WO1990010227A1

WO1990010227A1 - Method and diagnostic test kit for detection of anti-cardiolipin

Info

Publication number: WO1990010227A1
Application number: PCT/US1990/001028
Authority: WO
Inventors: Marcia Sterhan; Luis R. Lopez
Original assignee: Biostar Medical Products Inc
Current assignee: Biostar Medical Products Inc
Priority date: 1989-02-27
Filing date: 1990-02-26
Publication date: 1990-09-07
Anticipated expiration: 1991-08-27
Also published as: EP0460097A1; AU5262390A; CA2047742A1; EP0460097A4

Abstract

A method and apparatus optimized as a diagnostic sandwich assay test kit, which has immobilized cardiolipins onto a precoated support surface utilized for the purpose of detection of antibodies specific to such compounds by either ELISA (Enzyme Linked Immunosorbent Assay) or FIA (Fluorescent Immunoassay) techniques.

Description

METHOD AND DIAGNOSTIC TEST KIT FOR DETECTION OP ANTI-CARDIOLIPIN

BACKGROUND OF THE INVENTION

There exists three types of anti-phospholipid autoantibodies. The first anti-phospholipids were detected during the World War II mass screening for syphilis. In a number of patients with systemic lupus erythematosus (SLE) , 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. Although correlation studies show that all three types of anti-phospholipid antibodies associated with similar pathologies, the three antibodies are not identical and each is indicative of differing medical risks.

Research has been performed on all of the anti-phospholipids; unfortunately the tests developed for VDRL and lupus anti¬ coagulant have major limitations. Therefore, clinical testing and immunological studies have focused on the development of radioimmunoassays and ELISAs for detection of the third anti- phospholipid antibody, anti-cardiolipin. The anti-cardiolipin antibody has been linked with recurrent cerebral thrombosis, recurrent arterial thrombosis, recurrent abortion, thrombocytopenia, chorea, epilepsy, and idiopathic pulmonary hypertension. These medical problems have been identified in a subset of SLE patients, which have a common syndrome identified as the Anti-Cardiolipin Syndrome.

The reasons for the strong association between thrombosis and anti-cardiolipin antibodies has not been determined. Two hypotheses have been tendered: (1) a reaction between the phospholipids in endothelial cell membranes which effect the release of prostacyclin; or (2) an action of anti-cardiolipin antibodies (aCL) against the phospholipids in the platelet membrane. Regardless of the mechanisms which link aCL and thrombosis, the relationship between high levels of aCL and further occurrence of thrombosis is well established.

Although the biological mechanisms which trigger aCL to react causing the clinical manifestations of aCL syndrome to appear are not fully understood, the clinical significance of monitoring aCL is substantial. High levels of aCL appear to correlate well with renewed disease activity. 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. The development of a stable, reproducible anti-cardiolipin antibody test has become an international project. In April of 1986, scientists from thirty laboratories in Britain, the U.S.A., France, Italy, New Zealand, the Netherlands, and Sweden all participated in an international study to evaluate anti-cardiolipin (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.

A variety of different techniques have been developed in the search for a stable, reproducible, speedy method of detecting IgG and IgM aCL. Although each method has shown some promise, none of the methods has adequately met the requirements necessary to be a suitable assay method. Examples of the various limitations of the prior techniques follow.

Firstly, 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.

Secondly, aCL ELISAs have been developed for routine diagnostic testing in clinical laboratories. Unfortunately, ELISAs have not been able to detect low concentrations of IgG or IgM anti-cardiolipin antibodies. The scientific community has attempted to standardize results to avoid the lack of sensitivity in the low levels of positive (S. Loizon, 1985 Chem. Exp. Immunol. 62, 738-745). This lack of sensitivity has lead to a host of false negative test results, which renders these tests unsuitable for diagnosis or monitoring of a subclass of patients.

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. As a consequence, to overcome the limitation of insensitivity to low levels of aCL, repeat tests must be run increasing the cost to the patient and increasing the work time expended by lab personnel. However, 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.

It is therefore an objective of the present invention to provide 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.

BRIEF SUMMARY OF THE INVENTION

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

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or quantitation. The present invention utilizes in its broadest sense, anti-phospholids antibodies, which have a particular affinity for association with cardiolipin. As described herein, 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) . 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) . The mBSA provides a positively charged surface which enhances the adherence of the cardiolipin to all surface areas of the well. While methylated 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. In the practice of this invention 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.

The reproducibility of results from the present invention is associated with the mBSA and also with the next two steps of forming the pre-treated wells; the drying and the addition of a hydrolyzed casein blocker. A casein-type blocker has been used in various ELISA techniques (Robert F. Bogt [1987] J. Immunological Methods, 101, 43-50) to prevent through a protein-plastic interaction non-specific binding to plastic. It is an unexpected realization that a microwell coated with hydrolyzed casein blocker properly dried and stored at 4 degrees C in a sealed plastic bag maintains a consistent inhibition of non-specific binding over an extended period of time.

Although the direct mechanisms by which the drying process and the blocker increase the stability and shelf life of the microwells is not fully understood, it is clear that the storage time is increased by these two processes.

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.

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. Then 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.

Subsequent qualitative and quantitative detection of the anti- cardiolipin antibody is relatively simple if the format is either an ELISA or a FIA. Although horseradish peroxidase was used in the examples, numerous enzyme-conjugated antibodies and fluorescent marked antibodies specific for any of the immunoglobulin classes can be substituted to perform the assay. The quantitation of the anti-cardiolipin antibodies present is accomplished by the related instruments; the ELISA technique utilizes a spectrophotometer, and 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. Immunology 109, 129-135), and The Enzyme Linked Immunosorbent Assay (ELISA) by Voller, A. , Bidwell, D.E. and Bartlett, A., (1979) Dynatech Laboratories, Inc. , Alexandria Virginia, both of which are, in their totality, incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are supplied for the purpose of clarifying the invention and are not intended to limit the scope of the invention:

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) . 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) Solution: 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.

Conjugated Antibodies: For an ELISA, antibodies were chemically conjugated with horseradish peroxidase. For a FIA, antibodies were chemically conjugated with Fluorescein Isothiocyanate.

Immuno^globulin: 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. Such 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. Optical Density (OP) or Absorbance: A number which refers to the color absorbance of a solution. Optical density is related to the percent of light transmitted through the solution by the following formulate: OD = 2-log(percent transmittance) .

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:

1 vial (50 ml) Sample Diluent - green solution: contains 0.1% sodium azide.

1 vial (0.25 ml) Human Negative Control. Contains 0.1% sodium azide.

4 vials (0.25 ml) Human Positive Controls - anti-cardiolipin activity on the label. Contains 0.1% sodium azide. (Controls included for GPL and MPL) .

5 vials (0.25 ml) of Calibrator sera, standardized against

Nigel Harris' reference preparations.

12 pre-coated 8-well Microwell Strips with frame holder.

1 vial (8 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase-conjugated anti-human IgM. vial (8 ml) Conjugated Antibody Working Solution containing horseradish peroxidase-conjugated anti-human IgG.

bottle (8 ml) TMB Substrate Solution A - containing 3,3',5,5', tetramethylbenzidine in buffer.

bottle (8 ml) TMB Substrate Solution B - containing hydrogen peroxide

1 bottle (12 ml) Stop Reagent: contains 2.5N H2S04 ** l N HCL may also be used as stop reagent.

1 packet - Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters of 0.01 M PBS, pH 7.4.

Plate template

Two sets of Calibrator sera have been included in order to generate a standard curve, one set for IgM antibodies (MPL) , and one set for IgG antibodies (GPL) . These have been standardized against the Reference Sera proposed by Dri Nigel Harris. The amounts supplied and the levels of anti- cardiolipin activity in the controls and calibrators can be varied without affecting the performance of the assay. This kit is designed to be used in clinical laboratories to measure levels of anti-cardiolipin in approximately 45 minutes. The results of the kit's measurement are adequately reproducible within and between assays. Due to the stability of the pre-coated wells and of the various reagents used in the assay,, the shelf life of the kit is greater than six months. What follows is a description of a preferred embodiment of the pre-coated wells of the present invention, along with the preferred method for preparation of and utilization of the various elements of the kit to detect anti- cardiolipins in the sample of body fluid.

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.

Next, 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.

This completes the coating of the microtiter wells and each kit is then supplied with 96 coated wells. The shelf life of the pre-treated wells stored at 4 degrees C in a sealed plastic bag is up to one year.

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. 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.

100 microliters of the diluted sample is then placed into the appropriate wells. Adherence of the anti-cardiolipin from the body fluid to the pre-coated wells is enhanced by room temperature incubation for fifteen minutes.

Following incubation to achieve antibody adherence, 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. For the examples described hereinafter, 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. For the examples described hereinafter, 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. To prepare the 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, as previously described, 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.

As with any enzyme-linked immune assay, the resultant color of the reaction product is proportional to the number of conjugated antibodies which have bound to the anti- cardiolipin. For most cases, the number of bound conjugated antibodies is linearly related to the number of anti- cardiolipins. Hence, as the amount of conjugated antibodies fixed on the film increases, so does the optical density, or absorbance of the enzyme reaction.

CARDIOLIPIN

EXAMPLE 1

In a preferred embodiment polystyrene microtiter wells were coated with a film of methylated bovine serum albumin (mBSA) by the following procedure:

1. 20 ug/ml of methylated bovine serum albumin in powdered form (available commercially from Sigma) was dissolved in water.

2. 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.

3. After the incubation, the solution was emptied from the wells and they were inverted to drain.

4. 20 microgram/ml of cardiolipin from beef heart (commercially available from Sigma) was dissolved in 100% denatured ethanol.

5. 100 microliter of the cardiolipin solution was placed in each microwell, and incubated at room temperature for 18- 24 hours until the ethanol was completely evaporated. 6. 100 ml of 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. The casein blocker solution was buffered to a pH of 7.3 +/- .1. 200 microliters of blocker solution was dispensed into each microwell.

7. The wells containing casein blocker solution were incubated overnight at 4 degrees C, then the blocker solution was shaken from the wells and they were inverted and allowed to drain for 15 minutes.

8. The microwells were then uprighted and allowed to dry at room temperature for at least 24 hours.

9. The microwell plates were stored at 4 degrees.C in sealed plastic bags, or used in the next step.

The cardiolipin coated wells were then used to determine the presence of anti-phospholipid antibody in serum or plasma samples drawn from individuals with:

1. no apparent pathology; (normal)

2. anti-Cardiolipin positive plasma;

3. positive RPR (rapid plasma reagin) syphilis plasma. The sera and plasma had been drawn from patients known to have systemic lupus erythematosus. An anti-cardiolipin assay, using the cardiolipin coated wells, as previously described, with a sample from one of the three sample groups, was performed by the procedure summarized below:

1. A 1000 ml solution of 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.

2. Sera were prepared at a 1 to 50 ratio of sample to Sample Diluent.

3. 100 ul of the diluted sample or calibrator was transferred to the microwells in duplicate.

4. The wells were incubated at room temperature for 15 minutes. Following incubation the wells were washed four times with PBS and inverted to drain.

5. One set of duplicate microwells were exposed to a mixture containing horseradish peroxidase (HRP) conjugated goat anti-human IgM antibodies (hereinafter referred to as M conjugated) and conjugate diluent; the remaining wells were exposed to a mixture of HRP conjugated goat anti- human IgG antibodies (hereinafter referred to as G conjugate) and conjugate diluent. 100 ul of either conjugate solution was dispensed to each microwell.

6. 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.

7. 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 H₂S0₄ (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.

DETECTION OF CARDIOLIPIN IgG

Source GPL OP

Calibrator 1 6 .08

Calibrator 2 18 .17 Calibrator 3 35 .29

Calibrator 4 70 .52

Calibrator 5 105 .68 Refer to Figure 1

Source Mean Value GPL OP

Normal Serum < 2 .05

(positive) anti CL 46 moderate positive plasma .36 antibody plasma

RPR positive plasma 45 42

DETECTION OF CARPIOLIPIN IgM

Source MPL OP

Calibrator 1 5 .08

Calibrator 2 10 .11

Calibrator 3 20 .2

Calibrator 4 50 .45

Calibrator 5 100 .73

Re er to Figure 2

Source Mean Value MPL OP

Normal Serum < 3 .03

(positive) anti CL 80 high positive plasma .85 antibody plasma

RPR positive patient 59 .55

In each test sample, IgG and IgM aCL levels are reported in GPL and MPL units respectively. One GPL unit is defined as the cardiolipin binding activity of 1 ug/ml of an affinity purified IgG aCL preparations from a standard serum. One 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

To demonstrate the importance of the cardiolipin coating, a polystyrene well was uncoated and the test was run under the procedure described in Example 1. After the wells were assayed with horseradish peroxidase conjugated antibodies, there was only a slight selectivity observed, and a very high background was observed, in contrast to the background and selectivity of the coated wells.

Uncoated

Calibrators MPL Coated Wells O.D. Wells O.D.

100 MPL .85 .72

50 MPL .50 .44

20 MPL .20 .19

10 MPL .12 .13

5 MPL .07 .10 Uncoated

Calibrators GPL Coated Wells O.D. Wells O.D.

105 GPL .93 .85

70 GPL .76 .76

35 GPL .44 .53

18 GPL .28 .36

6 GPL .13 .29

Uncoated

Source MPL Coated Wells O.D. Wells O.D.

Normal Serum .04 .09

Positive #1 .65 .50

Positive #2 1.11 .44 Uncoated

Source GPL Coated Wells O.D. Wells O.D.

Normal Serum .08 .25

Positive #1 .63 .39

Positive #2 .53 .44

EXAMPLE THREE Test Performed with the Diagnostic Test Kit

The kit contained 96 pre-coated microwells with affinity for anti-cardiolipin and:

1 vial of (30ml) Sample Diluent (green solution) - containing 0.1% sodium azide, to be used for diluting sera samples, calibrators and controls.

1 vial of (0.25 ml) Human Negative Control.

2 vials of (0.25 ml) Human GPL Positive Control (two moderate positive controls.

2 vials of (0.25) ml Human MPL Positive Control (moderate positive control and high positive control) .

5 vials of (0.25 ml) calibrators for GPL.

5 vials of (0.25 ml) calibrators for MPL. 1 vial of (8 ml) Conjugated Antibody Working Solution containing horseradish peroxidase conjugated anti-human IgM; and a second similar vial of (8 ml) Conjugated Antibody Working solution containing horseradish peroxidase conjugated anti-human IgG.

One bottle of (8 ml) of TMB Substrate solution A containing 3,3',5,5', temtramethylbenzidine, and one (8 ml) bottle of Peroxidase Substrate Solution B containing hydrogen peroxide. Solution A and B are combined to form a substrate capable of generating a colored product.

l bottle of (12 ml) Stop Reagent containing 2.5N H2S04; (IN HCl may also be substituted) ;

A packet of Phosphate Buffered Saline (PBS) . This reconstitutes to 2 liters of 0.01 M PBS, pH 7.4.

1 packet of Phosphate Buffered Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash.

1 Plate template.

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.

100 ul of each diluted sample, control or calibrator was added to appropriate microwells. The plate was allowed to incubate for 15 minutes at room temperature. To perform the rinse step the contents of the PBS packet was added to 2 liters of reagent grade water, and the solution was mixed well, until all the crystals were dissolved. Then the buffer was used to wash the wells four times. The microwells were inverted between each wash to empty the fluid, and the wells were blotted on absorbent paper to remove residual wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.

100 ul of conjugated antibody (red solution) was added to each well, incubated 15 minutes at room temperature, and then the wells were again washed four times with PBS solution. Next, the wells were blotted on absorbent paper to remove residual wash fluid.

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.

Next 100 ul stop reagent was added to each veil to end the enzyme reaction and the O.D. of each well was read at 450 n against a water blank. The results were then calculated by plotting GPL or MPL calibrator values against O.D.s on linear graph paper. Sample results are read from the resulting "standard curve".

Normal Ranges and Degree of Positivity:

The following estimates have been established by Pr. Nigel

Harris:

Normal = < 5 GPL and < 3 MPL

Low Positive = < 15 GPL and < 6 MPL

Mod Positive = 15-100 GPL and 6-60 MPL

High Positive = > 100 GPL and > 60 MPL

PETECTION OF CARPIOLIPIN IgG and IgM Refer to Figures 3 and 4

Control results fell within predetermined ranges.

Mean Mean

Controls Value GPL O.P. Value MPL O.P.

Normal 2 .7 2 .02

Moderate positive 46 .76 16 .32

Moderate positive 78 .99 81 .96

High positive 78 .99 81 .96 Mean Mean

Source Value GPL O.D. Value MPL O.D.

Normal human serum 3 .08 2 .02 aCL (high positive) 92 1.15 70 .87

.ve) 44 .73 17 .34

EXAMPLE 4

Samples were drawn from 52 patients that tested VDRL positive. The test was run in accordance with Example 3. Approximately 17% of the samples showed positive for anti-Cardiolipin antbodies. The low positive IgG was determined to be 23 GPL, and the low positive IgM was determined to be 11 MPL. After converting the O.D. data, the following values were obtained:

Patient GPL MPL

1 5.5 4.7

2 7.8 1.3

3 23.1 6.7

4 2.9 6.7

5 16.0 8.0

6 9.3 3.3

7 65.2 6.0

8 6.5 3.3

9 39.9 12.7

10 9.3 7.3

11 7.8 10.7

12 4.6 4.7

13 18.3 12.0

14 12.9 2.0

15 28.5 6.0

16 13.2 16.0

17 14.8 4.0

18 12.6 6.7

19 9.5 4.0

20 6.8 7.3

21 10.3 2.7

22 3.3 2.7

23 4.1 4.0

24 7.7 6.0

25 5.5 5.3

26 19.9 8.0

27 7.1 1.7

28 8.7 5.8

29 7.7 5.0

30 4.2 2.9

31 15.5 10.8

32 4.1 6.7

33 13.4 9.6

34 7.4 4.2

35 3.7 1.3

36 31.6 6.7 37 13.0 1.7 38 9.2 4.6 39 5.7 2 40 4.8 2 41 7.1 5 42 2.5 2 43 18.7 7.9 44 48.2 30.0 45 8.1 1.7 46 47.5 6.7 47 15.3 3.3 48 5.2 1.7 49 6.4 1.7 50 11.8 6.7 51 2.7 8.3 52 8.5 1.3

EXAMPLE 5

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.

Patient IgG O.D. GPL IgM O.D. MPL

1 .10 6.6 .08 3.1

2 .28 18.3 .12 4.7

3 .44 *28.8 .16 6.2

4 .46 *30.1 .11 4.3

5 .52 *34.1 .18 7.0

6 .46 *30.1 .19 7.4

7 .51 *33.4 .16 6.2

8 .12 7.9 .10 3.9

9 .12 7.9 .08 3.1

10 .68 *44.5 .18 7.0

11 .20 13.1 .09 3.5

12 .13 8.5 .10 3.9

13 .20 13.1 .013 5.1

14 .31 20.3 .48 18.7

15 .12 7.9 .16 6.2

16 .15 9.8 .11 4.3 17 .15 9.8 .04 1.6

18 .18 11.8 .20 7.8

19 .60 *39.3 1.21 *47.2

20 .36 *23.6 .17 6.6

21 .22 14.4 .11 4.3

22 .38 *24.9 .12 4.7

23 .49 *32.1 .16 6.2

24 .21 13.8 .07 2.7

25 .30 19.7 .06 2.3

26 .023 15.1 .16 6.2

27 .21 13.8 .21 8.2

28 .19 12.4 .10 3.9

29 .15 9.8 .16 6.2

30 .31 20.3 .86 33.5

31 .20 13.1 .08 3.1

32 .15 9.8 .07 2.7

33 .30 19.7 .09 3.5

34 .56 *36.7 .12 4.7

35 .20 14.4 .12 4.7

36 .28 18.3 .11 4.3

37 .32 20.1 .14 5.5

38 .56 *36.7 .07 2.7

39 .28 18.3 .11 4.3

40 .27 17.7 .11 4.3

41 .41 *30.0 .12 5.1

42 .34 *24.9 .5 *21.1

43 .30 21.9 .12 5.1

44 .41 *30.0 .12 5.1 45 .32 *23.4 .09 3.8

46 .32 *23.4 .10 4.2

47 .33 *24.1 .20 8.4

48 .54 *39.5 .14 5.9

49 .21 15.4 .12 5.1

50 .32 *23.4 .17 7.2

51 .46 *33.6 .08 3.4

52 .23 16.8 .07 2.9

53 .05 3.7 .02 .8

54 .64 *46.8 .20 8.4

55 .44 *32.2 .07 2.9

56 .23 16.8 .11 4.6

57 .14 10.2 .12 5.1

58 .22 16.1 .09 3.8

59 .34 *24.9 .07 2.9

60 .61 *44.6 .12 5.1

61 .32 *23.4 .12 5.1

62 .51 *37.3 .09 3.8

63 .12 8.8 .10 4.2

64 .30 21.9 .10 4.2

65 .34 *24.9 .18 7.6

66 .37 *27.0 .10 4.2

67 .42 *30.7 .12 5.1

68 .55 *40.2 .19 8.0

69 .15 11.0 .10 4.2

70 .36 *26.3 .39 16.4

71 .18 13.2 .16 6.7

72 .20 14.6 .16 6.7 73 .06 4.4 .17 7.2 74 .30 21.9 .13 5.5 75 .48 *35.1 .10 4.2 76 .35 *25.6 .08 3.4 77 .22 16.1 .07 2.9 78 .15 11.0 .14 5.9 79 .21 15.4 .13 5.5 80 .16 11.7 .18 7.6 *Positive Result ^■hsSe-..

EXAMPLE 6

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:

PATIENT IgG O.D. GPL IgM O.D. MPL

1 .43 *26.4 .20 10.2

2 .11 7.9 .14 *12.4

3 .25 17.9 .04 3.6

4 .10 7.2 .09 8.0

5 .12 8.6 .13 *11.6

6 .07 5.0 .13 *11.6

7 .19 13.6 .13 *11.6

8 .13 9.3 .05 4.4

9 .17 12.2 .35 *31.1 10 .22 15.8 .13 *11.6 11 .15 10.8 .07 6.2 12 .48 *29.4 .01 .5 13 .09 6.5 .02 1.8 14 .51 *31.3 .08 .41 15 .18 12.9 .05 4.4 16 .56 *40.2 .02 1.8 17 .37 *26.5 .03 2.7 18 .50 *35.9 .10 8.9 19 .40 *28.7 .04 3.6 20 .29 20.8 .34 *30.2

*Positive Results.

EXAMPLE 7

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

1 .10 6.7 .07 2.9

2 .09 6.0 .10 4.2

3 .15 10.0 .11 4.6

4 .17 11.3 .02 .08

5 .19 12.7 .03 1.2

6 .41 *27.3 .22 9.2

7 .32 22.7 .10 2.1

8 .34 21.3 .05 2.1

9 .07 4.7 .04 1.7

10 .10 6.7 .04 1.7

11 .58 *38.7 .35 *14.6

12 .10 6.7 .04 1.7

13 .80 *53.4 .08 3.3

14 .05 3.3 .02 .08

15 .32 21.3 .14 5.8

16 .17 11.3 .17 7.1

17 .15 10.0 .02 .8

18 .20 14.0 .08 3.3

19 .20 13.3 .09 3.7

20 .46 *30.7 .11 4.6

* Positive results

Example 8

The prevalence of IgG and IgM a-Cl antibodies in a large series of serum samples obtained from 149 systemic lupus erthematosus (SLE) , 93 progressive sclerosis (PSS) , 80 rheu atoid arthritis (RA) , 24 osteoarthritis (OA) patients, was tested by the test kit in accordance with the protocol in Example 3. The results were compared to the results from the test kit generated from 202 serum samples obtained from healthy individuals. These patients were followed in rheumatology clinics and the diagnosis of SLE, PSS, and RA was based on ARA criteria. One hundred and two of the healthy individuals were screened and selected by the absence of any clinical manifestation related to the a-CL syndrome, medication, pregnancy or recent infection. The 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.

Mean GPL Prevalence Mean MPL Prevalence Serum +/- 1 s.d. GPL +/- 1 s.d. MPL (%)

Healthy 12 +/- 5.3 2 5 +/- 2.9 4

Healthy 7 +/" 3.0 5 6 +/- 3.1 4

(selected)

SLE* 16 +/-10.8 21 6 +/- 5.3 10

PSS 15 +/-H-0 13 4 +/" 3.0 3

RA** 21 +/-10.4 42 6 +/- 6.4 6

OA 11 +/- ⁹«⁷ 4 3 +/" 1.7 0

*No significant correlation between GPL (r = 0.19) or MPL (r = 0.04) with anti-dsDNA was found.

**No significant correlation between GPL (r = 0.01) or MPL (r = 0.09) with IgM-RF was found. In this study, the results indicate statistically significant higher serum levels of IgG a-CL antibody activity in patients with SLE (p < 0.001), PSS (p < 0.025), and RA (p < 0.001) when compared to healthy individuals and OA patients. The highest prevalence was found in patients with RA and SLE and this activity cannot be attributed to cross-reactivity with IgM RF or dsDNA antibodies. There was no statistical difference in IgG or IgM a-CL serum levels in SLE patients with active versus inactive disease. The selection process of the healthy group appears to significantly lower the IgG a-CL mean value (p < 0.001). IgG a-CL serum levels seem to be the most clinically significant in autoimmune disorders.

The foregoing examples serve to illustrate the efficiency and utility of the methylated BSA to provide a coating which inhibits non-specific binding and provides a coating capable of affixing cardiolipin antigen evenly over the solid support. Without being bound to the specific quantities given in the definitional section, it is possible to utilize a wide latitude of concentrations of mBSA or other similar, functionally equivalent substitutes which have the capability to evenly attach cardiolipin antigen to the support solid by providing a charged surface or by any other like mechanism to affix the cardiolipin and to continue to inhibit non-specific binding. Likewise, 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.

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. 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. The 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.

It is contemplated that the inventive concepts herein described may have differing embodiments and it is intended that the appended claims be construed to include all such alternative embodiments of the invention except insofar as they are limited by the prior art.

Claims

1. A solid article for selectively detecting anti-cardiolipin antibodies from solutions placed in contact with such article comprising:

* support means adapted to be contacted with solutions containing anti-cardiolipins;

* coating means comprising a composition containing methylated BSA capable of being affixed to said support;

* a coating means comprising a composition containing cardiolipin ligand capable of adhering anti-cardiolipin antibodies; and

* a blocking means capable of limiting non-specific binding to the solid support; whereby the coated solid article is capable of adhering any anti-cardiolipins exposed to the coating.

2. A method of performing an assay for anti-cardiolipin antibodies in serum comprising the steps of contacting the coated support article with a solution to be tested for the presence of anti-cardiolipin; facilitating adherence of the anti-cardiolipin present in the solution to the coating on the solid article; and detecting the anti-cardiolipin retained by said coating.

3. A diagnostic test kit comprised of a solid article for selectively detecting anti-cardiolipin antibodies from solutions placed in contact with such article comprising:

* a coating means comprising a composition containing cardiolipin ligand capable of adhering anti- cardiolipin antibodies; and

* a blocking means capable of limiting non-specific binding to the solid support; whereby the coated solid article is capable of adhering any anti-cardiolipins exposed to the coating;

and utilizing a method of performing an assay for anti- cardiolipin antibodies in serum comprising the steps of contacting the coated support article with a solution to be tested for the presence of anti-cardiolipin; facilitating adherence of the anti-cardiolipin present in the solution to the coating on the solid article; and detecting the anti- cardiolipin retained by said coating.