CN108267576B - Modified CCP antigen and use thereof, anti-CCP antibody detection kit and manufacturing method thereof - Google Patents
Modified CCP antigen and use thereof, anti-CCP antibody detection kit and manufacturing method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a modified CCP antigenThe polypeptide has a sequence shown in the following formula I:
wherein the carboxyhydroxy group of glutamic acid E in formula I is substituted with an R group selected from: c1‑10Alkoxy radical, C1‑10alkyl-CO-NH-groups and C1‑10alkyl-CO-NH-NH-yl; said C is1‑10Alkyl is optionally substituted by one or more groups selected from C1‑4Alkyl radical, C1‑4Alkyl oxygen, hydroxyl, amino, nitro and phenyl. The invention also relates to the use of the antigen for detecting anti-CCP antibody, an anti-CCP antibody detection kit comprising the antigen and a manufacturing method thereof. The advantages of the invention include that the antigen activity of the derivative is higher after the modified CCP antigen is connected with the protein carrier; the precision of the detection of the CCP antibody is higher; the accuracy of the anti-CCP detection is higher.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a modified CCP antigen, application of the antigen in detecting anti-CCP antibodies, an anti-CCP antibody detection kit comprising the antigen and a manufacturing method thereof.
Background
Rheumatoid Arthritis (RA) is a multi-system, inflammatory, autoimmune, teratogenic disease that mainly involves the peripheral joints. The disease incidence rate in European and American countries is 1%, and the disease incidence rate in China is 0.32% -0.36%. The disease can cause irreversible bone joint destruction within 2 years, and is one of the main reasons for the loss of the labor force of people.
Studies in Guo Da et al have found that anti-cyclic citrullinated peptide antibodies have predictive value for the onset of rheumatoid arthritis (Guo Da, Wang bright, Highua, etc.. anti-cyclic citrullinated peptide antibodies have predictive value for the onset of rheumatoid arthritis [ J ]. Chinese Experimental diagnostics, 2005, 9 (1): 13-14).
The anti-Cyclic Citrullinated Peptide (CCP) antibody is mainly of IgG type, the anti-CCP antibody is secreted spontaneously by B lymphocytes of RA patients, but the anti-CCP antibody is not secreted spontaneously by B lymphocytes of other disease patients and normal people, therefore, the anti-CCP antibody has higher specificity to RA, the detection of the anti-CCP antibody mostly adopts a solid phase synthesis method to artificially synthesize Citrullinated Peptide fragments, the initial use of a linear Citrullinated Peptide fragment containing 19 amino acids, the amino acid sequence of which is SHQESTGSGRSGRSGS, however, the research finds that the detection of the anti-Cyclic Citrullinated Peptide by the E L ISA method leads to failure because the Peptide fragment cannot be adsorbed on polystyrene, and the later use of the Peptide fragment for improving the antigenic activity of the Citrullinated Peptide chain, overcomes the shortage of the linear Citrullinated Peptide fragment, the Schellekens (science of Schelleken, Vistained, cysteine, serine, cysteine, serine, cysteine, serine, cysteine, serine, cysteine, serine, cysteine, serine.
The detection of Anti-CCP antibody has high specificity (specificity is more than 90%) for the diagnosis of RA, and can be used for early diagnosis of RA. Approximately 70% of RA patients develop Anti-CCP antibodies in the serum early in the onset, and antibody-positive patients develop osteoarticular lesions detectable by radiological methods more readily than antibody-negative patients. Tiercy JM (Rheumatology, 2002, 41 (7): 809) found when studying the significance of Anti-CCP antibodies, AKA and IgM-RF in distinguishing RA from other rheumatic diseases: IgM-RF was highly sensitive to RA diagnosis (75%), followed by Anti-CCP antibody (68%); while the Anti-CCP antibody had the highest specificity for RA diagnosis (96%), followed by AKA (94%); while IgM-RF is only 74% specific for RA diagnosis. Therefore, Anti-CCP antibodies have higher specificity than IgM-RF in diagnosing RA.
The current Anti-CCP diagnostic techniques mainly comprise enzyme-linked immunoassay (E L ISA), chemiluminescence (C L IA), immunochromatography (colloidal gold or latex particle method), time-resolved immunofluorescence assay (TRFIA) and immunodot assay, which have respective advantages and disadvantages.
C L IA is used in detecting CCP antibody, and has high detection sensitivity, good specificity and repeatability, wide detection range, stable reagent, no toxicity, no pollution, simple operation, short detection period, easy automation and detection of single sample.
In the conventional protocol for detecting anti-CCP antibody by chemiluminescence immunoassay, a solid phase carrier is coated with a derivative formed by linking CCP antigen and a protein carrier, and an anti-human IgG secondary antibody is labeled with a luminescent marker. When the Anti-CCP antibody is combined with a solid phase carrier through a CCP antigen, the Anti-CCP antibody is combined with an Anti-human IgG secondary antibody, and finally, the solid phase carrier separation technology is used, so that the aim of accurately detecting the Anti-CCP antibody in the serum sample is fulfilled.
CCP antigens are small cyclic polypeptides having the sequence: HQCHQEST-Cit-GRSRGRCGRSGS (expressed in three characters as NH 2-His-Gln-Cys-His-Gln-Glu-Ser-Thr-Cit-Gly-Arg-Ser-Arg-Gly-Arg-Cys-Gly-Arg-Ser-Gly), disulfide bond: c3 ═ C16. Citrulline (Cit) is an important immunogenic site among them. The CCP antigen is coupled to the protein carrier via a carboxyl group.
The prior art still has higher requirements on the antigen activity of the derivative after the CCP antigen is connected with the protein carrier, and has higher requirements on the precision and the accuracy of the anti-CCP antibody detection.
Disclosure of Invention
The inventors have conducted continuous studies in order to overcome one or more technical problems existing in the prior art. It has been unexpectedly found, through continuing experimentation, that a modified CCP antigen can achieve the technical objects of the present invention.
The CCP antigen has a sequence represented by formula I:
wherein the carboxyhydroxy (-OH) group of glutamic acid E (i.e. the sixth amino acid) in formula I is substituted with an R group selected from one or more of the following: c1-10Alkoxy radical, C1-10alkyl-CO-NH-groups and C1-10alkyl-CO-NH-NH-yl; said C is1-10Alkyl represents an alkyl group having 1 to 10 carbon atoms such as methyl, ethyl, isopropyl, tert-butyl, neopentyl, n-nonyl and the like; the oxy represents-O-; -CO-represents a keto group;
said C is1-10Alkyl groups are optionally substituted with one or more conventional substituents. The substituent can be selected from C1-4Alkyl (e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl), C1-4Alkyloxy (i.e. C)1-4Alkyloxy), hydroxyl (HO-), amino (NH2-), nitro (NO2-) and phenyl (C6H 5-).
Preferably, the R group is selected from one or more of: c1-5Alkoxy radical, C1-5alkyl-CO-NH-groups and C1-5alkyl-CO-NH-NH-groups.
Preferably, the R group is selected from one or more of: methyloxy, ethyloxy, tert-butyloxy, benzyloxy, benzhydryloxy ((C6H5)2CHO-), acetamido (CH3CHO-NH-) and acetylhydrazino (CH3 CHO-NH-NH-).
In another embodiment, the present invention relates to the use of the above-described modified CCP antigen for the preparation of a detection reagent for anti-CCP antibodies.
In another embodiment, the present invention relates to an anti-CCP antibody detection reagent comprising the modified CCP antigen described above.
In another embodiment, the present invention relates to a medicament comprising a modified CCP antigen as described above.
In another embodiment, the invention relates to an anti-CCP antibody detection kit comprising component a and component B, wherein component a comprises magnetic microspheres coated with the above-described modified CCP antigen coupled to a protein carrier, and component B comprises a luminescent marker solution labeled with a secondary mouse anti-human IgG antibody.
Preferably, the protein carrier is selected from animal-derived or human-derived proteins such as Bovine Serum Albumin (BSA), cationic bovine serum albumin (cBSA), hemocyanin (K L H), Ovalbumin (OVA), serum albumin HSA, bovine gamma globulin, and human gamma globulin.
Preferably, the magnetic microspheres, i.e. magnetic spheres, may be any commercially available magnetic spheres, including but not limited to: the surface of the magnetic ball is provided with an epoxy group, a sulfonyl group, an aldehyde group, an amido group, an amino group, a carboxyl group, a sulfydryl group and a hydroxyl group modified magnetic ball.
Preferably, the luminescent marker is selected from: luminol, isoluminol and its derivatives, Horseradish Peroxidase (HRP), alkaline phosphatase, and the like.
Preferably, the working concentration of the magnetic microspheres is 1-10mg/m L (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10mg/m L), and/or the working concentration of the modified CCP antigen is 50-100ng/m L (such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 96 or 100ng/m L), and/or the working concentration of the protein carrier is 10-40ng/m L (such as 10, 15, 20, 25, 30, 35 or 40ng/m L), and/or the working concentration of the murine anti-human IgG secondary antibody is 100ng/m L (such as 100, 150, 200, 250 or 300ng/m L) and/or the working concentration of the luminescent marker is 10-30ng/m L (such as 10, 15, 20, 25 or 30ng/m L).
Preferably, the kit further comprises a low concentration calibrator solution (such as 2, 3, 4, 5 or 6U/m L, specifically 3.91U/m L) and a high concentration calibrator solution (such as 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500U/m L).
Preferably, each of the components A and B in the kit contains BSA and a preservative, wherein the BSA concentration by mass percent is 0.1-0.5%, and the preservative is any one of potassium sorbate, sodium benzoate, sodium azide, sodium nitrite and Proclin series or a mixture thereof.
In another embodiment, the present invention relates to a method for manufacturing an anti-CCP antibody detection kit, the method comprising:
1) coupling the modified CCP antigen as described above to a protein carrier, preferably in a molar ratio of the modified CCP antigen to the protein carrier of from 20:1 to 80:1, such as 30: 1, 40: 1, 50: 1, 60: 1 or 70: 1;
2) coating the magnetic microspheres with the conjugate obtained in step 1) to obtain component A, wherein the weight ratio of the magnetic microspheres to the conjugate is 1 mg: 10 μ g-1 mg: 40 μ g, for example, 15, 20, 25, 30 or 35 μ g of the conjugate is used per 1mg of the magnetic microspheres;
3) reacting a mouse anti-human IgG secondary antibody with a luminescent marker to obtain a component B;
4) assembling component a and component B into a kit.
Preferably, the manufacturing method further comprises manufacturing the low-concentration calibrator solution and the high-concentration calibrator solution, and placing them in a kit or using them in combination.
Specifically, one embodiment of the present invention relates to a method for preparing a test kit for detecting anti-CCP antibodies, which comprises the following core preparation steps:
preparation 1: the modified CCP antigen (hereinafter referred to as novel CCP antigen) of the present invention is conjugated to a protein carrier
1) Reacting the novel CCP antigen with a cross-linking agent in the presence of a catalyst to obtain a novel CCP antigen reaction solution;
wherein the cross-linking agent includes but is not limited to glutaraldehyde, BS (PEG)5, BS (PEG)8, PEG400, PEG800, succinic anhydride, etc. in an amount of 1eq to 4 eq;
the catalyst comprises but is not limited to triethylamine, pyridine, potassium hydroxide and potassium carbonate, and the dosage of the catalyst is 1eq to 4 eq;
2) adding a protein carrier into the novel CCP antigen reaction solution obtained in the step 1) for reaction, and purifying to obtain a conjugate formed by the novel CCP antigen and the protein carrier;
wherein, the mol ratio of the novel CCP antigen to the protein carrier is 20:1-80: 1.
Preparation 2: preparation of novel CCP antigen conjugate coated magnetic sphere suspension
The conjugate obtained in preparation 1 is used for coating magnetic microspheres (produced by biomedical engineering Co., Ltd. of New Production of Shenzhen, 80% of particle size distribution is 1-5 μm, magnetization intensity is 4000), the concentration of the magnetic spheres is 100mg/m L, and the hydroxyl number is 95. the specific steps are as follows:
1) placing the magnetic ball in acetic acid buffer solution, ultrasonically stirring and cleaning, removing supernatant through magnetic separation, and repeating the step three times to obtain acetic acid buffer solution suspension magnetic ball;
2) adding CDC and novel CCP antigen-protein carrier conjugates into an acetic acid buffer suspension magnetic sphere by adopting a magnetic sphere connection CDC method (magnetic sphere-CDC-antigen/antibody), and placing the magnetic sphere in a constant-temperature oscillating water bath box for reaction to obtain magnetic sphere reaction liquid coated by the novel CCP antigen conjugates;
wherein the ratio of the magnetic ball to the novel CCP antigen-protein carrier conjugate is 1 mg: 10 mug to 1 mg: 40 mug;
3) removing supernatant of the reaction solution obtained in the step 2) through magnetic separation, adding a magnetic ball cleaning solution, stirring and cleaning, and repeating the step for four times;
wherein the magnetic ball cleaning solution is: adding 0.5% (w/v) BSA (produced by Roche) into 0.1M PBS buffer solution with pH7.4, and mixing;
4) after the washing is finished, adding a magnetic sphere suspension for suspension to obtain a magnetic sphere suspension coated by the novel CCP antigen conjugate;
wherein the suspension of the magnetic spheres is 0.1M PBS buffer solution with pH7.4.
Preparation 3: preparation of mouse anti-human IgG Secondary antibody labeled luminescent marker
1) Filling a carbonic acid buffer solution of a mouse anti-human IgG secondary antibody into a dialysis bag with a proper interception amount, and putting the dialysis bag into a dialysate for dialysis, wherein the dialysate is the carbonic acid buffer solution;
2) adding a luminescent marker into the dialyzed solution in the step 1) for reaction; wherein the reaction temperature is 25-37 ℃, and the reaction time is 8-12 h;
the luminescent marker may be selected from: luminol and its derivatives, isoluminol and its derivatives, horseradish peroxidase, alkaline phosphatase, etc.;
3) purifying the reaction solution obtained in the step 2) through a G-25 gel column, and collecting a solution with a peak value to obtain a luminescent marker solution marked by a mouse anti-human IgG secondary antibody;
4) adding the luminescent marker solution marked by the mouse anti-human IgG secondary antibody obtained in the step 3) into BSA protective solution for later use.
One embodiment of the present invention relates to a chemiluminescent immunological method for detecting anti-CCP antibodies comprising the steps of:
1) respectively adding a sample solution to be detected, a high-concentration calibrator solution and a low-concentration calibrator solution into different reaction cup holes;
2) respectively adding the special magnetic microspheres coated by the novel CCP antigen prepared by the invention into the step 1-) to combine with the substance to be detected;
3) adding a luminescent marker labeled by a mouse anti-human IgG secondary antibody into the step 2) respectively for incubation, so that the mouse anti-human IgG secondary antibody is combined with the magnetic microspheres coated by the novel CCP antigen and the substance to be detected;
wherein the incubation temperature is 25-37 deg.C, and the incubation time is 10-20 min;
the luminescent marker may be selected from: luminol and its derivatives, isoluminol and its derivatives, horseradish peroxidase, alkaline phosphatase, etc.;
4) performing magnetic separation to obtain a conjugate of a mouse anti-human IgG secondary antibody, the magnetic microspheres coated by the novel CCP antigen and a substance to be detected;
5) adding a luminescence excitation substrate into the combination obtained in the step 4), and detecting the intensity of a light signal;
when the luminescent label is isoluminol, the luminescence excitation substrate may be NaOH and H2O2(ii) a When other substances are adopted as the luminescent markers, the luminescent excitation substrate can be changed into other common substances correspondingly;
6) and automatically calculating according to the detection light intensity of the sample by the corrected working curve of the calibrator to obtain the concentration of the anti-CCP antibody of the sample to be detected.
The present invention also relates to a kit produced by the above-mentioned production method, a method for using the kit, a detection method using the kit of the present invention, a method for detecting anti-CCP antibodies using the modified CCP antigen of the present invention, and an apparatus for carrying out this method.
Compared with the prior art, the invention has the advantages that:
the antigen activity of the modified CCP antigen derivative connected with the protein carrier is higher; the precision of the detection of the CCP antibody is higher; the accuracy of the anti-CCP detection is higher.
Detailed Description
The invention is further described with reference to specific examples. The scope of the invention is not limited thereto.
The embodiments are directed to the following terms,
a marker: the term "label" as used herein refers to a substance that can be detected in an immunoassay. The label may be selected from any nanoparticle-based label, and may also be referred to as a "label complex" comprising the label.
Labeling antigen: the term "labeled antigen" as used herein refers to an antigen, also called a labeled protein, that binds to an antibody of interest to be detected in an immunoassay.
Ligand: the term "ligand" as used in the present invention is a molecule that can be specifically recognized by a tag, such as an antibody, avidin, etc.
Labeling: the term "tag" as used herein includes, but is not limited to, a polypeptide or protein, biotin, or a combination thereof with a peptide or protein, where the polypeptide or protein is a polypeptide or protein other than any portion of the antigen coated in the kit.
Alkoxylation: a reaction of introducing an alkoxy group into an organic compound molecule.
The following examples were examined using a Maglumi2000plus chemiluminescence immunoassay analyzer manufactured by Shenzhen, New Productivity, biomedical engineering, Inc.
The raw material sources are as follows:
novel sources of CCP antigens: shenzhen, a new product, biomedical engineering shares, Inc.; the novel CCP antigen substitutes the carboxyl hydroxyl of glutamic acid E into an R group on the basis of the traditional CCP antigen (HQCHQEST-cit-GRSRGRCGRSGS);
source of ABEI (N- (4-aminobutyl) -N-ethylisobutol): shenzhen, a new product, biomedical engineering shares, Inc.;
source of murine anti-human IgG secondary antibody: sigma company;
magnetic microspheres: shenzhen, a new product, produced by biomedical engineering, Inc., 80% of the particles have a size distribution of 1-5 μm, a precipitation time of 10-15 seconds when the magnetization is 4000 gauss, and a protein adsorption concentration of 0.8-1.2 mg when the BSA is 30 mg.
Preparation 1: preparation of novel CCP antigen and protein carrier forming derivatives
23.4mg of novel CCP polypeptide antigen is dissolved in dimethyl sulfoxide to prepare 100mg/m L reaction solution, 21.3mg of cross-linking agent BS (PEG)5(4eq, thermo) and 5.6 mu L catalyst triethylamine (4eq), the reaction solution is reacted for 2.5h at 25 ℃ and 250 rpm.
According to the ratio of 20:1 of the novel CCP antigen to the bovine IgG, 40 mu L of reaction liquid is added into 1M L of bovine IgG solution (10mg/M L and 0.1M NaHCO)3) At room temperature, 250rpm, the reaction was carried out for 2.5 h. G25 was gel purified in 0.01M PBS pH 7.4.
Preparation 2: preparation of novel CCP antigen derivative coated magnetic sphere suspension
The magnetic microsphere (produced by Shenzhen, New Production biomedical engineering Co., Ltd., 80% particle size distribution of 1-5 μm, magnetization of 4000), the concentration of 100mg/m L, and the hydroxyl number of 95 are coated with the novel CCP antigen derivative obtained by preparation 1. the specific steps are as follows:
the magnetic ball treatment process comprises the following steps:
1) preparation of 0.05 mol/L pH3.6 acetate buffer:
2.55g of sodium acetate trihydrate is weighed, dissolved by 4500m L purified water, then added with 14m L acetic acid and mixed evenly, and the volume is adjusted to 5000m L, thus obtaining 0.05 mol/L pH3.6 acetic acid buffer solution.
2) Magnetic sphere connection CDC method (magnetic sphere-CDC-antigen/antibody)
Adding an acetic acid buffer solution with the pH value of 3.6 and 0.05 mol/L which is 5 times of the coating volume into a small white bottle, putting the small white bottle into an ultrasonic instrument, stirring and cleaning the small white bottle for 2 to 3 minutes while carrying out ultrasonic, then placing the small white bottle on a magnet, pouring out the supernatant after the supernatant is clear, and repeating the step for three times.
3) Antigen/antibody loading and reaction
Adding equal amount of pH3.6 acetic acid buffer solution with coating volume to suspend magnetic spheres with concentration of 20mg/m L, adding CDC with concentration of 10mg/m L, adding purified novel CCP antigen-bovine IgG derivative according to the proportion of 1 mg: 12 μ g, placing in a constant temperature shaking water bath box, reacting at 37 deg.C for 24 hours
4) Cleaning the magnetic balls:
① arrangement of magnetic ball cleaning liquid
0.5% (w/v) BSA (produced by Roche) was added to 0.1M, pH7.4 PBS buffer and mixed for further use.
② pouring the magnetic ball into a beaker, placing on a magnet for precipitation, pouring out the supernatant, adding 5 times of magnetic ball cleaning solution, stirring, cleaning, placing on the magnet, pouring out the supernatant after the supernatant is clear, and repeating the cleaning step for four times.
5) Suspension of magnetic balls:
after washing, a coating volume of magnetic sphere suspension was added at a suspension concentration of 20mg/m L.
Preparation 3: preparation of mouse anti-human IgG Secondary antibody labeled ABEI
1) Preparation of dialysate (solution F):
na was added to a 5000m L beaker2CO314.31g，NaHCO326.46g of the solution is added with water to reach 4500m L, and the prepared F solution is placed on a magnetic stirrer for standby.
2) Selecting a dialysis bag with a proper interception amount (14000 is commonly used), measuring the dialysis bag with a proper size, tightening one end after wetting, and testing leakage of purified water for 3 times (without leakage).
3) 1mg of a mouse anti-human IgG secondary antibody was adjusted to 1m L with 0.1 mol/L of a carbonic acid buffer solution (F solution) having a pH of 9.5, and the other end was bound to the dialysate and dialyzed for 2 hours (stirring speed 400).
4) The dialyzed solution was filled into a small white bottle (1 m L per bottle), and 300. mu.g of an ABEI activated ester was added thereto and reacted at 37 ℃ for 2 hours.
5) The G-25 gel column was mounted, and after being washed clean with purified water, the column was equilibrated with PBS buffer at pH 7.4.
6) After the gel column is balanced and eluted, the marked ABEI is purified by passing through the column, and then the solution with the peak value is collected.
7) The collected protein solution was added to an equal volume of BSA protecting solution containing 50mg/m L.
Example 1:
the polypeptide sequence of the novel CCP antigen employed in this example is: HQCHQ (R) ST-cit-GRSRGRCGRSGS, disulfide bond: c3 ═ C16, in which the radical R is methoxy CH3O-。
The kit comprises the following components:
1) the component A is a novel CCP antigen derivative solution for coating a magnetic sphere, wherein the novel CCP antigen derivative is formed by coupling a novel CCP antigen and a bovine IgG protein carrier, the working concentration of the magnetic sphere is 1mg/m L, the working concentration of the novel CCP antigen is 50ng/m L, and the concentration of the bovine IgG protein carrier is 10ng/m L;
2) and the component B is an ABEI solution marked by a mouse anti-human IgG secondary antibody, wherein the working concentration of the ABEI is 30ng/m L, and the working concentration of the mouse anti-human IgG secondary antibody is 300ng/m L.
3) Calibrator solution-low concentration calibrator solution with concentration of 3.91U/m L and high concentration calibrator solution with concentration of 250.0U/m L.
The components all contain BSA and preservative, the mass volume percentage concentration of the BSA is 0.1%, and the preservative mainly contains sodium azide (NaN)3) The mass volume percentage concentration is 0.2%.
The preparation method of each component of the kit of the embodiment comprises the following steps:
preparing a novel CCP antigen-bovine IgG derivative formed by the novel CCP antigen and a bovine IgG protein carrier according to preparation 1 above;
preparing a magnetic sphere suspension coated with the novel CCP antigen-bovine IgG conjugate according to preparation 2 above;
a mouse anti-human IgG secondary antibody-labeled ABEI solution was prepared according to preparation 3 above.
The method comprises the following steps of preparing 10 parts of standard solutions with different concentrations by using an anti-CCP antibody standard and bovine serum as a solvent, and detecting the 10 parts of solutions by using the kit components prepared in the embodiment, wherein the detection steps are as follows:
a) respectively adding a 10 mu L sample solution to be detected, a high-concentration calibrator solution and a low-concentration calibrator solution into different reaction cup holes;
b) adding special magnetic microspheres coated by 20 mu L of novel CCP antigen respectively;
c) 100 mu L of mouse anti-human IgG secondary antibody labeled ABEI is added respectively;
d) bathing at 37 deg.C for 15min, and cleaning in magnetic environment for 3 times;
e) adding 200 mu L systematic lotion;
f) adding luminescent substrate 1(NaOH) and luminescent substrate 2 (H) respectively2O2) Detecting the intensity of the optical signal;
g) and automatically calculating according to the detection light intensity of the sample by the corrected working curve of the calibrator, and obtaining the anti-CCP antibody concentration of the sample to be detected from the working curve.
Example 2:
the R group of the novel CCP antigen employed in this example is pentoxy CH3(CH2)4O-the rest is the same as in example 1.
Example 3:
the R group of the novel CCP antigen used in this example is an acetamido group (CH3CHO-NH-), and the rest is the same as in example 1.
Example 4:
the R group of the novel CCP antigen used in this example is an acethydrazide group (CH3CHO-NH-), the rest being the same as in example 1.
Comparative example:
this comparative example used a conventional CCP antigen (HQCHQEST-cit-GRSRGRCGRSGS), i.e., the R group was a hydroxyl group, and the remainder was the same as in example 1.
Performance evaluation
The performance of the kits of examples 1, 2 and comparative example was evaluated as follows:
1 blank limit
The zero concentration calibrator was repeatedly assayed 20 times using the anti-CCP assay kit prepared in examples and comparative examples, and the relative luminescence intensity (R L U) was recorded for 20 tests.
1.1 data processing
Calculating the average value (M) and Standard Deviation (SD) of the R L U for 20 times to obtain M +2SD, substituting the value of M +2SD into the working curve of the kit (the corresponding concentration when the R L U value is M +2SD can be directly calculated by using the user software matched with the instrument), and obtaining the corresponding concentration value, namely a blank limit.
2 precision
2.1 internal precision of batch
Using low, medium and high 3 concentration samples prepared from zero-value serum (concentration selected as close to medically determined level as possible) and a kit quality control as assay samples, each sample was assayed 20 times in duplicate using the anti-CCP assay kit prepared in examples and comparative examples, and the assay results were recorded 20 times for each sample.
2.2 precision between batches
Using low, medium and high 3 concentration samples prepared from zero value serum (the concentration is selected as close to the medically determined level as possible) and a kit quality control as measurement samples, continuously repeating the measurement of each sample for 20 times by using three batches of reagents respectively, and recording the measurement result of each sample for 60 times.
2.3 data processing
The mean (M) and Standard Deviation (SD) of the intra-and inter-batch measurements were calculated, respectively, and the CV value was calculated according to equation (1).
CV＝SD/M×100％.....................(1)
3 degree of accuracy
3.1 test acceptance criteria
The relative deviation should be within + -10%.
3.2 test methods
The cyclic citrullinated peptide industry standard was prepared at a concentration of about 100U/m L (tolerance ± 10%), and used as a sample, and after repeating the measurement 3 times, the mean value of the results was recorded, and the measurement deviation was calculated according to the formula (2).
3.3 data processing
The deviation is calculated according to equation (2):
the measured deviation is (measured mean value-theoretical value)/theoretical value × 100% ·
Evaluating the blank limit of the kit, wherein the detection result is shown in table 1;
the results of the evaluation of the in-batch precision of the kit are shown in tables 2, 3, 4 and 5;
the inter-batch precision of the kit was evaluated, and the results are shown in table 6;
the measurement accuracy of the kit was evaluated, and the results are shown in table 7.
Table 1: margin limit
In conclusion, according to the blank limit detection results of the example 1, the example 2, the example 3, the example 4 and the comparative example, after the novel CCP antigen is used in the example 1, the example 2, the example 3 and the example 4, the value of the blank limit R L U is reduced by 40 percent relative to the blank limit value of the comparative example, and the blank limit performance of the antibody CCP antibody detection kit is remarkably improved.
Table 2: run-in-run (example 1vs. comparative)
Table 3: run-in-run (example 2vs. comparative)
Table 4: run-in-run (example 3vs. comparative)
Table 5: run-in-run (example 4vs. comparative)
And (4) conclusion: according to the results of the in-batch difference detection of the example 1, the example 2, the example 3, the example 4 and the comparative example, after the novel CCP antigen is used in the examples, the CV values of the low-value sample, the medium-value sample and the high-value sample are all smaller than those of the comparative example, so that the in-batch precision of the anti-CCP antibody detection kit can be remarkably improved by the novel CCP antigen.
Table 6-1: batch to batch variation (example 1)
Table 6-2: batch to batch variation (example 2)
Tables 6 to 3: batch to batch variation (example 3)
Tables 6 to 4: batch to batch variation (example 4)
Tables 6 to 5: batch to batch difference (comparative example)
In conclusion, according to the results of the batch-to-batch difference detection of the example 1, the example 2, the example 3, the example 4 and the comparative example, after the novel CCP antigen is used in the example, the CV values of the low-value sample, the medium-value sample and the high-value sample are all smaller than those of the comparative example after the three batches (L ot1, L ot2 and L ot3) are detected, so that the batch-to-batch precision of the anti-CCP antibody detection kit can be remarkably improved by the novel CCP antigen.
Table 7: accuracy of
And (4) conclusion: according to the accuracy detection results of the example 1, the example 2, the example 3, the example 4 and the comparative example, after the novel CCP antigen is used in the examples, the detection deviation values are respectively 0.12%, 0.73%, 0.38% and 4.44%, which are less than 8.41% of the comparative example, which indicates that the accuracy of the examples is better than that of the comparative example, so that the accuracy of the anti-CCP antibody detection kit can be improved by the novel CCP antigen provided by the invention.
The inventor finds out through the above examples that the novel CCP antigen can unexpectedly improve the blank limit performance, precision and accuracy of the anti-CCP antibody detection kit.
Without being limited by theory, the inventors attempt to explain the principles of the present invention as follows: the novel anti-CCP antigen of the invention uses modified glutamic acid to replace natural glutamic acid in the traditional CCP antigen, so that the glutamic acid in the synthesized novel CCP antigen no longer contains carboxyl. When the novel CCP antigen is connected with a protein carrier, the novel CCP antigen is well coupled through a carboxyl at a C terminal, and the influence of the protein carrier connected to the carboxyl of glutamic acid on the activity of the CCP antigen is avoided, so that a better technical effect is obtained, and the expected technical purpose of the invention is realized.
In conventional unmodified CCP antigens, the carboxyl group contained in glutamate is very close to citrulline, which is a key site for immune activity, and the two are separated by only 2 amino acids. When carrier proteins are connected through carboxyl of glutamic acid, citrulline in CCP antigen is forced to be too close to a protein carrier, so that the influence of steric hindrance effect is generated, the antigen activity of a derivative of CCP antigen connected with the protein carrier is reduced, and the precision and accuracy of detecting anti-CCP antibody are further reduced.
On the other hand, the derivative formed by the novel CCP antigen and the protein carrier is used for coating the magnetic sphere so as to carry out derivative amplification on the small molecule antigen, and the precision and the accuracy of the Anti-CCP antibody subsequent detection are improved. This also contributes to the achievement of the technical object of the present invention.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (10)
1. A modified CCP antigen, said CCP antigen having a sequence represented by the following formula I:
characterized in that the carboxyloxy group of glutamic acid E in formula I is substituted by an R group,
the R group is selected from one or more of the following: methyloxy, pentyloxy, acetamido and acethydrazide groups.
2. Use of a modified CCP antigen as defined in claim 1 for the preparation of an anti-CCP antibody detection reagent.
3. An anti-CCP antibody detection kit, which comprises a component A and a component B, and is characterized in that,
said component A comprising magnetic microspheres coated with a modified CCP antigen of claim 1 or 2 coupled to a protein carrier,
the component B comprises a luminescent marker solution labeled by a mouse anti-human IgG secondary antibody.
4. The kit of claim 3, wherein the luminescent marker is selected from the group consisting of: luminol, isoluminol and its derivatives, horseradish peroxidase and alkaline phosphatase.
5. The kit of claim 3, wherein the protein carrier is selected from the group consisting of Bovine Serum Albumin (BSA), cationic bovine serum albumin (cBSA), hemocyanin (K L H), Ovalbumin (OVA), Human Serum Albumin (HSA), bovine gamma globulin and human gamma globulin.
6. The kit of claim 3, wherein the kit satisfies one or more of:
the working concentration of the magnetic microspheres is 1-10mg/m L;
the working concentration of the modified CCP antigen is 50-100ng/m L;
the working concentration of the protein carrier is 10-40ng/m L;
the working concentration of the mouse anti-human IgG secondary antibody is 100-300ng/m L;
the working concentration of the luminescent marker is 10-30ng/m L.
7. A preparation method of an anti-CCP antibody detection kit is characterized by comprising the following steps:
1) coupling the modified CCP antigen of claim 1 or 2 to a protein carrier;
2) coating the magnetic microspheres with the conjugate obtained in the step 1) to obtain a component A;
3) reacting a mouse anti-human IgG secondary antibody with a luminescent marker to obtain a component B;
4) assembling component a and component B into a kit.
8. The method according to claim 7,
the molar ratio of the modified CCP antigen to the protein carrier is 20:1-80: 1; and/or
The weight ratio of the magnetic microspheres to the conjugate is 1 mg: 10. mu.g-1 mg: 40 μ g.
9. The method of claim 7, wherein step 1) comprises reacting said CCP antigen with a cross-linking agent in the presence of a catalyst, followed by addition of said protein carrier.
10. The method according to claim 9,
the cross-linking agent is selected from: glutaraldehyde, bs (PEG)5, bs (PEG)8, PEG400, PEG800 and succinic anhydride; and/or
The catalyst is selected from: triethylamine, pyridine, potassium hydroxide and potassium carbonate.
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