CN110845610B

CN110845610B - Detection antibody pair aiming at diphtheria toxoid and application thereof

Info

Publication number: CN110845610B
Application number: CN201911175895.1A
Authority: CN
Inventors: 陈雯; 胡业勤; 郑梦婷; 杨晓明; 段凯; 李新国; 张智
Original assignee: WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Current assignee: WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2021-06-15
Anticipated expiration: 2039-11-26
Also published as: CN110845610A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a detection antibody pair aiming at diphtheria toxoid and application thereof, wherein the monoclonal antibody is an antibody 2B17-B and 3K8, the amino acid sequence of a heavy chain variable region (VH) of the antibody 2B17-B is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region (VL) is shown as SEQ ID NO. 2; the heavy chain variable region (VH) amino acid sequence of 3K8 is shown in SEQ ID NO.3, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO. 4. The invention also relates to a nucleic acid sequence for encoding the monoclonal antibodies 2B17-B and 3K8 and a kit for detecting diphtheria toxoid by the nucleic acid sequence.

Description

Detection antibody pair aiming at diphtheria toxoid and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a pair of detection antibodies for diphtheria toxoid and application thereof.

Background

Diphtheria is a bacterium which is strictly parasitic to human beings, and patients and carriers are the only infection sources; people are generally susceptible to white larynx, and can obtain lasting immunity to white larynx toxin only through vaccination. Adverse reactions to vaccination with diphtheria toxoid are generally caused by non-toxin proteins in the preparation, and as the quality of diphtheria toxoid increases, the vaccination reactions decrease. Therefore, it is particularly important to monitor the quality of diphtheria toxoid during its production. According to the requirements of Chinese biological product regulation, two methods are adopted for measuring the titer of the commonly used diphtheria toxoid, wherein firstly, an immune animal generates specific immune response; second, flocculent unit assay; the animal experiment period is long, and the influence factors are many; the flocculent unit determination method is judged by naked eyes and has certain errors. It is therefore desirable to monitor the quality of diphtheria toxoid in a manufacturing process using a simple, rapid, sensitive, low-impact method. Enzyme-linked immunosorbent assay (ELISA) has the advantages of sensitivity, rapidness and strong tolerance, and can be used for quality control of toxoid in the production process of diphtheria toxoid.

Disclosure of Invention

The invention firstly relates to a group of monoclonal antibodies for detecting Diphtheria Toxoid (DT) individually and/or jointly, wherein the antibodies are:

(1) the monoclonal antibody 2B17-B,

the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO.1, SEQ ID NO. 1:

the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO.2, SEQ ID NO. 2:

(2) the monoclonal antibody 3K8 was obtained,

the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO.3, SEQ ID NO. 3:

the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO.4, SEQ ID NO. 4:

the invention also relates to a nucleic acid sequence for encoding the monoclonal antibodies 2B17-B and 3K 8.

The constant region (Fc) region of the 2B17-B and 3K8 monoclonal antibodies is murine Fc.

The monoclonal antibodies 2B17-B and 3K8 are all murine antibodies.

The invention also relates to application of the monoclonal antibodies 2B17-B and 3K8 in preparation of a diphtheria toxoid detection reagent.

The invention also relates to a test kit for the detection of diphtheria toxoid, said kit comprising:

(1) detecting an effective amount of 2B17-B antibody and/or 3K8 antibody;

(2) necessary chromogenic or quantitative reagents.

Preferably, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

More preferably, the

The enzyme linked immunosorbent assay kit is a double-antibody sandwich enzyme linked immunosorbent assay kit, wherein,

(1) monoclonal antibody 2B17-B was used as a coating antibody or a capture antibody;

(2) using 3K8 antibody labeled with quantitative label as detection antibody or secondary antibody;

the quantitative label is preferably horseradish peroxidase or biotin.

Drawings

FIG. 1, linear range in the detection of the double-antibody ELISA.

Detailed Description

The main reagents and sources used by the invention are as follows:

the amino acid sequences of the light chain and the heavy chain of the anti-DT-2B17-B monoclonal antibody are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequences of the light chain and the heavy chain of the anti-DT-3K8 monoclonal antibody are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;

the 96-well enzyme label plate is purchased from Xiamen Yunpeng science and technology Limited; diphtheria toxoid standards were purchased from NIBSC (No. 02/176);

other reagents are all domestic analytical purifications unless specified otherwise.

Example 1 establishment of the double antibody ELISA detection method

1. Determination of Linear Range and working antibody concentration

(1) Diluting the coated anti-DT monoclonal antibody (2B17-B) to 5 mu g/ml for coating according to the conventional coating conditions (coating is carried out for 16-24 hours at 2-8 ℃, and the formula of a coating solution is Na₂CO₃1.59g，NaHCO₃2.93g, adding purified water to 1L, adjusting pH to 9.5-9.6)

(2) Washing away unbound coating antibody by PBST the next day, and then adding 1% BSA for blocking (blocking for 1 hour at 37 ℃), thus obtaining an enzyme label plate coated with primary antibody;

(3) when the diphtheria toxoid reagent is used, a series of diluted diphtheria toxoid standard substances and a series of diluted horseradish peroxidase-labeled anti-DT monoclonal antibody (3K8-HRP) are sequentially added, reaction is carried out at 37 ℃, then a substrate A/B liquid of a horseradish peroxidase color development system is added for color development, and finally, a specific light absorption value is read after termination by 2M sulfuric acid.

Through experiments, the results show that the optimal working dilution factor of the enzyme is 4000 times; in the range of 0.00026-0.00425Lf/ml, the absorbance is highly linearly related to the detection concentration (Lf/ml) (R²＞0.98)。

Example 2 verification of the double antibody ELISA detection method

1. Verification of specificity

Pertussis Toxin (PT), pertussis Filamentous Hemagglutinin (FHA), pertussis adhesin (PRN), and Tetanus Toxoid (TT) were diluted to appropriate concentrations, and Diphtheria Toxoid (DT) was added as a positive control, and the detection kit prepared in example 1 was used to detect that none of the above antigens other than diphtheria toxoid could be detected, indicating that the detection method was highly specific. The results are shown in Table 1.

TABLE 1 verification of specificity

Cutoff is 2.1 times the blank OD mean; where blank OD values below 0.05 were calculated according to a 0.05 shift.

2. Verification of accuracy

Diphtheria toxoid was diluted to three concentrations, high, medium and low, and detected using the detection kit prepared in example 1, and each concentration was repeated 6 times. And counting the detection recovery rates of the high, medium and low concentrations, and finding that the recovery rate is between 90 and 115 percent, which indicates that the detection method has high accuracy. The results are shown in Table 2.

TABLE 2 verification of accuracy

Recovery was calculated as:

3. verification of precision

(1) Precision-repeatability

Diphtheria toxoid was diluted to three concentrations, high, medium and low, and detected using the detection kit prepared in example 1, and each concentration was repeated 6 times. And (3) counting the variation degree of 6 times of detection at different concentrations, and finding that the coefficient of variation CV is lower than 5%, which indicates that the detection method has good repeatability. The results are shown in Table 3.

TABLE 3 precision-repeatability

(2) Precision-intermediate precision

The diphtheria toxoid is diluted to high, medium and low concentrations, and the diphtheria toxoid is detected by the same detector on different days; three testers detect the test at the same time on the same day; and (3) counting the variation degree of the same detector on different detection dates and the variation degree of different detectors, and finding that the coefficient of variation CV is lower than 10%, which shows that the detection method has good precision. The results are shown in Table 4.

TABLE 4 precision-intermediate precision

(3) Precision-linearity and range validation

Diluting diphtheria toxoid standard substance to each concentration within linear detection range, repeatedly measuring for 5 times, counting correlation coefficient R2 of each detection, and finding out correlation coefficient R²All are larger than 0.98, which shows that the detection method has good linearity. The results are shown in Table 5.

TABLE 5 Linear and Range validation

Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of the present invention.

SEQUENCE LISTING

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Claims

1. A panel of monoclonal antibody pairs for use in combination to detect Diphtheria Toxoid (DT), said antibody pairs being:

(1) the heavy chain variable region (VH) amino acid sequence of the monoclonal antibody 2B17-B is shown in SEQ ID NO.1, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO. 2;

(2) the heavy chain variable region (VH) amino acid sequence of the monoclonal antibody 3K8 is shown in SEQ ID NO.3, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO. 4.

2. The monoclonal antibody pair of claim 1, wherein the constant region (Fc) of antibody 2B17-B, antibody 3K8 is murine Fc.

3. The pair of monoclonal antibodies of claim 1 or 2, wherein the antibodies 2B17-B, antibody 3K8 are murine antibodies.

4. A polynucleotide encoding the monoclonal antibody pair of any one of claims 1-3.

5. Use of a monoclonal antibody according to any one of claims 1 to 3 in the preparation of a test reagent for the detection of diphtheria toxoid.

6. A test kit for the detection of diphtheria toxoid, the kit comprising:

(1) detecting an effective amount of monoclonal antibody 2B17-B and monoclonal antibody 3K8 of claim 1;

(2) necessary chromogenic or quantitative reagents.

7. The kit of claim 6, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit, wherein,

(1) monoclonal antibody 2B17-B was used as a coating or capture antibody;

(2) monoclonal antibody 3K8 labeled with a quantitative tag was used as the detection or secondary antibody.

8. The kit of claim 7, wherein the quantitative label is selected from horseradish peroxidase or biotin.