Detection antibody pair for diphtheria toxoid and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a detection antibody pair for diphtheria toxoid and application thereof. The detection antibody pair comprises monoclonal antibodies 2B17-B and 3K8, an amino acid sequence of a heavy chain variable region (VH) of 2B17-B is shown as SEQ ID NO.1 while that of a light chain variable region (VL) of the same is shown as SEQ ID NO.2, and an amino acid sequence of a heavy chain variable region (VH) of 3K8 is shown as SEQ ID NO.3 while that of a light chain variable region (VL) of the same is shown as SEQ ID NO.4. Theinvention further relates to a nucleic acid sequence coding the monoclonal antibodies 2B17-B and 3K8 and a kit prepared thereby and for detecting diphtheria toxoid.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a detection antibody pair for diphtheria toxoid and application thereof. Background technique

[0002] Diphtheria bacillus is a bacterium that strictly parasitizes humans, and patients and carriers are the only source of infection; people are generally susceptible to diphtheria, and only through vaccination can the population obtain lasting immunity to diphtheria toxin. Adverse reactions to inoculation with diphtheria toxoid are generally caused by non-toxin proteins in the product. As the quality of diphtheria toxoid increases, the inoculation reaction will decrease. Therefore, it is particularly important to monitor the quality of diphtheria toxoid during its production. According to the requirements of the "China Biological Products Regulations", two methods are used to measure the potency of diphtheria toxoid commonly used, one is to immunize animals to produce a spe...

Examples

Embodiment 1

[0031] Example 1. Establishment of double antibody ELISA detection method

[0032] 1. Determination of linear range and antibody working concentration

[0033] (1) According to conventional coating conditions, dilute the coated primary anti-DT monoclonal antibody (2B17-B) to 5 μg / ml for coating (2-8°C for 16-24 hours, coating solution formula : Na 2 CO 3 1.59g, NaHCO 3 2.93g, add purified water to 1L, adjust pH to 9.5-9.6)

[0034] (2) The next day, the unbound coated antibody was washed away with PBST, and then 1% BSA was added for blocking (blocked at 37°C for 1 hour), and the primary antibody-coated ELISA plate was prepared;

[0035] (3) When in use, add serially diluted diphtheria toxoid standard, serially diluted horseradish peroxidase-labeled anti-DT monoclonal antibody (3K8-HRP), and add horseradish peroxide after reacting at 37°C The substrate A / B solution of the enzyme chromogenic system develops color, and finally reads the specific absorbance value after termin...

Embodiment 2

[0037] Example 2, verification of double antibody ELISA detection method

[0038] 1. Verification of specificity

[0039] Dilute pertussis toxin (PT), pertussis hemagglutinin (Filamentousheamagglutinin, FHA), pertussis adhesin (pertactin, PRN) and tetanus toxoid (TT) to an appropriate concentration, diphtheria toxoid ( diphtheria toxoid, DT) was added as a positive control substance, and detected with the detection kit prepared in Example 1, it was found that all the above-mentioned antigens except diphtheria toxoid could not be detected, indicating that the detection method has good specificity. The results are shown in Table 1.

[0040] Table 1. Verification of specificity

[0041]

[0042]

[0043] *cutoff is 2.1 times the mean OD value of the blank control; where the OD value of the blank control is lower than 0.05, it is calculated as 0.05.

[0044] 2. Verification of accuracy

[0045] Diphtheria toxoid was diluted to three concentrations of high, medium and low...

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a detection antibody pair for diphtheria toxoid and application thereof. Background technique

[0002] Diphtheria bacillus is a bacterium that strictly parasitizes humans, and patients and carriers are the only source of infection; people are generally susceptible to diphtheria, and only through vaccination can the population obtain lasting immunity to diphtheria toxin. Adverse reactions to inoculation with diphtheria toxoid are generally caused by non-toxin proteins in the product. As the quality of diphtheria toxoid increases, the inoculation reaction will decrease. Therefore, it is particularly important to monitor the quality of diphtheria toxoid during its production. According to the requirements of the "China Biological Products Regulations", two methods are used to measure the potency of diphtheria toxoid commonly used, one is to immunize animals to produce a spe...

Examples

Embodiment 1

[0031] Example 1. Establishment of double antibody ELISA detection method

[0032] 1. Determination of linear range and antibody working concentration

[0033] (1) According to conventional coating conditions, dilute the coated primary anti-DT monoclonal antibody (2B17-B) to 5 μg / ml for coating (2-8°C for 16-24 hours, coating solution formula : Na 2 CO 3 1.59g, NaHCO 3 2.93g, add purified water to 1L, adjust pH to 9.5-9.6)

[0034] (2) The next day, the unbound coated antibody was washed away with PBST, and then 1% BSA was added for blocking (blocked at 37°C for 1 hour), and the primary antibody-coated ELISA plate was prepared;

[0035] (3) When in use, add serially diluted diphtheria toxoid standard, serially diluted horseradish peroxidase-labeled anti-DT monoclonal antibody (3K8-HRP), and add horseradish peroxide after reacting at 37°C The substrate A / B solution of the enzyme chromogenic system develops color, and finally reads the specific absorbance value after termin...

Embodiment 2

[0037] Example 2, verification of double antibody ELISA detection method

[0038] 1. Verification of specificity

[0039] Dilute pertussis toxin (PT), pertussis hemagglutinin (Filamentousheamagglutinin, FHA), pertussis adhesin (pertactin, PRN) and tetanus toxoid (TT) to an appropriate concentration, diphtheria toxoid ( diphtheria toxoid, DT) was added as a positive control substance, and detected with the detection kit prepared in Example 1, it was found that all the above-mentioned antigens except diphtheria toxoid could not be detected, indicating that the detection method has good specificity. The results are shown in Table 1.

[0040] Table 1. Verification of specificity

[0041]

[0042]

[0043] *cutoff is 2.1 times the mean OD value of the blank control; where the OD value of the blank control is lower than 0.05, it is calculated as 0.05.

[0044] 2. Verification of accuracy

[0045] Diphtheria toxoid was diluted to three concentrations of high, medium and low...

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