KIT FOR MEASURING ANTI-CYCLIC CITRULLINATED PEPTIDE ANTIBODY, APPLICATION THEREOF, AND TEST METHOD

Abstract

A kit for measuring an anti-cyclic citrullinated peptide antibody, an application thereof, and a test method is provided. The kit includes a biotinylated antigen formed by coupling a plurality of branched peptide units having branched peptide chains using lysine, and the branched peptide units each is formed by coupling a plurality of citrullinated peptides using lysine. The kit and method have high test sensitivity and good specificity; compared with a mixed antigen, citrulline being coupled and then used as a single-component antigen, reduces the complexity of components of the antigen and the difficulty of quality control of the reagent, and improves inter-batch consistency, and quantitative test can be performed and the time needed to complete all procedures to obtain a result is 45 min.

Claims

1. A kit for measuring an anti-cyclic citrullinated peptide antibody, comprising a biotinylated antigen formed by coupling a plurality of branched peptide units having branched peptide chains using lysine, wherein each of the branched peptide units are formed by coupling a plurality of citrullinated peptides using lysine.

2. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 1, wherein the citrullinated peptides are selected from the group consisting of the following amino acid sequences and combinations thereof: TABLE-US-00009 SEQ ID NO. 1: SHQESTRGRSRGXSGRSGS; SEQ ID NO. 2: SHQESTRGRSRGRSGXSGS; SEQ ID NO. 3: SHQESTXGXSRGRSGRSGS; and SEQ ID NO. 4: SHQESTXGRSXGRSGRSGS.

3. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 2, wherein a plurality of citrullinated peptides as shown in SEQ ID NO. 1 form a first branched peptide unit, a plurality of citrullinated peptides as shown in SEQ ID NO. 2 form a second branched peptide unit, a plurality of citrullinated peptides as shown in SEQ ID NO. 3 form a third branched peptide unit, a plurality of citrullinated peptides as shown in SEQ ID NO. 4 form a fourth branched peptide unit, and the biotinylated antigen comprises the first branched peptide unit, the second branched peptide unit, the third branched peptide unit, and the fourth branched peptide unit.

4. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 3, wherein the lysine of the first branched peptide unit and the lysine of the second branched peptide unit are respectively coupled with one lysine, and the lysine of the third branched peptide unit and the lysine of the fourth branched peptide unit are respectively coupled with another lysine.

5. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 1, wherein each of the branched peptide units respectively has 4 to 6 citrullinated peptides, and the biotinylated antigen has 4 to 6 branched peptide units.

6. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 1, wherein in the biotinylated antigen, each of the citrullinated peptides is coupled with biotin.

7. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 1, wherein the kit further comprises a magnetic particle separation reagent, an alkaline phosphatase-labeled anti-human IgG antibody, and a chemiluminescence substrate.

8. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 7, wherein magnetic particles in the magnetic particle separation reagents have a diameter of 0.1 to 0.5 μm, have superparamagnetism, and have streptavidin groups on surfaces thereof.

9. The kit for measuring an anti-cyclic citrullinated peptide antibody according to claim 1, wherein the biotinylated antigen is prepared by the following steps: Step 1: coupling the citrullinated peptides with lysine to form branched citrullinated peptides; Step 2: mixing 2 to 5 mg of synthesized branched citrullinated peptides and 0.5 to 0.8 mg of biotin activated by N-hydroxysuccinimide evenly, and reacting at 22 to 25° C. for 25 to 40 min; Step 3: adding 15 to 20 μL of tris(hydroxymethyl)aminomethane buffer with a concentration of 0.04 to 0.06 mol/L, mixing and reacting at 28 to 32° C. for 15 to 30 min, then adding 550 to 650 μL of glycerol to obtain biotinylated CCP branched peptide antigen, which is stored at −20° C. for future use; and Step 4: diluting the biotinylated citrullinated branched peptide antigen into a mixed solution with a concentration of 1 to 5 μg/ml using a phosphate buffer of pH 7-7.5 and a concentration of 0.01 mol/L, to give a biotinylated antigen working fluid.

10. An application of the kit according to claim 1 for testing the content of anti-cyclic citrullinated peptide antibody.

11. A method for testing the content of anti-cyclic citrullinated peptide antibody using the kit according to claim 1, the method comprising: reacting a sample to be tested with a magnetic particle separation reagent and biotinylated antigen at 36 to 38° C. for 10 to 25 min to obtain a first complex, washing and then adding alkaline phosphatase-labeled anti-human IgG antibody, and reacting at 36 to 38° C. for 10 to 25 min to obtain a second complex, washing and then adding a chemiluminescent substrate and reacting at 36 to 38° C. for 5 to 10 min, and testing.

Description

BRIEF DESCRIPTION

[0038] Some of the embodiments will be described in detail, with references to the following Figures, wherein like designations denote like members, wherein:

[0039] FIG. 1 is schematic structure diagrams of the respective branched peptide units and biotinylated antigen;

[0040] FIG. 2 is a test principle diagram; and

[0041] FIG. 3 is a linear regression diagram of the actual test concentration tested, according to the test method of Embodiment 6, using the test kit of anti-cyclic citrullinated peptide antibody IgG of Embodiment 5 and the theoretical concentration.

DETAILED DESCRIPTION

[0042] In the following, the present disclosure is further explained in detail combining with specific embodiments, but the present disclosure is not limited to the following embodiments. The implementation conditions employed by the embodiments may be further adjusted according to specific requirements, undefined implementation conditions usually are conditions in conventional experiments, and the reagents in the present disclosure are all commercially available.

Embodiment 1: Preparation of Biotinylated Antigen

Synthesis of 16-Branched Citrullinated Peptide

[0043] According to the structural characteristics of citrulline and arginine, their spatial structure changes were predicted using bioinformatics related software, the known citrullinated antigens were analyzed, classified to screen out a citrulline-containing polypeptide library. The citrulline-containing polypeptide library and rheumatoid arthritis serum were screened to obtain a group of citrullinated peptides from which four citrullinated peptides with the highest combined test sensitivity were screened. Utilizing the characteristics that a lysine has two amino groups, the citrullinated peptides were coupled into a molecule with four identical branched peptide chains, and finally four kinds of four-branched citrullinated peptides were obtained. Then these four different branched peptide molecules were coupled into one molecule with lysine, which was 16-branched citrullinated peptide, wherein the method of coupling the branched peptides with lysine was a conventional method.

[0044] The amino acid sequences of citrulline selected are:

[0045] SEQ ID NO. 1: SHQESTRGRSRGXSGRSGS, namely Ser His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Xaa Ser Gly Arg Ser Gly Ser, wherein, X and Xaa respectively represent citrulline, as shown in Sequence 1 of the sequence listing;

[0046] SEQ ID NO. 2: SHQESTRGRSRGRSGXSGS, namely Ser His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Arg Ser Gly Xaa Ser Gly Ser, wherein, X and Xaa respectively represent citrulline, as shown in Sequence 2 of the sequence listing;

[0047] SEQ ID NO. 3: SHQESTXGXSRGRSGRSGS, namely Ser His Gln Glu Ser Thr Xaa Gly Xaa Ser Arg Gly Arg Ser Gly Arg Ser Gly Ser, wherein, X and Xaa respectively represent citrulline, as shown in Sequence 3 of the sequence listing;

[0048] SEQ ID NO. 4: SHQESTXGRSXGRSGRSGS, namely Ser His Gln Glu Ser Thr Xaa Gly Arg Ser Xaa Gly Arg Ser Gly Arg Ser Gly Ser, wherein, X and Xaa respectively represent citrulline, as shown in Sequence 4 of the sequence listing;

[0049] The specific preparation method of the biotinylated antigen:

I. Materials and Instruments:

[0050] Materials: synthesized 16-branched citrullinated peptides, biotin activated by N-hydroxysuccinimide, tris(hydroxymethyl)aminomethane buffer, glycerol, and phosphate buffer;

[0051] Instruments: a reagent cryopreservation box, and a biochemical incubator.

II. Preparation Steps:

[0052] Step 1: 2 mg of synthesized 16-branched citrullinated peptides and 0.5 mg of biotin activated by N-hydroxysuccinimide were mixed evenly and reacted at 25° C. for 30 min;

[0053] Step 2: 20 μL of tris(hydroxymethyl)aminomethane buffer with a concentration of 0.05 mol/L was added, mixed and reacted at 30° C. for 30 min, then 600 μL of glycerol were added to obtain biotinylated CCP branched peptide antigen, which was stored at −20° C. for future use;

[0054] Step 3: The biotinylated citrullinated branched peptide antigen was diluted into a mixed solution with a concentration of 2 μg/ml using a phosphate buffer of pH 7.5 and a concentration of 0.01 mol/L, to give a biotinylated antigen working fluid.

[0055] The structure of the biotinylated antigen is shown in FIG. 1, wherein, citrullinated peptide 1 is citrullinated peptide shown in SEQ ID NO. 1, citrullinated peptide 2 is citrullinated peptide shown in SEQ ID NO. 2, citrullinated peptide 3 is citrullinated peptide shown in SEQ ID NO. 3, and citrullinated peptide 4 is citrullinated peptide shown in SEQ ID NO. 4.

Embodiment 2: Preparation of Alkaline Phosphatase-Labeled Anti-Human IgG Antibody

Materials and Instruments:

[0056] Materials: anti-human IgG antibody, alkaline phosphatase, coupling reagent 2-iminothiolane hydrochloride, glycine, and tris(hydroxymethyl)aminomethane buffer.

[0057] Instruments: a G-25 gel column, a reagent cryopreservation box, a Supperdex200 gel purification column, an analytical balance, and a biochemical incubator.

Operation Steps:

[0058] Step 1: 3 mg of anti-human IgG antibody was added into 40 mL of a coupling reagent 2-iminothiolane hydrochloride with a concentration of 10 mg/mL, and the system stood at 20° C. for 20 min;

[0059] Step 2: 2 mL of 0.08 mol/L glycine solution was added, the system stood at 20° C. for 4 min and was desalted through the G-25 gel column, and the activated anti-human IgG antibody was collected and stored at 5° C. for future use;

[0060] Step 3: 3 mg of an alkaline phosphatase solution was added into 4 mg/mL N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate solution, the system stood at 25° C. for 30 min and was desalted through the G-25 gel column, and the activated alkaline phosphatase was collected and stored at 5° C. for future use;

[0061] Step 4: The activated anti-human IgG antibody and the activated alkaline phosphatase were mixed, and the mixture stood at 5° C. for 20 h, purified through Supperdex200 gel purification column to obtain a concentrated connected material solution, which was stored at 5° C. for future use;

[0062] Step 5: The concentrated connected material solution of Step 4 was diluted into a concentration of 1 μg/mL of the alkaline phosphatase-labeled anti-human IgG antibody using a mass ratio of 1% of bovine serum albumin and tris(hydroxymethyl)aminomethane buffer of pH 7.8 to 8.0 and a concentration of 0.05 mol/L, namely the alkaline phosphatase-labeled anti-human IgG antibody.

Embodiment 3: Preparation of Calibrator: for Calibration Curve

I. Materials and Instruments:

[0063] Materials: anti-citrullinated peptide antibody, phosphate buffer, and standard substance;

II. Preparation Steps:

[0064] The anti-citrullinated peptide antibody was selected, diluted according to a certain proportion using a phosphate buffer of pH 7.5 and a concentration of 0.01 mol/L, and with reference to the standard substance, prepared to be calibrators respectively with concentrations of 20 RU/ml and 200 RU/ml.

Embodiment 4: Preparation of Quality Control Materials

I. Materials and Instruments:

[0065] Materials: anti-citrullinated peptide antibody, phosphate buffer, and standard substance;

II. Preparation Steps:

[0066] The anti-citrullinated peptide antibody was selected, diluted according to a certain proportion using a phosphate buffer of pH 7.5 and a concentration of 0.01 mol/L, and with reference to the standard substance, prepared to be quality control materials respectively with concentrations of 10 RU/ml and 80 RU/ml.

Embodiment 5: Test Kit of Anti-Cyclic Citrullinated Peptide Antibody IgG

[0067] A test kit of anti-cyclic citrullinated peptide antibody IgG of the present embodiment, comprises:

[0068] biotinylated antigen (concentration: 2 μg/mL) prepared according to the method of Embodiment 1, 5 mL;

[0069] alkaline phosphatase-labeled anti-human IgG antibody (concentration: 1 μg/mL) prepared according to the method of Embodiment 2, 30 mL;

[0070] magnetic particle separation reagent purchased from Thermo Fisher, 5 mL;

[0071] calibrator prepared according to the method of Embodiment 3, 1 mL;

[0072] quality control material prepared according to the method of Embodiment 4, 1 mL;

[0073] chemiluminescence substrates prepared according to Formula II of APSH-1 in Table 1.2 disclosed in Patent Application No. CN201510359183.0, an enzyme-promoting chemiluminescence substrate using alkaline phosphatase, namely:

TABLE-US-00002 2-amino-2-methyl-1-propanol 200 mM AMPPD 0.5 g/L HOBEP-1 0.5 g/L mgSO.sub.4 5.0 Mm ZnCl.sub.2 1.0 mM ProClin300 0.1 g/L.

Embodiment 6: Test of Kit

[0074] The kit of Embodiment 6 adopts an automatic chemiluminescence analyzer for testing, which specifically comprises the following steps:

[0075] Step 1: the test kit of Embodiment 5 was used in conjunction with a suitable automatic chemiluminescence analyzer. The kit was placed in the corresponding position of the reagent storehouse of the automatic chemiluminescence analyzer, and the kit information was input into the instrument system through the barcode scanner or set through the instrument supporting software;

[0076] Step 2: the calibrator was placed in the sample storehouse of the instrument, scanned through the barcode scanner to identify the calibrator information, and assigned to the position of the calibrator in the instrument system;

[0077] Step 3: the quality control material/sample to be tested were placed in the sample storehouse of the instrument, and the corresponding test information was edited through the instrument supporting software;

[0078] Step 4: start running program, and all the processing steps of the calibrator/quality control material/sample to be tested were automatically performed, wherein, the sample to be tested and magnetic particle separation reagent as well as biotinylated antigen were reacted at 37° C. for 15 min to obtain a first complex, the first complex was washed and the alkaline phosphatase-labeled anti-human IgG antibody was added, and the system was reacted at 37° C. for 15 min to obtain a second complex, the second complex was washed and then the chemiluminescent substrate was added, and the system was reacted at 37° C. for 5 min, and tested.

[0079] When the test kit is used in conjunction with an automatic chemiluminescence analyzer, the steps of dilution, sample addition, incubation, washing, and testing are fully automated, and unattended flow operation is possible. The fully automatic closed operating system not only has simple and convenient operation, high reliability, good stability, and good reproducibility of test results, but also avoids result deviation caused by human operation, and effectively improves test efficiency and saves labor costs.

Embodiment 7: Implementation of Test and Evaluation of Test Effect

[0080] (1) Sample Comparison

[0081] Negative and positive coincidence rate: The test kit of Embodiment 5 of the present disclosure was used to test the content of CCP in 250 clinical sera, and compared with the CCP antibody ELISA test kit of EURO DIAGNOSTICA company (Table 1). The results show that, compared with the ELISA test kit, the negative coincidence rate of the CCP kit of the present disclosure is 99.0% (198/200), and the positive coincidence rate is 94.0% (48/50), indicating that the kit and the existing anti-CCP antibodies testing reagents on the market have a high degree of consistency. The data are shown in Table 1.

TABLE-US-00003 TABLE 1 Sample comparison Kit of Embodiment 5 Clinic Sample Negative Positive Total ELISA Negative 198 2 200 testing kits Positive 2 48 50 Total 200 50 250

[0082] (2) Sensitivity: The LOD of the test kit of Embodiment 5 of the present disclosure is 0.039 RU/ml, while the sensitivity of the CCP antibody ELISA test kit of EURO DIAGNOSTICA is 2 RU/ml. The kit of Embodiment 5 of the present disclosure was used to test the minimum detectable quantity reference substances (L1, L2, L3) of the testing company, and the test was repeated 3 times. The operation was according to the kit instructions and the test results was evaluated.

[0083] L1 reference substance concentration: the concentration was 60 RU/ml, the concentration error was not more than 10%;

[0084] L2 reference substance concentration: the concentration was 40 RU/ml, the concentration error was not more than 10%;

[0085] L3 reference substance concentration: the concentration was 10 RU/ml, the concentration error was not more than 10%.

[0086] (3) Linearity: a high-value serum (theoretical concentration 400 RU/ml, measured concentration 449.4 RU/ml) was diluted according to 1/1, 1/2, 1/8, 1/20, 1/80, 1/200, the diluted samples were tested through the kit of Embodiment 5 of the present disclosure, and a regression curve based on the dilution ratio and the detection concentration was drawn. The squared value of the correlation coefficient R was found out. The results are shown in FIG. 3, which shows that the linear correlation coefficient of the CCP kit is 0.9998, the result is greater than 0.9900, and the slope is 1.0924.

[0087] (4) Accuracy: the accuracy of the kit of Embodiment 5 of the present disclosure is evaluated through sample recovery. A high-value serum H (measured concentration is about 300 RU/ml), a median-value serum M (measured concentration is about 100 RU/ml), and a low-value serum L (measured concentration is about 50 RU/ml)) were added to the corresponding 3 parts of basal serum (measured concentration <10 RU/ml) according to 1:9, and the concentrations were calculated. Results: The sample recovery of serum was between 85% and 115%. The data are shown in Table 2.

[0088] Note: Sample recovery=Measured concentration of sample after addition/(0.1*Measured concentration of Sample A+0.9*Measured concentration of Sample B)*100%

TABLE-US-00004 TABLE 2 Accuracy Measured Measured Measured concen- concen- concen- tration tration tration Average of of of of Sample Sample Sample Sample sample Sample sample A A B B after recovery recovery High- 281.87 Basal 4.03 37.65 111.97% 102.75% value 290.99 serum 5.14 34.14  98.59% serum 298.45 1 4.27 33.06  97.69% H Median- 102.04 Basal 7.28 18.59 112.30% 108.55% value 105.25 serum 6.40 17.50 111.06% serum 106.52 2 6.66 16.36 102.28% M Low- 44.01 Basal 5.49 9.15  92.06%  96.97% value 53.79 serum 5.39 9.42  95.60% serum 57.67 3 4.40 9.25 103.25% L

[0089] (5) Precision: the test kit of Embodiment 5 of the present disclosure was used to test the quality control materials at three concentrations, twice a day in the morning and afternoon, with 4 repetitions each time for a total of 10 days, a total of 80 measurements for each concentration were made to calculate the coefficient of variation (coefficient of variation CV=concentration mean/standard deviation*100%). The results show that the coefficient of variation is within 10%.

TABLE-US-00005 TABLE 3 Precision Concentration Test Inter-assay (RU/ml) repetition CV (%) 10 80 5.2 20 80 3.1 100 80 6.4

[0090] (6) Stability: the kit of Embodiment 5 of the present disclosure was placed at 4° C. for 7 days and 37° C. for 7 days, respectively, and measured the quality control signal retention rate of 3 kinds of concentration, namely high concentration (measured concentration was about 200 RU/ml), medium concentration (measured concentration was about 80 RU/ml), and low concentration (measured concentration was about 10 RU/ml). The results were all >90%, indicating that the kit is stable and meets clinical requirements. The data are shown in Table 4.

TABLE-US-00006 TABLE 4 Stability Re- Sam- 4°C. D7 37° C. D7 tention ple RLU AVER CV RLU AVER CV rate 10 125347 128684 3.67% 124066 123823 0.28%  96.22% RU/ 132020 123580 ml 80 932437 889415 6.84% 916876 901594 2.40% 101.37% RU/ 846392 886311 ml 200 2377448 2398137 1.22% 2316782 2380701 3.80%  99.27% RU/ 2418826 2444620 ml

[0091] (7) Specificity: different concentrations of bilirubin, hemoglobin, rheumatoid factor, triglyceride, and human anti-mouse antibody were added to serum with different concentrations, namely high concentration (300 RU/ml), medium concentration (100 RU/ml) and low concentration (20 RU/ml), and the test results showed: the added substances had no effect on the test result of the kit of Embodiment 5 of the present disclosure. The data are shown in Table 5.

TABLE-US-00007 TABLE 5 Specificity results Interferent Addition concentration Cross reaction rate (%) Bilirubin 20 mg/dL 0.89 Hemoglobin 1000 mg/dL 1.52 Triglyceride 2000 mg/dL 3.01 Human anti-mouse 2000 ng/mL 2.64 Rheumatoid factor 1000 IU/mL 1.86

[0092] The results in Table 5 show that the above added substances have no effect on the test results of the anti-cyclic citrullinated peptide antibody test kit of Embodiment 5.

[0093] (8) Inter-batch variation: three different batches of the kits of Embodiment 5 of the present disclosure were used to test the internal precision reference substances of Haooubo (RP1:10 RU/ml, RP2: 20 RU/ml, RP3: 100 RU/ml), each with 10 repetitions, the mean (M), standard deviation (SD) and inter-batch coefficient of variation (CV) of the 30 test results were calculated, and the results should meet the requirements CV<15%. The calculation formula is:

[00001]$\mathrm{CV}=\frac{\mathrm{SD}}{M}\times 100.Math.\%$

[0094] wherein:

[0095] CV—Coefficient of variation;

[0096] SD—Standard deviation of 30 test results

[0097] M—Mean of 30 test results

TABLE-US-00008 TABLE 6 Inter-batch variation RP1: RP2: RP3: 10 RU/ml 20 RU/ml 100 RU/ml LOT Name Dose Dose Dose 1 1 10.68 20.19 105.25 2 10.54 20.20 104.33 3 10.77 19.42 100.15 4 9.79 21.62 100.19 5 9.79 20.03 96.84 6 9.91 20.37 90.88 7 9.64 18.31 92.58 8 9.49 20.60 87.30 9 10.51 20.03 94.73 10 10.35 20.78 99.34 2 11 11.25 22.69 106.32 12 10.96 23.48 111.36 13 10.11 20.37 104.09 14 10.45 21.90 103.03 15 9.98 20.12 105.74 16 10.20 20.60 104.41 17 11.72 24.12 109.19 18 11.36 20.20 119.89 19 10.93 22.55 99.78 20 11.30 23.73 99.68 3 21 11.90 21.55 101.03 22 9.99 22.01 90.05 23 10.59 21.12 101.15 24 10.23 22.13 110.73 25 9.58 20.84 99.34 26 9.32 21.33 92.99 27 9.98 19.61 98.38 28 10.16 21.41 97.99 29 10.04 18.75 90.63 30 10.28 20.83 93.20 AVER 10.39 21.03 100.35 SD 0.65 1.38 7.23 CV 6.30% 6.56% 7.20%

[0098] It can be seen from Table 6 that the inter-batch variation is less than 10%, and the inter-batch variation is small, which meets the industry requirements.

[0099] Although the present invention has been disclosed in the form of preferred embodiments and variations thereon, it will be understood that numerous additional modifications and variations could be made thereto without departing from the scope of the invention.

[0100] For the sake of clarity, it is to be understood that the use of ‘a’ or ‘an’ throughout this application does not exclude a plurality, and ‘comprising’ does not exclude other steps or elements.