Preparation method and application of anti-human alpha fetoprotein (AFP) single-chain antibody and fusion antigen peptide
A single-chain antibody and inclusion body technology, applied in the field of bioengineering, can solve the problems of poor diffusion into tumors, limited target presentation and tumor antagonism, and weak vascular permeability.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0028] Example 1: Animal immunization
[0029] Human alpha-fetoprotein was dissolved in normal saline, mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and inoculated into the abdominal cavity of BALB / c mice (provided by the Comparative Medicine Center of Yangzhou University, SCXK (Su) 2007-0001). 10 μg AFP protein / 200 μL / body; after the basic immunization, the same antigen dose was mixed with an equal volume of Freund’s incomplete adjuvant for booster immunization at intervals of two weeks; one week after the second booster immunization, the immune system was measured by indirect ELISA method Mouse antiserum titers. The normal mouse serum was used as the negative control, and the determination was carried out with the judgment standard of (measurement well A value-blank value) / (negative control well A value-blank value)≥2.1. The end point titers of mouse antiserum were all above 1:12,8000.
[0030] The mouse with the highest serum hAFP-specific an...
Embodiment 2
[0031] Embodiment 2: Preparation of monoclonal antibody
[0032] 1. Cell Fusion
[0033] Cell fusion adopts the polyethylene glycol method: aseptically take the spleen of the mouse immunized as described in Example 1, make a cell suspension, centrifuge, and count the cells; divide 1×10 8 splenocytes with 2 x 10 7 Mix SP2 / 0 cells, centrifuge at 1000rpm for 10min, discard the supernatant, and place the centrifuge tube in a 37°C water bath; take 1ml of 50% PEG preheated to 40°C and slowly add it dropwise to the cell pellet, and suspend the cells with the basal medium ; Centrifuge at 1000rpm for 10min, discard the supernatant, add HAT medium to resuspend the cells, aliquot them into 96-well cell culture plates, and place the culture plates at 37°C and 5% CO 2 Culture in a humidified cell incubator, replace 1 / 2 of the culture medium in the culture plate well with fresh HAT after 5 days, and replace the HAT with HT medium after 10 days.
[0034] 2. Screening and cloning culture o...
Embodiment 3
[0037] Example 3: Construction of anti-human alpha-fetoprotein single-chain antibody
[0038] 1. Cell culture and identification: The hybridoma cells secreting anti-human alpha-fetoprotein monoclonal antibody were cultured in complete RPMI1640 medium containing 10% calf serum. The mixed gas containing 5% CO2 in the incubator was used to identify the specificity and affinity of the monoclonal antibody in the culture supernatant by ELISA method.
[0039]2. Cloning of the light chain and heavy chain variable region genes of the anti-human alpha-fetoprotein monoclonal antibody: Take a well-grown hybridoma cell line, extract total RNA with Trizol reagent, reverse transcribe to synthesize cDNA, and use PCR technology in the reaction system Add cDNA and a set of antibody antibody light chain or heavy chain variable region primers, 10×PCR reaction buffer, final concentration of 2.5nM dNTP, 0.5μL primeStar polymerase, and the total reaction system is 50uL. Perform 30 circular reactions...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More 
PatSnap Eureka turns technology decisions into work you can execute. Powered by our Innovation Knowledge Graph, it runs expert workflows across engineering, life sciences, materials and intellectual property. Get your review-ready output in minutes.
