Antibody of mature brain-derived neurotrophic factor and its application and diagnostic kit
A neurotrophic factor, diagnostic kit technology, applied in application, disease diagnosis, anti-growth factor immunoglobulin, etc., can solve problems such as lack of specific antibodies and differences in processing methods
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Embodiment 1
[0048] Screening of anti-mBDNF antibodies
[0049] The amino acid sequence (SEQ ID NO. 11) of the antigen mBDNF used is as follows:
[0050] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR.
[0051] The above mBDNF protein was used as the antigen for immunization and the antigen for screening. The immunized animals were 6-8 week old female Balb / c mice, injected with antigen, and rapidly immunized with special adjuvant for about two weeks. After immunizing the animals, the antibody titer was detected, and when the antibody titer was high enough, the animals were anesthetized with isoflurane and then sacrificed, and the lymph nodes were harvested and fused with myeloma after digestion. Sp2 / 0 myeloma cells were cultured in DMEM, and when the cells were in good condition and the viable cell rate was over 80%, they were fused with lymphocytes through polyethylene glycol. The cells were then cultured in HAT s...
Embodiment 2
[0065] Purification of anti-mBDNF antibodies
[0066] The antibody gene sequence measured above was codon-optimized, synthesized and inserted into the vector of pcDNA3.4, and the Fc region adopted the sequence of the original antibody subtype IgG2b. The constructed expression vector was transferred into Escherichia coli for amplification and expression, and the expression plasmid was purified. The plasmid was electrotransfected into 293 cells. After 2 days of culture, the antibody protein in the supernatant was extracted, and the protein A affinity column was used for Antibodies were purified and characterized using HPLC-SEC and SDS-Page to determine their concentration and purity. After testing, the antibody concentration was 0.862 mg / ml, and the purity reached 98%.
Embodiment 3
[0068] Specific detection of anti-mBDNF antibodies
[0069] Antigen sample: mBDNF (amino acid sequence shown in SEQ ID NO.11) or ProBDNF is detected by ELISA method:
[0070] 1) Coating: Coat a 96-well plate with 50 μl of mBDNF or proBDNF protein at a concentration of 2 μg / ml at 4°C overnight, the concentration is shown in Table 2, then wash the plate three times with PBST (Tween 0.05%) and pat dry.
[0071] 2) Blocking: Add 200 μl of blocking solution (PBST solution containing 1% BSA) to a 96-well plate with the configured blocking solution, block for 1 hour at room temperature, wash the plate four times, and pat dry.
[0072] 3) Add 50 μl of sample (purified antibody of Example 2 above), incubate at room temperature for 1 hour, wash the plate four times, and pat dry.
[0073] 4) Secondary antibody incubation: Secondary antibody (1:2000) was prepared with blocking solution, 100 μl of secondary antibody was added to each well, incubated at room temperature for 1 hour, washed ...
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