Preparation of monoclonal antibodies against phosphopeptides.
Preparation of monoclonal antibodies against phosphopeptides.
This experiment provides steps starting from inoculation of candidate hybridoma clones into a 96-well culture plate all the way through to isolation of a hybridoma clone with the desired activity. Content from Compact Molecular Biology Laboratory Guide (5th Edition)
Operation method
basic program
Materials and Instruments
Fused candidate hybridoma cell line BSA-crosslinked homophosphorylated peptide BSA-crosslinked homophosphorylated nonphosphorylated peptide BSA-crosslinked nonhomophosphorylated tyrosine peptide BSA-crosslinked homophosphorylated peptide Move 1. Inoculate the fused hybridoma cells at a low density (preferably one cell per three wells) into multiple 96-well polystyrene tissue culture plates and culture in HT medium. After approximately 2 weeks the number of clones was recorded using a 96-grid-grid paper sheet to check and identify all monoclonal hybridoma cell culture wells. The wells containing one clone are labeled for easy identification. 2. 2. Transfer a portion of the supernatant from each potential candidate well (e.g., 100 μl from 200 μl of culture medium) to another 96-well polystyrene plate ("Screening Plate") using aseptic technique. Record the number and location of the wells of the initial culture plate and the plate number and location of the wells of the corresponding screening plate (e.g., record the number and location of the wells of the initial plate from which the supernatant originated on a piece of grid paper representing the screening plate). Add 100 μl of fresh HT medium at 37°C to each well of the initial plate. 3. Add 100 μl of fresh HT medium at 37°C to each well of the screening plate. 3. Add 150 μl of Screening Diluent to the supernatant from each well of the Screening Plate. 4. Take a small portion (e.g., 50 μl) of the supernatant of each candidate hybridoma from the screening plate and screen it by ELISA using the homologous phosphopeptide-BSA cross-link as the antigen. Record the screening plate number and well location corresponding to each positive sample, as well as the number and well location of the corresponding initial culture plate. Pre-immunization serum (negative control) and post-immunization serum (positive control) of mice used for fusion were tested simultaneously, and both sera were diluted 1:100 in a mixture of HT medium and screening diluent (1:1) before measurement. Re-incorporate an ELISA containing only the Screening Diluent (additional negative control). 5. Take a small portion of the positive samples screened in Step 4 and perform an ELISA against the homologous non-phosphopeptide-BSA cross-links to remove clones with independent phosphorylation reactivity. One copy of the positive sample screened against an unrelated phosphotyrosine peptide-BSA crosslink is subjected to ELISA to remove clones that are not selective for phosphotyrosine but are both able to react. In addition, if cross-reactivity with homologous phosphoproteins is predicted, perform an ELISA screen against any homologous phosphoprotein-BSA cross-links that may cross-react. 6. 6. Clones that meet all screening criteria are amplified, partitioned and partially frozen, and the remainder are subcloned by limited dilution. 7. Culture supernatants from the subclones are screened for continued antibody production and specificity as in steps 4 and 5. 8. 8. To further characterize the subclones and identify the most useful clones, the culture supernatants are tested for reactivity to homologous all-phosphate proteins using various immunoassays (e.g., immunoprecipitation or immunoblotting assays). Caveat All solutions and equipment that will come into contact with live cells must be sterile and proper aseptic handling techniques should be used. For more product details, please visit Aladdin Scientific website.
Screening dilutions Negative control (pre-immunization serum for mice preparing hybridoma lines) Positive control (post-immunization serum for mice preparing hybridoma lines)
HT medium 96-well polystyrene tissue culture plates Grid-labeled paper sheets