CN106644985B

CN106644985B - Marker, application thereof, kit and detection method of marker

Info

Publication number: CN106644985B
Application number: CN201611248659.4A
Authority: CN
Inventors: 楼建荣
Original assignee: Guangzhou Leide Biotechnology Co ltd
Current assignee: Guangzhou Leide Biotechnology Co ltd
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2021-02-05
Anticipated expiration: 2036-12-29
Also published as: CN106644985A

Abstract

The invention relates to the technical field of biology, in particular to a marker, application thereof, a kit and a detection method of the marker. The invention utilizes the principle that endogenous citrullinated antigen and ACPA can form antigen-antibody immune complex, finds and searches a detection method of citrullinated antigen-antibody immune complex and a related ELISA kit, can reflect the level of ACPA and the level of citrullinated antigen-antibody immune complex, and the citrullinated antigen-antibody immune complex is associated with disease state, and the change of the amount can directly reflect the progress of disease and the evaluation of treatment effect, so as to realize the purpose of rapid, accurate and convenient diagnosis and evaluation of RA.

Description

Marker, application thereof, kit and detection method of marker

Technical Field

The invention relates to the technical field of biology, in particular to a marker, application thereof, a kit and a detection method of the marker.

Background

Rheumatoid Arthritis (RA) is a chronic autoimmune disease that is pathologically altered primarily by the synovial inflammatory response of joints. The pathological process of RA is closely related to the generation of a plurality of autoantibodies in a patient body and the formation of antigen-antibody Immune Complexes (ICs) with corresponding antigens, and part of circulating ICs can be repeatedly deposited on joint synovial tissues to cause synovial inflammation through activation of a complement system-linked reaction, so that joint damage is caused. ICs in joint fluid and serum of RA patients can promote growth of chondrocytes, generation of NO and apoptosis, and indicate that the ICs play an important role in the pathogenesis of RA. The sensitivity of the ICs in the RA detection is higher, and the continuous increase of the ICs usually indicates that the prognosis of RA patients is poorer.

Only complexes containing antibodies of the IgG and IgM classes activate the classical pathway of complement and cause inflammation. The two types of antibodies have high content in serum and are main antibodies forming immune complexes; the larger immune complex formed by the soluble antigen and the corresponding antibody has stronger capability of activating complement. The IgG antibody is bivalent and can be interactively combined with a corresponding multivalent antigen to form a larger immune complex; IgM molecules can form larger immune complexes. Antigen-antibody affinity is high, and large and stable immune complexes are easily formed. Immune complexes formed by citrullinated peptide and ACPA exist at higher level in joint fluid and serum of RA patients, normally, antigen-antibody complexes are phagocytosed and cleared in the body, and ICs can induce inflammatory response under the condition of case.

The onset of RA is accompanied by the activation of various immunocompetent cells such as T cells, B cells, macrophages, synovial Fibroblasts (FLS), neutrophils and the like and the release of cytokines (IL6, TNF- α), chemokines and inflammatory mediators. RA FLS proliferation is closely related to local joint inflammation, pannus formation and bone destruction, and FLS proliferation and cytokine secretion abnormality play a key role in the pathogenesis of RA. The presence of ICs containing citrullinated protein and ACPA in RA serum can promote FLS proliferation, stimulate FLS to secrete more cytokines and participate in RA pathogenesis.

Autoimmunity is a major feature of rheumatoid arthritis, best exemplified by the high titers of autoantibodies detectable in plasma, of which Rheumatoid Factors (RF) are the best studied, but RF is present not only in RA but also in normal humans and is elevated in several other diseases. In recent years, anti-citrullinated protein antibodies (ACPA) have become the first choice for diagnosing RA, although they have not completely replaced RF in routine diagnosis of RA. Overall, ACPA is similar to RF sensitivity, but more specific. These antibodies recognize a variety of proteins that are citrullinated after transcription, including filaggrin (filaggrin), vimentin (vimentin), alpha and beta fibrin (alpha-and beta-fibrin), alpha-enolase (alpha-enolase), and Type I and Type ii collagen polypeptides (Type I and Type ii collagen).

ACPA was assayed by measuring the response of serum to Cyclic Citrullinated Polypeptide (CCP) by ELISA. The initial clinical test was based on the detection of a response of cyclic polypeptides against citrullinated filaggrin, since the cyclic citrullinated polypeptides are closer to the native conformational epitope, thereby increasing the accuracy of the experimental results. This experiment has now been replaced by a second generation of more accurate results-anti-CCP 2 Elisa-using citrullinated polypeptides selected from a library of citrullinated polypeptides, all of which are artificially synthesized. Recent studies have shown that citrullinated residues can be expressed in lung and synovial membranes, and that ACPA production may result from the deimination of proteins in lung and synovial membranes mediated by Peptidyl Arginine Deiminase (PAD). These proteins can be taken up by specific antigen presenting cells or immune complexes formed by B cells and presented to T cells, facilitating activation of ACPA-reactive B cells.

Currently, the detection specificity with respect to RF is low; the use of artificially synthesized citrullinated polypeptides for detection of ACPA may only reflect levels of autogenous antibodies, and ACPA levels are not necessarily associated with disease states.

Disclosure of Invention

In view of the above, the present invention provides a marker, applications thereof, a kit, and a method for detecting the marker. The invention aims to find and search a citrullinated antigen-antibody immune complex detection method and a related ELISA kit by utilizing the principle that an endogenous citrullinated antigen and ACPA can form an antigen-antibody immune complex, which can reflect the level of ACPA and the level of the citrullinated antigen-antibody immune complex, and the citrullinated antigen-antibody immune complex is associated with the disease state, and the change of the amount of the citrullinated antigen-antibody immune complex can directly reflect the progress of the disease and the evaluation of the treatment effect, so as to realize the purposes of quickly, accurately and conveniently diagnosing and evaluating RA.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides a marker (ECP) which is an immune complex of an endogenous citrullinated antigen with an anti-citrullinated antibody.

The result of detecting ACPA by using the endogenous RA specific citrullinated protein antigen can not only reflect the level of ACPA in the serum of a patient, but also further reflect the level of the endogenous citrullinated antigen and ACPA antigen-antibody complex of the patient or the capability of immune complex reaction, and ECP in circulation can be repeatedly deposited on joint synovial tissue to cause synovial inflammation and joint injury. Therefore, ECP can be used as a biological marker for RA diagnosis and prognosis judgment, can play a role in disease progression, and has important significance for current theoretical research and clinical practice.

Citrullinated protein with native conformational epitope in the serum of RA patient is easier to form antigen-antibody immune complex with ACPA, and no method for detecting ACPA by using endogenous citrullinated protein antigen is available at present.

The invention utilizes the principle that endogenous citrullinated antigen and ACPA can form antigen-antibody immune complex, finds and searches a detection method of citrullinated antigen-antibody immune complex and a related ELISA kit, can reflect the level of ACPA and the level of citrullinated antigen-antibody immune complex, and the citrullinated antigen-antibody immune complex is associated with disease state, and the change of the amount can directly reflect the progress of disease and the evaluation of treatment effect, so as to realize the purpose of rapid, accurate and convenient diagnosis and evaluation of RA.

In some embodiments of the invention, the endogenous citrullinated antigen in the marker is a type of endogenous citrullinated protein.

In some embodiments of the invention, the anti-citrullinated antibody in the marker is an IgG and/or IgM class antibody.

The invention also provides application of the anti-citrullinated antibody corresponding to the marker in preparation of a diagnostic reagent or a diagnostic tool for rheumatoid arthritis.

In some embodiments of the invention, the diagnostic tool in said use provided by the invention is a diagnostic kit.

The invention also provides a kit, which comprises the anti-citrullinated antibody and an acceptable reagent in detection; the anti-citrullinated antibody and an endogenous citrullinated antigen in a sample to be detected form the marker.

In some embodiments of the present invention, the reagent in the kit provided by the present invention further comprises one or a mixture of two or more of a carrier, a blocking solution, a washing solution, a sample diluent, a labeled antibody, a substrate, a developing solution, and a stop solution.

The invention relates to a detection method of citrullinated antigen-antibody immune complex and a related ELISA kit, which comprises a carrier pre-coated with an anti-citrullinated antibody, a labeled anti-human IgG/IgM antibody, and related sample diluent, washing liquid, developing liquid and stop solution.

In some embodiments of the present invention, the anti-citrullinated antibody is an anti-citrullinated antibody produced by immunizing an animal with PAD-catalyzed Bovine Serum Albumin (BSA) forming BSA with multiple citrullinated sites as an antigen. The specific sites are all arginine sites of the BSA protein which can be citrullinated; the antibody can be purchased from the market.

In some embodiments of the invention, anti-human citrullinated antibodies can be obtained by immunizing animals with citrullinated protein antigen formed from other proteins, particularly human synovial membrane specific proteins, catalyzed by PAD or the like. Specifically, two enzymes PAD4/PAD2 can catalyze the formation of citrulline from arginine sites on the protein.

In some embodiments of the present invention, the anti-citrullinated antibody pre-coated carrier is a conventional coated microplate prepared by diluting the anti-citrullinated antibody to 0.5-100ug/ml with a coating buffer.

In some embodiments of the present invention, the labeled anti-human IgG antibody is an anti-human IgG labeled with an enzyme, a chemiluminescent substance, a fluorescein or a gold by a conventional method, and the species source thereof is required to be consistent with the species source for preparing the citrullinated antibody.

The invention also provides application of the kit in detecting rheumatoid arthritis.

The invention also provides a detection method of the marker, which comprises the following steps:

step 1: coating the anti-citrullinated antibody with a carrier, mixing with a sample to be detected, and incubating to obtain the carrier containing the sample liquid to be detected;

step 2: coating the anti-citrullinated antibody with a carrier, mixing with a negative reference sample, and incubating to obtain a carrier containing a negative reference sample to-be-detected liquid;

and step 3: and (3) respectively taking the carrier containing the sample to-be-detected liquid prepared in the step (1) and the carrier containing the negative reference sample to-be-detected liquid prepared in the step (2), washing, mixing with a labeled secondary antibody respectively, incubating, washing, developing, detecting the light absorption value of the to-be-detected sample and the light absorption value of the negative reference sample, and obtaining the concentration of the marker.

In some embodiments, the concentration of the marker obtained in step 3 is equal to the light absorption value of the sample to be detected/the light absorption value of the negative reference sample.

The invention also provides a detection method of rheumatoid arthritis, and a detection result is obtained according to the concentration of the marker.

In some embodiments, the detection method provided by the present invention is a semi-quantitative detection result, and the detection result is expressed by an S/N value.

The marker detection result S/N is positive when the S/N is more than 3.2;

the marker detection result S/N is less than or equal to 3.2, and the result is negative.

Compared with the prior art, the invention provides a citrullinated antigen-antibody immune complex (ECP) detection method, which has the following advantages:

1. compared with rheumatoid factor detection, the invention has higher specificity (more than 90 percent) and higher sensitivity.

2. Compared with ACPA detection, the method does not use artificially synthesized citrullinated polypeptide, and avoids the difference between the ACPA detection methods caused by the difference of the types of artificially synthesized citrullinated peptide.

3. According to the invention, an immune complex is formed by endogenous citrullinated protein and ACPA, so that not only ACPA but also an immune complex of citrullinated antigen and antibody can be detected.

4. The citrullinated antigen-antibody immune complex can be detected by using a conventional ELISA method, and the method is simple and short in time consumption.

5. The circulating ECP can be repeatedly deposited on joint synovial tissue, and can cause synovial inflammation by activating complement system-linked reaction, thereby causing joint damage.

The ability and level of citrullinated antigen antibodies in RA patients to form immune complexes have important guiding effects and significance for RA diagnosis, disease evaluation and pathogenesis research.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 illustrates the detection principle of the present invention; wherein FIG. 1(A) shows a schematic diagram of the formation of a citrullinated antigen antibody immune complex (ECP) between an endogenous citrullinated protein and ACPA; FIG. 1(B) shows a schematic diagram of the ECP Elisa detection principle;

FIG. 2 shows the ECP levels detected in serum of samples at different dilution times;

figure 3 shows ECP levels in normal versus RA patients.

Detailed Description

The invention discloses a marker, application thereof, a kit and a detection method of the marker, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The principle of the technical method of the invention is as follows: the method comprises the steps of taking an anti-human citrullinated antibody as a primary antibody coating, forming an immune complex (ECP) by citrullinated protein in a sample and ACPA during sample adding and incubation, binding citrullinated protein in the ECP immune complex with the coated primary antibody, removing unbound protein by washing, adding a labeled anti-human IgG/IgM antibody as a secondary antibody, binding the labeled secondary antibody with ACPA in the ECP immune complex, removing the unbound labeled secondary antibody by washing, adding a substrate corresponding to the labeling of the secondary antibody for reaction, and detecting the reaction result by using a related instrument, wherein the content level of ECP in the sample is reflected by the result (see figure 1).

Wherein the anti-citrullinated antibody is generated by immunizing animals by using BSA (cit-BSA) which is formed by catalyzing Bovine Serum Albumin (BSA) by PAD and has a plurality of citrullinated sites as an antigen. The specific sites are all arginine sites of the BSA protein which can be citrullinated; the antibody can be purchased from the market.

Wherein, the cit-BSA can be used for other proteins, particularly human joint synovial membrane specific protein, to form citrullinated protein antigen through enzyme catalysis such as PAD and the like so as to immunize animals to obtain the anti-human citrullinated antibody. Specifically, two enzymes PAD4/PAD2 can catalyze the formation of citrulline from arginine sites on the protein.

The carrier pre-coated with the anti-citrullinated antibody is an enzyme label plate which is prepared by diluting the anti-citrullinated antibody to 0.5-100ug/ml with a coating buffer solution and is coated conventionally.

The labeled anti-human IgG antibody is anti-human IgG labeled by enzyme, chemiluminescent substance, fluorescein or gold in conventional method, and its species source requirement is identical to that of citrullinated antibody.

The specific technical scheme is as follows:

1) antibody preparation

Adopting PAD to catalyze Bovine Serum Albumin (BSA) to form BSA (cit-BSA) with various citrullinated sites, taking healthy immune animals, using the cit-BSA as an antigen to carry out immunization, uniformly grinding the antigen, a BCG vaccine and Freund's adjuvant for the first time, injecting animal lymph nodes or subcutaneous muscle tissues, immunizing for 4-5 times every two weeks, increasing and decreasing the antigen amount according to the weight of the animals, taking blood after the last two weeks of immunization, carrying out secondary precipitation on animal antiserum by using saturated ammonium sulfate, then loading the animal antiserum onto an ion exchange chromatographic column to obtain the anti-human citrullinated antibody of the animals, measuring the titer, and freezing for later use. The antibody is purchased from the market, and can also be prepared according to the preparation method.

2) Antibody labeling

The animal anti-human IgG/IgM antibody is labeled by enzyme, chemiluminescent substance or fluorescein by conventional method.

3) Preparation of coated antibody polystyrene microporous plate

Diluting anti-human citrullinated antibody to 0.5-100ug/ml with 0.01M PBS buffer solution with pH7.4, adding 100 microliter of anti-human citrullinated antibody into the bottom of a polystyrene micropore plate, and incubating for 8-48 hours at 2-8 ℃; taking out, washing with 0.01-0.5% Triton X-100 for 4 times, adding 0.5-5% skimmed milk powder, sealing at room temperature for 0.5-3 hr, pouring off the sealing solution, oven drying at 37 deg.C for 0.5-3 hr, and sealing and packaging with aluminum foil bag.

The polystyrene micropore plate can be replaced by magnetic beads or other media with antibody adsorption capacity.

4) Other applied reagents:

(1) coating buffer solution: PBS buffer (0.01M, pH 7.4);

(2) sealing liquid: BSA, skim milk powder, casein (concentration in the range of 0.1% to 5%);

(3) diluting liquid: BSA, skim milk powder, casein (concentration in the range of 0.1% to 5%);

(4) washing liquid: tween20, Triton X-100 (concentration ranging from 0.05% to 1%);

(5) color development liquid: TMB

(6) Stopping liquid: sulfuric acid

(7) And (3) the other: the substrate and the related reagent are used for enzyme labeling, chemiluminescent labeling or fluorescein labeling.

5) The specific operation method of the ECP diagnostic kit comprises the following steps:

A. diluting the serum or plasma sample 5-100 times with 0.5-5% skimmed milk powder, and dripping 100ul of the sample into the sample hole of the ELISA reaction plate; selecting other holes and adding a negative reference substance and a positive reference substance;

B. placing the reaction plate at room temperature for incubation for 0.5-3 hours, so that citrullinated protein in serum or plasma is crosslinked with the antibody on the reaction plate, and ACPA in serum or plasma is crosslinked with citrullinated protein to form an antigen-antibody immune complex;

C. washing the plate with 0.01-0.5% Triton X-100 for 4 times, each time for 0.5-5min, and washing off non-crosslinked protein;

D. diluting horseradish peroxidase (HRP) -labeled anti-human IgG antibody by 5% skimmed milk powder by 1:500-1:20000 times, dripping 100ul of antibody into a sample hole of the reaction plate, and incubating the plate at room temperature for 0.5-3h to combine the anti-human IgG antibody with the ACPA adsorbed on the reaction plate;

E. washing the plate with 0.01-0.5% Triton X-100 washing solution for 4 times, each time for 0.5-5min, and washing off non-crosslinked anti-human IgG antibody;

F. dripping 100ul of color development liquid containing a color development agent TMB, and carrying out color development reaction for 30min at room temperature;

G. 100ul of stop solution of 1M sulfuric acid is dripped to terminate the reaction;

H. and (3) detecting the light absorption value by using an enzyme-labeling instrument or a spectrophotometer under the condition of selecting the optimal absorbance within the wavelength range of 400-500nm, simultaneously determining any wavelength of 600-650nm as background reference, and representing the concentration level of the ECP in the sample by using the ratio of the absorbance of the sample to be detected to the absorbance of the negative reference.

Wherein, the anti-human IgG/IgM antibody can be enzyme label, chemiluminescence substance label or fluorescein label;

wherein the substrate is corresponding to enzyme label, chemiluminescence material label or fluorescein label.

(1) The citrullinated antigen-antibody immune complex (ECP) can be used as a biomarker for in vitro diagnosis of Rheumatoid Arthritis (RA).

(2) Aiming at the biomarker ECP in (1), an Elisa method for detecting the biomarker is invented. The method can simultaneously detect citrullinated antigen-antibody immune complexes containing IgG and IgM antibodies, and can also respectively detect ECP containing IgG antibodies and ECP containing IgM antibodies.

(3) The Elisa kit for detecting ECP comprises a coated enzyme label plate, a confining liquid, a washing liquid, a sample diluent, an enzyme labeled antibody, a substrate and a stop solution.

(4) The coated enzyme label plate in the step (3) is an enzyme label plate which is prepared by coating with an anti-citrullinated antibody, washing, sealing, drying and packaging.

(5) The citrullinated antibody described in (4) is an anti-citrullinated antibody produced by immunizing an animal with citrullinated protein formed in vitro as an antigen.

(6) The coating concentration of the anti-citrullinated antibody in the step (4) is 0.5-100 ug/ml; the coating volume is 50-200ul, and the coating buffer solution is conventional PBS or carbonate buffer solution; the coating condition is coating for 8-48 hours at 2-8 ℃ or coating for 1-8 hours at 37 ℃;

(7) the washing solution in (4) is 0.01-0.5% Triton X-100 or 0.01-0.5% Tween20, is prepared by adopting a conventional PBS buffer solution, and is used for washing plates manually or mechanically.

(8) And (4) the sample diluent is 0.1% -5% BSA, skimmed milk powder or casein, and is prepared by adopting a conventional PBS buffer solution.

(9) The ELISA plate in (4) is made of polystyrene material, polyvinyl chloride material or magnetic beads or other materials which can be stably combined with antibodies.

(10) And (3) the blocking solution is 0.1% -5% BSA, skimmed milk powder or casein, and is prepared by adopting a conventional PBS buffer solution.

(11) The enzyme-labeled antibody in (3) is an anti-human IgG/IgM antibody, and is labeled by enzyme, a chemiluminescent substance or fluorescein.

(12) The substrate in (3) is used in correspondence with the label used for the enzyme-labeled antibody.

(13) The ECP detection specificity is high, more than 90%, and the sensitivity is high;

(14) the ECP assay can reflect both the level of ACPA and the ability and level of antigen-antibody complexes.

(15) The circulating ECP can be repeatedly deposited on joint synovial tissue, and can cause synovial inflammation by activating complement system-linked reaction, thereby causing joint damage. ECP is associated with a disease state and changes in the amount can directly reflect the progression of the disease and the assessment of the efficacy of the treatment.

The marker, the application thereof, the kit and the raw materials and reagents used in the detection method of the marker are all commercially available.

The invention is further illustrated by the following examples:

example 1 measurement of citrullinated antigen antibody immune Complex (ECP) levels at various dilutions in serum of one example of RA patients

1) Serum preparation

Collecting fresh blood of RA patient with blood collecting tube without adding any anticoagulant, centrifuging at 3000 r for 10min with centrifuge, collecting supernatant as serum, and storing at-20 deg.C to-80 deg.C for use.

2) Preparation of ECP Elisa detection kit

Coating an enzyme label plate; sample dilutions (5% skim milk powder); washing (0.5% Triton X-100); enzyme-labeled antibody; a color developing liquid (TMB); stop solution (1M sulfuric acid).

3) Experimental procedure

A. Diluting the serum sample by 10 times, 20 times, 50 times and 100 times by using a sample diluent; respectively adding the mixture into sample holes of a reaction plate, wherein the volume of each sample hole is 100 ul;

B. incubation at room temperature (25 ℃) for 1 hour;

C. 200ul of washing liquid is added into each hole, and the plate is washed for 4 times, 2min each time;

D. diluting the enzyme-labeled antibody with a sample diluent according to a ratio of 1:10000, and adding 100ul of the diluted enzyme-labeled antibody into each hole;

E. incubation at room temperature (25 ℃) for 1 hour;

F. 200ul of washing liquid is added into each hole, and the plate is washed for 4 times, 2min each time;

G. 100ul of color development liquid is added into each hole; color reaction is carried out for 30min at room temperature (25 ℃);

H. adding 100ul of stop solution into each hole;

I. the absorbance of each well was measured at 450nm using a microplate reader (see FIG. 2 and Table 1 for the results).

TABLE 1 citrullinated antigen antibody immune complex (ECP) levels at different dilutions of serum

Dilution factor	OD
			10 times of	1.078
20 times of	0.619
		50 times of	0.490
100 times of	0.300

The result shows that the dilution factor of the sample has an influence on the detection result, and the dilution factor and the OD value are in a linear relation.

The invention can simultaneously detect citrullinated antigen-antibody immune complexes containing IgG and IgM antibodies, and also can detect ECP containing IgG antibodies and ECP containing IgM antibodies respectively, and ECP of different antibodies is detected according to the detection method, and the results are shown in Table 2.

TABLE 2 detection of ECP with different antibodies using different three antibodies

Three antibodies used	Detection of ECP containing different antibodies	Test results (S/N)
			Labeled anti-human IgG antibodies	ECP containing IgG class antibody	16.89
Labeled anti-human IgM antibodies	ECP containing IgM antibodies	14.84
			Labeled anti-human IgG and IgM antibodies	ECP containing IgG and IgM antibodies	24.77

The result shows that the marked anti-human IgG antibody or the marked anti-human IgM antibody respectively or the marked anti-human IgG and IgM antibodies can detect positive results (the S/N is positive than 3.2), so the invention can simultaneously detect citrullinated antigen-antibody immune complexes containing the IgG and IgM antibodies and can also respectively detect ECP containing the IgG antibodies and ECP containing the IgM antibodies.

Example 2 comparison of the levels of citrullinated antigen antibody immune complexes (ECP) in serum of 35 normal persons and 45 RA patients

1) Serum preparation

Collecting fresh blood of normal people and RA patients by adopting a blood collecting tube without adding any anticoagulant, centrifuging for 10min at 3000 r.p.m. by using a centrifuge, collecting supernatant fluid which is serum, and storing at-20 ℃ to-80 ℃ for later use.

2) Preparation of ECP Elisa detection kit

3) Experimental procedure

A. Diluting the serum sample by 10 times with a sample diluent; respectively adding the mixture into sample holes of a reaction plate, wherein the volume of each sample hole is 100 ul; selecting the rest wells, adding 100ul of each of the negative reference sample and the positive reference sample

B. Incubation at room temperature (25 ℃) for 1 hour;

E. incubation at room temperature (25 ℃) for 1 hour;

H. adding 100ul of stop solution into each hole;

I. detecting the light absorption value of each hole by using a microplate reader at 450nm and 630nm, and expressing the light absorption value of the sample by using the difference value of A450-A630;

J. and (4) calculating a result:

absorbance ratio (light absorption value of sample hole to be measured/light absorption value of negative reference sample hole)

The results are shown in Table 3, Table 4 and FIG. 3. Table 3 shows the absorbance ratios measured for 35 normal samples; table 4 shows the ratio of absorbance in the serum assay of 45 cases of RA patients

TABLE 3 absorbance ratio of normal sample detection

TABLE 4 absorbance ratio of serum assay for RA patients

As can be seen from tables 3 and 4:

1. feasibility of the invention: the average value of ECP level in the serum of normal human is lower; the mean ECP level in the serum of RA patients is obviously increased (P is less than 0.05), which is 3 times higher than that of normal people.

2. Sensitivity and specificity: the sensitivity was 66.7% at a specificity of 92.5%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A kit comprising an anti-citrullinated antibody and a reagent acceptable for detection; also included are markers that are immune complexes of endogenous citrullinated antigens with anti-citrullinated antibodies;

the anti-citrullinated antibody is generated by immunizing an animal by using BSA (cit-BSA) with various citrullinated sites formed by catalyzing Bovine Serum Albumin (BSA) by using PAD as an antigen;

the reagent also comprises one or a mixture of more than two of a carrier, a confining liquid, a washing liquid, a sample diluent, a labeled antibody, a substrate, a developing liquid and a stopping liquid;

the use method of the kit comprises the following steps:

step 1): coating the anti-citrullinated antibody with a carrier, sealing, mixing with a sample to be detected, and incubating to obtain a carrier containing a sample liquid to be detected;

step 2): coating the anti-citrullinated antibody with a carrier, sealing, mixing with a negative reference sample, and incubating to obtain a carrier containing a negative reference sample to-be-detected liquid;

step 3): respectively taking the carrier containing the sample to-be-detected liquid prepared in the step 1 and the carrier containing the negative reference sample to-be-detected liquid prepared in the step 2, washing, respectively mixing with a labeled secondary antibody, incubating, washing, developing, detecting the light absorption value of the to-be-detected sample and the light absorption value of the negative reference sample, and obtaining the concentration of the marker;

obtaining the concentration of the marker in the step 3), namely the light absorption value of the sample to be detected/the light absorption value of the negative reference sample;

the endogenous citrullinated antigen is a type of endogenous citrullinated protein;

the anti-citrullinated antibody is an IgG and/or IgM antibody.