CRYAB antibody for acute kidney injury detection and application thereof
An acute kidney injury and antibody technology, applied in the field of biomedicine, can solve the problem of lack of biomarkers for early detection of acute kidney injury, and achieve the effect of improving detection efficiency
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Embodiment 1
[0014] Example 1 Preparation of mouse anti-human CRYAB polyclonal antibody
[0015] PCR amplification of the gene fragment encoding the amino acid sequence 67-149 of αB-crystallin (CRYAB core antigen), the nucleotide sequence shown in SEQ ID No. 2, is 249 bp in length. The recombinant plasmid pcDNA3.1-CRYAB-His-Ampicillin was constructed by double digestion with KpnI and NotI. The verification of the construction of the recombinant plasmid is as follows figure 1 As shown, the DNA Marker was obtained from TAKARA Corporation (Cat. No. DL2000). The successfully constructed recombinant plasmid was transfected into 293F cells, and the CRYAB antigen protein expressed in the supernatant was purified to obtain a protein concentration of 5.29 mg / mL. The 6-8 week old BALb / c mice were immunized at a dose of 100 μg / time / mice, three times in total, with an interval of two weeks between each immunization. For the first immunization, complete Freund's adjuvant and protein ratio were mixed ...
Embodiment 2
[0016] Example 2 Antigen-binding site verification of mouse anti-human CRYAB polyclonal antibody
[0017] The gene fragment encoding the full length of αB-crystallin (full length of CRYAB antigen protein) was amplified by PCR, and the nucleotide sequence shown in SEQ ID No. 3 was 528 bp in length. At the same time, PCR was used to amplify the gene fragments encoding the N-terminal, core region, C-terminal, C-terminal+core region of the CRYAB protein, and then the following recombinant plasmids were constructed: pEBG-CRYAB full-length (containing the nucleosides shown in SEQ ID No.3) nucleotides 1-528 of the acid sequence), pEGB-CRYAB N-terminal (containing the nucleotide sequence 1-198 of the nucleotide sequence shown in SEQ ID No. 3), pEGB-CRYAB core region (containing SEQ ID No. 3 nucleotides 199-447 of the nucleotide sequence shown), pEGB-CRYAB C-terminal (containing nucleotides 448-528 of the nucleotide sequence shown in SEQ ID No. 3), pEGB-CRYAB C End + core region (cont...
Embodiment 3
[0021] Example 3 Determination of the titer of mouse anti-human CRYAB polyclonal antibody
[0022] Add 5 μg / mL CRYAB recombinant protein antigen (prepared in Example 1) to a 96-well ELISA plate, add 100 μL to each well, coat overnight at 4°C, and wash 3 times with PBST for 5 min each; then add 200 μL of 5% nonfat milk powder to each well , blocked at 37°C for 2h; using the antibody to prepare pre-immune serum as a negative control, the mouse anti-human CRYAB polyclonal antibody prepared in Example 1 was diluted with PBS, and the dilution ratios were 1:1000, 1:10000, and 1:100000, respectively. , add 100 μL to each well, incubate at 37°C for 2 h, and wash 3 times with PBST for 5 min each; add 100 μL of goat anti-mouse HRP-labeled secondary antibody diluted 10,000 times in PBS to each well, incubate at 37°C for 2 h, and wash 3 times with PBST, each time 5min; add 100μL of TMB chromogenic substrate solution, incubate at 37°C for 15min in the dark, read the OD450 value on the micr...
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