CN115902240A - Anti-phospholipid antibody spectrum detection kit and application thereof - Google Patents

Abstract

The invention relates to preparation of an anti-phospholipid antibody spectrum detection reagent and application of the reagent in a kit for diagnosing anti-phospholipid syndrome diseases, wherein the kit contains a reagent for detecting antibody level, and the reagent comprises at least one of a reagent for detecting anti-phosphatidylserine antibody (IgG, igM and IgA) level, a reagent for detecting anti-phosphatidylethanolamine antibody (IgG, igM and IgA) level, a reagent for detecting anti-phosphatidylglycerol antibody (IgG, igM and IgA) level, a reagent for detecting anti-phosphatidic acid antibody (IgG, igM and IgA) level and a reagent for detecting anti-phosphatidylinositol antibody (IgG, igM and IgA) level. The invention also provides an antiphospholipid antibody spectrum detection kit and a modified covalent coating microsphere. The detection of the invention is beneficial to the diagnosis of phospholipid-resistant syndrome (APS), improves the diagnosis sensitivity and reduces missed diagnosis.

Description

Anti-phospholipid antibody spectrum detection kit and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an antiphospholipid antibody spectrum detection kit and application thereof.

Background

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, mainly manifested by arteriovenous thrombosis and morbid pregnancy (abortion, fetal death and premature birth), with persistent antiphospholipid antibody positivity. Anti-phospholipid antibodies (aPL) are autoantibodies with high heterogeneity, mainly using phospholipids or phospholipid binding proteins as target antigens, and mainly include anti-cardiolipin antibodies (aCL), anti- β 2 glycoprotein 1 antibodies (β 2-GP 1), lupus Anticoagulants (LA), and the like. The laboratory standards revised by sydney 2006 stipulate that at least one medium to high titer aPL (LA, igG or IgM type aCL, igG or IgM type β 2-GP 1) is detected in plasma, 2 or more times and at least 12 weeks apart. The actual incidence of APS is not clear. According to current estimates, the incidence is approximately 5 per 1,000,000 people per year, with a prevalence of 40-50 per 1,000,000 people.

In practice, patients are often encountered whose clinical manifestations suspect APS but LA, aCL and β 2-GP1 are consistently negative, termed "serologically negative" APS. For obstetrics and gynecology, early treatment is the key to abortion prevention, and serological markers in the existing classification standards have insufficient sensitivity, so that the aPL spectrum needs to be widened urgently. In addition, there are many clinical manifestations of APS that are not currently included in the diagnostic standard, including thrombocytopenia, reticulocytosis, kidney involvement, valvular heart disease, etc., and this subset of patients is referred to as non-standard diagnostic APS.

Initially, aPLs were thought to recognize only negatively charged phospholipids, and as research has progressed over the years, aPLs specific for different antigens have been discovered. The antigen specificity can be divided into the following categories: (1) negative phospholipids: such as cardiolipin, phosphatidylserine, phosphatidic acid, phosphatidylinositol, etc.; (2) neutral phospholipid: such as phosphatidylcholine; (3) amphoteric phospholipid: such as phosphatidylethanolamine; (4) phospholipid-binding proteins: such as beta 2 glycoprotein 1, prothrombin, annexin A5, protein S and protein C, etc. Although only LA, and also the IgG or IgM type aCL, igG or IgM type β 2-GP1 are included in diagnostic standards for APS, several studies indicate that aPLs in addition to these antibodies contribute to the diagnosis of "seronegative" APS patients. The positive rate of some antiphospholipid antibodies in APS is shown in Table 1.

Table 1 partial antiphospholipid antibodies and positive rates (— from journal of chinese test medicine 2015

With respect to the definition of recurrent pregnancy (RSA), the american society for reproductive medicine is standardized to 2 or more pregnancy failures; the Royal College of Obstetricians and gynecology in the united kingdom (RCOG) is defined as the fetal loss occurring 3 or more times in succession with the same partner and before 24 weeks of gestation; while the fetal loss before 28 weeks of gestation for 3 or more times is usually called recurrent abortion in China, most experts think that 2 continuous abortions should be considered and evaluated, and the risk of recurrent abortion is similar to that of 3 patients. RSA patients account for approximately 1% -5% of women in childbearing age, and have a complex etiology, with approximately 50% of the etiologies being unknown. Clinically, 10-15% of RSA women are combined with APS, if no intervention is given, the fetal loss rate of APS patients can reach 91%, and if positive treatment intervention is carried out, the live birth rate can be greatly improved to 84%, so that the APS is considered as one of curable causes of the RSA, and the improvement of the diagnosis sensitivity of the APS is very important. However, the sensitivity of diagnosing APS using diagnostic standard antiphospholipid antibodies (anticardiolipin antibodies, anti- β 2-GPI antibodies and lupus anticoagulant) is not high, and particularly in obstetrical APS patients, APS that are "serologically negative" are more likely to appear. A large number of researches show that the detection of the non-diagnostic standard antiphospholipid antibody is beneficial to improving the APS diagnostic efficiency, and has a very wide application prospect. The national and international RSA related guidelines conditionally support the increase of non-diagnostic standard anti-phospholipid antibody detection in RSA etiology screening.

The flow microsphere technology (CBA) is a new technology developed on the basis of flow cytometry. The method takes polystyrene microspheres as a reaction interface, and can capture some small molecular substances after the microspheres are coated by a specific monoclonal antibody, so that a detection signal is amplified, and the corresponding small molecular substances can be detected by detecting the microspheres. In recent years, CBA has been increasingly used for detection of soluble proteins and cytokines in various liquid phases such as serum, plasma, and culture cell supernatant.

Chinese patent document CN108226534A discloses application of an antibody of MYO5A protein as a biomarker in judging anti-phospholipid syndrome. Antibodies to MYO5A protein include at least one of IgG, igM, or IgA. Can be used for identifying primary and secondary phospholipid-resistant syndromes. The application of the antibody of the MYO5A protein as a biomarker in judging the phospholipid-resistant syndrome provides a novel marker for judging the phospholipid-resistant syndrome, provides a novel application direction, and has important significance in judging the phospholipid-resistant syndrome.

However, no report is found about a novel anti-phospholipid antibody spectrum detection kit and application at present.

Disclosure of Invention

The first purpose of the present invention is to provide an application of an anti-phospholipid antibody spectrum detection reagent to overcome the defects in the prior art.

The second purpose of the invention is to provide an antiphospholipid antibody profile detection kit.

It is a third object of the present invention to provide a modified covalently coated microsphere.

In order to realize the first purpose, the invention adopts the technical scheme that:

an application of an anti-phospholipid antibody spectrum detection reagent in preparing a kit for diagnosing recurrent abortion of an anti-phospholipid syndrome is disclosed, wherein the kit contains a reagent for detecting antibody level, and the reagent comprises at least one of a reagent for detecting anti-phosphatidylserine antibody (IgG, igM, igA) level, a reagent for detecting anti-phosphatidylethanolamine antibody (IgG, igM, igA) level, a reagent for detecting anti-phosphatidylglycerol antibody (IgG, igM, igA) level, a reagent for detecting anti-phosphatidic acid antibody (IgG, igM, igA) level and a reagent for detecting anti-phosphatidylinositol antibody (IgG, igM, igA) level.

As a preferable example, the kit contains a reagent for detecting the level of antibodies, and the reagent comprises a reagent for detecting the level of anti-phosphatidylserine antibodies (IgG, igM and IgA), a reagent for detecting the level of anti-phosphatidylethanolamine antibodies (IgG, igM and IgA), a reagent for detecting the level of anti-phosphatidylglycerol antibodies (IgG, igM and IgA), a reagent for detecting the level of anti-phosphatidic acid antibodies (IgG, igM and IgA), and a reagent for detecting the level of anti-phosphatidylinositol antibodies (IgG, igM and IgA), wherein the total number of the reagents is fifteen.

As a preferable example, the kit contains modified covalent coating microspheres, and is characterized in that the microspheres are prepared by modifying phospholipid antigen into water-soluble antigen with amino groups and combining the water-soluble antigen with fluorescence coding microspheres through covalent bonds, the modified phospholipid antigen is coated on the fluorescence coding microspheres modified by carboxyl, and the antigen is phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and phosphatidylinositol.

In order to achieve the second object, the invention adopts the technical scheme that:

the reagent kit comprises a reagent for detecting the antibody level, wherein the reagent for detecting the antibody level comprises at least one of a reagent for detecting the anti-phosphatidylserine antibody (IgG, igM, igA) level, a reagent for detecting the anti-phosphatidylethanolamine antibody (IgG, igM, igA) level, a reagent for detecting the anti-phosphatidylglycerol antibody (IgG, igM, igA) level, a reagent for detecting the anti-phosphatidic acid antibody (IgG, igM, igA) level and a reagent for detecting the anti-phosphatidylinositol antibody (IgG, igM, igA) level.

Preferably, the kit contains a reagent for detecting the level of antibodies, and the reagent comprises a reagent for detecting the level of anti-phosphatidylserine antibodies (IgG, igM and IgA), a reagent for detecting the level of anti-phosphatidylethanolamine antibodies (IgG, igM and IgA), a reagent for detecting the level of anti-phosphatidylglycerol antibodies (IgG, igM and IgA), a reagent for detecting the level of anti-phosphatidic acid antibodies (IgG, igM and IgA), and a reagent for detecting the level of anti-phosphatidylinositol antibodies (IgG, igM and IgA), wherein the total number of the reagents is fifteen.

As a preferable example, the kit contains modified covalent coating microspheres, and is characterized in that the microspheres are prepared by modifying phospholipid antigen into water-soluble antigen with amino groups and combining the water-soluble antigen with fluorescence coding microspheres through covalent bonds, the modified phospholipid antigen is coated on the fluorescence coding microspheres modified by carboxyl, and the antigen is phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and phosphatidylinositol.

As a preferable example, the sample detected by the kit comprises serum, plasma and other body fluids.

In order to achieve the third object, the invention adopts the technical scheme that:

a modified covalent coated microsphere is prepared by modifying phospholipid antigen into water-soluble antigen with amino groups and combining the phospholipid antigen with a fluorescent coding microsphere through covalent bonds.

As a preferred example, the modified phospholipid antigen is coated with a carboxyl-modified fluorescent-encoded microsphere, and the antigen is phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol.

The invention has the advantages that:

the invention discloses a kit for jointly detecting an antiphospholipid antibody based on a flow fluorescent microsphere technology and application thereof. The profile of anti-phospholipid antibodies detected included anti-phosphatidylserine antibodies (IgG/IgM/IgA), anti-phosphatidylethanolamine antibodies (IgG/IgM/IgA), anti-phosphatidylglycerol antibodies (IgG/IgM/IgA), anti-phosphatidic acid antibodies (IgG/IgM/IgA) and anti-phosphatidylinositol antibodies (IgG/IgM/IgA). Specifically, the modified phospholipid antigen is coated on the carboxyl modified fluorescent coding microsphere, and the antigen is phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and phosphatidylinositol. Further, reacting with a sample to be detected, and capturing an antibody to be detected; washing to remove the unbound antibody to be detected, and adding a fluorescence-labeled anti-human IgG, igM or IgA antibody; and detecting the fluorescence intensity of the microspheres by using a flow cytometer so as to qualitatively and quantitatively analyze the antibody to be detected in the sample. The kit for detecting the anti-phospholipid antibody spectrum by the flow-type fluorescent microsphere method has the advantages of high flux, simple and convenient operation, accurate and reliable result and the like. The positive rate of at least one type of the 5 novel 15-type antiphospholipid antibodies in RSA combined APS patients is 85 percent; at least one type of antibody positive rate was 62.67% in serologically negative RSA. The invention can obviously improve the sensitivity of the laboratory diagnosis of the antiphospholipid syndrome (APS), and particularly provides evidence-based medical evidence for the clinical diagnosis of 'seronegative' APS. The early discovery and the definition of the cause are key measures for preventing adverse events such as APS common thrombus and/or pathological pregnancy.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications can be made to the present invention by those skilled in the art after reading the present specification, and these equivalents also fall within the scope of the invention defined by the appended claims.

Example 1: preparation of the kit

1. Test materials and reagents

(1) Phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol were purchased from Sigma

(2) Fluorescent-encoded microspheres were purchased from Luminex, USA

(3) The fluorescently labeled secondary antibody is a FITC-labeled polyclonal antibody, namely goat anti-human IgG-FITC, goat anti-human IgM-FITC, goat anti-human IgA-FITC purchased from Jackson of America

(4) Coating liquid: 0.01mol/L PBS, pH =7.4.

(5) Sealing liquid: 2% BSA-PBS (0.01 mol/L, pH = 7.4) buffer.

(6) Washing solution: 0.05% Tween-20/PBS (0.01 mol/L, pH = 7.4) buffer.

(7) Sample diluent: 1% gelatin/PBS (0.01 mol/L, pH = 7.4) buffer.

(8) Secondary antibody dilution: 0.01mol/L PBS,0.05% thimerosal, pH =7.4.

(9) Positive quality control serum: adding appropriate amount of antiseptic for human serum with anti-phospholipid antibody positive effect.

(10) Negative quality control serum: adding proper amount of preservative into the anti-phospholipid antibody negative human serum.

(11) Flow cytometry (BD FACSCalibur, usa).

(12) Dichloromethane, m-chloroperoxybenzoic acid, petroleum ether, ethyl acetate and the like are all chemically pure.

2. Production process

2.1 Pre-coated microspheres

2.1.1 phospholipid modification

Under nitrogen protection, 1mmol of phospholipid (phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol) was added to a 20mL glass flask containing 5mL of dichloromethane, and magnetically stirred at room temperature until completely dissolved. 3.5mmol of m-chloroperoxybenzoic acid was added thereto, and the reaction was maintained at room temperature for 36 hours. The reaction solution was poured into an ice-water bath, and the organic compounds therein were extracted with petroleum ether. After the petroleum ether is evaporated to dryness under reduced pressure and dried, 5mL of ammonia water with the mass fraction of 15% is added, and the temperature is continuously raised until the reflux reaction is carried out for 8 hours. After cooling, the mixture was poured into ethyl acetate and washed 3 times with water. Drying to obtain colorless oily substance, and separating and purifying by liquid chromatography to obtain modified phospholipid with side chain having amino group.

2.1.2 coating

Taking the fluorescent coded microspheres marked as B4, B6, B8, C4 and C6, carrying out vortex oscillation on the microspheres for 2 minutes to resuspend the microspheres, respectively taking 50 mu L of the microspheres, placing the microspheres in a 1.5m LEppendoff tube, carrying out ultrasonic treatment on the microspheres in an ultrasonic cleaner for 10 minutes, and standing the microspheres at room temperature for later use. The modified phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol) were dissolved in coating buffer at a concentration of 1ug/mL, 50 μ L of the treated microspheres B4, B6, B8, C4, C6 were added, respectively, incubated with shaking at room temperature for 6 hours, centrifuged at 13000g for 5 minutes, and washed with 1000 μ L of washing solution 3 times.

2.1.3 sealing

Then 200. Mu.L of blocking buffer is added, shaking incubation is carried out for 2 hours at room temperature, 13000g is centrifuged for 5 minutes, washing is carried out for 3 times by 1000. Mu.L of washing solution, 100. Mu.L of blocking solution is added for resuspension, and 5 kinds of coupled microspheres which are B4-phosphatidylserine, B6-phosphatidylethanolamine, B8 phosphatidylglycerol, C4-phosphatidic acid and C6 phosphatidylinositol respectively are obtained. The coated fluorescent microspheres can be stored separately or mixed together, and can be stored stably for 1 year in a refrigerator at 4 ℃.

2.2 preparation of the second antibody working solution

The used secondary antibody is a polyclonal antibody marked by FITC, namely a goat anti-human IgG-FITC, a goat anti-human IgM-FITC and a goat anti-human IgA-FITC, which are directly purchased, and are respectively diluted into working solution for standby by a second antibody diluent according to the mass ratio of 1.

2.3 Standard preparation

Human positive mixed serum. Specifically, the positive sera to be detected were mixed with anti-phosphatidylserine antibody (IgG/IgM/IgA), anti-phosphatidylethanolamine antibody (IgG/IgM/IgA), anti-phosphatidylglycerol antibody (IgG/IgM/IgA), anti-phosphatidic acid antibody (IgG/IgM/IgA) and anti-phosphatidylinositol antibody (IgG/IgM/IgA), respectively, and centrifuged at 3000rpm for 30min to obtain the supernatant. The positive human mixed serum is diluted into a 4-degree concentration standard substance by a sample diluent according to a proportion gradient of 1. The unit is defined as U/mL.

TABLE 2 concentration values of standards

2.4 Positive quality control serum preparation

Diluting positive human serum, and adding antiseptic.

2.5 preparation of negative quality control serum

And adding preservative to treat negative human serum.

3. The kit comprises the following components:

example 2: method of using kit

1. Diluting the sample diluent with distilled water according to a ratio of 1; diluting the concentrated washing solution with distilled water according to the ratio of 1; the standard was diluted with sample dilutions in a 1.

2. Serum samples were diluted with serum dilutions at 1.

3. Taking three subtypes IgG, igM and IgA to be detected for 5 antibodies at the same time as an example, the reaction is carried out at room temperature for 2 hours according to the following table 3, 500ul of cleaning solution is added to each tube, centrifugation is carried out at 2000rpm for 5 minutes, and the reaction is repeated for 3 times. The positive quality control and the negative quality control are set for each detection, 50ul of diluted serum is taken to be respectively mixed with the positive quality control and the negative quality control, and the reaction conditions are the same as those of a standard substance and a sample tube. The reaction is carried out in a flow sample introduction pipe or a micropore plate.

TABLE 3 sample application program Table (sample application amount unit: μ L)

4. 100ul secondary antibody, either goat anti-human IgG-FITC or goat anti-human IgM-FITC or goat anti-human IgA-FITC was added to each tube as shown in Table 3. The reaction was carried out at room temperature for 30 minutes, 500. Mu.l of the washing solution was added to each tube, and the mixture was centrifuged at 2000rpm for 5 minutes, and the reaction was repeated 3 times.

5. Add 500ul of wash resuspension microspheres per tube and test on a flow cytometer.

6. And drawing a standard curve, and calculating the content of the antibody to be detected according to the fluorescence intensity of the sample tube. Standard curve plotting and analysis were performed using FCAPArray v3.0 software.

7. According to different subtypes of the added secondary antibody, the content of the antibody to be detected of the corresponding subtype can be obtained. Such as

IgG class: anti-phosphatidylserine antibody-IgG, anti-phosphatidylethanolamine antibody-IgG, anti-phosphatidylglycerol antibody-IgG, anti-phosphatidic acid antibody-IgG;

IgM class: anti-phosphatidylserine antibody-IgM, anti-phosphatidylethanolamine antibody-IgM, anti-phosphatidylglycerol antibody-IgM, anti-phosphatidic acid antibody-IgM, and anti-phosphatidylinositol antibody-IgM;

IgA class: anti-phosphatidylserine antibody-IgA, anti-phosphatidylethanolamine antibody-IgA, anti-phosphatidylglycerol antibody-IgA, anti-phosphatidic acid antibody-IgA, and anti-phosphatidylinositol antibody-IgA

8. Quality control standard: the curve fitting degree r is more than 0.99, the fluorescence signal of the positive quality control tube is strong, and the fluorescence signal of the negative quality control tube is weak.

Example 3 comparison of preparation Process of Pre-coated microspheres: the effect of two modes of covalent coating and traditional direct coating after phospholipid modification is compared

Covalent coating: for the scheme adopted by the invention, the specific process is shown in 2.1. Direct coating: dissolving a certain amount of phospholipid in an organic solvent (generally methanol or chloroform) to be mixed with the polystyrene microspheres, washing after 4 degrees of liquid passing, and carrying out covalent coating in the sealing process. Two batches of coated microspheres were produced in two coating modes, respectively, and the intra-and inter-batch precision was evaluated as in example 2.

The specific experimental scheme is as follows: the test was performed in 2 batches per day, and the same sample was measured in duplicate for 20 days for each batch. There were 40 pairs, i.e., 80 test results, at the end of the evaluation. The intra-batch precision was determined from the difference of duplicate results in the 40-batch measurements. Batch-to-batch precision was calculated from all 80 data. The results show that the in-and inter-batch precision of the assay using the modified covalently coated microspheres is superior to that of the directly coated microspheres, see table 4.

TABLE 4 precision of directly coated microspheres and precision of modified covalently coated microspheres

Example 4: diagnostic value analysis of the kit of the invention

1. Sample source

270 patients with recurrent abortion in the first gynecological, infant and reproductive and immunologic department in Shanghai are collected, 120 patients with combined antiphospholipid syndrome (RSA combined with APS) and 150 patients with non-APS (RSA). The diagnosis and etiology screening of recurrent abortion refer to the consensus of experts made in 2016 (1), the age is 20-40 years, and patients with abortion caused by couple chromosome abnormality, infection, dissection, endocrine and the like are excluded. The diagnosis of antiphospholipid syndrome (APS) was made in 1999 and was modified in 2005 by the international conference on thrombo-hemostasis [ 2 ]. In addition, 330 healthy women in the childbearing age are collected, the bad pregnancy history such as infertility and spontaneous abortion does not exist, and the physical examination does not have abnormality.

【1】 The Chinese medical society, the department of obstetrics and gynecology, the consensus of diagnosis and treatment of recurrent abortion [ J ]. J.J. China J.J.J.J.J.2016, 51 (1): 3-9).

【2】Miyakis S,Lockshin MD,Atsumi T,et al.International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome(APS).J Thromb Haemost.2006；4(2):295-306.

2. Detection method the kit prepared in example 1 and the kit using method described in example 2 of the present invention were used to detect 15 subtypes of 5 autoantibodies in 330 healthy control groups, 120 RSA-pooled APS groups and 150 RSA groups.

3. Analysis of results

3.1 clinical Limit determination

The upper limit of normal is defined as the Mean fluorescence signal value +2SD (Mean +2 SD) of the healthy control group, the positive is Mean +3SD, the gray scale range is between the two, and Mean +5SD is strong positive. The FCAP Array v3.0 software was used to plot standard curves and convert fluorescence signal values to concentration values (U/mL) with clinical limits as shown in Table 5.

TABLE 5 clinical values (U/mL)

3.2 comparison of the Positive rates of the novel anti-phospholipid antibodies in RSA

The positive rate of at least one type of the 5 types of 15 novel antiphospholipid antibodies in RSA combined APS patients is 85 percent; at least one type of antibody positive rate was 62.67% in serologically negative RSA. The novel anti-phospholipid antibody detection is helpful for APS diagnosis, improves the diagnosis sensitivity and reduces missed diagnosis. The positive rate is shown in Table 6.

TABLE 6 Positive rate

Detecting items	RSA merging APS (n = 120)	RSA(n＝150)
			Anti-phosphatidylethanolamines-IgG,％	3.33(4)	30.67(46)
Anti-phosphatidylethanolamines-IgM,％	5.0(6)	27.33(41)
			Anti-phosphatidylethanolamines-IgA,％	1.7(2)	20.0(30)
Total,％	8.33(10)	41.33(62)
			Anti-phosphatidylserine-IgG,％	17.5(21)	20.67(31)
Anti-phosphatidylserine-IgM,％	63.33(76)	22.67(34)
			Anti-phosphatidylserine-IgA,％	21.67(26)	19.33(29)
Total,％	80.0(96)	30.67(46)
			Anti-phosphatidylinositol-IgG,％	14.17(17)	6.67(10)
Anti-phosphatidylinositol-IgM,％	35.0(42)	33.33(50)
			Anti-phosphatidylinositol-IgA,％	16.67(20)	9.33(14)
Total,％	49.17(59)	40.67(61)
			Anti-phosphatidy-DL-Glycerol-IgG,％	9.17(11)	10.0(15)
Anti-phosphatidy-DL-Glycerol-IgM,％	22.5(27)	27.33(41)
			Anti-phosphatidy-DL-Glycerol-IgA,％	7.5(9)	13.33(20)
Total,％	25.83(31)	30.0(45)
			Anti-phosphatidicacid--IgG,％	13.33(16)	20.67(31)
Anti-phosphatidicacid--IgM,％	22.5(27)	32.67(49)
			Anti-phosphatidicacid--IgA,％	14.17(17)	12.0(18)
Total,％	27.5(33)	40.0(60)
			Total,％	85.0(66)	62.67(94)

The kit for detecting the anti-phospholipid antibody spectrum by the flow-type fluorescent microsphere method has the advantages of high flux, simple and convenient operation, accurate and reliable result and the like. The invention can obviously improve the sensitivity of laboratory diagnosis of the antiphospholipid syndrome (APS), and particularly provides evidence-based medical evidence for clinical diagnosis of serological negative APS.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (9)

1. The application of the anti-phospholipid antibody spectrum detection reagent in preparing the kit for diagnosing the recurrent abortion of the anti-phospholipid syndrome is characterized in that the kit contains a reagent for detecting the antibody level, and the reagent comprises at least one of a reagent for detecting the anti-phosphatidylserine antibody (IgG, igM, igA) level, a reagent for detecting the anti-phosphatidylethanolamine antibody (IgG, igM, igA) level, a reagent for detecting the anti-phosphatidylglycerol antibody (IgG, igM, igA) level, a reagent for detecting the anti-phosphatidic acid antibody (IgG, igM, igA) level and a reagent for detecting the anti-phosphatidylinositol antibody (IgG, igM, igA) level.

2. The use according to claim 1, wherein said kit comprises reagents for detecting antibody levels, said reagents comprising a reagent for detecting anti-phosphatidylserine antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidylethanolamine antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidylglycerol antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidic acid antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidylinositol antibody (IgG, igM, igA) levels, and fifteen reagents in total.

3. The use of claim 1 or 2, wherein the kit comprises modified covalently coated microspheres, wherein the microspheres are prepared by modifying phospholipid antigen to water-soluble antigen with amino groups and combining the antigen with fluorescent-encoded microspheres through covalent bonds, the modified phospholipid antigen is coated with carboxyl-modified fluorescent-encoded microspheres, and the antigen is phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid or phosphatidylinositol.

4. The anti-phospholipid antibody spectrum detection kit is characterized by comprising an antibody level detection reagent, wherein the antibody level detection reagent comprises at least one of an anti-phosphatidylserine antibody (IgG, igM, igA) level detection reagent, an anti-phosphatidylethanolamine antibody (IgG, igM, igA) level detection reagent, an anti-phosphatidylglycerol antibody (IgG, igM, igA) level detection reagent, an anti-phosphatidic acid antibody (IgG, igM, igA) level detection reagent and an anti-phosphatidylinositol antibody (IgG, igM, igA) level detection reagent.

5. The kit of claim 4, wherein said kit contains reagents for detecting antibody levels, said reagents comprising a reagent for detecting anti-phosphatidylserine antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidylethanolamine antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidylglycerol antibody (IgG, igM, igA) levels, a reagent for detecting anti-phosphatidic acid antibody (IgG, igM, igA) levels, and a reagent for detecting anti-phosphatidylinositol antibody (IgG, igM, igA) levels, totaling fifteen.

6. The kit according to claim 4 or 5, wherein the kit comprises modified covalently coated microspheres, wherein the microspheres are prepared by modifying phospholipid antigens into water-soluble antigens with amino groups and combining the water-soluble antigens with fluorescence-encoded microspheres through covalent bonds, the modified phospholipid antigens are coated with carboxyl-modified fluorescence-encoded microspheres, and the antigens are phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and phosphatidylinositol.

7. The kit according to claim 4 or 5, wherein the sample detected by the kit comprises serum, plasma, or other body fluids.

8. The modified covalent coated microsphere is characterized in that the microsphere is prepared by modifying phospholipid antigen into water-soluble antigen with amino groups and combining the phospholipid antigen with a fluorescent coding microsphere through covalent bonds.

9. The modified covalently coated microsphere of claim 8, wherein said modified phospholipid antigen is coated with a carboxyl-modified fluorescently encoded microsphere, and said antigen is phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol.