A kind of monoclonal antibody and kit for liver cancer cell metastasis detection
A monoclonal antibody and kit technology, applied in the field of detection, can solve the problem that the binding activity of the antibody needs to be further improved, and achieve the effect of good binding ability and specificity
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Embodiment 1
[0022] Example 1 Expression of RRM2B Specific Protein Fragments
[0023] According to the sequence of the RRM2B protein fragment that has been confirmed to be related to diagnosis and prognosis, the codon and GC content are optimized through specific optimization means, such as Figure 1A and Figure 1B As shown, the optimized sequence can significantly increase the expression of the gene after adjusting various parameters, and the optimized sequence is shown in SEQ ID NO: 1; design specific primers, respectively at the 5' end of the primers Add protective bases and restriction endonucleases, the primer sequence is as follows:
[0024] F:5'-CGC GGATCC ATGGGCGACCCGGAAAGGCCGG-3' (SEQ.ID.NO.3):
[0025] R:5'-CCG GAATTC TTAAAAAATCTGCATCCAAGG-3' (SEQ.ID.NO.4):
[0026] Using the optimized RRM2B gene fragment synthesized through the whole gene as a template (Shanghai Sangong Synthetic), PCR amplification was performed with the above-mentioned specific primers.
[0027] The ...
Embodiment 2
[0033] Example 2 Preparation of Monoclonal Antibody
[0034]Three 8-week-old BALB / c female mice received recombinant RRM2B protein 100 μg / (mouse·time). For the first and second immunizations, respectively, the protein was emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant for subcutaneous multi-point injection, and the final booster immunization was directly injected with pure protein solution through the tail vein with an interval of 28 days. 3 times. On the 3rd day after the last immunization, the spleens of the mice were taken out to make a single cell suspension and used for fusion immediately.
[0035] 10-14 days before fusion, release the cryopreserved myeloma cells SP2 / 0. After thawing frozen cells in a warm water bath at 37 °C, immediately add RPMI1640 culture medium, centrifuge at 900 × g for 10 min, and discard the supernatant. Use 20% FBS-RPMI 1640 culture medium (containing 1 mmol / L L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomyci...
Embodiment 3
[0040] Example 3 Preparation of ascites and determination of its titer, subtype and specificity
[0041] (1) The monoclonal cells prepared in Example 2 were continuously subcultured in vitro for 30 generations and frozen for 6 months, and the antibody titer in the cell supernatant was basically unchanged. The in vivo induction method was used to prepare ascites, and the collected ascites was serially diluted for indirect ELISA detection. The D450nm value of ascites was regarded as P, the D450nm value of negative control was N, and P / N>2.0 was regarded as positive. Depend on image 3 It can be seen that the titer of the monoclonal antibody is 1:25600.
[0042] (2) Monoclonal antibody subtype and specificity identification results
[0043] Use the mouse antibody typing kit to detect the antibody subtypes in the culture supernatant and ascites of monoclonal cells, see the results Figure 4 . Depend on Figure 4 It can be seen that the antibody secreted by the monoclonal anti...
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