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WO1990010227A1 - Procede et kit de test de diagnostic pour la detection d'anti-cardiolipine - Google Patents

Procede et kit de test de diagnostic pour la detection d'anti-cardiolipine Download PDF

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Publication number
WO1990010227A1
WO1990010227A1 PCT/US1990/001028 US9001028W WO9010227A1 WO 1990010227 A1 WO1990010227 A1 WO 1990010227A1 US 9001028 W US9001028 W US 9001028W WO 9010227 A1 WO9010227 A1 WO 9010227A1
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WIPO (PCT)
Prior art keywords
cardiolipin
solution
antibodies
wells
coating
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PCT/US1990/001028
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English (en)
Inventor
Marcia Sterhan
Luis R. Lopez
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Biostar Medical Products, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biostar Medical Products, Inc. filed Critical Biostar Medical Products, Inc.
Publication of WO1990010227A1 publication Critical patent/WO1990010227A1/fr

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    • 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
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the first anti-phospholipids were detected during the World War II mass screening for syphilis.
  • SLE systemic lupus erythematosus
  • the test for syphilis yielded a false positive.
  • This false positive test for syphilis eventually led to the discovery of the three types of anti-phospholipid antibodies responsible for biological false positive reactions (BFP) : VDRL (Venereal Disease Research Laboratory) , lupus anti-coagulant, and anti-cardiolipin antibodies.
  • ACL levels should be monitored in SLE patients undergoing anti-coagulant therapy, in patients with previous thrombosis, in patients under 45 who have had myocardial infarctions, in patients with venous thrombosis, or placental infarctions or recurrent incidence of intra-uterine death and spontaneous abortions, and in patients electing to take oral contraceptives to determine the status of their aCL levels.
  • SLE patients undergoing anti-coagulant therapy in patients with previous thrombosis, in patients under 45 who have had myocardial infarctions, in patients with venous thrombosis, or placental infarctions or recurrent incidence of intra-uterine death and spontaneous abortions, and in patients electing to take oral contraceptives to determine the status of their aCL levels.
  • the development of a stable, reproducible anti-cardiolipin antibody test has become an international project.
  • aCL tests At least three types of (aCL) tests have been developed: a diagnostic kit based on the ELISA method (by Cheshire Diagnostics, Cheshire, UK) , a solid phase radioimmunoassay method (Harris, et. al., The Lancet, 1211-1214, [Nov. 26, 1983]), and a sandwich ELISA technique (E.N. Harris, et. al., Clin. Exp. Immunol. 68, 215-225 [1987]).
  • Anti-phospholipid syndrome can cause a patient to have a high level of IgG aCL alone, or a high level of IgM aCL; therefore, these methods are designed to measure the concentration of both IgG and IgM anti-cardiolipins.
  • RIA techniques have been developed for anti- cardiolipin concentration measurements. RIAs provide sensitive and accurate measurements of high and medium .levels of IgM and IgG concentrations. However, RIAs are plagued with the hazards and expense associated with radioactive material.
  • One specific RIA develped by E.N. Harris for the detection of anticardiolipin antibodies is 400 times more sensitive than the preceding test, which was the precipitation method used in the Venereal Disease Reference Laboratory test. Although this RIA was more sensitive than the previous test, it unfortunately had drawbacks of its own. The run-time of this test is twenty-two hours, which makes this test impractical in clinical laboratories.
  • An ELISA for aCL has been produced as a diagnostic kit by Cheshire Diagnostics Limited.
  • a disadvantage is that this test kit has a shelf life of only 12 weeks from the date of manufacture, and problems with variations in results, as evidenced by the kits Interpretation of Results section which requires a repeat of tests with results in the IgG and IgM bordering positive range.
  • repeat tests must be run increasing the cost to the patient and increasing the work time expended by lab personnel.
  • the test kit's run time of 2 1/2 hours is substantially less than most other aCL assays. Development of a stable, speedy ELISA capable of accurately measuring low levels of anti-cardiolipin antibodies is essential to surmount the problems of the prior methods.
  • compositions, methods, and articles for the detection of aCL antibodies at low, medium, and high levels of concentration It is the further objective of this invention to overcome the aforementioned length of run time, and stability and sensitivity disadvantages inherent in other methods of detection of anti-cardiolipin antibodies. It is furthermore an objective of this invention to provide a novel, highly adaptable, readily utilizable means for quantitative detection of anti-cardiolipins, and further to provide test kits which use such a technique for the purpose of clinical detection, or any other purpose associated with human or animal medical testing.
  • the present invention provides novel compositions and novel methods for performing a sandwich assay for .the detection of IgM or IgG (aCL) antibody, for the purpose of identification. 90/10227
  • the present invention utilizes in its broadest sense, anti-phospholids antibodies, which have a particular affinity for association with cardiolipin.
  • the anti-cardiolipin antibodies in human serum are employed to directly affix to the pre-coated cardiolipin, and furthermore are specifically selected to have an affinity for an enzyme conjugated goat anti-human IgM, or an enzyme conjugated goat anti-human IgG antibody, which produce, through enzymatic action with the substrate, a colored by-product which is detectable and quantitatable using standard photometric instrumentation.
  • This color change which is generally measured as optical density, is in direct relationship to the concentration of IgG or IgM present in the sample solution.
  • the anti-human antibody can be obtained from a variety of differing animal species.
  • the REAADS R test kit is a sandwich ELISA that employs pre- coated suitable solid support, such as test tubes, plates or wells (hereinafter referred to as wells or microwells) .
  • suitable solid support such as test tubes, plates or wells (hereinafter referred to as wells or microwells) .
  • the coating which is advantageously utilized to allow adherence of the cardiolipin to the side walls and to the bottom of the wells is methylated bovine serum albumin (mBSA) .
  • mBSA methylated bovine serum albumin
  • the mBSA provides a positively charged surface which enhances the adherence of the cardiolipin to all surface areas of the well.
  • methylated bovine serum preparations have frequently been utilized in anti-DNA ELISA methods, it is unique and highly surprising to find that a coating of methylated bovine serum on a solid support affixes cardiolipin in an even concentration throughout the well without the typical problem of the formation of globules of cardiolipin on the bottom of the well.
  • protamine sulfate as well as functionally equivalent substitutes have been found to be capable of forming a positively charged surface; however, due to cross reactivity of some of these substrates, mBSA is the preferred coating.
  • the utilization of a methylated bovine serum underlying the cardiolipin antigen coating provides for pre-treated wells which have a shelf life of six months to one year; furthermore, the wells also provide a high level of reproducibility of results.
  • a variety of differing blocking agents could be utilized which are functionally equivalent to or chemically related to the casein blocking agent; for example, BSA and porcine thyroglobulin, dried milk, whole goat serum, etc. , however the most preferable is the hydrolyzed casein (commercially available by Sigma) due to its high level of inhibition of non-specific binding and its storage stability.
  • the pre-treated wells are then used to detect the presence of anti-cardiolipin antibody in the samples.
  • the plasma or serum samples are prepared with a sample diluent and are then assayed 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.
  • 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 to allow the binding process 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. If the enzyme format is utilized, the substrate is added and 10 minutes is allotted for the production of the color. If a fluorescent marker was used on the anti-human antibody then no substrate is needed, therefore the run time is shortened by 10 minutes reducing it to 35 minutes.
  • Methylated Bovine Serum Albumin Solution Unless otherwise specified, is intended to mean a solution with 20 micrograms of methylated bovine serum albumin (mBSA) dissolved in water at a ratio of 1 ml of distilled water to 20 micrograms of (mBSA) .
  • mBSA methylated bovine serum 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.
  • PBS Solution A .01 molar solution of buffer containing 1.43 g potassium phosphate, dibasic, .25 g potassium phosphate, monobasic, and 8.5 g sodium chloride in one liter of water.
  • the pH is 7.3 +/- •!•
  • Cardiolipin from beef heart
  • Purified cardiolipin (Sigma) is dissolved in 100% denatured ethanol at 20 micrograms cardiolipin per ml of solution.
  • Alternative sources of antigen includes, but are not limited to, phosphatidylserine, phos-phatidylcholine, phosphatidylethano- lamine, phosphatidylglycerol, phosphatidylinositol.
  • Casein Blocker Solution 2 ml glyercol, 10 grams sucrose and 15 milligrams of hydrolyzed casein added to TEN buffer for a total volume of 100 ml. Adjust pH to 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 HCL to 900 ml deionized water. Adjust pH to 7.3 and add deionized water sufficient to give 1000 milliliter total volume.
  • Anti-phospholipid Antibody Circulating autoantibodies directed against complex lipid antigens such as cardiolipin.
  • Double Antibody Sandwich ELISA or FIA An assay utilizing a solid support that 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. The binding of the conjugated antibody to the original antibody makes the sandwich. If the sandwich assay is an ELISA then the second antibody is conjugated with an enzyme and a 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.
  • Serum Is intended to mean the fluidic component of any body fluid remaining after cells and coagulable proteins such as fibrin which may be present in such body fluidic components have been removed by appropriate physical, chemical, or physicochemical means. Typically, this term refers to the residual watery fluid remaining after clotting of blood and removal of the clot, but in its broad sense is intended to include the fluidic component of cerebrospinal fluid, urine, interstitial fluid, cellular cytoplasm, and the like.
  • Sample Diluent A liter solution containing 100 mis of native bovine serum, 1.42 g of potassium phosphate (dibasic), .26 g of potassium phosphate (monobasic) , 1 g of sodium azide, and 8.6 g of sodium chloride dissolved in 900 mis of water. 1 ml of stock green dye is added to the solution, if a FIA format is used then the dye is unnecessary. The solution is then filtered through a .2 micron filter.
  • Sample Diluent Solution 10 microliters of sera or plasma dissolved in 500 microliters of sample diluent.
  • Conjugate Diluent A phosphate buffer, and protein stabilizer, plus .02% thimerosal adjusted to a pH of 7.5, (commercially available from Medix) 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 l volume of concentrated conjugated antibodies/3000 volume of conjugate diluent. The dilution is subject to change based on the concentration level of the conjugated antibody.
  • Immuno g lobulin Any member of the gammaglobulin fraction of serum possessing the ability to bind another agent.
  • Antibody A class of serum proteins which specifically bind to an antigen which induced the formation of the antibody.
  • Antigen Molecules (from whatever source nature or man-made) which induce an immune reaction when recognized by the host's immune system.
  • Immunoglobulin Classes Antibodies separated by electrophoretic mobility specifically IgG and IgM.
  • Substrate Solution To quantitate the conjugated antibody, a buffered solution containing (3,3',5,5') Tetramethylbenzidine/ hydrogen peroxide, (commercially available from Kirkegaard Perry) was used. The substrate solution will vary according to the enzyme used or the test format used.
  • Labelled Antibodies Any antibody substance which has been covalently or otherwise combined with a molecule or ion for the purpose of selectively identifying that group of antibodies.
  • adduct molecules or ions include enzymes, fluorescent substances, radionuclides, and the like.
  • 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. Such 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-cardiolipins in sera is a diagnostic test kit.
  • the optimized kit contains:
  • Sample Diluent - green solution contains 0.1% sodium azide.
  • Stop Reagent contains 2.5N H2S04 ** l N HCL may also be used as stop reagent.
  • PBS Phosphate Buffered Saline
  • Step 1 Preparation of the Pre-Coated Microtiter Wells: 20 ug/ml methylated bovine serum albumin is dissolved in water. This solution is used to render the surface of the microtitre wells slightly positively charged. To affix mBSA to the surface of the wells, 100 microliters of the prepared solution is placed in wells that have been rinsed with deionized water and thoroughly drained. Thus, 2 micrograms of mBSA is placed in each individual well. Examples of microwells that have been used are Dynatech Immulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The wells are incubated at room temperature for two hours, and when removed the excess solution is shaken from the plate and the wells are inverted to drain thoroughly.
  • the ligand or antigen is diluted and coated to the receiving surface of the wells.
  • Cardiolipin is added to 100% denatured ethanol with the final concentration of cardiolipin in the resultant solution being 20 micrograms/ml weight per volume. 100 microliters of this solution is placed in contact with each well and the solution is allowed to completely evaporate at room temperature. This evaporation process lasts about 18 to 24 hours.
  • the casein blocking step decreases the non-specific binding that can occur due to protein-plastic interaction.
  • Hydrolyzed casein can be commercially obtained from Sigma, and the blocking solution is prepared by mixing 2 ml glycerol, 10 g sucrose, and 15 milligrams of hydrolyzed casein, and adding sufficient TEN buffer to make 100 ml of solution. The pH is adjusted to 7.3 +/- .1. Next, 200 ul of casein blocking solution is dispensed into each well, and the wells are incubated at 4 degrees C overnight. Following incubation the wells are inverted, shaken to remove excess solution, and allowed to drain for 15 minutes. The wells are turned upright and allowed to dry at room temperature for at least 24 hours.
  • Step 2 Adhering anti-cardiolipins antibodies to the prepared wells in Step 1: Sample Diluent is supplied in the kit as 50 ml of a green solution. To prepare a 1000 ml solution of sample diluent 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, 1 ml of stock green dye are dissolved in demineralized water sufficient to make 1000 milliliters of solution. This solution is then filtered through a .2 micron filter and stored at 4 degrees C.
  • Sample Diluent is supplied in the kit as 50 ml of a green solution. To prepare a 1000 ml solution of sample diluent 100 milliliters of native bovine serum, 1.42 g of Potassium Phosphate (dibasic), .26 g of Potassium
  • the serum Prior to contacting the body fluid with the prepared wells, the serum is diluted by adding an aliquot of serum to the Sample Diluent in a 1:50 ratio (volume of serum:volume of sample diluent) , although the dilution is not critical and depends upon the nature of the body fluid and the assay techniques employed.
  • the excess solution is shaken from the wells removing nonbound antibodies that are present in the sample. This is done since there are many more free antibodies than there are bound to the cardiolipin on the wells and residual free antibodies would elevate background absorbance values.
  • the wells are then washed four times with phosphate-buffered saline, and drained.
  • Step 3 Assay for Anti-Cardiolipin Antibodies Affixed to the Plate: Standard enzyme-linked assay techniques, previously described, are used for this assay, although any suitable means of detection such as radioactive labeling, fluorescence, or the like can be employed.
  • anti-human IgG and IgM induced in goats was used to ascertain whether anti-cardiolipin IgG and IgM antibodies were present.
  • 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 two 8 ml vials of previously diluted conjugated antibody, one contains anti-human IgM antibody conjugated to horseradish peroxidase, and the other contains anti-human IgG similarly conjugated.
  • a conjugate diluent used to dilute the conjugated antibody a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4 (commercially available from Medix) was mixed with protease inhibitor (commercially available from Miles Pentex) at a .01% ratio of inhibitor to volume of buffer. This diluent solution enhances the stability of the conjugated antibody.
  • the working conjugated antibody solution is prepared at a ratio of 1:3000; one part of concentrated conjugated IgM or IgG antibodies is aliquoted into 3000 parts conjugate diluent. This dilution ratio will vary depending on concentration of concentrated conjugated antibody.
  • the enzyme conjugated goat anti-human antibody solution, prepared as described, is then added to each well in 100 microliter increments. Binding of these antibodies to the anti-cardiolipin is permitted for at least 15 minutes at room temperature. The wells are emptied of their contents, and washed four times with PBS and allowed to drain.
  • the presence of a labeled antibody is determined by incubating the plates with a solution of (3,3',5,5') Tetramethylbenzidine and buffered hydrogen peroxide.
  • This solution is supplied in the kit in two 8 ml vials; one contains (3,3',5,5') Tetramethylbenzidine; the other bottle 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 prior to 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 100 microliters of 2.5 normal sulfuric acid to each well and the intensity of color (the optical density, or OD or absorbance) is read by a spectrophotometric device such as 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- cardiolipin.
  • the number of bound conjugated antibodies is linearly related to the number of anti- cardiolipins.
  • polystyrene microtiter wells were coated with a film of methylated bovine serum albumin (mBSA) by the following procedure:
  • the methylated bovine serum albumin solution was placed in the microwells in aliquots of 100 microliters per well, and incubated at room temperature for two hours.
  • casein blocker solution is comprised of 2 ml glycerol, 10 g of sucrose and 15 milligrams of hydrolyzed casein diluted to 100 ml by the addition of TEN (Tris, EDTA, NaCL) buffer.
  • TEN Tris, EDTA, NaCL
  • the casein blocker solution was buffered to a pH of 7.3 +/- .1. 200 microliters of blocker solution was dispensed into each microwell.
  • microwells were then uprighted and allowed to dry at room temperature for at least 24 hours.
  • microwell plates were stored at 4 degrees.C in sealed plastic bags, or used in the next step.
  • cardiolipin coated wells were then used to determine the presence of anti-phospholipid antibody in serum or plasma samples drawn from individuals with:
  • Sample Diluent was comprised of 100 mis of native bovine serum; 1.42 g of potassium phosphate dibasic, .26 g of potassium phosphate monobasic, 1 gram of sodium azide, and 8.5 g of sodium chloride, 1 ml of stock green dye, and 900 mis of distilled water.
  • the diluent solution is then filtered through a .2 micro Nalzene filter and stored at 4 degrees C.
  • Sera were prepared at a 1 to 50 ratio of sample to Sample Diluent.
  • the wells were then incubated at room temperature for 15 minutes, to allow attachment of the conjugate. After incubation, the wells were washed four times with PBS to remove the unbound enzyme conjugated antibodies. The wells were inverted between each wash to empty the excess fluid. After the final wash the wells were inverted to drain excess fluid.
  • Each well was assayed for horseradish peroxidase activity by adding 100 microliters of (3,3',5,5') Tetramethylbenzidine/ buffered hydrogen peroxide solution to each microwell. The wells were allowed to incubate at room temperature for 10 minutes, after which 100 ul of 2.5 N H 2 S0 4 (1.0 N HCL can be substituted) was added to terminate the color reaction. The presence of anti- cardiolipin was detected by the presence of color. The color was quantitated at 450 nm using a Dynatech MR600 plate reading spectophotometer which was calibrated against a water blank. Reagent controls were wells which were not contacted with sample.
  • GPL and MPL units are reported in GPL and MPL units respectively.
  • GPL unit is defined as the cardiolipin binding activity of 1 ug/ml of an affinity purified IgG aCL preparations from a standard serum.
  • MPL unit is defined as the cardiolipin binding activity of 1 ug/ml of an affinity purified IgM aCL preparation from a standard serum. The units have been established by Dr. Nigel Harris. EXAMPLE TWO
  • the kit contained 96 pre-coated microwells with affinity for anti-cardiolipin and:
  • TMB Substrate solution A containing 3,3',5,5', temtramethylbenzidine
  • 8 ml Peroxidase Substrate Solution B containing hydrogen peroxide.
  • Solution A and B are combined to form a substrate capable of generating a colored product.
  • PBS Phosphate Buffered Saline
  • PBS Phosphate Buffered Saline
  • the plate template was labelled for sample placement in the microwells.
  • a 1:50 dilution of the controls, calibrators and patient samples were 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 was prepared just before using according to the kit instruction. 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. 100 ul of working substrate solution was added to each well and the wells were incubated for ten minutes at room temperature.
  • Example 3 The following test was run according to the protocol outlined in Example 3. The samples were taken from patients known to have rheumatoid arthritis, thus some anti-cardiolipin antibodies are expected to be present. A low positive was determined to be 23 GPL and 11 MPL based on the calibrators used. There were approximately 42% positive for IgG anti- cardiolipin antibodies and 6% positive for IgM anti- cardiolipin antibodies. The following data was generated using the present invention.
  • Example 3 The following test was run according to the protocol outlined in Example 3. The samples were suspected of having anti- cardiolipin antibodies as the patients were all SLE patients. The low positive was determined to be 23 GPL and 11 MPL. Seven patients were GPL positive and seven were MPL positive. The following data was gathered by the test kit procedure:
  • Example 3 The following test according to the protocol in Example 3 was performed on 20 patients having progressive systemic sclerosis. Some anti-cardiolipin antibodies were expected to be present in the samples. Using 23 GPL and 11 MPL as the low positive cut-off number, 4 samples were found positve for IgG and 1 sample was positive for IgM. The following data' was generated by the test kit: PATIENTS IgG O.D. GPL IgM O.D. MPL
  • test kit used include controls and calibrators that are standardized against reference samples from EN Harris. The results were expressed as GPL or MPL units. Each sample was assayed in duplicate and the normal ranges were established by the mean value of the unselected healthy individuals + 2 s.d. The prevalence rate is reported as the percent of positive samples above the normal range.
  • 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 anti-cardiolipin antibodies.
  • the detection of anti-cardiolipin 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.
  • 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.

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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, comportant des cardiolipines immobilisées sur une surface de support pré-enduite utilisée pour la détection d'anticorps spécifiques auxdits composés, à l'aide de techniques soit ELISA (Analyse d'immunosorbant à liaison enzymatique) sont FIA (immunoanalyse fluorescente).
PCT/US1990/001028 1989-02-27 1990-02-26 Procede et kit de test de diagnostic pour la detection d'anti-cardiolipine WO1990010227A1 (fr)

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US31556689A 1989-02-27 1989-02-27
US315,566 1989-02-27

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0460102A4 (en) * 1989-02-27 1992-01-15 Biostar Medical Products Incorporated Method and diagnostic test kit for detection of autoimmune antibody
US5506110A (en) * 1989-10-19 1996-04-09 Yamasa Shoyu Kabushiki Kaisha Carrier for binding of anti-phospholipid antibodies, and immunoassay method using the same and a kit therefor
US6177282B1 (en) 1997-08-12 2001-01-23 Mcintyre John A. Antigens embedded in thermoplastic
WO2007061793A3 (fr) * 2005-11-18 2007-08-09 Us Gov Health & Human Serv Cardiolipine modifiee et ses utilisations
CN103185783A (zh) * 2011-12-30 2013-07-03 深圳市亚辉龙生物科技有限公司 一种检测抗ssb抗体的试剂装置及其方法
CN103185785A (zh) * 2011-12-30 2013-07-03 深圳市亚辉龙生物科技有限公司 一种检测抗心磷脂抗体的试剂装置及其方法
US8778619B2 (en) 2005-11-18 2014-07-15 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Oxidized cardiolipin and uses to detect cardiolipin antibodies
CN117288948A (zh) * 2023-06-19 2023-12-26 无锡嘉润诊断技术有限公司 抗凝血酶原抗体检测试剂盒

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EP0460102A4 (en) * 1989-02-27 1992-01-15 Biostar Medical Products Incorporated Method and diagnostic test kit for detection of autoimmune antibody
US5506110A (en) * 1989-10-19 1996-04-09 Yamasa Shoyu Kabushiki Kaisha Carrier for binding of anti-phospholipid antibodies, and immunoassay method using the same and a kit therefor
US6177282B1 (en) 1997-08-12 2001-01-23 Mcintyre John A. Antigens embedded in thermoplastic
US8906603B2 (en) 2005-11-18 2014-12-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Modified cardiolipin and uses therefor
WO2007061793A3 (fr) * 2005-11-18 2007-08-09 Us Gov Health & Human Serv Cardiolipine modifiee et ses utilisations
US7888043B2 (en) 2005-11-18 2011-02-15 The United States Of America As Represented By The Department Of Health And Human Services, Centers For Disease Control And Prevention Modified cardiolipin and uses therefor
US9081009B2 (en) 2005-11-18 2015-07-14 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Oxidized cardiolipin and uses to detect cardiolipin antibodies
US8778619B2 (en) 2005-11-18 2014-07-15 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Oxidized cardiolipin and uses to detect cardiolipin antibodies
CN103185785A (zh) * 2011-12-30 2013-07-03 深圳市亚辉龙生物科技有限公司 一种检测抗心磷脂抗体的试剂装置及其方法
CN103185785B (zh) * 2011-12-30 2015-01-07 深圳市亚辉龙生物科技有限公司 一种检测抗心磷脂抗体的试剂装置及其方法
CN103185783B (zh) * 2011-12-30 2015-01-07 深圳市亚辉龙生物科技有限公司 一种检测抗ssb抗体的试剂装置及其方法
CN103185783A (zh) * 2011-12-30 2013-07-03 深圳市亚辉龙生物科技有限公司 一种检测抗ssb抗体的试剂装置及其方法
CN117288948A (zh) * 2023-06-19 2023-12-26 无锡嘉润诊断技术有限公司 抗凝血酶原抗体检测试剂盒
CN117288948B (zh) * 2023-06-19 2024-06-04 无锡嘉润诊断技术有限公司 抗凝血酶原抗体检测试剂盒

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AU5262390A (en) 1990-09-26

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