Application of Protein Chip Technology in Preparation of High-throughput Monoclonal Antibodies v1
Authors: Smitu Bello 
Published: November 2018

Aiming at a large amount of human consumption during the specific screening process in the preparation of traditional monoclonal antibodies, a method for the preparation of monoclonal antibodies using a protein chip was established.N immunized with recombinant proteins were Ha2 mice, on the basis of conventional cell fusion, based on the cloning, protein microarray, prepared positive cell cloned by limiting dilution of positive cells mixed N antigens after immunization associated hybridomas antibody. With the deepening of functional genomics and proteomics research, there is an increasing demand for antibodies, especially monoclonal antibodies. It is expected that based on classical antibody recognition, high-coverage antibody sets will be established to identify all human expressions. protein. Hybridoma technology and phage display peptide library technology are mainly used in high-throughput antibody preparation technology, and the use of classical monoclonal antibody technology is still the most widely used, and it is easy to obtain high-priced, high-affinity antibodies. Materials and Methods Experimental materials and instruments Experimental animals: 6-week-old BALA/c female mice。 Immune protein: 7 human recombinant proteins and 1 schistosomiasis recombinant protein。 Reagents and instruments: fusion with polyethylene glycol (molecular weight 4000, containing 10% dimethyl sulfoxide), HAT and 8-azaguanine, horseradish peroxidase-labeled goat anti-mouse monoclonal antibody, biotin labeling Sheep anti-mouse monoclonal antibody, cross-linked streptavidin phycoerythrin, TMB chromogenic reagent, slide matrix for protein chip preparation. Preparation and detection of microplate protein chips The microplate was activated by 70% methanol according to Millipore's experimental manual. After PBS equilibration, the chip preparation was performed using a spotting instrument. The chip array 4X4, 25nL of each protein sample was sprayed, and the chip was dried at 37 ° C for 1 h, 4 Save in degrees Celsius. The positive reference point was serum of unimmunized mice, the negative reference point was pH 7.0, 0.05 mmol/L PBS, and the concentration of 8 recombinant proteins was 0.2-0.6 mg/mL. 50 μl of the cell culture solution to be tested was directly added to the chip, and incubated for 1 hour at room temperature with shaking, and the chip was washed 3 times with PBST for 3 minutes each time; Add HRP-labeled goat anti-mouse antibody diluted with PBS at pH 7.0, 0.050 mmol/L, incubate at room temperature for 0.5H, and wash with PBST 3 times for 3 min each time; add 30 μl of TMB color solution per well. Color, color development at room temperature for 1 minute, visual or scanner scan to record the experimental results. Preparation and detection of slide matrix protein chip The slide matrix protein chip was prepared according to the reference. The chip array format was 12x2, 10 arrays per chip, divided by a fence, and the chip was dried at room temperature. Take 20 μl of the cell culture solution to be tested and add the slide matrix protein chip. After incubating for 1 h at room temperature, the chip was washed 3 times with PBST for 5 min each time; biotin labeling diluted with PBS diluted at pH 7.0, 0.50 mmol/L was added. The goat anti-mouse antibody was incubated for 0.5 h at room temperature, washed 3 times with PBST for 5 min each time; 10 μl of cross-linked streptavidin phycoerythrin was added to each well, incubated for 10 min at room temperature, and washed three times with PBS. 5 min, the chip scanner records the experimental results. Immunization and antibody preparation Immunization, cell fusion and screening: BALB/c female mice were immunized with 8 recombinant proteins according to the classical method. The protein used for each immunization was 40 μg, boosted after 3 immunizations, and aseptically taken spleen 3 days later, SP2/0 mouse myeloma cells were fused and cultured in HAT medium. On the 10th day after cell fusion, the recombinant protein was used as antigen, and the antibody in the culture supernatant was detected by indirect ELISA. The positive hybridoma cell wells with higher absorbance values were selected, and the liquid nitrogen was frozen after culture, and each antigen was frozen. Save more than 4 tubes. Hybridoma cell mixing and cloning: 1 tube of frozen hybridoma cells was taken from each antigen. After resuscitation, the cells were counted. Each hybridoma took about 60 cells, and a total of 480 cells were mixed for cloning. Culture, a total of 10 96-well culture plates. Screening and cloning of microplate protein chips: Hybridoma cells mixed and cloned were screened with microplate protein chip, positive cell well expanded culture, liquid nitrogen frozen, single antigen positive well limited dilution method for clone preparation antibody; Single antigen-positive well cloning was detected by indirect ELISA. Each recombinant protein was coated with 96-well microtiter plate using 50 ng/well (diluted in pH 9.6 carbonate buffer), blocked overnight at 4 ° C, and 100 μL per well. Ascending cell culture solution, the negative control is a cell-free growth medium, and the OD value of 492 nm is read by a microplate reader according to a routine operation. Related services: high throughput monoclonal antibody generation high throughput antibody production recombinant antibody