WO1990010229A1 - Procede et kit de test de diagnostic pour la detection d'anticorps autoimmun - Google Patents

Procede et kit de test de diagnostic pour la detection d'anticorps autoimmun Download PDF

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Publication number
WO1990010229A1
WO1990010229A1 PCT/US1990/001029 US9001029W WO9010229A1 WO 1990010229 A1 WO1990010229 A1 WO 1990010229A1 US 9001029 W US9001029 W US 9001029W WO 9010229 A1 WO9010229 A1 WO 9010229A1
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coating
antigen
rnp
antibody
sera
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PCT/US1990/001029
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English (en)
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Luis R. Lopez
Marcia Sterhan
Tammy Peterson
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Biostar Medical Products, Inc.
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Publication of WO1990010229A1 publication Critical patent/WO1990010229A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • Antibodies generated by the immune system to diverse tissue and cellular components have been used to diagnose and monitor autoimmune disease activity. These antibodies include anti-dsDNA, anti-RNP (ribonucleoprotein), anti-DNP (Deoxynucleoprotein), anti-Cardiolipin, anti-histones (symptomatic of drug induced lupus), anti-Sm (Smith), anti-E A (extractable nuclear antigen) , which are often found in patients with Systemic Lupus Erythematosus (SLE) , anti-RNP, and anti-ENA are also symptomatic of Mixed Connective Tissue Disease (MCTD).
  • MCTD Mixed Connective Tissue Disease
  • anti-RNA ribonucleic acid
  • anti-Scl-70 Scleroderma-70
  • PSS Progressive Systemic Sclerosis
  • anti-SS-A and B Sjogren's Syndrome Antibody A and B
  • SLE a type of autoimmune disease
  • one of the antibodies produced reacts with DNA that is found widely distributed in cell nuclei in a multitude of body tissues. Formation of antibodies to double-stranded or native deoxyribonucleic acid (anti-dsDNA), is relatively specific to SLE.
  • MCTD Mixed Connective Tissue Disease
  • DIL Drug Induced Lupus
  • Rheumatoid Arthritis Scleroderma, PSS, and Sjogren Syndrome
  • detecting anti-dsDNA is useful in specifically diagnosing SLE. Anti-dsDNA levels correlate well with the disease activity of the patient; thus making it a good monitoring tool.
  • one of the antibodies produced reacts with Sm antigen that is found idely distributed in cell nuclei in a multitude of body tissues. Formation of antibodies to Sm antigen is relatively specific to SLE. Detecting anti-Sm antibodies which are specific for SLE is useful in diagnosing SLE. Anti-Sm antibodies are frequently accompanied by another antibody anti-RNP. Antibodies directed against the RNP antigen are found in the absence of anti-Sm antibodies in MCTD, which is an "overlap syndrome" that combines features of SLE, PSS, and Polymyositis. Therefore, detecting anti-Sm/RNP antibodies is useful in diagnosing two different autoimmune diseases, SLE and MCTD.
  • the RIA technique has been developed in a variety of formats to measure levels of anti-dsDNA in sera.
  • a RIA diagnostic test kit (using the Farr technique) has been developed for commercial sale to clinical laboratories. This test precipitates the bound labelled DNA which is then retained for counting. This test is sensitive to high levels of anti-DNA but has less specificity in lower levels of anti-dsDNA activity, thus resulting in false negatives. Unfortunately, this test has a run time of approximately two hours and fifteen minutes, and a limited stable shelf life.
  • this test kit has the same draw-backs which are inherent in all RIAs; the expense associated with radioactive material, which also have a limited shelf life and can be potentially dangerous.
  • Crithidia luciliae immunofluorescence tests have been developed as diagnostic test kits for the detection of anti-dsDNA.
  • This test kit is based on a staining method associated with the kinetoplast of the protozoan. A positive reaction is demonstrated by detection of specific fluorescence in the kinetoplast of most cells.
  • the kinetoplast, within the protozoan, contains many other components besides dsDNA which can cross react with anti-dsDNA or other antibodies, thus rendering false positive results.
  • This test is extremely subjective, as it is based on an individual's ability to recognize a positive fluorescent pattern.
  • this method which has a run time of approximately two hours, has a lack of sensitivity in low levels of concentration of anti-dsDNA resulting in false negatives.
  • the false negative and false positive results make this method more useful when used together with other tests in clinical practice.
  • the immunological community has developed diagnostic test kits for the determination of anti-dsDNA levels in the standard ELISA format. Although a variety of these kits have been developed, the same limitations of stability, cross reactivity with ssDNA, and length of run time plague each assay.
  • kits claim a low level of cross reactivity with ssDNA which should render good sensitivity to low, medium, and high levels of anti- dsDNA; however, as evidenced by the kit instructions, there is a lack of sensitivity in the sera sample which are borderline positives. As a consequence, these kits cannot be used to accurately monitor patients who are borderline positives. In fact, most kits require that low positives must be retested to assure confidence in the results, thereby increasing the cost to the patient. Development of an assay which is sensitive to all levels of anti-dsDNA can only be achieved by reducing non-specific binding by such components as ssDNA.
  • the immunodiffusion technique has a long incubation time, typically 24 hours or more.
  • the immunological community has developed diagnostic test kits for the determination of anti-Sm antibody levels in the standard ELISA format. These ELISAs have shown certain limitations in stability, cross reactivity, and length of run time.
  • compositions, methods, articles, and a diagnostic test kit for the selective adsorption, or affixing of dsDNA, Sm antigen, or Sm/RNP antigen, or any of the various antigens to the autoimmune antibodies previously listed, to a pre-coated plate for the effective detection of the specific autoimmune antibodies present in sera or plasma. It is a further objective of this invention to overcome the aforementioned sensitivity, subjectivity, stability, and shelf life disadvantages inherent in many of the previously described assays. It is, moreover, also an objective of this invention to provide in the form of a kit a novel, readily utilizable means for quantitative and qualitative detection of anti-dsDNA, anti-Sm or anti-Sm/RNP.
  • the present invention includes novel compositions, novel methods and articles for the direct selective absorption, adsorption or attachment, by whatever mechanism, of the anti-dsDNA, or the anti-Sm or the anti-Sm/RNP antibody, or any of the listed autoimmune antibodies, from the body fluid sample to the dsDNA, the Sm-antigen, or the Sm-RNP antigen, or possibly for any of the autoimmune antigens coated onto any suitable solid support, such as test tubes, plates or wells (hereinafter referred to as wells or microwells), for the purpose of quantitative and qualitative identification.
  • any suitable solid support such as test tubes, plates or wells (hereinafter referred to as wells or microwells)
  • the present invention utilizes immobilized native DNA, Sm, Sm- RNP, or possibly for any listed autoimmune antigen from whatever source, which has an affinity for attachment of anti-dsDNA, anti-Sm, or anti-Sm/RNP, or the other antibodies of the listed autoimmune antibodies.
  • This invention utilizes a sandwich ELISA format which includes pre-coated wells made of any suitable material such as plastic, glass, etc., and the various reagents and antibodies necessary to run a highly sensitive, 45 minute assay for the presence of anti-dsDNA, or for the presence of anti-Sm, or for the presence of anti-Sm/RNP.
  • the various reagents and the method of coating these reagents to the microwells have been employed advantageously in the practice of the present invention to formulate a test which has minimized or eliminated the problems associated with previous assays.
  • the coating protocol has advantageously utilized methylated Bovine Serum Albumin (mBSA) for two main purposes; namely, the mBSA provides a positively charged surface which enhances the adherence of the antigen such as dsDNA, Sm, and Sm/RNP to the polystyrene wells, and second to eliminate the binding of anti-histone antibodies, which can create false positive results.
  • mBSA methylated Bovine Serum Albumin
  • mBSA as a coating prior to the application of DNA antigen for use in solid phase assays for anti-dsDNA was first explored by R. Rubin (J. Immunology 63, 359-366 [1983]).
  • protamine sulfate as well as other functionally equivalent substitutes have been found to be capable of forming a positively charged surface; however, due to the reactivity of these coatings with other components such as ssDN A and anti-histone antibodies, mBSA is preferred.
  • a second factor which contributes to the lack of sensitivity and non-specificity is the cross reactivity. This is particularly a problem when detecting antibodies to dsDNA, because of the cross reactivity between ssDNA and anti-dsDNA.
  • S endonuclease
  • nuclease which is an enzyme specific for the' reakdown degradation of ssDNA and has no affect on dsDNA.
  • the shelf life and the reproducibility of results from the ELISA is associated not only with the mBSA pre-coating, and the S, nuclease treatment (in the anti-dsDNA kit), but also with the next two steps of forming the pre-treated wells; and the addition of a hydrolyzed casein blocker and the drying.
  • a casein-type blocker has been used in various ELISA techniques (Robert F. Bogt; J. Immunological Methods, 101 , 43-50 [1987]) to block non-specific binding to plastic through a protein-plastic interaction. It is an unexpected realization that a coating of hydrolyzed casein blocker maintained a consistent inhibition of non-specific binding over an extended period of time, when the wells were stored at 4 degrees C in a sealed plastic bag.
  • a variety of differing blocking agents could be utilized which are functionally equivalent to or chemically related to the casein blocking agent.
  • BSA and porcine thyroglobulin, dried milk, whole goat serum, etc. the most preferable is the hydrolyzed casein (commercially available from Sigma) due to its high level of inhibition of non-specific binding and its storage stability.
  • the pre-coated wells are then used to detect the presence of anti-dsDNA antibody, anti-Sm antibody, or the anti-Sm/RNP antibody in the sample.
  • the plasma or serum samples are prepared with a sample diluent and are then assayed for their components by an immunoassay technique, the ELISA and the fluorescent immunoassay (FIA) formats being the preferred methods, though it is possible to perform a RIA or a luminescent assay with little modification.
  • the assays depicted in the following examples have an approximate run time of 45 minutes.
  • the wells, when exposed to the samples, are provided with approximately 15 minutes at room temperature to allow the binding of the antibody to the antigen to go to completion.
  • the labelled goat anti-human antibodies are exposed to the wells and a similar 15 minute incubation at room temperature is provided for. If the enzyme is utilized a substrate can be added (although this is not necessary) and 10 minutes is allotted for the production of the color. If a fluorescent marker is used on the goat anti-human antibody then no substrate is needed, therefore the run time is shortened by 10 minutes reducing it to 35 minutes. Subsequent qualitative and quantitative detection of the antibody is relatively simple if the format is either an ELISA or a FIA.
  • the ELISA technique utilizes a spectrophotometer
  • the FIA technique utilizes a microfluorometer. Use of the ELISA techniques were first described by Engvall and Perlman ([1971] Immunochemistry 8, 871-874 and [1972] J.
  • FIG. 3 This graph depicts a standardized curve based on arbitrary READS® units/ml. This curve was generated by using the ELISA format of this invention.
  • Figure 4 This graph depicts the range of negative and positive human sera control values expected for the anti-Sm test kit.
  • Figure 5 This graph depicts a standardized curve based on arbitrary READS ⁇ units/ml. This curve was generated by using the ELISA format of this invention.
  • the Center for Disease Control (CDC) ANA Human Reference Lot #82-001 #5 Serum (for Sm antigen) and the ANA Human Reference Lot #82-011 #4 Serum (for RNP antigen) was run on an immunodiffusion assay with median titer of 1 :64.
  • An equal volume of reference sera #4 and #5 are combined to form the standard.
  • This standard was serially diluted with normal sera and graphed. The 1:1 or neat dilution was assigned arbitrarily a value of 1400 READS units.
  • Figure 6 This graph depicts the range of negative and positive human sera control values expected for the anti-Sm/RNP test kit.
  • FIG. 7 This chart shows autoimmune disease and their associated antigens which could possibly be detected by the present invention.
  • Methylated Bovine Serum Albumin Solution Unless otherwise specified, is intended to mean a solution of 1 milliliter of water or PBS with 20 micrograms of methylated bovine serum
  • mBSA Albumin
  • a substitute for mBSA is protamine sulfate or any other chemical or chemical process capable of producing a slightly positively charged coating which is evenly distributed over the surface of the microtitre well. (It is believed that the anti- histone test may not employ methylated bovine serum coating on the microtiter plate.)
  • PBS Solution A .01 molar solution of buffer containing 1.43 g potassium phosphate, dibasic,
  • dsDNA Solution Purified dsDNA solution from calif thymus was utilized.
  • Alternative sources of DNA include, but are not limited to, native linear DNA from E. Coli, and circular DNA from plasmid, virus, crithidia, and synthetic polynucleotides (poly (dA.dT)).
  • Sm Solution Purified Sm antigen solution from calf thymus was utilized. Alternative source can be employed.
  • dsDNA Casein Blocker Solution 15 milligrams of hydrolyzed casein blocker (Sigma), 2 ml glyercol, 10 grams sucrose was dissolved in TEN buffer sufficient to bring the final volume to 100 mis. The solution is adjusted to pH 7.3 +/- .1.
  • TEN Buffer Is made by adding 6.1 g TRIS, .38 g EDTA, 8.8 g NaCL, 3.8 mL of concentrated
  • Anti-dsDNA Antibody Circulating autoantibodies directed against dsDNA.
  • Anti-Smith Antibody Circulating autoantibodies directed against Sm antigen.
  • Anti-Sm/RNP Antibody Circulating autoantibodies directed against Sm/RNP antigen.
  • Anti-histone Antibody Circulating autoantibodies directed against histone antigen.
  • Anti-DNP Antibody Circulating autoantibodies directed against DNP antigen.
  • Anti-RNA Circulating autoantibodies directed against RNA antigen.
  • Anti-Scl-70 Circulating autoantibodies directed against Scl-70 antigen.
  • Anti-SS-A Circulating autoantibodies directed against SS-A antigen.
  • Anti-SS-B Circulating autoantibodies directed against SS-B antigen.
  • Nuclease An endonuclease (enzyme) which is capable of digestion of ssDNA antigen.
  • Double Antibod y Sandwich ELISA or FIA A solid support is coated with material which detects and binds the antibody of interest to the coated surface. To render a signal, a second conjugated antibody with an affinity for the previously bound antibody is exposed to the coated surface. This antibody binding to the original antibody makes the sandwich. If the sandwich assay is an ELISA then the second antibody is conjugated with an enzyme and substrate is used to produce a color. If the assay is an FIA then the second antibody is marked with a fluorescent tag and a substrate is unnecessary.
  • Buffer for S Nuclease Digestion: 95 mis of acetate/acetic acid buffer, pH 4.6, is mixed with 5 mis glycerol, .29 g NaCL, .029g ZnS04, for a final volume of 100 mis.
  • S Nuclease Buffer Solution: Buffer for S, nuclease digestion plus 100 units (Sigma) S1 nuclease per ml of buffer.
  • One unit of S, nuclease is defined as: Causing 1.0 microgram of ssDNA per minute to become perchloric acid soluble at pH 4.6 and 37 degrees C.
  • Sample Diluent A 1 liter solution has 100 mis of native bovine serum, 1.42 grams of potassium phosphate (dibasic), .26 g of potassium phosphate (monobasic), 1 gram of sodium azide, and 8.6 grams of sodium chloride dissolved in 900 mis of water. If an ELISA is run, then 1 ml of stock green dye is added to the solution. If the FIA format is used then the dye is unnecessary. The solution is then filtered through a .2 micron filter and stored at 4 degrees C.
  • Sample Solution 10 microliters of sera dissolved in 490 microliters of sample diluent.
  • Con j u g ate Diluent A phosphate buffer, and protein stabilizer, plus .02% thimerosal adjusted to a pH of 7.5 (commercially available from Medix Biotech Inc.) into which is added a protease inhibitor, aprotinin, (commercially available from Miles Pentex) at .01% of the volume of the buffer.
  • Working Conjugated Antibody Solution 1 volume of concentrated conjugated antibodies/3000 volumes of conjugate diluent. The dilution is subject to change based on the concentration level of the conjugated antibody (dsDNA).
  • the dilution is subject to change based on the concentration level of the conjugated antibody.
  • ImmunoQlobulin Any member of the gammaglobulin fraction of serum possessing the ability to bind another agent.
  • Antigen Molecules (from whatever source, nature or man-made) which induce an immune reaction when recognized by the host's immune system.
  • Antibody A class of serum proteins which specifically bind to an antigen which induced the 15 formation of the antibody.
  • Immunoolobulin Classes Antibodies separated by electrophorectic mobility specifically, IgG and IgM.
  • Substrate Solution To quantitate the horseradish peroxidase, 100 microliters of buffered (3,3',5,5') Tetramethylbenzidine/ hydrogen peroxide (commercially available from Kirkegaard 20 Perry) was used.
  • Labelled Antigens Any antigen substance which has been covalently or otherwise combined with a molecule or ion for the purpose of selectively identifying that group of antigens.
  • adduct molecules or ions include enzymes, fluorescent substances, radionuclides, and the like.
  • the preferred embodiment of the method and apparatus for the detection of anti-dsDNA in sera is a diagnostic test kit.
  • the optimized kit contains:
  • TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
  • TMB Substrate Solution B - contains hydrogen peroxide.
  • Stop Reagent contains 2.5 N H2SO4. (1.0 N HC1 can be used as a substitute)
  • PBS Phosphate Buffered Saline
  • This kit for the measurement of anti-dsDNA in serum samples has been designed for use in a clinical laboratory. To determine the AU/ml of the anti-dsDNA present in the sample the kit includes calibrators and controls for generating a standard curve. The selection of the 5 levels of anti-dsDNA activity in these controls and calibrators can be varied without affecting performance of the assay.
  • kits with pre-coated test wells and reagents which produce low levels of variation between assays and within assays. Furthermore, these reagents and test wells are stable for extended periods of time.
  • This kit has been optimized 10 to have a 45 minute run time at room temperature, which is significantly shorter than other test kits, presently marketed for anti-dsDNA detection.
  • mBSA mBSA
  • PBS distilled water or PBS at a ratio of 20 ug/ml.
  • An aliquot of 100 micrograms of this prepared solution is placed in each microwell, to produce a surface which is slightly positively charged.
  • the mBSA serves two important functions; one is to provide a stable evenly coated microwell surface, and second is to inhibit non-specific binding such
  • Unbound mBSA solution is shaken from the wells, and they are rinsed with PBS solution, pH 7.3, and drained thoroughly.
  • the ligand or antigen is prepared and exposed to the receiving surfaces of the
  • dsDNA (calf thymus) is dissolved into .01 M PBS, pH 7.4 in a ratio of 5 micrograms/ml (weight per volume). The buffered ligand is then dispensed into each microwell, 100 microliters of buffered ligand per well. The binding is enhanced by an incubation period of 18-24 hours at 4 degrees C. The excess solution is shaken from the wells.
  • a buffer is prepared for S, nuclease digestion.
  • .03M acetic acid was titrated together with .03M sodium acetate in a ratio of 1.1 part acetic acid to 1 part sodium acetate to a final pH of 4.6.
  • 95 mis of the prepared acetate/acetic acid buffer is mixed with 5 mis of glycerol, .29 g NaC1 , .029 g ZnS04, for a final volume of 100 mis.
  • S nuclease
  • S nuclease buffered solution which is dispensed in 100 microliter increments into each individual receiving well.
  • 10 units of S nuclease is contacted with each individual well.
  • This solution is of sufficient concentration to digest the unwanted ssDNA in the previously coated ligand material after a two hour incubation period at 37 degrees C.
  • S nuclease eliminates ssDNA which is a source of false positives in many assays.
  • the wells are inverted to remove excess solution, then the wells are thoroughly rinsed twice with PBS solution. The wells are again inverted on a paper towel and allowed to drain.
  • the next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots of 200 microliters. This step decreases the non-specific binding that can occur due to protein-plastic interaction.
  • the hydrolyzed casein used in the blocker solution can be commercially obtained from Sigma.
  • the casein blocker solution is prepared by mixing 2 ml glycerol, 10 g sucrose, and 15 mg of hydrolyzed casein, and adding sufficient TEN buffer to make 100 ml of solution.
  • the wells containing casein blocker solution are again incubated at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15 minutes. Then the wells are uprighted and allowed to dry at room temperature for at least 24 hours.
  • Step 2 Adhering Anti-dsDNA to the Prepared Wells From Step 1 :
  • the sample diluent is supplied in the kit as a 30 ml green solution.
  • 100 milliliters of native bovine serum, 1.42 g of Potassium Phosphate (dibasic), .26 g of Potassium Phosphate (monobasic), 1 gram of sodium azide, and 8.6 g of sodium chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1000 milliliters of solution. This solution is then filtered through a .2 micron filter.
  • the Sample Diluent acts as a blocking agent similar to the casein blocker which was previously coated on the wells.
  • the principle blocking component in the Sample Diluent is the native bovine serum which acts to inhibit the binding of any BSA-reactive antibodies to the mBSA coating on the surface of the well.
  • the serum Prior to contacting the body fluid with the prepared plate, the serum is diluted by adding aliquots of sera to the sample diluent in a 1:50 ratio (volume of serum:volume of sample diluent), although this exact dilution is not critical and depends upon the nature of the body fluid and the assay techniques employed.
  • the body fluid is aliquoted in 10 microliter proportions into 500 microliters of Sample Diluent. In an individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the anti-dsDNA is enhanced by 15 minutes incubation at room temperature.
  • the wells are thoroughly washed four times with PBS to remove the free unbound antibodies that are present in the sample, which if allowed to remain, would elevate the background absorbance.
  • Step 3 Assay for the Anti-dsDNA Affixed to the Wells: Standard enzyme-linked immunoassay techniques, previously described, are used forthis assay, although any suitable means of detection such as radioactive labeling, fluorescence, or the like can be employed.
  • anti-human IgG and anti-human IgM induced in goats were used to ascertain whether anti-dsDNA IgG and IgM antibodies were present. Please note that other species of animal can be used to produce anti-human antibodies..
  • These antisera were linked to horseradish peroxidase, an enzyme which yields a colored product whenever one of its substrates is present together with hydrogen peroxide.
  • the substrate should be chosen to be consistent with the enzyme conjugated to the antibody.
  • the substrate was (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
  • the kit contains one 15 ml vial of conjugated antibody solution with anti-human IgM and IgG antibody conjugated to horseradish peroxidase.
  • a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4 (commercially available from Medix) was mixed with aprotinin, a protease inhibitor (commercially available from Miles Pentex) at a .01% ratio of inhibitor to volume of buffer.
  • microtiter well (prepared as described) is added to each microtiter well. Binding of these antibodies to the anti-dsDNA is permitted for at least 15 minutes at room temperature, then the microtiter wells are emptied of their contents, washed four times with PBS, and allowed to drain before the next step.
  • the presence of a label and antibody is determined by incubating the wells with a solution of buffered (3,3' ,5,5') Tetramethylbenzidine and hydrogen peroxide.
  • This solution is supplied in the kit in two 8 ml vials; one contains (3,3' ,5,5') Tetramethylbenzidine; the other vial contains hydrogen peroxide.
  • the separate vials are necessary due to the interaction between the two solutions.
  • the two solutions are mixed in a one to one ratio just before use, and 100 microliters of the mixed solution is dispensed into each microwell.
  • the reaction is permitted to continue for 10 minutes at room temperature, or until sufficient color appears to be read on the spectrophotometric device used.
  • the reaction is subsequently stopped through the addition of an equal volume of 2.5 normal sulfuric acid, and the intensity of color (the optical density, "OD", or absorbance) is read by a spectrophotometric device such as a Dynatech MR600 or the like.
  • the resultant color of the reaction product is proportional to the number of conjugated antibodies which have bound to the anti-dsDNA.
  • the number of bound conjugated antibodies is linearly related to the number of anti-dsDNA antibodies. Hence, as the amount of anti-dsDNA bound to the wells increases, so does the optical density, or absorbance of the enzyme reaction.
  • This preferred embodiment of the method and apparatus for the detection of anti-Sm antibodies in a sera is a diagnostic test kit.
  • the preferred embodiment of the method and apparatus for the detection of anti-Sm/RNP antibodies in sera is a diagnostic test kit, with the following components (qualitative format).
  • TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
  • TMB Substrate Solution B - contains hydrogen peroxide.
  • Stop Reagent contains 2.5 N H2S04. (1.0 N HC1 can be used as a 10 substitute) (not supplied).
  • PBS Phosphate Buffered Saline
  • This kit for the measurement of anti-Sm antibodies in serum samples has been designed for use in a clinical laboratory.
  • the anti-Sm/RNP antibodies present in the sample kit can include calibrators and controls for generating a standard curve. The selection of the levels of anti-Sm or anti-Sm/RNP antibody activity in these controls and calibrators can be varied without affecting performance of the assay.
  • Unbound mBSA solution is shaken from the wells and drained thoroughly.
  • the ligand or antigen is prepared and exposed to the receiving surfaces of the microwells.
  • Purified Sm antigen or the purified Sm/RNP antigen (calf thymus) is dissolved into .01 M PBS, pH 7.4 in a ratio of 5 units/ml, and for Sm/RNP 1 unit/ml.
  • the buffered ligand is then dispensed into each microwell, 100 microliters of buffered ligand per well. The binding is enhanced by an incubation period of 18-24 hours at 4 degrees C.
  • the next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots of 200 microliters. This step decreases the non-specific binding that can occur due to protein-plastic interaction.
  • the hydrolyzed casein used in the blocker solution can be commercially obtained from Sigma.
  • the casein blocker solution is prepared by mixing 2 ml glycerol, 10 g sucrose, and 25 mg of hydrolyzed casein, and adding sufficient TEN buffer to make 100 ml of solution.
  • the wells containing casein blocker solution are again incubated at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15 minutes. Then the wells are uprighted and allowed to dry at room temperature for at least 24 hours.
  • Step 2 Adhering Anti-Sm or Anti-Sm/RNP antibodies to the Prepared Wells From Step 1 :
  • the sample diluent is supplied in the kit as a 40 ml green solution.
  • 100 milliliters of native bovine serum, 1.42 g of Potassium Phosphate (dibasic), .26 g of Potassium Phosphate (monobasic), 1 gram of sodium azide, and 8.6 g of sodium chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1000 milliliters of solution. This solution is then filtered through a .2 micron filter.
  • the serum Prior to contacting the body fluid with the prepared plate, the serum is diluted by adding aliquots of sera to the sample diluent in a 1 :50 ratio (volume of serum:volume of sample diluent), although this exact dilution is not critical and depends upon the nature of the body fluid and the assay techniques employed.
  • the body fluid is aiiquoted in 10 microliter proportions into 490 microliters of Sample Diluent. In an individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the anti-Sm or anti-Sm/RNP antibody is enhanced by 15 minutes incubation at room temperature.
  • the kit contains one 12 ml vial of conjugated antibody solution with anti-human IgM and IgG antibody conjugated to horseradish peroxidase.
  • a working conjugated antibody solution a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4
  • the solution is mixed at a ratio of 1/4000 for IgG and 1/1500 for IgM; one part of concentrated conjugated IgM and IgG antibodies is aiiquoted into 4000 and 1500 parts conjugate diluent, respectively.
  • the ratio of concentrated conjugated antibody to conjugate diluent is subject to wide latitudes of dilutions based on the manufacturer's concentration of conjugated antibody used in the assay.
  • the presence of a label and antibody is determined by incubating the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
  • This solution is supplied in the kit in two 8 ml vials; one contains (3,3',5,5') Tetramethylbenzidine; the other vial contains hydrogen peroxide.
  • the separate vials are necessary due to the interaction between the two solutions.
  • the two solutions are mixed in a one to one ratio just before use, and 100 microliters of the mixed solution is dispensed into each microwell.
  • the reaction is permitted to continue for 10 minutes at room temperature, or until sufficient color appears to be read on the spectrophotometric device used.
  • the reaction is subsequently stopped through the addition of an equal volume of 2.5 normal sulfuric acid, and the intensity of color (the optical density, OD", or absorbance) is read by a spectrophotometric device such as a Dynatech MR600 or the like.
  • the resultant color of the reaction product is proportional to the number of conjugated antibodies which have bound to the antibody.
  • the number of bound conjugated antibodies is linearly related to the number of bound antibodies.
  • dsDNA double stranded deoxyribonucleic acid
  • Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolved at a ratio of 20 micrograms/ml in distilled water or PBS.
  • S-1 nuclease buffer solution .03M sodium acetate and .03M acetic acid in a 1.1 to 1 ratio at pH 4.6 was prepared. Then 95 mis of this buffer was mixed with 5 mis
  • Casein blocker solution (pH 7.3) was dispensed in 200 microliter increments into each well.
  • the casein blocker solution consists of 2 ml glycerol, 10 g sucrose, 15 mg casein and sufficient TEN buffer to bring the volume to 100 ml.
  • the Sample Diluent was a solution consisting of 100 mis of native bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate (monobasic), 8.6 g sodium chloride, .1% sodium azide, 1 ml stock green dye, and distilled water added to bring the volume to 1000 mis.
  • the Sample Diluent was then filtered through a .2 micron filter.
  • Each well was assayed for horseradish peroxidase activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and dispensing 100 microliters of this substrate solution into each well.
  • the presence of the anti-dsDNA was detected by a blue color appearing after the 10 minute room temperature incubation.
  • 100 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color.
  • the yellow color was quantitated at 450 nm using a Dynatech MR600 plate reading spectrophotometer.
  • polystyrene wells were left totally uncoated but exposed to the sera and the standards as described in the previous example.
  • the wells were then exposed to horseradish peroxide conjugated IgG and IgM antibodies, and assayed for peroxidase activity.
  • IgG and IgM antibodies horseradish peroxide conjugated IgG and IgM antibodies
  • the kit contains pre-coated microwells and,
  • PBS Phosphate Buffered Saline
  • the various reagents of the kit were utilized to perform the assay.
  • the directions for the method of assaying for anti-dsDNA were included in the kit and these directions were followed precisely during this experiment.
  • the plate templates were labelled for sample placement in the microwells.
  • a 1 :50 dilution of the standards, controls and patient samples was prepared in Sample Diluent (green solution). 10 ul of sample was added to 500 ul sample diluent in a one volume to 50 volume sample dilution. 1 :50 dilutions of the assay standards and controls were diluted in the same manner.
  • the working substrate solution was prepared just before use, according to the kit instructions. Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate. The kit instructed that if properly combined this substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless working substrate solution was added to each well and the wells were incubated for ten minutes at room temperature.
  • Polystyrene wells were coated with Sm antigen by the following procedure:
  • Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolved at a ratio of 20 micrograms/ml in distilled water or PBS.
  • Casein blocker solution (pH 7.3) was dispensed in 200 microliter increments into each 15 well.
  • the casein blocker solution consists of 2 ml glycerol, 10 g sucrose, 25 mg casein and sufficient TEN buffer to bring the volume to 100 ml.
  • the coated Sm antigen wells were then used to determine the presence of anti-Sm antibody in seven CDC reference sera for anti-nuclear antibodies. The results show that only anti-Sm antibodies react in the assay.
  • the Sample Diluent was a solution consisting of 100 mis of native bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate (monobasic), 8.6 g sodium chloride, .1% sodium azide, 1 ml stock green dye, and distilled water added to bring the volume to 1000 mis.
  • the Sample Diluent was then filtered through a .2 micron filter.
  • the wells were exposed to a working conjugated antibody solution consisting of 1 part per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01% by volume a protease inhibitor (commercially available from Miles Pentex).
  • a working conjugated antibody solution consisting of 1 part per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01% by volume a protease inhibitor (commercially available from Miles Pentex).
  • Each well was assayed for horseradish peroxidase activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and dispensing 100 microliters of this substrate solution into each well.
  • the presence of the anti-Sm antibodies was detected by a blue color appearing after the 10 minute room temperature incubation.
  • 100 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color.
  • the yellow color was quantitated at 450 nm using a Dynatech MR600 plate reading spectrophotometer.
  • the mean absorbance reading of the reagent blank should be less than 0.05. Readings greater than 0.05 may indicate possible contaminated of the Substrate Solution.
  • the mean absorbance (O.D.) of negative control x 3.2 negative cut-off absorbance, which is equal to 20 READS units of anti-Sm activity. Samples with P.D. values below the cut-off have less than 20 READS units of anti-Sm activity, and should be scored as "negative”. Samples with P.D. values equal to or higher than the cut-off have greater than 20 READS units anti-Sm activity, and should be scored as "positive”. Samples with P.D. values close to the cut ⁇ off may warrant further testing (see Figure 4).
  • the cut-off P.D. was determined by running 200 normal sera samples and taking the mean of the P.D.s plus three standard deviations. This cut-off was equal to 3.2 x the P.D. of the standardized negative control.
  • the kit contains pre-coated microwells (qualitative format) and,
  • PBS Phosphate Buffered Saline
  • the various reagents of the kit were utilized to perform the assay.
  • the directions for the method of assaying for anti-Sm antibodies were included in the kit and these directions were followed precisely during this experiment.
  • the plate templates were labelled for sample placement in the microwells.
  • a 1 :50 dilution of the controls and patient samples was prepared in Sample Diluent (green solution) (no calibrators were used in this example). 10 ul of sample was added to 490 ul sample diluent in a one volume to 50 volume sample dilution.
  • the working substrate solution was prepared just before use, according to the kit instructions.
  • TMB Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate.
  • the kit instructed that if properly combined this substrate solution would be colorless and it was colorless.
  • a 100 ul of the colorless working substrate solution was added to each well and the wells were incubated for ten minutes at room temperature.
  • the sample P.D. is compared to the cut-off P.D. value calculated for the assay (see Figure 4). For semi- quantitative results the P.D. value can be converted to READS units by multiplying the sample
  • 6.2 READS units is the anti- Sm activity of the negative control, which was standardized against the CDC reference serum.
  • the cut-off is 20 READS units. o-.r. oBid ctm ⁇ iet u.u. READS Units
  • the resulting O.D.s were obtained.
  • the cut-off value for the present invention was 0.33 P.D.
  • the cut-off value of the competitive kit was the given by the kit instructions.
  • Polystyrene wells were coated with Sm/RNP antigen by the following procedure:
  • Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolved at a ratio of 20 micrograms/ml in distilled water or PBS.
  • Casein blocker solution (pH 7.3) was dispensed in 200 microliter increments into each well.
  • the casein blocker solution consists of 2 ml glycerol, 10 g sucrose, 25 mg casein and sufficient TEN buffer to bring the volume to 100 ml.
  • the wells were exposed to a working conjugated antibody solution consisting of 1 part per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01% by volume a protease inhibitor (commercially available from Miles Pentex).
  • a working conjugated antibody solution consisting of 1 part per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01% by volume a protease inhibitor (commercially available from Miles Pentex).
  • Each well was assayed for horseradish peroxidase activity by mixing equal volumes (3,3', 5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and dispensing 100 microliters of this substrate solution into each well.
  • the presence of the anti-Sm/RNP antibodies was detected by a blue color appearing after the 10 minute room temperature incubation.
  • 100 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color.
  • the yellow color was quantitated at 450 nm using a Dynatech MR600 plate reading spectrophotometer.
  • the mean absorbance reading of the reagent blank should be less than 0.05. Readings greater than 0.05 may indicate possible contamination of the substrate solution.
  • the mean absorbance (O.D.) of negative control x 2.8 negative cut-off absorbance, which is equal to 17.5 READS units of anti-SM/RNP activity.
  • Samples with O.D. values below the cut-off have less than 17.5 READS units of anti-SM/RNP activity, and should be scored as "negative”.
  • O.D. values equal to or higher than the cut-off have greater than 17.5 READS units anti-SM/RNP activity, and should be scored as "positive". Samples with O.D. values very close to the cut-off may warrant further testing (see Figure 6) .
  • the kit contains pre-coated microwells and,
  • PBS Phosphate Buffered Saline
  • the various reagents of the kit were utilized to perform the assay.
  • the directions for the method of assaying for anti-Sm/RNP antibodies were included in the kit and these directions were followed precisely during this experiment.
  • the plate templates were labelled for sample placement in the microwells.
  • a 1 :50 dilution of the controls and SLE patient samples was prepared in Sample Diluent (green solution). 10 ul of sample was added to 500 ul sample diluent in a one volume to 50 volume sample dilution.
  • the working substrate solution was prepared just before use, according to the kit instructions.
  • TMB Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate.
  • the kit instructed that if properly combined this substrate solution would be colorless and it was colorless.
  • a 100 ul of the colorless working substrate solution was added to each well and the wells were incubated for ten minutes at room temperature.
  • test kit was used in accordance with the protocol shown in Example 10.
  • Nine CDC reference for nuclear antigen were tested. All were negative except for the anti-Sm and anti-RNP sera which were positive.
  • Sm/RNP antigen coated microwells were treated with the optimized protocol, then duplicate microwells were stored at 37 degrees C and 4 degrees C, and each set of wells was tested at on day 1, 4, 6, 8, and 12, using a high anti-Sm/RNP sera sample, a moderate anti- Sm/RNP sera sample, and a negative anti-Sm/RNP sera sample.
  • the results of this experiment are shown in the following table:
  • the blocker utilized in the preferred embodiment of this invention to stabilize the shelf life and eliminate non-specific binding cannot be limited to the compounds or the ingredients or the concentrations thereof listed in the definitional section.
  • a variety of functional equivalent blocking agents are known to those skilled in the art. A partial listing of some materials which could be utilized to perform a similar function is found in (Robert F. Bogt; J. Immunological Methods, 101, 43-50 [1987]) and is hereby incorporated herein by reference.
  • a third element in the anti-dsDNA test kit which plays a function in eliminating non ⁇ specific binding is the S 1 nuclease used to degrade the ssDNA and thus to avoid any cross reactivity between the anti-dsDNA and the undesired ssDNA.
  • the described method of limiting cross reactivity is not limited to the definition given but the method can be performed with varying concentrations of S, nuclease or other similar, endonuclease enzymes, or similar functional equivalents which are capable of eliminating problems of cross reactivity without adversely affecting the antigen present in the invention.
  • the treatment of the solid support which can be any of a variety of formats, i.e. test tubes, plates, wells, etc., made of various suitable materials, i.e. glass, plastics, etc. with the aforementioned technology affords many important and useful approaches to the detection of the specific antibodies.
  • the detection of antibodies need not be limited to the conjugation of enzymes. Addition of fluorescent chemicals such as fluorescence or the like to the antibody will impart fluorescence to the assay if the antibody is present. Similarly, conjugation of the antibody with a radionuclide will impart radioactivity to the assay if the antibodies are present in the assay. Many other methods of detection of antibodies also exist, and these methods will yield positive results provided that the antibody exists in the assayed sera and is affixed according to the methods described herein.
  • test kit and the underlying coating and detection methods herebefore described are not intended to be limited by the assay format described or by the volumes or the concentrations or specific ingredients given for the various reagents, controls, and calibrators. It should be understood that similar chemical equivalents or other functional equivalents of the components found in the coatings, or in any of the various reagents, controls, and calibrators can be utilized within the scope of this invention.

Abstract

On a mis au point un procédé et un appareil optimisés se présentant sous la forme d'un kit de test d'analyse en sandwich, de diagnostic autoimmun, capable d'immobiliser un antigène sur des puits de support pré-enduits, permettant la détection d'anticorps autoimmuns spécifiques auxdits composés, à l'aide de techniques soit ELISA (analyse d'immunosorbant à liaison enzymatique) soit FIA (immunoanalyse fluorescente).
PCT/US1990/001029 1989-02-27 1990-02-26 Procede et kit de test de diagnostic pour la detection d'anticorps autoimmun WO1990010229A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013276A1 (fr) * 1991-01-22 1992-08-06 Akzo N.V. Procede pour la detection d'anticorps anti-arn
WO1995032430A2 (fr) * 1994-05-25 1995-11-30 Oklahoma Medical Research Foundation Analyse du pouvoir pathogene d'anticorps anti-adn
AU676683B2 (en) * 1992-07-08 1997-03-20 Vincent Agnello Chemiluminescent assay for dsDNA antibodies
US6280944B1 (en) 1994-05-25 2001-08-28 Oklahoma Medical Research Foundation Assay for pathogenicity of anti-DNA antibodies
EP1130398A2 (fr) * 2000-03-02 2001-09-05 Ortho-Clinical Diagnostics, Inc. Elements analytiques
CN113917136A (zh) * 2021-10-18 2022-01-11 北京和杰创新生物医学科技有限公司 抗双链脱氧核糖核酸抗体免疫球蛋白g的检测方法

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CN103575907B (zh) * 2013-10-30 2015-12-02 浙江理工大学 一种古代胶结剂文物材料中卵蛋白的检测方法

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US4784942A (en) * 1984-11-05 1988-11-15 The Board Of Regents For The University Of Oklahoma Monoclonal antibodies against autoimmune RNA proteins

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WO1990010227A1 (fr) * 1989-02-27 1990-09-07 Biostar Medical Products, Inc. Procede et kit de test de diagnostic pour la detection d'anti-cardiolipine

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Clinical and Experimental Immunology, Volume 62 issued 1985, S. LOIZOU, et al. "Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results", 738-745, see the Abstract. *
Clinical and Experimental Rheumatology, Volume 5, issued 1987, A.G. TZIOUFAS, et al. "Enzyme immunoassays for the detection of Ig G and Ig M anti-ds DNA antibodies: clinical significance and specificity", 247-253, see entire document. *
Journal of Immunological Methods, Volume 101 issued 1987, R.F. VOGT, JR. et al., "Quantitative differences among various proteins as blocking agents for ELISA microtier plates", 43-50. See the Abstract. *
Journal of Immunological Methods, Volume 63, issued 1983, R.L. RUBIN et al. "An improved ELISA for anti-native DNA by elimination of interference by anti-histone antibodies" 359-366, see materials and methods section. *
See also references of EP0460102A4 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013276A1 (fr) * 1991-01-22 1992-08-06 Akzo N.V. Procede pour la detection d'anticorps anti-arn
US5385824A (en) * 1991-01-22 1995-01-31 Akzo N.V. Method for detection of anti-RNA-antibodies
AU676683B2 (en) * 1992-07-08 1997-03-20 Vincent Agnello Chemiluminescent assay for dsDNA antibodies
WO1995032430A2 (fr) * 1994-05-25 1995-11-30 Oklahoma Medical Research Foundation Analyse du pouvoir pathogene d'anticorps anti-adn
WO1995032430A3 (fr) * 1994-05-25 1996-03-07 Oklahoma Med Res Found Analyse du pouvoir pathogene d'anticorps anti-adn
US5681700A (en) * 1994-05-25 1997-10-28 Oklahoma Medical Research Foundation Assay for pathogenicity of anti-DNA antibodies
US6280944B1 (en) 1994-05-25 2001-08-28 Oklahoma Medical Research Foundation Assay for pathogenicity of anti-DNA antibodies
EP1130398A2 (fr) * 2000-03-02 2001-09-05 Ortho-Clinical Diagnostics, Inc. Elements analytiques
EP1130398A3 (fr) * 2000-03-02 2002-07-24 Ortho-Clinical Diagnostics, Inc. Elements analytiques
US6620628B2 (en) 2000-03-02 2003-09-16 Ortho-Clinical Diagnostics, Inc. Analytical elements having a surface charge
CN113917136A (zh) * 2021-10-18 2022-01-11 北京和杰创新生物医学科技有限公司 抗双链脱氧核糖核酸抗体免疫球蛋白g的检测方法

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EP0460102A1 (fr) 1991-12-11
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CA2050340A1 (fr) 1990-08-28

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