Recovery______________________________________ 0 0.23 -- --0.5 0.38 0.15 30% 5 2.3 2.1 42% 50 29 29 58% 500 230 230 46% 5,000 3,500 3,500 70%50,000 45,000 45,000 90% Mean Recovery - 56%______________________________________
TABLE II______________________________________Recovery of rBPI Spiked into Pooled Citrated Human PlasmaAmountSpiked Amount Measured Amount Recovered Percentng/mL) (ng/mL) (ng/mL) Recovery______________________________________ 0 0.2 -- -- 3 3.1 2.9 97% 30 25.4 25.2 84%300 195 195 65%3,000 2,540 2,540 85% Mean Recovery - 83%______________________________________
COMPARATIVE IMMUNOREACTIVITY OF rLBP, rBPI AND rBPI23
In this example, the immunoreactivity of rLBP, rBPI and rLBP23 were compared in the BPI sandwich ELISA to determine possible immunologic cross-reactivity. Despite considerable sequence homology between LBP and BPI (see Schumann et al., Science, 249:1429 (1990), the results illustrated in FIG. 3 show that, on a mass basis, rLBP produced a signal which was approximately 5 orders of magnitude lower than that of rBPI23 and 4 orders of magnitude lower than that of rBPI. For example, a concentration of 100,000 ng/mL (100 μg/mL) of rLBP generated a signal which was equal to that produced by 3 ng/mL of rBPI or 0.6 ng/mL of rBPI23. At rLBP concentrations below 3,125 ng/mL, no quantifiable signal was detected in the BPI sandwich ELISA. These results demonstrate minimal cross-reactivity of the antibody with LBP confirming the specificity of the assay for BPI.
In this example, the effects of processing time and centrifugal force on the measurement of BPI in citrated plasma were also investigated. In general, increasing the processing time for citrated plasma increased the amount of endogenous BPI measured by the BPI sandwich ELISA when centrifugation was performed at either 400 g (cross-hatched bars) or 1300 g (solid bars) (FIG. 6). The lowest endogenous BPI levels were measured in plasma samples processed within 30 minutes of collection and at a centrifugal force of approximately 1300 g.
In this example, SDS-PAGE and Western Blot analyses were conducted on serum and plasma samples. Specifically, serum and plasma samples were diluted with an equal volume of PBS-BSA/Tween containing 10 units/mL of sodium heparin. The diluted serum and plasma samples were added in a volume of 50 μL to 6 replicate wells of a microtiter plate which had been coated with affinity purified rabbit anti-rBPI23 antibody and blocked with non-fat milk in the same manner as for the BPI sandwich ELISA described above. After a 1 hour incubation at 37� C., wells were washed 9 times with wash buffer. Captured immunoreactive material was solubilized by the sequential incubation and transfer (3 minutes per well while shaking at room temperature) of 60 μL of SDS-PAGE sample buffer (0.125M Tris-HCl, pH 6.8 containing 4% SDS, 10% glycerol, 0.004% bromphenol blue and 0.02% NAN3) in the six replicate wells for each sample. Final volume of the captured and solubilized immunoreactive material for each sample was approximately 50 μL.
Fifteen microliters of each solubilized sample were run in non-reducing 10% gels under the conditions of Laemmli, Nature, 227:680 (1970). Proteins were transferred to nitrocellulose by standard techniques (Towbin, et al., 1979). Blotted proteins were probed for immunoreactivity with biotin-labeled rabbit anti-BPI23 antibody (diluted 1/2000 in 0.025M Tris-HCl, pH 7.2, containing 0.2M NaCl and 0.3% Tween 20 (TBST)) or rabbit anti-rBPI antisera diluted 1/1000 in TBST. The biotin labeled rabbit anti-BPI23 antibody was followed by alkaline phosphatase-conjugated streptavidin (Zymed Laboratories Inc., San Francisco, Calif.) diluted 1/4000 in TBST. For the unlabeled anti-rBPI antisera, an incubation with biotin labeled goat anti-rabbit IgG (Zymed Laboratories Inc., San Francisco, Calif.) diluted 1/2000 with TBST preceded the incubation with alkaline phosphatase-conjugated streptavidin. After each incubation, the blots were washed four times with TBST. Blots were immersed in a 50 μg/mL solution of the substrate 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, Mo.) in 0.12M veronalacetate buffer, pH 9.8 containing 0.01% (w/v) nitro blue tetrazolium and 4 mM MgCl2. Color development was allowed to proceed for 1 hour at room temperature. Lanes of the Western blot were scanned with a densitometer (Shimadzu Model CS9000U, Shimadzu Corp., Kyoto, Japan) in reflectance mode and quantified by area integration.
Western blot analysis was performed on plasma and serum samples utilizing two different antibody probes to ascertain whether the ELISA immunoreactivity was due to the presence of holo-BPI, a fragment of BPI, or unrelated immunoreactive material. For these experiments, microtiter plates coated with the affinity-purified anti-BPI23 antibody were used to capture immunoreactive material directly from plasma and serum. The affinity captured material was then eluted in sample treatment buffer, electrophoretically separated under non-reducing conditions, blotted onto nitrocellulose and then probed with anti-BPI antibodies. Referring to FIG. 7, numbers on the top of each blot identify the lane while the numbers on the right side indicate the molecular weight. Blot A. lane 1 Blank, lane 2 rLBP (100 μg/mL), lane 3 rBPI (100 ng/mL), lanes 4-14 human serum samples, lane 13 native BPI. Blot B. lanes 1-3 same as blot A, lanes 4-14 human serum samples, lane 15 native BPI. (Weakly visible bands were detected at 62 and 64 kDa on the nitrocellulose blot for lanes 7, 10 and 12 on blot A and lanes 4, 5, 6, and 14 on blot B, but the bands did not reproduce well in the figure. Bands were also detected at 69.5 and 114.4 kDa on the nitrocellulose blot for lane 2 on both blots A and B, but reproduced poorly in the figures.) When the blots were probed with affinity purified anti-BPI23 antibody, the ELISA immunoreactivity appeared to correlate with the presence of two bands with apparent molecular weights of 62,000 and 64,000 Da. Native BPI extracted from human neutrophils also migrated as two bands at 62,000 and 64,000 Da. A faint band at 50 kDa was detected in both the BPI extracted from neutrophils and in the BPI derived from a few human serum samples (this is not visible in FIG. 7). This band may represent a non-glycosylated form of BPI (Mr 50,659) and was less than 7 % of the total BPI detected. rBPI migrated as single band at 64,000 Da. No other immunoreactive bands were visible on the Western blots for these plasma or serum samples. rBPI23 spiked into human plasma was also detected by these methods and was readily distinguishable from rBPI and native BPI (data not shown). When rLBP was added to anti-BPI23 coated wells at 100,000 ng/mL and processed as described above, two weakly immunoreactive bands were discernible with an apparent molecular weight of 69,500 and 114,400 Da (this is not visible in FIG. 7). The combined intensities (measured as integrated peak area) of the rLBP bands were approximately 5,000 fold lower than the rBPI peak area, on a mass basis.
When these same serum samples were probed with the anti-rBPI antisera, the ELISA immunoreactivity again correlated with the presence of the protein doublet at 62,000/64,000 Da and a significant relationship (R2 =0.807, p=0.0001) was observed between the ELISA signal and the Western blot integrated peak areas for the two BPI bands (FIG. 8). rLBP spiked at 100 μg/mL was not detectable when probed with the anti-rBPI antisera. These data suggest that the endogenous immunoreactivity of serum and plasma detected by ELISA is due to holo-BPI and not due to fragments of BPI or other cross-reactive material.
In this example, endogenous BPI immunoreactivity in human plasma and serum was determined. Plasma and serum samples were collected from 20 different healthy human donors. Utilizing the BPI sandwich ELISA and rBPI as the standard, BPI levels (ng/mL) were determined for each of these samples. For all individuals tested, the results shown in FIG. 4 show that BPI levels were consistently higher in the serum samples (cross-hatched bars) compared to the matched plasma samples (solid bars). The mean BPI concentration was 0.8 ng/mL in plasma and 27.1 ng/mL in serum. The concentration range of BPI was <0.2 to 2.1 ng/mL in plasma and 4.9 to 72.1 ng/mL in serum.
The experiment was then repeated for plasma and serum samples collected from 20 sepsis patients. The results shown in FIG. 5 show that the plasma and serum levels are not identical for the same patient. Moreover, the BPI levels are different from those of the healthy patients where the plasma BPI levels were always low. Statistical analysis of BPI levels in the serum of normal and septic patients shows that there is no statistically significant difference between the two (p=0.10). A scattergram showing BPI levels in the serum of healthy and septic individuals is shown in FIG. 9a. In contrast, the differences in BPI levels in the plasma of normal and septic patients is highly statistically significant (p=0.0014) as shown by the scattergam of FIG. 9b.
It is contemplated that BPI plasma levels will correlate with symptoms of sepsis including gram negative and gram positive bacteremia, DK, ARDS, ever APACHE II scores and/or survival. As an additional aspect of the invention, it has been found that there is no correlation of BPI plasma levels with numbers of white blood cells or neutrophils in septic patients.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the an upon consideration of the foregoing description of the presently preferred embodiments thereof. Consequently, the only limitations which should be placed upon the scope of the present invention are those which appear in the appended claims.
|Cited Patent
|Filing date
|Publication date
|Jun 16, 1989
|Sep 14, 1993
|Richard Ulevitch
|Acute phase protein modulating endotoxic activity of lipopolysaccharides, assay methods and polypeptides
|Mar 21, 1994
|Sep 29, 1994
|Incyte Pharmaceuticals, Inc.
|Use of bactericidal/permeability increasing protein and lipopolysaccharide binding protein levels and ratios thereof in diagnosis
|Dofferhoff, A. S. M. et al. Netherlands Journal of Medicine 39: 45 62, 1991.
|Dofferhoff, A. S. M. et al. Netherlands Journal of Medicine 39: 45-62, 1991.
|Elsbach et al., "Separation and Purification of a Potent Bactericidal/Permeability-Increasing Protein and a Closely Associated Phospholipase A2 from Rabbit Polymorphonuclear Leukocytes," J. Biol. Chem., 254:11000-11009 (1979).
|Elsbach et al., Separation and Purification of a Potent Bactericidal/Permeability Increasing Protein and a Closely Associated Phospholipase A 2 from Rabbit Polymorphonuclear Leukocytes, J. Biol. Chem. , 254:11000 11009 (1979).
|Erwin, A. L., et al. Laboratory Investigation 65(2): 138 144, 1991.
|Erwin, A. L., et al. Laboratory Investigation 65(2): 138-144, 1991.
|Gazzano Santoro et al., High Affinity Binding of the Bactericidal/Permeability Increasing Protein and a Recombinant Amino Terminal Fragment to the Lipid A Region of Lipopolysaccharide, Infection and Immunity , 60:4754 4761 (1992).
|Gazzano-Santoro et al., "High-Affinity Binding of the Bactericidal/Permeability-Increasing Protein and a Recombinant Amino-Terminal Fragment to the Lipid A Region of Lipopolysaccharide," Infection and Immunity, 60:4754-4761 (1992).
|Leturcq et al, "Generation of Monoclonal Antibodies to Human LBP and their Use in the Detection of LBP Protein in Serum," J. Cell. Biochem., 16C:161 (1992).
|Leturcq et al, Generation of Monoclonal Antibodies to Human LBP and their Use in the Detection of LBP Protein in Serum, J. Cell. Biochem. , 16C:161 (1992).
|Marra, M. N. et al. Journal of Immunology. 148:532 537, 1992.
|Marra, M. N. et al. Journal of Immunology. 148:532-537, 1992.
|Pereira et al, "Quantitation of a Cationic Antimicrobial Granule Protein of Human Polymorphonuclear Leukocytes by ELISA," J. Immunol. Methods, 117:115-120 (1989).
|Pereira et al, Quantitation of a Cationic Antimicrobial Granule Protein of Human Polymorphonuclear Leukocytes by ELISA, J. Immunol. Methods , 117:115 120 (1989).
|Pesce et al., "Cationic Antigens--Problems Associated with Measurement by ELISA," J. Immunol. Methods, 87:21-27 (1986).
|Pesce et al., Cationic Antigens Problems Associated with Measurement by ELISA, J. Immunol. Methods , 87:21 27 (1986).
|Schindler, R. et al. Clinical Nephrology 40(6):346 351, 1993.
|Schindler, R. et al. Clinical Nephrology 40(6):346-351, 1993.
|Spitznagel, J. K. Journal of Clinical Investigation 86:1381 1386, 1990.
|Spitznagel, J. K. Journal of Clinical Investigation 86:1381-1386, 1990.
|Spitznagel, R. et al. Clinical Nephrology 40(6): 346 351, 1993.
|Spitznagel, R. et al. Clinical Nephrology 40(6): 346-351, 1993.
|Taber, C. W. Taber s Cyclopedic Medical Dictionary. Philadephia: F. A. Davis Co., 1985. p. 545.
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|von der Mohien, et al., Bactericidal Permeability Increasing Protein Levels Predict Survival in Patients with Gram Negative Sepsis, Abstract presented at 13th International Symposium on Intensive Care and Emergency Medicine, (Brussels, Belgium) (1993).
|Weiss and Olsson, "Cellular and Subcellular Localization of the Bactericidal/Permeability-Increasing Protein of Neutrophils," Blood, 69:652-659 (1987).
|Weiss and Olsson, Cellular and Subcellular Localization of the Bactericidal/Permeability Increasing Protein of Neutrophils, Blood , 69:652 659 (1987).
|Weiss et al., "Human Bactericidal/Permeability-Increasing Protein and a Recombinant NH2 -Terminal Fragment Cause Killing of Serum-Resistant Gram-Negative Bacteria in Whole Blood and Inhibit Tumor Necrosis Factor Release Induced by the Bacteria," J. Clin. Invest., 90:1122-1130 (1992).
|Weiss et al., "Purification and Characterization of a Potent Bactericidal and Membrane Active Protein from the Granules of Human Polymorphonuclear Leukocytes," J. Biol. Chem., 253:2664-2672 (1978).
|Weiss et al., Human Bactericidal/Permeability Increasing Protein and a Recombinant NH 2 Terminal Fragment Cause Killing of Serum Resistant Gram Negative Bacteria in Whole Blood and Inhibit Tumor Necrosis Factor Release Induced by the Bacteria, J. Clin. Invest. , 90:1122 1130 (1992).
|Weiss et al., Purification and Characterization of a Potent Bactericidal and Membrane Active Protein from the Granules of Human Polymorphonuclear Leukocytes, J. Biol. Chem. , 253:2664 2672 (1978).
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|White M. L. et al. J. Immunol. Methods 167:227-235, 1994.
|Citing Patent
|Filing date
|Publication date
|May 10, 1996
|Apr 21, 1998
|Xoma Corporation
|Antithrombotic materials and methods
|Sep 10, 1997
|May 26, 1998
|Xoma Corporation
|Therapeutic uses of BPI protein products in humans with hemmorhage due to trauma
|Jun 7, 1995
|Jan 5, 1999
|Xoma Corporation
|Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof
|Mar 21, 1996
|Jan 12, 1999
|Xoma Corporation
|Anti-fungal peptides
|Aug 9, 1996
|Mar 30, 1999
|Xoma Corporation
|Anti-chlamydial uses of BPI protein products
|Sep 10, 1997
|Mar 30, 1999
|Giroir; Brett P.
|Therapeutic uses of BPI protein products for human meningococcemia
|Apr 20, 1998
|Aug 10, 1999
|Xoma Corporation
|Antithrombotic materials and methods
|May 23, 1997
|Aug 31, 1999
|Xoma Corporation
|Therapeutic uses of BPI protein products in humans with hemorrhage due to trauma
|Dec 1, 1998
|Nov 23, 1999
|Xoma Corporation
|Therapeutic uses of BPI protein products for human meningococcemia
|Jun 19, 1998
|Jan 11, 2000
|Xoma Corporation
|Bactericidal/permeability-increasing protein (BPI) deletion analogs
|Jun 18, 1999
|Jul 11, 2000
|Xoma Corporation
|Bactericidal/permeability-increasing protein (BPI) deletion analogs
|Jun 20, 1997
|Jul 25, 2000
|Three-dimensional structure of bactericidal/permeability-increasing protein (BPI)
|Apr 26, 1999
|Aug 22, 2000
|Xoma Corporation
|Antithrombotic materials and methods
|Mar 29, 1999
|Dec 19, 2000
|Xoma Corporation
|Uses of BPI protein products
|Aug 3, 1999
|Jun 5, 2001
|Xoma Corporation
|Therapeutic uses of BPI protein products for human meningococcemia
|Feb 22, 1999
|Nov 19, 2002
|Xoma Corporation
|Therapeutic uses of N-terminal BPI protein products in ANCA-positive patients
|Oct 30, 2000
|Jun 24, 2003
|Xoma Corporation
|Anti-chlamydial methods and materials
|Sep 14, 2001
|Jul 8, 2003
|University Of Zurich
|Polysome display in the absence of functional ssrA-RNA
|Nov 30, 2000
|Jul 22, 2003
|Xoma Corporation
|Therapeutic uses of BPI protein products for human meningococcemia
|May 25, 2000
|Jul 29, 2003
|Xoma Corporation
|Bactericidal/permeability-increasing protein (BPI) deletion analogs
|Jul 6, 2000
|Jul 29, 2003
|Xoma Corporation
|Antithrombotic materials and methods
|Nov 19, 2002
|Jan 1, 2004
|Xoma Technology Ltd.
|Therapeutic uses of N-terminal BPI protein products in ANCA-positive patients
|Oct 19, 2004
|Apr 20, 2006
|Renovar Incorporated
|Reagents for urine-based immunological assays
|Dec 28, 2006
|Nov 8, 2007
|Toru Yoshimura
|Homocysteine immunoassay
|Jan 28, 2010
|Toru Yoshimura
|Homocysteine immunoassay
|Aug 12, 2008
|Feb 18, 2010
|Ez Bio Inc.
|Reagents, Methods and Kits for the Universal Rapid Immuno-Detection
|Oct 19, 2005
|Apr 27, 2006
|Renovar, Inc.
|Reagents for urine-based immunological assays
|U.S. Classification
|435/7.1, 435/7.32, 436/518, 435/7.95, 435/7.94, 435/7.9
|International Classification
|Cooperative Classification
|European Classification
|Nov 15, 1993
Owner name: XOMA CORPORATION A CORPORATION OF DELAWARE, CALIF
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WHITE, MARK L.;CARROLL, STEPHEN F.;MA, JEREMY KAM-KUEN;REEL/FRAME:006763/0373
Effective date: 19931008
|Dec 10, 1996
|Certificate of correction
|May 3, 1999
|Fee payment
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|Apr 23, 2003
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|Fee payment
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text:
Hair of all cuts and colors has been offered up for a good cause.
The Senior High Youth Group from New Tripoli has presented a check for more than $1,200 to the Prager Cancer Fund at Lehigh Valley Health Network.
The group hails from New Life Evangelical Lutheran Church.
The kids raised the money through a hair donation fundraiser last month.
The group and their teacher, Dr. Jennifer Mesker, organized the event.
While the hospital received the money, "Locks of Love" received 42 ponytails.
Students donate money to LVH through hair-cutting
Published On: Jul 10 2013 08:00:00 PM EDT Updated On: Jul 12 2013 06:17:51 AM EDT
Hair of all cuts and colors has been offered up for a good cause.
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Dorney Park upgrades old favorite for new season
Dinosaurs come to life in Reading
Students dance to raise money for pediatric cancer
On-and-off rain and drizzle tonight
After more than a week, '16 Mile Fire' 100 percent contained
FAA begins early inspection of Allegiant Air operations
Whitehall Twp. Police seek to ID identity theft suspect
Teddy bears get their check-ups at the YMCA
Police search for knife-wielding robber in Warren County, NJ
Deceased man found in Target parking garage


text:
About i_wuz_nver_here : I was bored so I made this account. Hi.
Photographer, student, lover of life.
About i_wuz_nver_here : I was bored so I made this account. Hi.
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Today, I had to patiently listen as a customer nattered on and on about how incompetent I was for not stocking the movie she was looking for. It took nearly 20 minutes to get her to calm down long enough for me to explain that there is no such movie as "Hobbits With Shotguns". FML
by Anonymous / 03/09/2012 at 5:36pm / United States / Miscellaneous
Today, I was the 10th caller on a radio show. I answered the question correctly. The DJ informed I won a free air guitar of my choosing. I yelled with excitement over the air. The DJ then instructed me how to use my new air guitar. FML
by h4rdy / 03/09/2012 at 11:55am / United States (Texas) / Miscellaneous
Today, I was at my girlfriend's house for the first time. I cracked a joke that offended her, so she gave me the silent treatment. I had to pee, and since she wouldn't tell me where the bathroom was, I went to look for it. I walked in on her parents making love. FML
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Today, a stuffed ferret was the latest addition to the list of weird items my colleagues have found in our rubbish tip, and that they put in my office. The list also includes explicit fetish porn playing cards, live ammo and dead pheasants, to name a few. I need a new job. FML
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Today, I got more turned on by the idea of sex with my boyfriend than actually doing it. FML
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text:
Local Notes
5 km Pink Rock run in memory of James Lawlor draws large crowd
A large crowd of runners travelled to the Pink Rock on Friday evening for the James Lawlor Memorial Race.
Local Notes
A large crowd of runners travelled to the Pink Rock on Friday evening for the James Lawlor Memorial Race.
The freezing cold of last week couldn't keep loyal New Ross Standard readers away from shops across the district and five lucky individuals were rewarded for their efforts with €20 notes.
David Tucker Professionals from Social Care Ireland, Youth Work Ireland, KARE, Saplings, Carlow Regional Youth Services and Mental Health Services are among the community, educational and healthcare providers participating in IT Carlow's forthcoming Youth, Social & Community Career Development Seminar.
Amy Lewis Cushinstown NS pupils ditched their books for the ballot box recently when the Parent's Association staged a mock election in the school.
A proposal to convene a meeting on the morning of Easter Sunday by Cathaoirleach Cllr Michael Sheehan was rejected as several councillors are away on the day at national commemoration events.
Visitors to Wexford General Hospital are being asked to remember that new visiting hours have been introduced at the hospital.
David Tucker The Wexford Food Family, the not-for-profit network of food producers established in 2011 to promote Wexford as a food brand locally, nationally and internationally, has announced the dates for the third annual Wexford Food Summit.
The monthly Tea Dance will take place in Carrig Community Centre on Friday 19th February from 9.30pm, music by John Hogan Band. Refreshments served a night not to be missed.
Transition Year students at New Ross CBS Secondary School participated in a songwriting workshop in the school recently, recording two original songs in the process.
Paddy Ronan was an enterprising man who, throughout his life, had many businesses and passions, from horseracing to politics.
Catherine Byrne spent her life helping and supporting others and the affection in which she was held was reflected in the huge crowd in attendance at her funeral mass in the church of the Immaculate Conception Rowe Street, Wexford.
The GAA community in Adamstown gathered on Saturday night to donate money to six deserving charities following a Kube fundraiser run by the club before Christmas.
Cards: The weekly '30 Card Game continues in the Community Centre this Friday 29th January at 9pm. Catering on the night will be: Siobhan Hynes, Rosie Siggins & Maura Barden.
Thank you: Enable Ireland wish to thank all who purchased charity Christmas cards in aid of Enable Ireland Wexford branch. The support was much appreciated.
The death has occurred of Richard Sutton, more fondly known as 'The Yank', formally of 26 Cross Street, New Ross.
Vera was the third youngest of eleven children born to the late Jimmy and Bridie Purcell of Clongeen.
Esther Hayden Wexford Naturalists Field Club will to host a talk by two distinguished members on Thursday, February 4.
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Alma Jobs
Looking for jobs in Alma? Snagajob has great opportunities with the area's leading companies. And applying for Alma jobs has never been easier. Look at the job listings below, click on a job title that interests you and hit the "apply now" button. Part-time jobs, teen jobs, student jobs and summer jobs are now just a click away. Start your job search now.
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Photo Gallery
It looks like we don't have any photos for Jack Esformes yet. You can add or change photos at IMDb Pro. You can also browse other available content for Jack Esformes on IMDb such as filmography, trivia, personal details, etc.
Jack Esformes is an actor, known for Wag the Dog (1997), Defense Play (1988) and Breaking In (1989). See full bio »


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Dance Clothes
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John E. Gruber Sr. 1922 - 2011 Eudora
Paid family tribute
Eudora — Mass of Christian Burial for John E. Gruber Sr., 89, Eudora, will be at 10 a.m. Wednesday at Holy Family Catholic Church in Eudora. Burial will be in Holy Family Catholic Cemetery in Eudora. Mr. Gruber died Sunday, December 18, 2011, in the home he built. Mr. Gruber was born March 17, 1922 in Overbrook, the son of William J. and Martha Bergerhaus Gruber.
Mr. Gruber grew up in Northeast Kansas. In 1940 he graduated from Williamsburg High School where he excelled in athletics and academics. He lettered in baseball, football, and basketball. During the Second World War, as part of this country's greatest generation, Mr. Gruber served in Europe as a machinist and blacksmith in the U.S. Army Air Force. On the G.I. Bill he went to Kansas State College of Agriculture and Applied Science to become a High School teacher. Upon graduation he taught in Paxico and Wellsville. He went on to work at the Sunflower Army Ammunition Plant in De Soto and at the University of Kansas. He retired from The University of Kansas' Facilities Operations in 1987.
A man of many interests including agriculture and horticulture, travel, and carpentry, Mr. Gruber was an avid gardener, traveled with his family throughout most of the fifty states and built his own home. He leaves a wife of 20 years, Rita, a daughter Margaret, two sons John Jr. and Mark, daughter-in-law Mary Ann as well as grandchildren Tammy, Mark Jr., Heather, Matthew, and Ryan. He is survived by his sister Mary. His brother Paul preceded him in death.
A rosary will be said at 5 p.m. Tuesday, December 20, 2011 at Holy Family Catholic Church in Eudora followed by a visitation until 7 p.m.
The family requests all donations be sent to Holy Family Catholic Church in Eudora, Kansas and may be sent in care of the mortuary, 120 W. 13th St., Lawrence, KS 66044.
Condolences may be delivered to Warren-McElwain Mortuary in Eudora or sent to www.warrenmcelwain.com.
This information was provided to the Journal-World as a paid tribute.
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