An elisa kit for detecting anti-chromatin antibodies and a detection method thereof

Abstract

The application discloses an ELISA kit for detecting anti-chromatin antibodies. The kit contains purified chromatin monomers with stripped H1 histone components in a natural structure. The application also discloses a detection method for the ELISA kit. The chromatin structure in the eukaryotic cell nucleus is complex, and is a natural complex composed of 40% DNA, 40% histone, 20% non-histone, a small amount of RNA and other macromolecules. The purification technology is high in requirement, and the price of the natural purified chromatin monomers is high. The application purifies the chromatin monomers with stripped H1 histone components according to the structure of the natural chromatin, retains the effective antigen components, and greatly reduces the cost. The kit can be used for detecting the anti-chromatin antibodies, fills the blank of the domestic anti-chromatin antibody detection method, and has the characteristics of large detection flux, accurate result, high sensitivity and simple operation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibody detection, and particularly relates to an ELISA kit for detecting anti-chromatin antibody and a detection method thereof. BACKGROUND

[0002] Systemic lupus erythematosus is a kind of diffuse connective tissue disease mediated by an autoimmune mechanism, with multiple organ and multiple system involvement as main features. The clinical manifestations are complex and diverse, and the disease course is prolonged and repeated. Most patients will have fever, fatigue and other symptoms at the initial stage of the disease. Butterfly erythema is also often common in patients with systemic lupus erythematosus. Kidney, nervous system, blood system, lung and digestive system can also be involved in patients with systemic lupus erythematosus. Once the patient is diagnosed with systemic lupus erythematosus, long-term treatment with glucocorticoids is required, and some immunosuppressive agents are also needed to be selected according to the patient's condition.

[0003] Anti-chromatin antibody plays a key role in the pathogenesis of systemic lupus erythematosus. Anti-chromatin antibody and anti-ds-DNA antibody are diagnostic markers for SLE. Not only is the level of this antibody highly positively correlated with the severity of SLE, but also is related to the incidence of lupus nephritis. Dynamic observation of the level of this antibody in serum helps to predict the evolution of the disease and guide clinical treatment.

[0004] The traditional chromatin extraction method is to separate chromatin from calf thymus cell nuclei. The antigen components obtained by this method are relatively mixed, and it is difficult to use them as a high-specificity antigen source. At present, there is no commercial anti-chromatin antibody detection kit on the domestic market. The performance of imported similar products (ANA spectrum screening kit of American Bio-Rad Company) is poor in correlation with systemic lupus erythematosus. Therefore, in view of the importance of detecting anti-chromatin antibody, it is particularly important to develop a widely applicable, efficient, sensitive and specific anti-chromatin antibody detection kit and the corresponding detection method for predicting the evolution of the disease and guiding clinical treatment. SUMMARY

[0005] In order to overcome the deficiencies in the prior art, the purpose of the present application is to provide an ELISA kit for detecting anti-chromatin antibody.

[0006] In order to achieve the purpose of the present application, the technical solution adopted is:

[0007] An ELISA kit for detecting anti-chromatin antibody, the kit contains chromatin monomers with H1 histone components stripped after purification;

[0008] The chromatin monomers with H1 histone components stripped are from the following method:

[0009] Step one, preparation of cell nucleus;

[0010] Collect peripheral venous blood, add ACD for anticoagulation;

[0011] Then the anticoagulated blood is subjected to red blood cell lysis until the red blood cells are completely dissolved to obtain white precipitate;

[0012] After adding buffer to the white precipitate and mixing, pure nuclear product is obtained by using proteinase K100 and 20% SDS water bath extraction;

[0013] Step two, extraction of chromatin:

[0014] The pure nuclear product is suspended in Tris-HCl buffer solution with pH 7.6 and homogenized by using a homogenizer, centrifuged, and the supernatant is discarded, and the obtained precipitate is crude chromatin;

[0015] The crude chromatin is suspended in a mixed solution of urea and PBS with pH 7.6, mixed, centrifuged, and the supernatant is discarded, and the precipitate is washed and centrifuged again, and the supernatant is discarded, and the precipitate is reserved;

[0016] After adding 6.5% ethanol solution to the precipitate and washing, centrifuging and discarding the supernatant, the chromatin monomer is obtained after several times of washing to remove urea salt;

[0017] H1 protein removal:

[0018] After the natural chromatin is digested by micrococcal nuclease, it is suspended in NH4Ac-HAc buffer solution with pH 4.5, and is subjected to multiple extractions;

[0019] The products of multiple extractions are respectively centrifuged and precipitated, and the purified chromatin monomer with stripped H1 histone components is collected.

[0020] In a preferred embodiment of the present application, the kit is provided with an anti-chromatin antibody detection strip, which is a 96-well enzyme-labeled plate coated with the chromatin monomer with stripped H1 histone components.

[0021] In a preferred embodiment of the present application, the kit is further provided with negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate developing solution, and termination solution.

[0022] In a preferred embodiment of the present application, the negative control serum is SLE patient immune method detection anti-chromatin antibody negative serum, the positive control serum is SLE patient immunoprecipitation method detection anti-chromatin antibody positive serum, and the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-human IgG;

[0023] The washing solution is PBST.

[0024] The substrate color developing solution is 3,3'5,5'-tetramethylbenzidine.

[0025] The termination solution is a 0.5% H2SO4 solution.

[0026] The second object of the present application is to provide a detection method for an ELISA kit for detecting anti-chromatin antibodies, comprising the following steps:

[0027] 1) stripping H1 histone component chromatin monomers to coat a 96-well plate, 4 degrees overnight;

[0028] After coating, wash and discard the coating solution, wash with PBST, repeat 3-5 times;

[0029] 2) add 5% BSA, 400ul / well BSA incubate at room temperature for 2h, then wash with PBST, repeat 3-5 times;

[0030] 3) add standard (known positive serum) and serum to be tested, 100ul per well, incubate at room temperature for 1h;

[0031] 4) standard dilution and blank control: 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, blank: 100ul PBS, serum to be tested 1:100 dilution, add 100ul respectively;

[0032] 5) wash with PBST, repeat 3-5 times;

[0033] 6) add enzyme-labeled secondary antibody 1:2000 (goat anti-human HRP), incubate at room temperature for 30 minutes, wash with PBST, repeat 3-5 times;

[0034] 7) add substrate solution and develop color for 15min;

[0035] 8) terminate the reaction, read on a 450 / 630nm microplate reader.

[0036] In a preferred embodiment of the present application, in step (1), the concentration of the stripped H1 histone component chromatin monomers is 0.5ug / ml, and 100ul is added to each reaction well.

[0037] The present application has the following main innovations:

[0038] The natural structure of chromatin is complex and requires high technology, and the price of purified chromatin is even more expensive. The present application uses stripped H1 histone component chromatin monomers, which not only improves the specificity of diagnosis, but also greatly reduces the cost.

[0039] The kit of the present application can be used for anti-chromatin antibody detection, fills the blank of domestic anti-chromatin antibody detection method, has the characteristics of large detection flux, accurate result, high sensitivity, simple operation, and is easy to popularize and apply. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a natural chromatin structure diagram;

[0041] Figure 2 It is a chromatin monomer structure diagram of stripping H1 histone component;

[0042] Figure 3 It is a diagram of coarse chromatin monomer and purified (H1 chromatin monomer) after stripping;

[0043] Figure 4 It is a diagnostic efficiency structure diagram of chromatin monomer of stripping H1 histone component;

[0044] Figure 5 It is a structure diagram of the correlation between anti-chromatin antibody and systemic lupus erythematosus disease activity;

[0045] Figure 6 It is a structure diagram of the correlation between anti-chromatin antibody and lupus nephritis. DETAILED DESCRIPTION

[0046] The present application is further illustrated by the following examples, but these examples shall not be used to explain the limitation of the present application.

[0047] Example 1

[0048] 1. Chromatin is a substance in the nucleus that can be stained by basic dyes, and it is a morphological structure that is transformed with chromosomes at different stages of the cell cycle. Chromatin refers to a linear complex structure composed of DNA, histone, non-histone protein and a small amount of RNA in interphase cells, which is the form of genetic material in interphase cells. The natural chromatin structure is very complex and contains many components. Among the main components of chromatin, DNA and histone are stably present, and the ratio of DNA to histone is about 1:1. However, the chromatin monomer stripped of H1 can be used as an antigen for ELISA platform detection and is related to diseases.

[0049] 2. Extract and separate the chromatin in the nucleus of eukaryotic cells, and purify it, including the following steps:

[0050] Preparation of cell nucleus;

[0051] Collect 10 ml of peripheral venous blood, add 1.7 ml of ACD (0.48 g of citric acid, 1.32 g of sodium citrate, 1.47 g of glucose, add water to 100 ml, 0.6 kg / cm2 sterilization for 30 min) anticoagulant;

[0052] Put blood into sterilized 50ml plastic centrifuge tube, add 30ml red cell lysate, main components are 0.32mol / L sucrose, 10mmol / l Tris-HCl (pH7.5), 5mmol / L MgCl2, 1% Triton-X 100, shake up and down gently until transparent, red cells are completely dissolved;

[0053] Ice bath for 10min, centrifuge, 3000rpm, 10min. Discard supernatant, red cell lysate is repeated for 1-3 times;

[0054] Discard supernatant, add 10ml buffer [0.1mol / L NaCl, 10mmol / l Tris-HCl (pH8.0), 1mmol / L EDTA (pH8.0)] to white precipitate, first a small amount, mix well, then add 10mg / ml proteinase K 100μl, 20% SDS 30μl, shake well, place in 37℃ water bath for 15-20h.

[0055] Extraction of chromatin

[0056] Suspend pure nucleus in 4℃ 10mmol / L Tris-HCl pH7.6 buffer, homogenize in glass homogenizer, after 2h, centrifuge at 1,800g x 15min, 4℃. Discard supernatant, precipitate is crude chromatin;

[0057] Suspend precipitate in 10ml cold 8mol / L urea 10.05mol / L PBS pH7.6 solution, mix well, centrifuge at 18,000g x 15min, 4℃. Discard supernatant, wash precipitate, discard supernatant after centrifugation, keep precipitate;

[0058] Add 20ml cold 6.5% ethanol solution to precipitate, centrifuge at 1,500g x 15min, 4℃. Discard supernatant, wash precipitate 5 times in similar way to remove urea. In this way, pure natural chromatin monomer is obtained.

[0059] H1 protein removal:

[0060] After natural chromatin is digested with micrococcal nuclease, suspend in 0.005mol / L NH4Ac-HAc pH4.5 buffer, extract in several times. For each extraction, use 25ml NH4Ac / HAch buffer, homogenize 5-6 times in ice bath with glass homogenizer, move into 100ml conical flask, extract for 4h in 4℃ cold room with electromagnetic stirring, centrifuge at 1,500g x 15min, 4℃. Re-extract precipitate, finally collect. Obtain large amount of purified H1-removed chromatin monomer.

[0061] 3. ELISA test with the antigen, comprising the following steps:

[0062] 3.1 The antigen concentration is 0.5 ug / ml, 55 ul of the chromatin monomer without H1 is used to coat a 96-well plate, 100 ul is added to each reaction well, and it is incubated at 4 degrees overnight;

[0063] 3.2 Washing: after the coating is finished, the coating solution is discarded, and PBST is used for washing, which is repeated for 3-5 times;

[0064] 3.3 Blocking: 5% BSA, 400 ul / well, incubated at room temperature for 2 hours, and then washed, same as 3.2;

[0065] 3.4 Adding standard and serum to be tested, 100 ul per well, incubated at room temperature for 1 hour;

[0066] 3.5 Diluting the standard (known positive serum):

[0067] Standard dilution: blank, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560;

[0068] Blank: 100 ul of PBS, 1:100 dilution of serum to be tested, and 100 ul is added respectively;

[0069] 3.6 Washing, same as 3.2;

[0070] 3.7 Adding secondary antibody 1:2000 (goat anti-human HRP), incubating at room temperature for 30 minutes, washing, same as 3.2;

[0071] 3.8 Adding substrate solution for color development for 15 minutes;

[0072] 3.9 Stopping the reaction, and reading with a microplate reader at 450 / 630 nm.

[0073] 4. Experimental data:

[0074] 4.1 The expression of anti-chromatin antibody in each group of people is detected, and compared with the detection of imported reagents, it can be seen that the sensitivity of the chromatin without H1 protein in the application in SLE is obviously improved, and the positive rate in other connective tissue diseases is significantly reduced. See Table 1.

[0075] Table 1 The expression of anti-chromatin antibody in various connective tissue diseases is shown in Table 1:

[0076]

[0077] 4.2 The diagnostic efficiency of the anti-chromatin antibody detection value in the application and the detection value of the imported similar product in SLE patients. The results showed that the diagnostic efficiency of the anti-chromatin antibody detection value in the serum of 141 patients with connective tissue disease other than systemic lupus erythematosus and 119 patients with systemic lupus erythematosus, the AUC area of the application (black) was 0.8197, while the detection value of the imported similar product (green) was only 0.6718, the detection specificity of the application was significantly increased, see Figure 3 .

[0078] 4.3 The correlation between the anti-chromatin antibody detection value in the application and the imported similar product and the activity of systemic lupus erythematosus. The SLEDAI scores of 119 patients with systemic lupus erythematosus were analyzed for their correlation with the expression of anti-chromatin antibody, the imported product detection value showed no significant correlation, while the anti-chromatin detection value in the application showed a positive correlation with the SLEDAI score, see Figure 4 .

[0079] 4.4 Comparison of the anti-chromatin antibody detection values of the two methods in 119 patients with systemic lupus erythematosus whether or not combined with lupus nephritis, the results showed that the anti-chromatin antibody detection value in the application was significantly increased in patients with systemic lupus erythematosus combined with lupus nephritis, see Figure 5 .

Claims

1. An ELISA kit for detecting anti-chromatin antibodies, characterized in that, The kit contains purified chromatin monomers stripped of H1 histone components; The chromatin monomers stripped of H1 histone components are from the following method: Step one, preparation of cell nucleus; Peripheral venous blood is collected and anticoagulated with ACD; The anticoagulated blood is then subjected to red blood cell lysis until the red blood cells are completely dissolved to obtain white precipitate; After adding buffer to the white precipitate and mixing, proteinase K 100 μl and 20% SDS are used to obtain pure nuclear product by water bath extraction; Step two, extraction of chromatin: The pure nuclear product is suspended in Tris-HCl buffer at pH 7.6 and homogenized by a homogenizer, centrifuged, and the supernatant is discarded, and the obtained precipitate is crude chromatin; The crude chromatin is suspended in a mixed solution of urea and PBS at pH 7.6, mixed, centrifuged, and the supernatant is discarded, and the precipitate is washed and centrifuged twice to discard the supernatant and retain the precipitate; After adding 6.5% ethanol solution to the precipitate and washing, centrifuging and discarding the supernatant, the chromatin monomers are obtained by washing several times to remove urea; Step three, removal of H1 protein: The natural chromatin is digested with micrococcal nuclease and suspended in 0.005 mol / L NH4Ac-HAc pH 4.5 buffer for fractionated extraction; The fractionated extraction products are centrifuged and precipitated to collect the purified chromatin monomers stripped of H1 histone components.

2. An ELISA kit for detecting anti-chromatin antibodies according to claim 1, characterized in that, The kit is provided with an anti-chromatin antibody detection strip, which is a 96-well enzyme-labeled plate coated with the chromatin monomers stripped of H1 histone components.

3. An ELISA kit for detecting anti-chromatin antibodies according to claim 1, wherein The kit also contains negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate developing solution, and termination solution.

4. An ELISA kit for detecting anti-chromatin antibodies according to claim 3, characterized in that, The negative control serum is SLE patient immune method detection anti-chromatin antibody negative serum, the positive control serum is SLE patient immunoprecipitation method detection anti-chromatin antibody positive serum, and the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-human IgG; The washing solution is PBST; The substrate developing solution is 3,3'5,5'-tetramethylbenzidine; The termination solution is 0.5% H2SO4 solution.

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