text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 9, 2010
|Filing date
|Jul 17, 2003
|Priority date
|Jul 17, 2002
|Fee status
|Publication number
|Sylvia Daunert, Michael Poon, Urvee Desai, Sapna K. Deo
|Original Assignee
|University Of Kentucky Research Foundation
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|BiBTeX, EndNote, RefMan
|Patent Citations (4), Non-Patent Citations (18), Classifications (25), Legal Events (2)
|External Links: USPTO, USPTO Assignment, Espacenet
This application claims the benefit of provisional application 60/396,122, filed Jul. 17, 2002, which is incorporated herein in its entirety.
This invention was made with government support provided by The National Institutes of Health.
The invention relates generally to methods of determining the level of prostacyclin in plasma using the conjugates. More particularly, the invention relates to a single step immunoassay for detection of the level of 6-keto-prostaglandin F1, which directly correlates to the level of prostacyclin in plasma.
Primary Pulmonary Hypertension (PPH) is a rare disease of the pulmonary vasculature, resulting in abnormally high pressure in the pulmonary artery, right ventricular failure, and death. While the pathogenesis of PPH is not clearly understood, the increased pulmonary vascular resistance observed is attributed to three main factors: 1) vasoconstriction, 2) thickening of the vessel wall due to vascular remodeling, and 3) in situ thrombosis. (Archer, S., Rich, S. Circulation 2000, 102, 2781-2791; Rich, S. Curr. Treat Options. Cardiovasc. Med. 2000, 2, 135-140). There is no cure for PPH and the treatment options are limited. (Naeije, R., Vachiery, J. L. Clin. Chest Med. 2001, 22, 517-527). The survival rate is less than a year in patients presented with New York Heart Association Functional Class IV symptoms. Although lung transplantation is a possible treatment option, there are obvious drawbacks such as shortage of donor organs and chronic rejection as well as the possibility of infection. (Franke, U., Wiebe, K., Harringer, W., Franke, T., Wittwer, T., Wahlers, T., Haverich, A. Eur. J. Cardiothorac. Surg. 2000, 18, 447-452). Long term intravenous treatment with epoprostenol (also known as prostacyclin or PGI2) has greatly increased the survival rate.
Prostacyclin or PGI2 (Prostaglandin I2) is a major cyclooxygenase metabolite of arachidonic acid, and is mainly produced by vascular endothelial cells. Prostacyclin is a prostaglandin, which occurs along with other eicosanoids like thromboxanes and leukotrienes. Prostacyclin is a potent vasodilator with platelet-inhibitory, anti-proliferative and fibrinolytic activities. (Moncada, S., Vane, J. R. Pharmacological Rev. 1979, 30).
The exact levels of prostacyclin in human plasma are unknown, however like most eicosanoids it occurs in very low amounts (pg/mL). Thromboxane B2 (metabolite of thromboxane A2, a vasoconstrictor) and its metabolites occur in very low amounts like 1-10 pg/mL in plasma. Leukotrienes generally function as vasoconstrictors and bronchoconstrictors and occur in varying amounts in plasma and urine. Leukotriene B4 occurs between 50 pg/mL-50 ng/mL, leukotriene E4 occurs between 80 pg/mL-1000 ng/mg of creatinine. Prostaglandin F2α concentrations are found between 10 pg/mL-100 pg/mL, prostaglandin D2 is generally found at 5 pg/mL-500 pg/mL, and prostaglandin F1α occurs at 1 ng/mL-100 ng/mL.
Currently intravenous epoprostenol is indicated for the chronic treatment of primary pulmonary hypertension (PPH) in New York Heart Association (NYHA) functional Class III and Class IV patients who do not respond to conventional therapy. (Krishnan, U. Indian J. Pediatr. 2000, 67, 523-527; Nauser, T. D., Stites, S. W. Am. Fam. Physician 2001, 63, 1789-1798). There is no set dose of prostacyclin for PPH and the starting dose varies between 4-8 ng/kg/min. Following the initiation of intravenous prostacyclin, most patients require an automatic dose increase of 2 ng/kg/min every two weeks until the rate of administration reaches 20 ng/kg/min. Further dose increase of 0.5 to 1 ng/kg/min per week is needed only upon the return of symptoms. Common side effects of intravenous prostacyclin include flushing, muscle pain, and abdominal cramps and these symptoms are not necessarily dose dependent. Thus, a method to quantify and correlate the plasma levels of prostacyclin with the onset of the untoward side effects or functional improvement would be useful in determining the optimal level of plasma prostacyclin for an individual patient.
A method to quantify the plasma levels of prostacyclin would be useful in determining the optimal level of plasma prostacyclin for an individual patient. Prostacyclin is very unstable with a half-life of only 60 minutes in plasma and 2-3 minutes in buffer. The immediate and stable product of hydrolysis of prostacyclin, 6-Keto-Prostaglandin F1α (6-keto-PGF1α), is quantifiable and can be used as a surrogate marker of the level of plasma prostacyclin. The detection method for prostacyclin or its breakdown product has to be extremely sensitive and accurate since either molecule is present in very low amounts (pg/mL) in the body. Moreover, the existing methods are time consuming mainly due to the necessity to extract 6-keto-PGF1α from biological samples. (Schramm, W., Smith, R. H., Jackson, T. M., Craig, P. A., Grates, H. E., and Minton, L. L. Clinical Chem. 1990, 36, 509-514; Tonai T., Y., K., Yano, T., Hayashi, Y., Yamamoto, S., Yamashita, K., and Miyazaki, H. Biochim. Biophys. Acta 1985, 836, 335-343). Thus there is a need for a rapid and sensitive detection method for prostacyclin. The present invention provides a single-step, solid phase immunoassay for 6-keto-PGF1α using the bioluminescent protein aequorin.
In one aspect of the invention there is provided an assay for determining amount of prostacyclin in a plasma sample comprising:
In a preferred embodiment of this aspect of the invention, the assay is performed in a microtiter format. Preferably, the secondary antibody is coated onto a surface and exposed to the plasma sample, primary antibody and 6-keto-PGF1-aequorin conjugate.
In another aspect of the invention there is provided a method of determining an appropriate dose of prostaglandin for the treatment of primary pulmonary hypertension in a patient comprising
In another aspect of the invention there is provide a kit for measuring amount of prostacyclin in plasma comprising
(1) a 6-keto-PGF1-aequorin conjugate;
(2) an anti-6-keto-PGF1 primary antibody; and
(3) a secondary anti-6-keto-PGF1 primary antibody coated plate.
Various types of detection methods have been reported in the literature for prostacyclin using its stable hydrolysis product, 6-keto-PGF1α, as a measure of prostacyclin levels. For example, the methods include radioimmunoassays, enzyme immunoassays, and detection using gas liquid chromatography-mass spectrometry (GLC-MS) or gas chromatography-selective ion monitoring (GC-SIM). (Tonai T., Y., K., Yano, T., Hayashi, Y., Yamamoto, S., Yamashita, K., and Miyazaki, H. Biochim. Biophys. Acta 1985, 836, 335-343; Fischer C, M. C. Biomed. Mass Spectrom. 1985, 12, 399404; and Maclouf, J., Pradel, M., Pradelles, P. and Dray, F. Biochim. Biophys. Acta 1976, 431, 139-146. Radioimmunoassay is a highly sensitive method but it has the disadvantages associated with the use of radioactivity. Moreover, all the immunoassays or chromatographic techniques reported for 6-keto-PGF1α, require extraction of 6-keto-PGF1α from plasma or plasma samples making the process laborious and time consuming. (Tonai T., Y., K., Yano, T., Hayashi, Y., Yamamoto, S., Yamashita, K., and Miyazaki, H. Biochim. Biophys. Acta 1985, 836, 335-343; Fischer C, M. C. Biomed. Mass Spectrom. 1985, 12, 399-404; and Maclouf, J., Pradel, M., Pradelles, P. and Dray, F. Biochim. Biophys. Acta 1976, 431, 139-146.
In contrast, the present invention provides a sensitive bioluminescent immunoassay for 6-keto-PGF1α where the plasma samples are analyzed directly without requiring the extraction of 6-keto-PGF1α from plasma. This results in a simpler, faster method for detection of this analyte in plasma samples. The present assay for 6-keto-PGF1α is preferably performed in a microtiter plate format using the photoprotein aequorin as the label. Colorimetric and fluorescent assays could possibly suffer from background interference limiting their use in direct plasma analysis. Aequorin, however, being a bioluminescent label has virtually no background associated with aequorin is a highly sensitive label.
Aequorin has proved to be a highly sensitive label in the development of binding assays for the determination of large and small biomolecules. (Zenno, S., Inouye, S. Biochem. Biophys. Res. Commun. 1990, 171, 169-174; Christopoulos, T. K., and Galvan, B. Anal. Chem. 1996, 68, 3545-3550; Zatta, P. F. J. Biochem. Biophys. Methods 1996, 32, 7-13; Stults, N. L., Stocks, N. F., Riviera H., Gray J., McCann R. O., O'Kane D., Cummings R. D., Cormier M. J., and Smith D. F. Biochemistry 1991, 31, 1433-1438). The bioluminescent protein aequorin is native to the jellyfish Aequorea victoria. Aequorin is a bioluminescent photoprotein that is composed of an apoprotein (apoaequorin, 189 amino acid residues), a chromophoric unit (coelenterazine) and molecular oxygen. Aequorin has three Ca2+ binding sites. When Ca2+ binds to aequorin, it undergoes a conformational change that results in the oxidation of coelenterazine to coelenteramide and this produces CO2 and emits a flash of light at 469 nm. The bioluminescent signal produced by aequorin has virtually no background signal and the quantum efficiency is between 11-17.5%, allowing it to be detected down to attomole levels. (Stults, N. L., Stocks, N. F., Riviera H., Gray J., McCann R. O., O'Kane D., Cummings R. D., Cormier M. J., and Smith D. F. Biochemistry 1991, 31, 1433-1438; Ramanathan, S., Lewis, J. C., Kindy, M. S., and Daunert, S. Anal. Chim. Acta. 1998, 369, 181-189; Shimomura, O. Biol. Bull. 1995, 189, 1-5; Lewis, J. C., Lopez-Moya, J. J., and Daunert S. Bioconjugate Chem. 2000, 11, 65-70).
In the present invention a cysteine-free mutant of aequorin is preferred because it shows enhanced activity. Lewis, J. C., Lopez-Moya, J. J., and Daunert S. Bioconjugate Chem. 2000, 11, 65-70, incorporated herein in its entirety. However, any suitable aequorin may be used as a label in the present methods and kits. Recombinant aequorin may be produced and purified for use in the present assay for 6-keto-PGF1α. For example, the compound 6-keto-PGF1α may be conjugated to pure aequorin by conventional covalent attachment to the free amine groups of the lysine residues in aequorin. Aequorin contains 15 lysine residues.
In developing the present assay, the effect of choosing different initial ratios of analyte to the apoprotein on the bioluminescence properties of the protein, and the binding ability of an anti-6-keto-PGF1α antibody was investigated. Further, the effect of an antibody for 6-keto-PGF1α on the luminescence signal was evaluated. Based on the results, the sensitive bioluminescent binding assay for 6-keto-PGF1α described herein was developed. The assay was then further optimized for detection of the analyte in plasma. Several plasma samples from PPH patients were analyzed by using them directly in the assay without any extraction of 6-keto-PGF1α.
In the present method, the detection and quantification of 6-keto-PGF1α directly in a plasma sample without extraction of the prostaglandin is achieved using a 6-keto-PGF1 antibody and a 6-keto-PGF-aequorin conjugate. The plasma sample may be diluted, for example about 200-fold with an appropriate medium, such as deionized water, and an effective amount of antibody and prostaglandin-conjugate added. An effective amount of antibody to be used in the assay dependent upon the particular antibody used and is selected to such that the light intensity generated therewith is sufficient to measure a signal above background, while keeping the amount of available binding sites to a minimum. In general, when the amount of antibody is increased, the sensitivity of the assay is improved but the detection limit of the assay is compromised. When a more diluted amount of antibody is used, the dynamic range is narrowed and sensitivity is reduced.
The amount of 6-keto-PGF-aequorin conjugate used in the present assay depends upon the specific conjugate used and can readily be determined by one of skill in the art. In general, the concentration range of conjugate is about 0.1�10−10 to about 9�10−10, preferably about 1�10−10.
In carrying out the present assay in a microtiter plate format, a plasma sample is obtained from a patient and applied to a surface, such as a surface of a microtiter plate, that has been precoated with a secondary antibody. An amount of anti-6-keto-PGF1α primary antibody and anti-6-keto-PGF1α-aequorin conjugate is then added to the plasma sample and the mixture incubated in the dark for a period of time ranging from about 1 to about 3 hours, preferably about 2 hours at room temperature. The surface is then washed to remove unbound primary antibody and conjugate, and the light intensity is determined and correlated with the amount of 6-keto-PGF1α present in the sample, which in turn directly correlates with the amount of prostacyclin in the sample.
The determination of amount of prostacyclin in a sample obtained from a patient may then be used to determine the appropriate dosage of prostglandin to administer to a patient being treated for PPH, for example.
It has been observed in the scientific literature that the drug, Viagra�, available from Pfizer Inc. is a potential candidate for therapy in patients suffering from PPH. Sayin et al., Can. J. Cardiol., 2002, 18(6):676-678. It has been demonstrated that Viagra� acts as an inhibitor of the enzyme, phosphodiesterase. Inhibition of this enzyme causes increase in levels of cGMP and cAMP, which control growth of the smooth muscle cell layer that surrounds pulmonary arterioles. Proliferation of these smooth muscle cells has been linked with PPH. Prostacylin also acts as a dilator of smooth muscle cells by increasing the levels of cyclic nucleotides. Furthermore, it has been found that phosphodiesterase inhibitor enhances vasodilatory efficacy of prostacyclin. Thus, the effects of Viagra� therapy on levels of prostacyclin in the blood can be determined using the assay for 6-keto-prostaglandin, described herein. Also, the appropriate dosage of Viagra� can also be determined by application of the assay for 6-keto-prostaglandin, described herein.
The present invention also provides unique aequorin mutants that are suitable for conjugation to 6-keto protaglandin F1 for use in the above-described assays or for conjugation to other biomolecules, such as those suspected of playing a pathologicaal role in PPH, e.g., endothelin, angiotensin II, vasopressin, and neurotransmitter. These mutant aequorins can be conjugated to the desired biomolecule to analyze the role of the biomolecule in PPH, to analyze the metabolism of the biomolecule, to determine any correlation of serum level with physiological effect, or for drug design, for example. Similarly, these conjugates can be used in assays to determine the effects of the conjugated biomolecule on drug therapy, for example
Aequorin- or mutant aequorin-biomolecule conjugates prepared either through non-specific, site-specific or genetic conjugation can be used in the assays of the invention. Aequorin mutants which have a unique cysteine at amino acid residue 69, 70, 74 or 76 (described in the examples) have been developed. These amino acid residues are close to the substrate-binding site in aequorin and therefore, binding of antibody to an analyte attached at any of these sites yields a homogeneous change in bioluminescence. Such results were demonstrated with an aequorin-thyroxine conjugate, where the thyroxine is bound to the sulfhydryl of a unique cysteine at position 71 of the aequorin molecule.
The present invention also contemplates kits for measuring amount of prostacyclin in a plasma sample. Kits preferably contain a primary antibody for 6-keto-PGF1α and a secondary antibody and a 6-keto-PGF1α-aequorin conjugate. Preferably, the conjugate is a cysteine-free mutant of aequorin conjugated to 6-keto-PGF1α. The kit may also include assay buffer, deionized water, and/or microtiter plates or other surfaces pre-coated with the secondary antibody or for coating with secondary antibody at the time of performing the assay.
Reagents used in the examples herein include: Luria Bertani (LB) broth and agar were purchased from DIFCO Laboratories (Detroit, Mich.). Sodium dodecyl sulfate (SDS), sodium hydroxide, sodium chloride, calcium chloride, bovine plasma albumin (BSA), kanamycin, ethylenediaminetetra acetic acid (EDTA) disodium salt and 6-keto-Prostaglandin F1α were obtained from Sigma Laboratories (St. Louis, Mo.). N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(Dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Pierce (Rockford, Ill.). HQH, quaternized polyethyleneimine anion exchanger was purchased from PerSeptive Biosystems (Cambridge, Mass.). The anti-6-keto-Prostaglandin F1α antibody and the 96-well microtiter plates coated with anti-sheep-IgG were purchased from Assay Designs Inc. (Ann Arbor, Mich.). Tris (hydroxymethyl)amino methane (Tris) was purchased from Research Organics (Cleveland, Ohio). Coelenterazine was purchased from Biosynth International (Naperville, Ill.). Bradford assay kits were obtained from Bio-Rad Laboratories (Hercules, Calif.). Patient samples as well as control samples were provided by Dr. Michael Poon (Mount Sinai School of Medicine, N.Y.). All solutions were prepared using deionized (Milli-Q Water Purification System, Millipore, Bedford, Mass.) distilled water. All chemicals were analytical reagent grade or better, with >95% purity and were used as received.
Cell cultures were grown in an orbital shaker, Form a Scientific (Marrietta, Ohio) and centrifuged using a Beckman J2-MI centrifuge (Palo Alto, Calif.). The fractions were lyophilized using a VirTis Bench Top three freeze dryer (Gardiner, N.Y.). Bioluminescence measurements for the assay were made on MLX Microtiter Plate Luminometer Dynex (Chantilly, Va.). For all the measurements four replicates were performed and three of the closest were selected. All luminescence intensities reported are the average of three replicates, and have been corrected for the contribution of the background.
Expression and Purification of Apoaequorin: The plasmid pSD110 containing the cysteine free mutant (mutant S) of aequorin was constructed and initially transformed in competent B. subtilis cells. For protocols for the expression and purification of apoaequorin and the conversion of apoaequorin to the photoprotein, see Lewis, J. C., Lopez-Moya, J. J., and Daunert S. Bioconjugate Chem. 2000, 11, 65-70, incorporated herein.
Conjugation of Aequorin to 6-keto-Prostaglandin F1α: A volume of 1 mL of 1�10−6 M. aequorin was allowed to incubate with coelenterazine for 3 h at 4� C. The N-hydroxysuccinimide ester of 6-keto-Prostaglandin F1α was prepared by reacting with the water-soluble analog of N-hydroxysuccinimide (NHS), sulfo-NHS, and the dehydrating agent EDC. Grabarek. Z. a. G., J. Anal. Biochem. 1990, 185. The analyte 6-keto-Prostaglandin F1α (1 mg/mL) was allowed to react with ˜5 mM sulfo NHS and ˜2 mM EDC (final concentrations) for 30 minutes at R.T. The reaction was quenched by adding β-mercaptoethanol to a final concentration of 20 mM. This NHS ester of the analyte was then conjugated to aequorin with bound coelenterazine in three different mole ratios, 1:50, 1:200, and 1:500 of aequorin: 6-keto-PGF1α The conjugates were passed through a size exclusion column in order to remove any unconjugated NHS ester of 6-keto-PGF1α and any residual reactants or reducing agent. The fractions were tested for activity of aequorin by taking 100 μL of each fraction, then adding 3 μL of 2.36�10−4 M coelenterazine and measuring the intensity of light emitted as described before. The fractions showing activity were combined and lyophilized. The lyophilized conjugates were resuspended in 30 mM Tris-HCl buffer, pH 7.0, containing 2 mM EDTA, 0.15 M NaCl and 1 mg/mL BSA (assay buffer) and stored in aliquots at −80� C.
Calibration curves were generated for each of the conjugates (1:50, 1:200, 1:500) by first converting the apoaequorin conjugate to aequorin with coelenterazine as described before. Standard solutions were prepared by serially diluting the stock solutions of the conjugates assay buffer. A volume of 100 μL of these solutions was placed in a microtiter plate and the luminescence activity of aequorin was measured. The luminescence signal was integrated over a 3 second time period. All points were obtained in triplicates.
For the binder dilution studies aliquots of 100 μL of different concentrations of serially diluted anti-6-keto-PGF1α antibody, produced in sheep and 50 μL of 1�10−10 M 6-keto-PGF1α-aequorin conjugate were placed simultaneously in microtiter plates pre-coated with a secondary antibody, donkey anti-sheep IgG. All dilutions were done in the assay buffer. The plates were incubated in dark at room temperature for 2 h with shaking at 300 rpm. Then the plates were washed with wash buffer (30 mM Tris-HCL buffer, pH 7.0, containing 2 mM EDTA, 0.15 M NaCl, and 0.1% Tween 20) three times to remove any unbound primary antibody or any unbound conjugate. Finally, the light intensity was measured as described earlier for the calibration plot.
Dose-Response Curve: A dose-response curve was generated for 6-keto-PGF1α by simultaneously incubating 100 μL of 6-keto-PGF1α antibody (1:10,000 dilution), 50 μL of different concentrations of the serially diluted analyte, 6-keto-PGF1α, and 50 μL of 1�10−10 M coelenterazine bound 6-keto-PGF1α-aequorin conjugate (1:500) on microtiter plates coated with the secondary antibody. The plate was incubated in dark at room temperature for two hours with shaking at 300 rpm. The plate was washed three times with the wash buffer to remove any unbound components. The light intensity was measured as described before.
A dose-response curve for 6-keto-PGF1α in plasma was generated by preparing serially diluted standard solutions of 6-keto-PGF1α in human plasma (previously diluted 1:200 times with deionized water) and adding them simultaneously with 6-keto-PGF1α-aequorin conjugate and the 6-keto-PGF1α antibody to the wells with the immobilized secondary antibody. The incubation and washing steps were performed in the same manner as previously described in the generation of dose-response curve. The bioluminescence was triggered and measured as described before.
Construction of Plasmids containing Aequorin Mutants. The gene of a mutant apoaequorin in which all three cysteines of the native aequorin were replaced by serines was used as a template for the PCR. A unique mutation was introduced on the purified apoaequorin gene (Ala 69 to cysteine 69) by employing PCR site-directed mutagenesis. The oligonucleotides used for PCR are AQ69-1 and AQ69-2 (see Table 1). The following two primers were used as outside primers to introduce the restriction endonuclease site for Hind III and Sal I restriction enzymes:
1) TCTTCTTCTTCTTCTAAGCTTGTCAAGCTTACATCAGACTTCGACAACCCAAGA (SEQ ID NO. 1); and 2) AGAAGAAGMGAGTCGACCAGATCTGGTACCCGGGAATT (SEQ ID No. 2).
The DNA obtained by PCR and the vector PFLAG-ATS which consists of the gene sequence for the octapeptide upstream of the multiple cloning site (MCS) were digested with the restriction enzymes Hind III and Sal I. Next, the digested DNA fragment was ligated into the plasmid pFLAG-ATS, to yield the pSD1004 vector. The apoaequorin mutants containing unique cysteine residues at position 70, 74 and 76 were prepared in the same manner (see Table 1) as described above. DNA sequencing was performed at the Macromolecular Center (University of Kentucky) to confirm the presence site-specific mutations.
Expression and Isolation of Aequorin Mutants. For expression of apoaequorin mutant 69 the plasmid pSD 1004 was transformed into Bacteria (E. coli, strain JM109). A 2 mL volume of LB broth containing 100 μg/mL of ampicillin was inoculated with a colony of bacteria containing plasmid pSD 1004. The cells were grown for 17 h at 37� C. in an aerated shaker. The cell culture was transferred to 400 mL LB broth containing 100 μg/mL ampicillin, and allowed to grow until it reached an absorbance of 0.6 at 600 nm. For induction of protein expression, 1 M IPTG stock solution was added such that the final IPTG concentration in the culture was 1.0 mM. The culture was allowed to grow for an additional 4.0 h at 37� C. in the shaker. The cells were centrifuged for 30 min at 27� C. and pelleted.
The apoaequorin mutant 69 thus expressed was released from the periplasm using an osmotic shock procedure. The pellets were first resuspended in 40 mL of 10 mM Tris-HCl/30 mM NaCl, pH 7.5. The cells were then collected by centrifuging for 15 min at 27� C., and were resuspended in 40 mL of 33 mM Tris-HCl, pH 7.5. The centrifugation and resuspension steps were repeated, but this time the cells were resuspended vigorously with 40 mL Stage I buffer. The resuspended cells were incubated at 37� C. for 10 min, with slow shaking. The cells were again collected by centrifuging for 10 min at 27� C. The pellets were finally resuspended rapidly with vigorous shaking for 10-15 min in 80 mL of chilled deionized-distilled water. After centrifuging for 10 min at 4� C., the supernatant was collected and lyophilized. The lyophilized sample was dissolved in deionized water and dialyzed three times against 30 mM Tris-HCl, pH 7.7, buffer containing 0.15 M sodium chloride, 2 mM EDTA, and 4 mM DTT. The dialyzed sample was then centrifuged for 10 min at 4� C. to remove any cell membrane fragments. The supernatant, which consisted of unpurified protein was filtered with a 0.2 μm syringe filter and was immediately purified. The apoaequorin mutants 70, 74 and 76 were expressed and isolated in the same way as described above by using bacteria containing plasmid pSD 1005, pSD 1006 and pSD 1007 respectively.
Purification of the Apoaequorin Mutants. The proteins were purified by using an anti-octapeptide M2 agarose affinity column. The column was equilibrated with 50 mM Tris-HCl, pH 7.4 containing 150 mM NaCl and 2 mM EDTA (TBS buffer). The solution containing protein was loaded on the column and the column was washed three times with 15 mL of TBS buffer. The proteins were eluted using 1 mL aliquots of 0.1 M glycine, pH 3.0, into tubes containing TBS buffer. The fractions containing the protein and their purity was determined by SDS-PAGE using 12.5% polyacrylamide gels, which were developed by silver staining (Pharmacia Biotech, Uppsala, Sweden). Fractions containing the fusion protein were pooled together and glucose was added to a final concentration of 30 mM followed by lyophilization. The lyophilized protein was resuspended in deionized distilled water. Excess salt was removed by dialyzing twice against 30 mM Tris-HCl, pH 7.4 containing 2 mM EDTA and 2 mM DTT. The protein concentration was determined by using the Bradford protein assay, with BSA as the standard.
Conversion of Apoaequorin Mutants to Photoproteins. A set amount of purified protein in 30 mM Tris-HCl, pH 7.4, buffer containing 2 mM EDTA and 2 mM DTT was mixed with a three time molar excess of coelenterazine in a glass tube. The mixture was briefly vortexed and then placed on ice for the required time.
Bioluminescence Emission Study. A volume of 10 μL of the mutant proteins at a concentration of 1 μg/mL was added to a glass tube, and the bioluminescence was measured by injecting 100 μL of luminescence triggering buffer (100 mM Tris-HCl, 100 mM CaCl2, pH 7.5). The bioluminescence signal was collected at 0.1 second intervals over a 4 second time period.
Bioluminescence Emission Spectra of Apoaequorin Mutants. Luminescence was triggered by careful addition of 1.0 mL of a 1.0�10−6 M solution of mutant aequorin using a constant-rate syringe pump into 0.5 mL of luminescence triggering buffer. Emission spectra were obtained using a Fluorolog-2 spectrofluorometer from Spex Industries (Edision, N.J.) without turning on lamp or chopper. The spectra were not corrected for changes in sensitivity of the photomultiplier tube to luminescence at different wavelengths.
Conjugation of Aequorin Mutant to Analyte: A volume of 1 mL of 1�10−6 M. aequorin was allowed to incubate with coelenterazine for 3 h at 4� C. Five-fold excess of a maleimide/iodoacetamide derivative of analyte was allowed to react with mutant aequorin for 2 h at R.T. The conjugates were passed through a size exclusion column in order to remove any unconjugated maleimide/iodoacetamide derivative of analyte. The fractions were tested for activity of aequorin by taking 100 μL of each fraction, then adding 3 μL of 2.36�10−4 M coelenterazine and measuring the intensity of light emitted as described before. The fractions showing activity were combined and lyophilized. The lyophilized conjugates were resuspended in 30 mM Tris-HCl buffer, pH 7.0, containing 2 mM EDTA, 0.15 M NaCl and 1 mg/mL BSA (assay buffer) and stored in aliquots at −80� C.
A comparative study between analysis of 6-keto-PGF1α with and without extraction from plasma was carried out. In order to extract 6-keto-PGF1α from plasma, a fixed volume of the plasma sample was first acidified with 2M HCl to pH 3.5. The sample was allowed to stand for 15 min at 4� C., after which it was centrifuged for 2 min to remove any precipitate. Meanwhile, a C18-reverse phase column was washed with 10 mL ethanol, followed by 10 mL deionized water. The sample was then applied under a slight positive pressure to obtain a flow rate of 0.5 mL/min. The column was washed with 10 mL of water, followed by 10 mL of 15% ethanol and finally 10 mL of hexane. The sample was eluted using 10 mL of ethyl acetate. Elution was carried out at a flow rate of 2 mL/min. The ethyl acetate in the samples was evaporated under a stream of nitrogen. The dried samples were first dissolved in 50 μL ethanol, and then in the same volume of the assay buffer as that of the plasma sample initially used for extraction. These samples were used in the dose response curve performed in buffer.
The analysis of 6-keto-PGF1α directly in plasma without extraction was performed by diluting the samples 1:200 times and using 50 μL in the dose-response curve along with 100 μL 6-keto-PGF1α of antibody and 50 μL of 6-keto-PGF1α-aequorin conjugate. The analysis was performed in the same manner as the dose-response curve described before. The amount of 6-keto-PGF1α in the samples was calculated from the standard dose response curve generated in plasma. The appropriate dilution factor was taken into consideration when calculating the amount of 6-keto-PGF1α present in each sample.
For in vivo analysis plasma samples were obtained from five patients with severe pulmonary arterial hypertension and 7 normal controls. Written informed consent was obtained from all patients; the protocol was approved by the Institutional Review Board at Mount Sinai School of Medicine. Of the five patients, three were on chronic intravenous prostacyclin. All patient samples were analyzed in the same manner as the dose response curve in plasma. The samples were diluted 1:200 times to minimize matrix effects and 50 μL of the sample was used in the assay. The amount of 6-keto-PGF1α in the samples was calculated as described before for sample analysis without extraction. All plasma samples were obtained and stored frozen at −20� C., in aliquots until use.
In order to obtain recovery data further analysis of the samples was performed. Two of the normal plasma samples and two patient plasma samples were first analyzed using the assay without extraction. Then the normal samples were spiked with 5 pg/mL of 6-keto-PGF1α and the patient samples were spiked with 10 pg/mL of 6-keto-PGF1α and the samples were used once again in the assay. In each case the samples were diluted 1:200 times to minimize matrix effects and 50 μL of the sample was used in the assay. The amount of 6-keto-PGF1α in the samples was calculated as described before for sample analysis without extraction. The samples were analyzed in triplicates.
The plasmid containing the cysteine free mutant of aequorin was previously constructed in our laboratory. Lewis, J. C., Lopez-Moya, J. J., and Daunert S. Bioconjugate Chem. 2000, 11, 65-70. The cysteine free mutant of aequorin was chosen for the development of this assay because it showed enhanced activity as compared to wild-type aequorin. The cysteine free mutant of aequorin was expressed from plasmid pSD110. Purification of aequorin was carried out using an HQH anion exchange column. The fractions containing bioluminescence activity were lyophilized with glucose and later resuspended in deionized water. SDS-PAGE analysis was carried out to check the efficiency of purification. A single band was observed at ˜20 kDa in the lyophilized fraction for pure aequorin. The yield of the protein was found to be 1.8 mg/L of culture.
Before conjugating 6-keto-PGF1α to aequorin, the lyophilized fraction was dialyzed against bicarbonate buffer to remove any residual Tris-HCl salt, which might interfere with the conjugation. The dialyzed aequorin was then charged with 3 times molar excess of coelenterazine so that the analyte would not conjugate in the coelenterazine binding site, which might cause loss in activity of aequorin.
The conjugation method used involved a two-step coupling method using EDC (1-Ethyl-3-(Dimethylaminopropyl) carbodiimide hydrochloride) and sulfo NHS, which is a water soluble analog of NHS (N-hydroxy succinimide). EDC, a dehydrating agent, reacts with the carboxyl group of 6-keto-PGF1α, and forms an amine-reactive unstable, O-acylisourea-intermediate of 6-keto-PGF1α. NHS reacts with the O-acylisourea-intermediate to form an NHS ester to stabilize this intermediate. The NHS ester then reacts with the amine groups on aequorin. The advantage of the two-step procedure is the prevention of the modification of the protein carboxyl groups due to exposure to EDC. Grabarek, Z. a. G., J. Anal. Biochem. 1990, 185. The reaction is quenched by a thiol-containing compound like β-mercaptoethanol. Three different conjugates were prepared (1:50, 1:200, 1:500) using this method, by increasing the initial molar concentration of the NHS ester of 6-keto-PGF1α while keeping the concentration of aequorin constant. The conjugates were passed through a size exclusion column to remove any unconjugated ester of 6-keto-PGF1α and unreacted β-mercaptoethanol. They were then lyophilized and later resuspended in the assay buffer.
In order to study the effect of conjugation on the bioluminescence activity of aequorin and to select an appropriate conjugate concentration for the assay, calibration plots were constructed for each of the conjugates (data not shown). The conjugates (1:50, 1:200 and 1:500) could be detected down to 1�10−12 M. A concentration of 1�10−10 M of the three conjugates was selected for the binder dilution study. This concentration was low enough to be able to detect aequorin at low levels with high sensitivity and at the same time it showed much higher activity than the background.
The next step was to study the interaction between the anti-6-keto-PGF1α antibody and the conjugates. For this, binder-dilution curves were generated for all three conjugates as shown in
Since the goal was to detect 6-keto-PGF1α in plasma, it was necessary to validate this assay in plasma and to study any possible matrix effects on the performance of the conjugate or the binding capabilities of the 6-keto-PGF1α antibody in plasma. The assay was, therefore, performed in 1:200 diluted human plasma spiked with known concentrations of 6-keto-PGF1α. The dose-response curve obtained (
The inter-assay precision was determined by generating the dose-response curve in plasma for five consecutive days in exactly the same manner as described earlier. The percent coefficient of variation for samples containing different concentrations of 6-keto-PGF1α, was calculated. The average coefficient of variation was found to be 4.5%. The intra-assay precision was determined by doing 4 replicate measurements of the same sample. The average coefficient of variation was found to be 1.25%.
Presence of various prostaglandins other than 6-keto-PGF1α in the blood can cause non-specific binding with the 6-keto-PGF1α antibody and interfere with the detection of 6-keto-PGF1α, giving extremely high values of 6-keto-PGF1α. Therefore, it becomes important to confirm that the amount of 6-keto-PGF1α obtained from our assay is only due to 6-keto-PGF1α and no other prostaglandin. The cross-reactivities of other prostaglandins similar in structure to 6-keto-PGF1α were determined by preparing standard solutions of the compounds in the assay buffer ranging from 100,000 to 100 pg/mL and using them in this assay for 6-keto-PGF1α. The measured 6-keto-PGF1α concentration at 50% B/B0 was then calculated. The percent cross-reactivities were calculated by comparison of measured cross-reactant concentration and the actual concentration (known amount) of the cross-reactant in the sample (Table 1). It must be noted that the prostaglandins occur at different times in the arachidonic acid pathway and in different concentrations. Also, 2,3-dinor-6-keto-Prostaglandin F1α is found in urine and does not occur in plasma, however since it is the immediate metabolite of 6-keto-PGF1α we decided to analyze the cross-reactivity of 2,3-dinor-6-keto-Prostaglandin F1α as well. From the table it is clear that the prostaglandins do not show any significant amount of cross-reactivity. This indicates that the assay is specific only for 6-keto-PGF1α and there is no interference from other prostaglandins, which might be present in the plasma samples. The assay can, therefore, be used to measure 6-keto-PGF1α directly from plasma.
Oligonucleotide sequence of primers
Cross-reactivities of related prostaglandins were measured by using varying concentrations of 50 μL of the cross reactant in the dose response curve instead of 6-keto-PGF1α. The percent cross-reactivities were calculated at 50% B/B0. The results are shown in Table 2.
Average Standard
Percent Bound
6-keto-Prostaglandin F1
Prostaglandin F1
Prostaglandin F2
Prostaglandin F1
2,3-dinor-6-keto-Prostaglandin F1
Prostaglandin D2
Prostaglandin E1
Prostaglandin F1
In vivo sample analysis was performed with the present assay. The PPH patients and control samples were provided by Dr. Michael Poon of the Mount Sinai Pulmonary Hypertension Program. Five PPH patient plasma samples were diluted (1:200) times with buffer and used directly in the assay. The concentration of 6-keto-PGF1α in the samples was measured using the standard dose response curve obtained in plasma by diluting plasma of a normal subject (1:200) to measure the amount of 6-keto-PGF1α. It was observed that the levels of 6-keto-PGF1α increased with increase in the prostacyclin dose. Three normal control samples were used to produce the standard dose-response curves, and the curves were found to be similar. The values of 6-keto-PGF1α in the samples of patients obtained with each of the standard curves were also similar. Table 2 shows the 6-keto-PGF1α values in normal and patients samples in vivo. Plasma samples of the normal subjects were used to produce standard dose-response curves and values of 6-keto-PGF1α in patient samples were obtained using those curves. The measurements were performed in triplicates. The results are shown in Table 3.
6-keto-Prostaglandin F1
concentration measured
Prostacyclin Dose
without extraction
Plasma Sample
Average of 3
normal samples
PPH Patient 1
PPH Patient 2
PPH Patient 3
PPH Patient 4
PPH Patient 5
In order to verify the concentration of 6-keto-PGF1α obtained from plasma using the assay of the present invention, 6-keto-PGF1α was extracted from the plasma samples of 7 normal subjects and the 5 PPH patients using the C18-Sep-Pak columns, and these values were compared with the values obtained when plasma samples were used directly in our assay without extraction. In the extraction method, the 6-keto-PGF1α was obtained in the ethyl acetate fraction. All ethyl acetate was evaporated under N2 and the dried 6-keto-PGF1α was dissolved first in ethanol followed by the assay buffer. This 6-keto-PGF1α was used in the assay performed in buffer. The concentrations of 6-keto-PGF1α obtained using the aequorin assay in plasma samples, without pretreatment were observed to be highly correlated with the measured 6-keto-PGF1α after extraction. The equation for the straight line can be given as y=0.79x+418, r=0.988, n=12.
Further in vivo analysis was performed in order to determine the recovery of 6-keto-PGF1α from samples using this assay. Two of the normal plasma samples and two patient plasma samples were first analyzed using the assay as described before and determining the concentration of 6-keto-PGF1α present in the samples by using the standard curve in 1:200 diluted plasma. A known amount of 6-keto-PGF1α was spiked into the samples and the samples were analyzed once again to determine the concentration of 6-keto-PGF1α. Percent recovery data were obtained using the concentration of 6-keto-PGF1α in unspiked samples and the concentration obtained after spiking. It is clear from the data (Table 3) that this assay for 6-keto-PGF1α gives recoveries within the range error. Percent recovery data of normal samples and patient samples are shown in Table 4. The measurements were performed in triplicate.
Concentration of
Amount of
Concentration of
found in unspiked
spiked into
found in spiked
Normal sample
Normal sample
PPH patient 1
PPH patient 2
In conclusion, this immunoassay, is the first direct assaying technique for quantification of plasma 6-keto-PGF1α without the process of extraction. The 6-keto-PGF1α concentrations obtained from patient samples by extraction and without extraction show good correlation. In addition, the antibody employed for the assay, and hence the assay itself, does not show significant binding to other related prostaglandins. Therefore, the possibility of obtaining falsely high values of 6-keto-PGF1α due to the presence of other similar prostaglandins is eliminated. The assay has a wide working range, and a low detection limit of 1 pg/mL, which enables the measurement of the creation of distribution curves of plasma prostacyclin levels in normal and disease states. This assay also characterizes the application of aequorin in quantification of important components present in the body by directly measuring them in the biological matrix without requiring any pre-treatment of the biological fluids. This assay may allow high throughput analysis of the levels of plasma prostacyclin in patients with primary pulmonary hypertension and the clinical implications of various levels of plasma prostacyclin in the pathogenesis and clinical management of this devastating disease.
The following abbreviations are used herein: 6-keto-PGF1α: 6-keto-ProstaglandinF1α; PPH: Primary Pulmonary Hypertension; EDC: 1-Ethyl-3-(Dimethylaminopropyl) carbodiimide hydrochloride; NHS: N-hydroxysuccinimide; BSA: Bovine plasma albumin; SDS-PAGE: Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis; Tris: Tris (hydroxymethyl)amino methane; EDTA: ethylenediaminetetraacetic acid.
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|U.S. Classification
|435/7.1, 435/975, 436/172, 435/971, 530/389.2, 435/7.93, 436/538, 435/69.1, 435/25, 436/518, 530/807, 435/8
|International Classification
|Cooperative Classification
|European Classification
|Apr 8, 2011
Owner name: UNIVERSITY OF KENTUCKY RESEARCH FOUNDATION, KENTUC
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|Publication number
|Publication type
|Publication date
|Oct 2, 1973
|Filing date
|Jul 29, 1968
|Priority date
|Jan 8, 1963
|Publication number
|Z Shapiro
|Original Assignee
|Nuclear Materials And Equip Co
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|Patent Citations (5), Referenced by (11), Classifications (19)
|External Links: USPTO, USPTO Assignment, Espacenet
1 1 Oct. 2, 1973 METHOD OF MAKING A HIGH TEMPERATURE BODY OF UNIFORM POROSITY [75] Inventor: Zalmarl M. Shapiro, Pittsburgh, Pa.
[73] Assignee: Nuclear Materials and Equipment Corporation, Apollo, Pa.
[22] Filed: July 29, I968 [21] Appl. No.: 751,680
Related'U.S. Application Data [6 Continuation of ser. No. 250,112, Jan. 8, 1963,
[52] US. Cl 29/420, 29/419, 29/530 [51] Int. Cl. B22f [58] Field of Search 29/419, 420, 527.1, 29/5272, 527.4, 423, 424
[56] References Cited UNITED STATES PATENTS 2,475,601 7/1949 Fink 117/107.2 3,049,799 8/1962 Breining et al. 29/420 3,135,044 6/1964 Mote et a1. 29/423 3,139,658 7/1964 Brenner et a1. 22/200 3,153,279 10/1964 Chessin 29/420 Primary Examiner-John F. Campbell Assistant ExaminerD. C. Reiley AttrneyI-Iymen Diamond [57] ABSTRACT A method of making a high temperature and stress resistant body of desired porosity in whose practice a green porous body (FIGS. 4, 5, 9, 10) of higher than the desired porosity is formed by compacting different size fractions of spherical particles (A, 23, 27 FIGS. 4, or by winding fine cold-drawn wire (53 FIG. 9) or mesh of fine cold-drawn wire (73 FIG. on a mandrel (51 FIG. 7; 71 FIG. 10). The green body is cemented into a rigid body and at the same time its porosity is decreased to the desired magnitude by depositing cementing material (H5 FIG. 11, 61 FIG. 9, 77 FIG. 10) in the pores FIGS. 5 and 6; FIG. 9) from a gas. Typically a body of tungsten is formed by reducing a tungsten halide in the pores with hydrogenv 9 Claims, 11 Drawing Figures Pmwrmm 3.T82.028
SHEET HP 2 METHOD F MAKING A IllIGII TEMPERATURE BODY OF UNIFORM POROSITY This application is a continuation of application Ser. No. 250,] 12, filed Jan. 8, 1963, and now abandoned.
This invention relates to the art of fabricating or forming bodies of materials which do not readily lend themselves to the usual forming operations such as machining, molding, casting, forging, spinning, rolling and the like. In one of its specific, but highly important aspects, this invention is applicable to bodies of tungsten. But in its broader aspects this invention applies to such materials as tantalum, niobium, zirconium, titanium, vanadium, silicon, rhenium, chromium and molybdenum. These materials may be characterized as highly refractory, high strength materials; that is, each of these materials has a high melting temperature and is capable of manifesting high strength.
Specifically, this invention deals with producing bodies of predeterrninable shape or form which have a predeterminable porosity. This invention also relates to the art of producing hightemperature and high-stress resistant materials and of bodies of such materials having predeterminable shape and porosity. This invention has particular relationship to the production of such materials and bodies which are capable of withstanding the environmental and operating conditions to which critical components of the nuclear and aerospace apparatus are subjected. Typical of such components are the nozzles of space rockets.
The temperature and pressure of the gases blasting through the nozzle of a space rocket operating with acceptable efficiency may exceed 6,000" F and 900 pounds per square inch of area. The abruptness with which the mechanical and thermal stresses imposed on the material of the nozzle chance enhances the severity of these conditions. On ignition, the temperature of the nozzle rises sharply and non-uniformity giving rise to high thermal and mechanical stresses which may cause the nozzle to spall and crack. In addition, the high temperature and reactive nature of the propellant gases as well as the possible presence of particulate matter tend to alter the original nozzle shape and size through the loss of material by melting, corrosion and erosion. Any of these deleterious effects may deteriorate the nozzle enough to prevent the rocket from accomplishing its mission and it is indispensable that the nozzle be fabricated of such material and in such manner as to minimize these changes.
It is an object of this invention to provide a method of producing a high temperature resistant body having a selectable form and porosity It is another object of this invention to provide a method of producing a material for a rocket nozzle or the like which shall be capable of meeting the above described rigorous conditions; it also is an object of this invention to provide a body such as a rocket nozzle of such a material.
In providing rocket nozzles and like bodies, the practice has been adopted of forming the body of a highmelting point substance. Such substantces as tantalum carbide (melting point 7,0l5 F) and hafnium carbide (meIting point 7,025 F) have high melting points but do not in their present form have the necessary physical attributes to lend themselves to the fabrication of such bodies. Tungsten (melting point 6,l70 F) which has a 6 In addition, with the advent and use of more advanced propellants, even tungsten must be impregnated with evaporative coolant materials such as silver or copper to prevent the surface from melting at the higher gas temperatures generated. For this purpose the body must be porous so that it can absorb the cooling metals during fabrication and release the cooling metals during the high temperature application. It is moreover essential that the pores be in communication and that the body have a mean porosity for the communicating pores which shall be reliably predictable and shall be constant, as required by the conditions of application. The higher gas pressures generated by these propellants also require stronger, tougher bodies to withstand the thermal shock and the resulting mechanical stresses. Conversely at the same gas pressures, stronger materials would permit reduction in weight.
In the practice of this art, porosity may be measured as a direct function of the purpose which it is to serve by measuring the quantity of a liquid which a porous body can absorb. in carrying out this measurement, mercury (or other dense liquids as carbon tetrachloride or acetylene tetrabromide) is injected in the pores under pressure and the quantity of mercury injected under preset conditions is determined. This procedure measures the mean open porosity which is of essential interest. The mean total porosity may be measured by determining the density of the body. The density may be expressed in terms of percent of the density of the solid substance (tungsten for example).
It is another object of this invention to provide a tough, strong body having a constant mean porosity which shall be capable of withstanding the thermal shock and stresses to which a rocket nozzle is subjected.
In accordance with the teaching of the prior art, refractory structure such as nozzles are made from tungsten powder by the isostatic pressing at high pressures (approximately 40 tons per square inch) and sintering at high temperatures (2,0002,200 C). The process is excessively costly and requires complicated equipment. In addition, the powder used is produced by chemically reducing a compound such as ammonium paratungstate. The resulting structure has relatively poor mechanical properties and must be massive.
The powder which is compressed usually is irregularly shaped and of a size distribution suited to achieve sintered densities between and theoretical from the green billets. Because of the nature of the powder and the fabrication process, the pores obtained are irregular in shape and random in distribution and size. A good percentage of the pores are totally closed and not interconnected with other pores and are not available as regards filling with cooling metals. Not only does the quantity of silver or copper which can be absorbed in such bodies vary somewhat from nozzle to nozzle, but the effusion and evaporation of the silver or copper during operation is dependent upon the nature of the pores from area to area and the protection afforded the nozzle varies in the same manner. Other shortcomings of this prior art practice are:
1. Die design and fabrication are costly, and heavy expensive presses and heating equipment are involved.
2. The practice is adaptable to fabrication only of relatively simple shapes, thus often forcing compromising of superior performance design to minimize fabrication difficulties.
3. Even though the starting powder may have been prepared carefully as to particle shape and particle size distribution, the pressed object invariably shows considerable variation from region to region in density and extent and nature of porosity (i.e., open versus closed porosity). Further, laminations and cracks are invariably present.
4. The high temperature sintering cycle is conducive to grain and crystal growth, thus leading to lowering of the mechanical strength below the poor strength which is at best available.
It is an object of this invention in its specific aspects to overcome the above described difficulties. Another object of this invention is to provide a low-cost method or a process for making a porous body of selectable shape and porosity and also to provide such a method for making a high temperature and high-stress resistant porous body such as a rocket nozzle, the pores of which shall communicate and the porosity of which shall be constant or reproducible within narrow limits. A further object of this invention is to provide such porous bodies.
A specific object of this invention is to provide a process or method for making tungsten and other refractory metal skeletal structures of reliably controllable density and porosity, which structures shall be capable of being infiltrated and/or consolidated with other metals such as silver, copper, silicon, hafnium, vanadium, chromium, niobium, zirconium, tantalum, tungsten, molybdenum rhenium and others as well as alloys of these and others or with refractory materials including ceramics, such as hafnium carbide, zirconium carbide, tantalum carbide, alumina and other carbides, borides, silicides and nitrides.
This invention in one of its aspects arises from the realization that the closer complete uniformity in pore size and characteristic is approached, the more uniform, predictable and reliable is the performance of the infiltrated nozzle. It was also realized that ideal uniformity might be closely approached by replacement of the reduced powder by spherical particles of specific size fractions, the quantity of each fraction and the particle dimensions being selected so as to provide the final required porosity. But such spherical powder is relatively inactive due to the low specific surface (surface area per unit mass) and previous preparation history and cannot readily be sintered. In accordance with this invention in one of its aspects a method is provided for cementing the spherical particles together without significant alteration in the geometry and uniformity of the pores and without weakening the green shape.
In accordance with this invention in one of its specific aspects one or more carefully selected size fractions of spherical tungsten particles are prepared by plasma jet fusion or other methods such as fluidizedbed decomposition or reduction of the halides and packed into appropriate shapes by mechanical packing and/or compaction in the manner described in R.K. McGeary's paper Mechanical Packing of Spherical Particles, Journal of Americans Ceramic Society 44 5l3-522 (1961). This paper is included herein by reference. For packings consisting of more than one fraction, the particles of each successive fraction would have a diameter of approximately 1/10 to 1/5 of the diameter of the preceding fraction. These particles dimensions may range between +400 U.S. standard mesh and 60 U.S. standards mesh; that is, the particles are of such dimensions as to pass through a 60 mesh sieve and be retained by a 400 mesh sieve.
The mass compacted as disclosed above is then consolidated or cemented together by reacting a tungsten halide such as pure tungsten hexafluoride or tungsten hexachloride with pure hydrogen obtained for example by diffusion through a silver-palladium membrane in the pores of the mass. Tungsten hexaiodide may also be used. This compound requires less or no hydrogen to effect the deposit. The reduction may also be effected by other reducing gases such as cracked ammonia. The mass may be precleaned to remove oxide or other contaminants prior to the cementing. This reaction produces uniform deposition of tungsten on the surface of the particles throughout the interstices of the porous body. As the tungsten deposits, it bridges the particles and connects them together. To assure uniformity of penetration and deposit, fine-grain structure, the reaction is best carried out slowly at relatively low temperatures up to approximately 900 F. Deposition at temperatures of about 480 to 900 F does not significantly alter the ductile-to-brittle transition temperature or the mechanical properties of the green mass. Because of the manner in which the spherical particles arrange themselves in a nested fashion during the compacting, the pores are in communication. The ultimate porosity is determined in the initial porosity and the amount of material deposited.
For example, assume that a final porosity ranging between 17 and 25 percent is desired. An initial packing density of approximately 60 percent would then allow for the deposition of adequate tungsten from the vapor phase to yield a strong body containing the desired pore volume. Such an initial packing density is readily achievable with spheres of a single size by the technique described in the McGeary paper cited above. Should it be desirable to reduce the amount of tungsten deposited from the vapor phase, higher initial green densities can be achieved by the utilization of a binary system consisting of spherical particles of two sizes. As cited above, use of ternary or quaternary systems of spherical particles can be used to obtain a range of initial green packed densities up to approximately 93 percent.
Another aspect of this invention arises from the realization that the tensile strength of substance such as tungsten increases and the ductile-brittle transition temperature decreases with the density of the substance and the fineness of its grain. Very dense, fine grained tungsten or other like material may be obtained by cold working (cold drawing or rolling) the material. Thus, the ultimate tensile strength of cold-drawn tungsten wire of 1 mil diameter wire at room temperature may exceed 600,000 pounds per square inch while that of sintered percent dense tungsten may just exceed 50,000 pounds per square inch. ln accordance with this aspect of the invention use is made of the high strength of tungsten wire for fabrication of complex shapes, particularly where additional reliability and weight reduction is very important, as for example, in the making of rocket nozzles. Conceivably the mass so formed may be cemented or bonded by plasma-jet flame spraying with tungsten. But more satisfactory bonding is achieved by reducing tungsten compounds in situ and thus depositing tungsten in the pores of the mass and this mode of bonding constitutes one of the important contributions of this invention.
In accordance with this specific aspect of the invention fine cold-drawn tungsten wire is wound on a mandrel with many lateral holes to allow the easy passage of gas through the holes. The mandrel has the internal shape of the desired object. Depending on the tension of the wire, a body is thus wound which typically may be 60-65 percent dense with respect to theoretical density. A tungsten halide, typically tungsten hexafluoride or tungsten hexachloride is then passed through the holes in the mandrel and through pores formed by the turns of the wire and reduced there by pure hydrogen. The deposited tungsten bridges the strands of wire and cements them together. The deposition takes place at a controlled temperature which may range from 480 to about 900 F, far below the recrystallization temperature of the wire and does not alter the mechanical properties of the wire. The wire in this case typically may be of a diameter of between 0.0001 inches and 0.020 inches. The diameter is determined by the porosity and mechanical properties desired. Successive layers after the first may be wound over the regions where successive turns of the lower layers are in contact. The winding is such that the pores of the structure are in communication. The wire can also be cross wound to achieve desired porosity, geometry, and mechanical properties.
The structure produced by winding wire on a mandrel as just described is stronger in a direction perpendicular to the axis of the turns than in a direction parallel to the axis of the turns. In accordance with a further aspect of this invention high strength in all directions of the structure (e.g., in the direction of the axis of the nozzle as well as in the direction perpendicular to the axis) is achieved by winding on a mandrel fine cold-drawn tungsten wire mesh or cloth of a variety of weaves, properly tailored to give the desired density or porosity and shape. This forms a green body of uniform pore size and distribution which is then cemented by the vapor phase reduction of the tungsten hexafluoride or chloride with high purity hydrogen. As in the other embodiments, the deposition is accomplished slowly at temperatures ranging from 480 F to approximately 900 F to minimize alteration in the favorable mechanical properties of the structure.
This invention in its specific aspects contemplates tungsten structures and tungsten is peculiarly suitable for the practice of this invention. In its broader aspects, this invention is not limited to tungsten, but includes structures of powder, wire or mesh of niobium, tantalum, zirconium, titanium, silicon, vanadium, hafnium, rhenium, chromium, and molybdenum. Further, the bonding or cementing agent may also include all of the aforementioned materials, and is not limited to tungsten or the base metal used to make the green shape.
Within its broader aspects this invention also includes deposition of refractory and ceramic materials within the interstices of cemented-bonded shapes prepared from particles, wires, and mesh to form cermetlike composite materials. Several such materials can be simultaneously co-deposited or several materials can be despoited in succession. Successive deposition is particularly suitable where it is desirable to deposit a barrier material to prevent interaction with the green mass or a layer on the green mass. In certain situations the green mass may be heated so that the outer layer fuses and cements the mass on cooling.
Metals may also be deposited to cement green masses of ceramic or glass particles, filaments or fibers including carbonaceous filaments or fibers. Deposition of these metals is accomplished by vapor phase reduction or decomposition or liquid phase infiltration followed where required by chemical reaction. As indicated previously, the materials which can be inserted into the porosities of the consolidated/cemented shapes include in addition to the metals the carbides of zirconium, tantalum, thorium, hafnium, niobium and the oxides of uranium, thorium, aluminum, magnesium, and zirconium as well as nitrides, silicides and borides. These materials have attractive properties, in that they combine the high strengths of the metal matrix with the additional refractory properties of the ceramic. Such materials have special use and attractiveness in high temperature applications where both high strength and good thermal shock properties are required.
The novel features considered characteristic of this invention are disclosed generally above. For a more thorough understanding of this invention both as to its organization and as to its operation, together with additional objects and advantages thereof, reference is made to the following description taken in connection with the accompanying drawings, in which:
FIG. 1 is a generally diagrammatic fragmental view in side elevation, highly enlarged, of a mass of spherical particles as they are packed in the practice of the aspect of this invention involving the compacting of particles;
FIG. 2 is a view taken in the direction of line IIII of FIG. 1;
FIG. 3 is a view in section taken along line III-III of FIG. 2;
FIG. 4 is a view in top elevation showing one layer of the largest particles of FIG. I and the smaller particles nested in the spaces between progressively larger particles;
FIG. 5 is a view in isometric of the layer shown in FIG. 4;
FIG. 6 is a view in section further enlarged taken along line VI--VI of FIG. 4;
FIG. 7 is a view in asymmetric illustrating apparatus for practicing the aspect of this invention involving the winding of wire into a mass;
FIG. 8 is a fragmental view in side elevation of a portion of a body produced with the apparatus shown in FIG. 8;
FIG. 9 is a view in section taken along line IX-IX of FIG. 8;
FIG. 10 is a fragmental view in section enlarged showing apparatus for practicing the aspect of this invention involving the formation of a body from wire mesh; and
FIG. 11 is a reproduction of a photomicrograph of about X magnification showing a portion of a body produced in the practice of this invention.
The article shown in FIGS. 1 through 6 is a portion of a porous body made up by mechanical packing or compacting a number of sizes of generally spherical particles. The largest particles as shown in FIGS. it and 2 are disposed in layers, the particles in the center layers being labelled A, the particles in the layer below A, B, and the particles in the layer above A, C. The particles A, B, C, should be compacted under such pressure that adjacent spheres are generally tangent. The pressure should not be so high that the spheres are deformed into a mass of appreciable solidity. Pressures of the order of 1.0 to 50 pounds per square inch are satisfactory.
With the particles A, B, C compacted so that they are tangent, there are regions 2i between the particles which are bounded by the adjacent spherical surfaces. These regions are in communication. Within these regions 21 smaller particles 23 may be nested. Typically the particles 23 of next smaller size than the particles A, B and C should have a diameter of HS to 1/10 the diameter of the particles A. There are spaces 25 defined by surfaces of the larger particles A, B, C and of the smaller particles 23. Within these spaces still smaller particles 27 having diameters of about 1/5 to 1/10 of the particles 23 are deposited.
In making the mass the spherical particles can be formed by feeding powder into a plasma jet torch and collecting the particles emitted by the plasma. The spherical particles are then assembled one fraction at a time while vibrated mechanically and tumbled thoroughly so that the distribution of the particles is relatively uniform. The loading may be effected by depositing the particles in the form under a piston on which a relatively small weight, of about 20 or 30 pounds, is placed. The second and next smaller size fraction is then added and caused to nest into the appropriate voids as shown in FIG. 3 by mechanical vibration with a load applied in the same way as to the largest fraction. The particles of the second size are deposited in the voids of the particles of the first size so that the volume of the mass remains unchanged. The position of the piston under the weight is also unchanged. This process is repeated for each successive smaller sized particles. The particles are thus compacted into a porous mass. The spaces between the particles are in communication.
The green compacted mass has a predetermined porosity. To reduce the porosity to the desired magnitude or to cement the particles, tungsten deposits 31 are produced in the pores by reducing a tungsten halide with pure hydrogen at controlled temperature in the range of 480 F to about 900 F. The spherical particles A, B, C, 23, and 27 are bridged by the deposit.
A body having a desired form and a desired porosity is readily fabricated in this way. The physical properties of this body can be set by proper selection of the green material and the cement and their relative properties and is determined primarily by the particle geometry.
In FIG. l I a photomicrograph of a body of tungsten in accordance with this invention is shown. This body includes particles M and 83 of two sizes. As can be seen from FIG. M, the deposited tungsten 45 cements the particles together.
The apparatus shown in FIGS. 7, 8, 9 includes a mandrel H on which a fine, cold drawn, tungsten wire 53 is wound. The mandrel 51 is rotatable by a suitable drive (not shown) to effect the winding of the wire. Between the turns of wire 511 there are spaces 54. Because of these spaces the green wound structure is porous and the pores 55 are in communication. The mandrel S11 is hollow and has holes 59 in its surface. A tungsten halide and pure hydrogen are conducted through the pores and produces tungsten deposits 61 in the pores. The deposit at is discontinuous as can be seen from FIG. 8.
In accordance with a further specific aspect of this invention, the wire 53 may be wound on the mandrel 51 under tension and may be maintained in tension while the tungsten is deposited from the halide. After the deposit is completed, the tension may be relaxed. A body formed in this way is prestressed and its strength is increased.
The embodiment of the invention shown in FIG. 110 includes a perforated mandrel 71 on which a mesh 73 of fine, cold-drawn, tungsten wire is deposited. A tungsten halide and pure hydrogen are conducted through the holes 75 in the mandrel. The mass is maintained at a temperature which can range from 480 to 900 F. Reduced tungsten deposits 77 are produced in the body wound on the mandrel. The deposits start at the junctions of the wires and spread out from these junctions. The quantity deposited depends on the density of the gas flow and the duration of the reducing process. Usually the deposited material is so small a proportion of the body volume as not to affect the strength. The desirable tensile properties of the wire is not affected by heating to 900 F during the time required for cementation. The mesh may be pretensioned in different directions during the winding and the cementing to produce a prestressed structure.
In accordance with a further aspect of this invention a green body consisting of some one or more flat layers of mesh or filaments or wires, prestressed, may be cemented by deposition as disclosed to produce a plate.
While as a rule uniform deposition from the halide is desirable, situations may arise where the cementing substance should be concentrated in one part or another of the green mass to achieve variable porosity or other properties. Variable deposition is achieved by localizing the flow of the halide, varying its concentration or varying the temperature of the green mass.
While preferred embodiments have been disclosed herein, many modifications thereof are feasible. This invention then is not to be restricted except insofar as is necessitated by the spirit of the prior art.
I claim:
1. The method of producing a body of predetermined substantially uniform communicative porosity throughout, which comprises compacting more than one selected size fractions of generally spherical particles, assembled one size fraction at a time, to produce a porous mass having a porosity greater than said predetermined porosity, so that the distribution of the particles is relatively unifonn and the particles of smaller size fractions are effectively nested throughout in the volumes defined between the particles of larger size fractions, the particles being selected from one or more materials of the class of materials consisting of tungsten, rhenium, niobium, tantalum, silicon, zirconium, titanium, vanadium, hafnium, chromium and molybdenum, and reacting in the pores of said mass one or more of the halides of said one or more materials with hydrogen to deposit said one or more materials in the pores of said mass to cement said mass into a rigid body, the quantity of said one or more materials deposited being such that said body has said predetermined substantially uniform porosity.
2. The method of forming a porous body resistant to high temperatures typically exceeding 6,000F which comprises compacting more than one selected size fractions of generally spherical particles of tungsten, assembled one size fraction at a time, to produce a porous mass, each of said size fractions being mechanically treated so that the distribution of particles is relatively uniform and mixtures of different size fractions being mechanically treated so that the smaller size fractions nest throughout in the voids of the larger fractions whereby a porous mass of substantially uniform porosity is produced, and thereafter reacting within the pores of said mass at a temperature which does not significantly alter the mechanical properties of said mass, one or more of the class of compounds consisting of tungsten hexafluoride and tungsten hexachloride with hydrogen to deposit tungsten from said compounds in the pores of said mass to cement said mass into a rigid hightemperature resisting body, the said reacting taking place at a rate and temperature as to assure uniformity of penetration and deposit and fine-grain structure of the deposited tungsten.
3. The method of forming a body of substantially uniform communicating porosity throughout and of predetermined density comprising compacting into a mass of a density smaller than said predetermined density, more than one selected size fractions of a powder of one or more materials of the class of materials consisting of tungsten, niobium, tantalum, zirconium, titanium, vanadium, silicon, rhenium, hafnium, chromium, and molybdenum, the particles of said powder having substantially spherical form, the particles being assembled one size fraction at a time and each of said size fractions being mechanically treated so that the distribution of particles is relatively uniform and mixtures of different size fractions being mechanically treated so that the smaller particles nest throughout in the voids of the larger fractions, whereby a porous mass of substantially uniform porosity is produced, said size fractions being selected so that said mass has said smaller density; and reacting within the pores of said mass one or more of the halides of said one or more materials with hydrogen or the like to deposit, without significant alteration in the geometry and uniformity of the pores, said one or more materials in said mass to cement said mass into a rigid body, the amount of said material deposited being such that said body has said predetermined density.
4. The method of claim 1 wherein the deposited material is the same as the material of the mass.
5. A method for the manufacture of an element of construction resisting thermal shocks and mechanical stresses, e.g. a rocket nozzle or nose by depositing of a binding metal from the gas phase in the interior of a preformed skeleton-like body, characterized in this that a green body is produced by compacting of generally spherical particles of more than one size fractions of a high temperature resisting and mechanically resisting material, e.g. tungsten, to a porous body of corresonding dimensions in such a way that a contiguous, homogeneous pores-space of substantially uniform porosity throughout is formed, the said size fractions being assembled one size fraction at a time, so that the particles of smaller size fractions are effectively nested throughout in the volumes defined between the particles of larger size fractions, and further characterized in this that a gaseous compound of the binding metaL' e.g., tungsten halide, with a reducing agent, e.g., hydrogen, is brought to reaction within the pores of the formed body in such manner that, without significant alteration of the geometry and uniformity of the pores, the binding metal is deposited in the pores and will cement the particles together.
6. The method of claim 5 wherein the material of the green body is a metal which is the same as the binding metal.
7. The method of claim 1 wherein the tungsten halides and the hydrogen are substantially pure, the hydrogen having been obtained by diffusion through a silver palladium membrane or the like.
8. The method of claim 5 wherein the porous mass is formed of at least two size fractions of the particles of generally spherical form, the smaller particles having a diameter of 1/10 to US of the diameter of the larger particles.
9. The method of claim 8 wherein each size fraction is mechanically treated so that the distribution of the size fractions is relatively uniform, and after being so treated one size fraction is then compacted by relatively small pressure and thereafter the other size fraction is added and the particles of the smaller size fraction are caused to nest in the voids of the larger size fraction substantially uniformly by mechanical treatment of the mixture of size fractions and the mixture is compacted by small pressure and thereafter the halides and hydrogen are reacted in the pores of the mixture. t
|Cited Patent
|Filing date
|Publication date
|Apr 26, 1946
|Jul 12, 1949
|Ohio Commw Eng Co
|Bonding of metal carbonyl deposits
|Jul 28, 1958
|Aug 21, 1962
|Union Carbide Corp
|Method of gas plating
|Jun 4, 1959
|Jun 2, 1964
|United Aircraft Corp
|Lightwight porous structures and methods of making same
|Dec 8, 1961
|Jul 7, 1964
|Abner Brenner
|Production of tungsten objects
|May 29, 1959
|Oct 20, 1964
|Horst Corp Of America V D
|Heat resistant solid structure
|Citing Patent
|Filing date
|Publication date
|Aug 26, 1974
|Oct 14, 1975
|Trimble Lyne S
|Method of fabricating magnetochemical particles
|Oct 18, 1974
|Aug 10, 1976
|Deschamps Laboratories, Inc.
|Method of manufacturing a heat exchanger core
|Jul 27, 1990
|Jan 1, 1991
|The United States Of America As Represented By The Secretary Of The Army
|Methods are producing composite materials of metal matrix containing tungsten grain
|Nov 20, 1986
|Jul 23, 1991
|Permelec Electrode Ltd.
|Process for providing titanium composite having a porous surface
|Mar 26, 1997
|Apr 1, 2003
|Roedhammer Peter
|Process for producing a composite material
|Aug 18, 2009
|Nov 12, 2013
|Richard W. Kauppila
|Cooling arrangement for conveyors and other applications
|May 14, 2009
|Mar 4, 2014
|Onera (Office National D'etudes Et De Recherche Aerospatiales)
|Method for preparing a cellular material based on hollow metal beads
|Aug 18, 2009
|Mar 11, 2010
|Kauppila Richard W
|Cooling arrangement for conveyors and other applications
|May 14, 2009
|Jul 14, 2011
|Alain Rafray
|Method for preparing a cellular material based on hollow metal beads
|Jun 6, 1997
|Apr 9, 1998
|Allied Signal Inc
|Method for making a rhenium rocket nozzle
|Dec 12, 2012
|Dec 27, 2013
|Ceratizit Austria Gesellschaft M.B.H.
|Permeable, porous article
|U.S. Classification
|419/1, 29/530, 29/896.6
|International Classification
|Cooperative Classification
|European Classification

subset: commoncrawl


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"Back In Care Chiropractic is committed to supporting optimum health and well-being through exceptional chiropractic care, while promoting a healthier lifestyle for everyone in the community," explains Dr Ron Zukerman. We tailor our recommendations to each patient's specific health issues and goals, with a focus on fixing the cause of the problem.
Dr Ron Zukerman (Chiropractor) has been in practice for over 20 years and has a wealth of knowledge and experience in his field. He is internationally renowned and practices with care, precision and passion. With the latest and most modern diagnostic approach, the Doctor will aim to locate the cause of your problem. He utilises a diversity of techniques based on your personal...
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Back in Care Chiropractic services the Melbourne CBD area and surrounding suburbs including Melbourne CDB, East Melbourne, Carlton, Carlton North, North Melbourne, Fitzroy, Brunswick, Collingwood, Parkville, Southbank, South Melbourne, Brunswick East, Brunswick West, Coburg, Preston, Essendon, South Yarra, Northcote and Prahan.Go to Profile
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Congratulations in joining millions of families around the world who are interested in improving their wellbeing with safe and natural Chiropractic Care. Our approach to health care in finding the underlying cause of your health concerns and then educating and empowering you with the tools and inspiration to stay well, means we love what we do.
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ChiroHealth Family and Sports Clinic
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Almond Chiropractic
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At Ringwood Family Chiropractic clinic we help you relive pain and improve your overall health and wellbeing by providing gentle and non-invasive chiropractic treatment in Melbourne eastern suburbs such as Ringwood, Croydon, Donvale, Mitcham, Blackburn, Nunawading, Vermont, Bayswater, Boronia, Wantirna, etc.
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Our chiropractor Dr. Rogan Sun also treats acute resulting from sport injuries, motor vehicle accidents and work related injuries. He is Workcover, TAC approved practitioner. Most health fund rebate available on site in the clinic (Hicaps ready). Medicare rebate available for patient with Enhanced Primary Care Program (EPC).Go to Profile
Dr Benjamin Apap
(03) 9312 0824
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- Yea


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3 Classes Tips from Someone With Experience
Do You Want Your Kids to Enroll in Performing Arts Class?
If you find your kids to have inclination in performing arts, let them develop their crafts. You would soon be proud of them when you see them performing on stage. Your kids would like to become actors and actresses so better hone their talents. As a parent, it is important for you to give your full support to your kids. If there are schools in your area that offer performing arts courses, you better enroll your kids there. If the kids develop to their fullest potential, you will surely see it later on.
What you need to do is to find the best school for sure. Hence, you need to look for some parents who also enrolled their children in performing arts classes. Those parents are your neighbors so you can easily chat them. It is important for you …