Grow your parental IgG3 antibody in the hybridoma cell line. Start with the IgG3 subclass to get all four subclasses. Subclass switching happens in the order the IgG genes occur in the DNA. Once the cell line switches from one subclass to another, the first (parental) subclass gene is lost from the DNA. Subclass switching cannot happen in the opposite order.
Plate the IgG3 cell line into a sterile 96-well cell culture plate, 1,000 cells per well in 200 mcl. Grow it for one week. Harvest 100 mcl of media and test it with an ELISA assay.
Coat the 96-well ELISA plates with an unlabeled goat anti-mouse IgG1 antibody in phosphate buffered saline --- PBS. Add the media and incubate 1 1/2 hours. Wash the plates with PBS and add the secondary antibody --- HRP-labeled goat anti-mouse IgG1 antibody. Incubate for 1 1/2 hours. Wash afterward with PBS. Add HRP substrate and incubate for 30 minutes. Add stop solution and read with an ELISA plate reader at 450 nm.
Match any IgG1-positive well to the original cell culture plate. Harvest the cells from these wells and plate 100 cells per well to a new 96-well cell culture plate. Plate five to 10 plates total. Grow for one week and repeat the ELISA assay.
Plate cells from the positive well to a new 96-well cell culture plate at one cell per well. Plate five to 10 plates total and grow for two weeks. Screen every well on a daily basis for cell growth and mark the wells that show a single-cell colony. This will ensure that the IgG1-producing cell line has come from a single cell and is identical. Repeat the ELISA assay.
Repeat the one-cell-per-well plating from positive wells. Repeat the ELISA assay.
Harvest IgG1-positive cells and grow them in a 24-well plate. Inoculate to a 25-ml flask, 75 ml flask and finally to a 125 ml flask. Once you have a stable cell population, grow in a hybridoma bioreactor for bulk production.
Redo the entire protocol starting with the IgG1-producing cells to get a IgG2b-producing cell line. Once you have the IgG2b cell line, repeat the entire protocol one more time to get the IgG2a-producing cell line. Do the ELISA assays with a anti-mouse IgG2b or IgG2a antibodies instead of IgG1, depending on which subclass you are isolating.