Vasculitis antibody joint inspection kit

Abstract

The invention discloses a vasculitis antibody joint detection kit. The kit comprises a detection membrane strip, an enzyme-linked substance, a substrate solution, a stop solution and a concentrated washing solution, the detection membrane strip is coated with a parallel detection line composed of natural antigens PR3, MPO and GBM, the kit can detect three autoantibodies at the same time, has the advantages of being high in anti-interference and anti-cross reaction capacity, is good in specificity and high in sensitivity, and can be suitable for manual or instrument automatic detection application scenes.

Description

Technical Field

[0001] The present invention relates to the technical field of vasculitis antibody detection, in particular to a vasculitis antibody joint detection kit. Background Art

[0002] Systemic vasculitis is a group of inflammatory diseases with vascular inflammation and necrosis as the main pathological changes. It often affects multiple systems in the body and causes organ dysfunction. The affected parts include the skin, kidneys, lungs, and nervous system. Systemic vasculitis is mainly divided into primary vasculitis and secondary vasculitis. Among them, primary vasculitis refers to those without a clear cause, which is divided into large, medium, and small vasculitis; secondary vasculitis refers to those with a clear cause, such as infectious diseases, autoimmune diseases such as SLE and RF, and drug-induced vasculitis. The main antibodies related to systemic vasculitis are anti-neutrophil cytoplasmic antibodies (ANCA). The main antigen components discovered so far are proteinase 3 (PR3), myeloperoxidase (MPO), histone protease (CG), lactoferrin (LF), bactericidal / permeability-increasing protein (BIP), human leukocyte elastase (HLE), lysosomal membrane glycoproteins, α-enolase, etc., and PR3 and MPO are more clearly studied. PR3 is a serine protease in the azure granules of neutrophils, with a molecular weight of about 29KD and a positive charge. It can be implanted in the glomerular basement membrane through the action of charge; it can also inhibit lysosomal enzymes. After PR3-ANCA binds to PR3, on the one hand, it can form immune complexes locally in the kidney, activate complement, and cause tissue damage; on the other hand, lysosomal enzymes are de-inhibited, and various enzymes widely dissolve the cells themselves, causing serious and lasting damage. The latter is the main cause of PR3-ANCA pathogenicity, which can also partially explain why PR3-ANCA-positive diseases are often multi-organ and multi-system damage, and the condition is more serious. For example, about 56% of patients with Wegner's granulomatosis (WG) are PR3-ANCA-positive, and its specificity can reach 96%, often invading the upper and lower respiratory tract, lungs, kidneys, skin, joints, muscles, eyes, etc. PR3 is more expressed in new segmented neutrophils (PMNS), so infection can induce WG recurrence.

[0003] MPO is also one of the neutrophil granule antigen components, with a molecular weight of 150KD, positively charged, and can be implanted on the glomerular basement membrane. MPO is an important negative feedback inhibitory enzyme in the activation process of neutrophils (PMNS), which can inactivate MPNS chemokines and inhibit the formation of hydroxyl free radicals. Therefore, when MPO-ANCA combines with MPO, the function of MPO is inhibited, PMNS is activated in large quantities, respiratory bursts, degranulation, and a large number of reactive oxygen species are produced, causing local tissue damage. MPO-ANCA is mainly seen in microscopic polyangiitis (MPA) and necrotizing crescentic nephritis (NCGN). About 90% of NCGN patients are MPO-ANCA positive.

[0004] Anti-glomerular basement membrane antibodies are a type of antibody in the serum that "attacks" the basement membrane. Patients with positive anti-GBM antibodies account for 5% of autoimmune nephritis, with an 80% positive detection rate in glomerulonephritis and anti-GBM antibody-related diseases, a 20% to 70% positive detection rate in patients with meniscus-forming glomerulonephritis, and can also be detected in proliferative nephritis.

[0005] Based on this, PR3, MPO, and GBM are of particular importance as specific antibodies for the diagnosis of vasculitis.

[0006] There are many marker antibodies that cause vasculitis. Single-item testing can no longer meet clinical needs. If it is not possible to test multiple autoantibody indicators in one tube of blood, there are problems of low testing efficiency and high cost. Summary of the invention

[0007] The invention provides a vasculitis antibody joint detection kit, which is used to solve the defects of the prior art detection kits, such as high cost and low detection efficiency.

[0008] In view of this, the scheme of the present invention is:

[0009] A vasculitis antibody joint detection kit comprises a detection membrane strip, a blocking solution, an enzyme conjugate, a substrate solution, a stop solution and a concentrated washing solution; the detection membrane strip is composed of a carrier sheet and three parallel antigen detection lines fixed on the carrier sheet and coated, the antigen detection lines are formed by natural antigens being independently drawn on a carrier membrane and coated; the natural antigens are PR3, MPO and GBM; the antigen GBM is type IV collagen α-3 chain, separated and purified from bovine kidney by an acetone method.

[0010] Furthermore, the vasculitis is an autoimmune vasculitis, including systemic vasculitis, Wegener's granulomatosis, microscopic polyangiitis, necrotizing crescentic nephritis, and glomerulonephritis.

[0011] Furthermore, the antigens PR3 and MPO are separated and purified from human neutrophils.

[0012] Furthermore, the carrier membrane is a nitrocellulose membrane or a nylon membrane.

[0013] Furthermore, the detection membrane strip is also provided with a weak positive quality control line and a positive quality control line.

[0014] Furthermore, the enzyme conjugate includes anti-human IgG-6#-HRP and HRP conjugate stabilizer / diluent in a ratio of 1:10000.

[0015] Furthermore, the blocking solution includes 8.0 g / L sodium chloride, 0.2 g / L potassium chloride, 1.42 g / L disodium hydrogen phosphate, 5.0 g / L polyethylene glycol 8000, 5.0 g / L bovine serum albumin and a preservative.

[0016] Furthermore, the substrate solution is 1.2 mmol tetramethylbenzidine; and / or the stop solution is 0.1 mol / L sulfuric acid.

[0017] Furthermore, the concentrated washing solution includes 36.72 g / L sodium chloride, 3.73 g / L potassium chloride, 18.3 ml / L Tween-20, 20.0 ml / L preservative, 7.8 ml / L concentrated hydrochloric acid and preservative.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The kit provided by the present invention can detect three autoantibodies at the same time, reducing the detection cost. It also has the advantages of strong anti-interference and anti-cross-reaction capabilities, good specificity and high sensitivity, and can be applied to manual or instrument automated detection application scenarios.

[0020] The kit provided by the present invention can reduce the concentration of PR3, MPO and GBM coated on the solid phase carrier by adjusting the concentration of the secondary antibody HRP (horseradish peroxidase) with high affinity in the immunoblotting method to enhance the secondary antibody binding signal, and solve the HOOK effect of high-concentration antibody samples through multi-step washing, thereby improving the accuracy of diagnosis.

[0021] The antigens used in the kit provided by the present invention are extracted from animal tissues or human cells, purified and separated, and then coated on a carrier of a nitrocellulose membrane, which makes up for the disadvantage that the antigens used in the immunoblotting diagnostic method do not have a natural structure, and improves the sensitivity, specificity and accuracy of the diagnosis of autoimmune diseases.

[0022] The composite quality control serum provided in the kit of the present invention can be used as a reference for the accuracy of clinical diagnosis results, thereby better improving the correctness of clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1It is a schematic diagram of the detection membrane strip in the vasculitis antibody joint detection kit of the present invention. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] At present, the coating concentrations of the three natural antigens in the vasculitis antibody detection kit of the blotting method are relatively high. The coating concentrations of the natural antigens of vasculitis proteinase 3 (PR3), myeloperoxidase (MPO) and glomerular basement membrane (GBM) are all 200-300μg / ml, while those of other autoimmune indicators are all around 50μg / ml, resulting in excessively high costs. In addition, when testing patients with high concentrations of vasculitis antibodies, the HOOK effect is easily produced, affecting the accuracy of clinical test results.

[0026] Example 1 Preparation of natural antigens

[0027] MPO: natural myeloperoxidase, isolated and purified from human neutrophils.

[0028] PR3: Natural proteinase 3, isolated and purified from human neutrophils.

[0029] GBM: Native type IV collagen α-3 chain, containing GBM antigen-related antigenic determinants, isolated and purified from bovine kidney.

[0030] 1. Isolation Method of Human Neutrophils

[0031] 1) Bring all reagents to room temperature;

[0032] 2) Collect 1-2 ml of whole blood in a centrifuge tube;

[0033] 3) Add an equal volume of Hanks solution to whole blood;

[0034] 4) Add 2 ml of neutrophil separation solution to the centrifuge tube;

[0035] 5) Add whole blood and Hanks solution to the separation solution;

[0036] 6) Centrifuge at 4℃, 900r / min for 8-10min. The blood should be separated into 6 layers: plasma, mononuclear cell layer, separation fluid, neutrophils, other separation fluid, and red blood cell sedimentation layer. If the layers are not very obvious, the separated cells will be impure and the separation needs to be repeated;

[0037] 7) Carefully remove the top three layers (plasma, monocytes, and separation fluid) with a pipette;

[0038] 8) Carefully aspirate the neutrophil layer and the separation solution layer below it and transfer them to a clean centrifuge tube.

[0039] 9) Add 3 ml of Hanks solution, centrifuge at 4°C, 900 rpm for 8-10 min, discard the supernatant and keep the precipitate, repeat twice;

[0040] 10) If the cells are used to extract RNA, 1 ml of Trizol can be added for preservation.

[0041] 2. Isolation method from bovine kidney

[0042] The natural protein was extracted from bovine kidney tissue using the acetone method. The specific extraction method is as follows: the bovine kidney was frozen in an ultra-low temperature refrigerator at -80°C. First, it was extracted with phosphate buffered saline (PBS) and Tris-HCl buffer at 4°C, respectively. The GBM was extracted into dry powder using acetone precipitated at -20°C. It was then subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and vertical transfer (Western blot) together with rabbit thymus acetone powder from Sigma. The powder was then transferred to a nitrocellulose membrane and anti-GBM antibodies were detected using ID and IBT. Secondly, the pig heart acetone powder antigen was precipitated step by step with ammonium sulfate at different saturation concentrations, and the bovine kidney acetone powder antigen was precipitated step by step with PEG6000 at different concentrations.

[0043] 3. Purification and separation of PR3, MPO and GBM antigens

[0044] Antigens were identified using positive standard serum (developed by Omon anti-myeloperoxidase, proteinase 3, and glomerular basement membrane antibody IgG detection kit); PR3, MPO, and GBM antigens were separated and purified using ammonium sulfate precipitation-dialysis-ion exchange column-dextran gel column-immunoaffinity chromatography.

[0045] Example 2 Preparation of the kit

[0046] 1. Preparation of test strips: Human IgG (antibody) and antigens (PR3, MPO and GBM) were coated in parallel on nitrocellulose membranes, dried at 37°C for 16-18 hours, assembled, cut into strips, and made into strips of 4 membrane blocks with a length of 10.8 cm and a width of 2 mm.

[0047] 2. Preparation of blocking solution: sodium chloride (NaCl) 8.0g / L, potassium chloride (KCl) 0.2g / L, disodium hydrogen phosphate (Na2HPO4) 1.42g / L, polyethylene glycol 8000 (PEG8000) 5.0g / L, bovine serum albumin (BSA) 5.0g / L, preservative (proclin300) 0.5ml / L.

[0048] 3. Preparation of enzyme conjugate: anti-human IgG-6#-HRP: HRP conjugate stabilizer / diluent I = 1:10000, preservative (proclin300) 200 μl / L.

[0049] 4. Preparation of substrate solution: 1.2 mmol tetramethylbenzidine (TMB);

[0050] 5. Preparation of stop solution: 0.1 mol / L sulfuric acid;

[0051] 6. Preparation of concentrated washing solution: sodium chloride (NaCl) 36.72 g / L, potassium chloride (KCl) 3.73 g / L, Tween-20 (Tween-20) 18.3 ml / L, preservative (Neolone M-10) 20.0 ml / L, concentrated hydrochloric acid 7.8 ml / L.

[0052] 7. Preparation of Quality Control Products

[0053] 7.1 Single Quality Control

[0054] 1) Whole blood, let stand, centrifuge to remove red blood cells and fibrin, and obtain serum;

[0055] 2) Inactivate serum / plasma at 56°C for 30 minutes;

[0056] 3) Conduct biosafety testing;

[0057] 4) Preparation of quality control products:

[0058] Single quality control products are divided into two categories: ① positive and negative quality control products (quality control products used for positive and negative compliance rate: serum sample volume ≥ 200μL; ② repeatability quality control products (quality control products used for repeatability / batch difference and detection limit): serum sample volume ≥ 1000μL.

[0059] Preparation of single quality control product: ①. Pipette a single serum sample with known test results into a clean EP tube or test tube, add 1% proclin 300, and mix well; ②. Test the single serum in ① above using the self-produced kit and the reference kit. If the test results of the two reagents are consistent, this quality control product meets the requirements.

[0060] 7.2 Mixed Quality Controls

[0061] 1) Whole blood, let stand, centrifuge to remove red blood cells and fibrin, and obtain serum;

[0062] 2) Inactivate serum / plasma at 56°C for 30 minutes;

[0063] 3) Conduct biosafety testing;

[0064] 4) Preparation of quality control products:

[0065] Mixed quality control products are divided into two categories: ① positive and negative quality control products (quality control products used for positive and negative compliance rate): serum sample volume ≥ 200μL; ② repeatability quality control products (used for repeatability / batch difference): serum sample volume ≥ 1000μL.

[0066] Preparation of mixed quality control products: ① Pipette multiple serum samples with known test results into the same clean EP tube or test tube and mix them evenly; ② Test the mixed serum in ① above with the self-produced kit. If the test result of the self-produced reagent meets the requirements, there is no need to continue to add serum samples with known test results. Otherwise, it is necessary to continue to add serum samples with known test results until the test result of the self-produced reagent meets the requirements; ③ Add 1% proclin 300 to the mixed serum that meets the requirements in ② above, and then filter it with a 0.22μm filter head; ④ Test the mixed serum filtered in ③ above with the self-produced kit and the reference kit. If the test results of the two reagents are consistent, this quality control product meets the requirements.

[0067] 7.3 Detection Limit Quality Control

[0068] Detection limit quality control products: ① It is composed of a single quality control product or a single quality control after serial dilution; ② The detection limit quality control products must contain negative, positive and weak positive; ③ The detection limit quality control products of weak positive can be traced back to the marketed quantitative products to verify their accuracy. The quantitative value of the weak positive detection limit quality control product should not exceed ±10% of the critical value specified in the quantitative kit.

[0069] Inspect the product according to the technical requirements. If all performance indicators meet the requirements, the product is qualified.

[0070] 7.4 Composition and standards of quality control products

[0071] 7.4.1 The composition of quality control products is shown in Table 1.

[0072] Table 1: Prepared autoimmune vasculitis antibody spectrum quality control products

[0073]

[0074]

[0075] Note: The detection limit quality control product contains three levels, namely positive (defined as L1), weak positive (can be judged as negative or positive, defined as L2), and negative (defined as L3).

[0076] 7.4.2 Quality control product standards

[0077] Negative coincidence rate (specificity)

[0078] ①. No less than 10 negative control samples;

[0079] ② When testing negative quality control products provided by the manufacturer, no false positives should occur.

[0080] Positive coincidence rate (sensitivity)

[0081] ① No less than 6 copies of positive quality control products;

[0082] ② When testing positive quality control products provided by the manufacturer, no false negatives should occur.

[0083] Precision

[0084] ① Intra-batch precision (repeatability): The results of 10 tests should be consistent.

[0085] Detection limit

[0086] ① The detection limit quality control products of each antibody should be no less than 9 copies. The positive detection limit reference product should detect positive, the weak positive detection limit reference product can detect negative or positive, and the negative detection limit reference product should detect negative;

[0087] ② Due to the different quantitative and qualitative detection systems, there may be certain differences, and the concentration unit detected by the quantitative kit is a custom unit and is not comparable;

[0088] ③The detection limit quality control product can be obtained by dilution. The dilution ratio of the detection limit quality control product is only for this batch of quality control products.

[0089] 7.4.3 Storage conditions and shelf life of quality control products: Store below -20°C and avoid repeated freezing and thawing. It can be stable for 18 months. After thawing, it can be stable for 1 month at 2-8°C.

[0090] 7.4.4 Usage: The quality control product is liquid and can be used directly, and the usage method is the same as that of the sample.

[0091] Example 3 Test results

[0092] 1 Specificity and accuracy verification

[0093] A total of 125 patients with vasculitis were selected, including 25 with systemic vasculitis (SV), 25 with Wegener's granulomatosis (WG), 25 with microscopic polyangiitis (MPA), 25 with necrotizing crescentic nephritis (NCGN), and 25 with glomerulonephritis (GN); and 75 healthy subjects were selected as the control group.

[0094] Sample collection: 3 ml of venous blood was drawn from all subjects on an empty stomach. Serum was separated and stored in a refrigerator at -20°C. The kit prepared in Example 2 was used to detect anti-PR3, anti-MPO and anti-GBM antibodies.

[0095] SPSS11.5 statistical software was used for statistical analysis. The results showed that the positive coincidence rate of PR3, MPO and GBM was 100%, the negative coincidence rate was 100%, and the specificity was 100%.

[0096] It can be seen that the kit of the present invention can accurately detect or diagnose autoimmune vasculitis diseases (including systemic vasculitis, Wegener's granulomatosis, microscopic polyangiitis, necrotizing crescentic nephritis, glomerulonephritis) through one tube of blood, showing many advantages such as good accuracy, strong specificity, high sensitivity, convenient operation, and low sample consumption.

[0097] 2. Quality Control Product Verification

[0098] When using the detection limit reference test kit, the level 1 (L1) test result must be positive for the corresponding antibody, the level 2 (L2) test result can be positive or negative for the corresponding antibody, and the level 3 (L3) test result must be negative for the corresponding antibody;

[0099] Repeatability: The results of 10 tests of the positive quality control product were consistent and positive;

[0100] Negative compliance rate: The negative control products were tested, and the compliance rate of negative control products of each antibody was not less than 95%;

[0101] Positive compliance rate: The positive quality control products were tested, and the compliance rate of each antibody positive quality control product was not less than 90%.

[0102] According to the quality control product batch number in Table 1 and referring to the corresponding test results, the test results of the reference reagent for the autoimmune vasculitis antibody spectrum quality control product with batch number V3S190800 are shown in Table 3.

[0103] Table 3:

[0104]

[0105]

[0106] From the results in Table 3 combined with Table 1, we can conclude that:

[0107] 1. Positive quality control product number 01-06, positive compliance rate 100%, in line with the "positive compliance rate ≥ 90%" in the preparation of quality control products;

[0108] 2. The detection limit quality control product numbers 19, 22, and 25 can detect negative or positive, the detection results of quality control product numbers 18, 21, and 24 are positive, and the detection results of quality control product numbers 20, 23, and 26 are negative, which meets the "detection limit quality control product standard" in the preparation of quality control products;

[0109] 3. Negative quality control product number 07-16, negative compliance rate 100%, in line with the "negative compliance rate ≥ 95%" in the preparation of quality control products;

[0110] 4. For the mixed quality control product No. 17, each antibody was able to detect positive results, and the repeatability test was repeated 10 times with consistent results, which met the "repeatability quality control product standard" in the preparation of quality control products;

[0111] Conclusion: The prepared autoimmune vasculitis antibody spectrum quality control product meets the requirements for quality control product preparation. From the test results, it can be seen that the kit and quality control product of the present invention can jointly diagnose the relevant indicators of autoimmune vasculitis such as systemic vasculitis, Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), necrotizing crescentic nephritis (NCGN), glomerulonephritis, etc., and have the advantages of high sensitivity, strong specificity, and simple operation.

[0112] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A vasculitis antibody joint detection kit, characterized in that: The invention comprises a detection membrane strip, a blocking solution, an enzyme conjugate, a substrate solution, a stop solution and a concentrated washing solution; the detection membrane strip is composed of a carrier sheet and three parallel antigen detection lines fixed on the carrier sheet and coated thereon, wherein the antigen detection lines are formed by natural antigens being independently drawn on a carrier membrane and then coated thereon; the natural antigens are PR3, MPO and GBM; the antigen GBM is type IV collagen α-3 chain, which is separated and purified from bovine kidney by an acetone method.

2. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The vasculitis is an autoimmune vasculitis, including systemic vasculitis, Wegener's granulomatosis, microscopic polyangiitis, necrotizing crescentic nephritis, and glomerulonephritis.

3. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The antigens PR3 and MPO are separated and purified from human neutrophils.

4. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The carrier membrane is a nitrocellulose membrane or a nylon membrane.

5. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The detection membrane strip is also provided with a weak positive quality control line and a positive quality control line.

6. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The enzyme conjugate includes anti-human IgG-6#-HRP and HRP conjugate stabilizer / diluent, with a ratio of 1:10000.

7. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The blocking solution includes 8.0 g / L sodium chloride, 0.2 g / L potassium chloride, 1.42 g / L disodium hydrogen phosphate, 5.0 g / L polyethylene glycol 8000, 5.0 g / L bovine serum albumin and a preservative.

8. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The substrate solution is 1.2 mmol tetramethylbenzidine; and / or the stop solution is 0.1 mol / L sulfuric acid.

9. The vasculitis antibody joint detection kit according to claim 1, characterized in that: The concentrated washing liquid includes 36.72 g / L of sodium chloride, 3.73 g / L of potassium chloride, 18.3 ml / L of Tween-20, 20.0 ml / L of preservative, 7.8 ml / L of concentrated hydrochloric acid and preservative.

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