Completely humanized monoclonal neutralizing antibody for tetanus toxin and application of neutralizing antibody
A monoclonal antibody and tetanus toxin technology, applied in the field of biomedicine, can solve the problems of follow-up research reports without functional test data
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Embodiment 1
[0066] Example 1 Preparation of fully human anti-tetanus toxin neutralizing antibody
[0067] 1. Sorting cells
[0068] A healthy volunteer was injected with 1500 IU tetanus toxoid (vaccine), and blood samples were collected on day 7. Then isolate mononuclear cells (PBMC); then use a BD FACSria flow cytometer to sort out plasma cells from PBMC, and place the single cells with good morphology in a 96-well PCR plate so that each well contains a memory B cell, Store in -80°C refrigerator for later use.
[0069] 2. Isolation of antibody variable region genes
[0070] Add 0.5 μM constant region primers of heavy and light chains of each subtype to a 96-well plate containing a single B cell and Superscript III reverse transcriptase, and incubate at 37°C for 1 hour; carry out PCR amplification according to the following parameters: 95°C for 15 minutes ; 95°C 1min, 55°C 1min, 72°C 1min, 30cycles; 72°C 10min; 4°C 5min. The product cDNA was stored at -20°C.
[0071] Using the above ...
Embodiment 2
[0089] Example 2 Detection of binding activity of TRN0010 antibody
[0090] Use the same ELISA method mentioned above to detect the binding activity of the expressed and purified antibody: use tetanus toxin as the antigen, and coat the 96-well ELISA plate after diluting the antigen 10 times with the coating solution, and coat each well with 100 μl overnight at 4°C. were blocked with blocking solution at room temperature for 2 h. Incubate 3-fold diluted TRN0010 antibody as the primary antibody at room temperature for 2 hours, and incubate with HRP / anti-His-tag (1:2000 dilution) as the secondary antibody at room temperature for 1 hour, add substrate chromogenic solution 100 μl / well, and keep away from light at room temperature After standing for 5 minutes, stop the reaction with 2M sodium sulfate, perform colorimetric detection with a wavelength of 450nm, and analyze the results.
[0091] The result is as figure 2 As shown, after diluting the expressed and purified TRN0010 an...
Embodiment 3
[0092] Example 3 Affinity detection of TRN0010 antibody
[0093] First, the CM5 chip was coupled with the capture molecules, and then the dextran surface of the chip was activated. The coupling amount was determined by the injection time, and finally the CM5 chip was used to capture the molecule capture ligand: the prepared fully human anti-tetanus neutralizing antibody was used as Ligand. The tetanus toxoid was diluted with HBS-EP buffer as the analyte, and the analyte flowed through the chip in sequence with increasing concentration, and the signal curves were respectively obtained. Each concentration was regarded as one cycle, and after completing one cycle, the chip was regenerated with 10 mmol / L glycine-hydrochloric acid to return to the original state of unbound antigen. BiaCore X-100 System software was used to analyze the affinity and kinetics of monoclonal antibody binding to tetanus toxoid (antigen).
[0094] The result is as image 3 As shown in Table 1, the diss...
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