This method can help answer key questions in the immunology field about antigen immunization induced dependent and independent antibody responses as well as B cell activation and germinal center reactions. The main advantage of this technique is that it provides reliable and quantitative Ig isotype response measurements with a direct comparison of B cell activation and humoral immunity in mice. For TNP-polysaccharide immunization load one milliliter insulin syringe per mouse with 100 microliters of freshly prepared antigen solution and insert the needle into the lower right quadrant of each animal at an approximately 30 degree angle to avoid lower organ injection.
When all the mice have been injected, return the animals to their cages for housing in a specific pathogen-free facility. At the appropriate experimental time points collect 150 to 200 microliters of blood from each immunized animal by retro-orbital bleeding and allow the samples to coagulate at room temperature for one to two hours. At the end of the incubation, centrifuge the coagulated blood samples and transfer the clear serum from the top of each blood clot into a sterile 1.5 milliliter microcentrifuge tube.
After a second centrifugation to remove any residual blood clot, transfer 50 microliters of serum into individual sterile 1.5 milliliter microcentrifuge tubes for storage at minus 80 degrees Celsius. Before plating, coat individual 96 well immunoplates with the appropriate concentration of mouse Ig isotype specific capturing antibodies for total serum Ig isotype TNP-38BSA antigen for TNP-specific Ig isotype or TNP-3BSA antigen for high affinity TNP-specific Ig isotype and incubate the coated plates overnight at four degrees Celsius. The next morning wash the plates two times with 200 microliters of wash buffer per well.
Discarding the wash buffer and patting the plates dry on a stack of paper towels after each wash. Next, block the plates with 200 microliters of blocking buffer per well for a one hour incubation at room temperature. While the plates are being blocked prepare the appropriate standard and serum dilutions and blocking buffer in a separate untreated 96 well plate at 150 microliters per well as indicated.
Then wash the ELISA plate with wash buffer as demonstrated and add 100 microliters of each diluted Ig isotype standard and serum sample to the appropriate wells of the ELISA plate. After an overnight incubation at four degrees Celsius wash the plates as demonstrated and add 100 microliters of an appropriate alkaline phosphatase conjugated isotype-specific antibody diluted in blocking buffer to each well for a one to two hour incubation at room temperature. At the end of the incubation, wash the plates and add 100 microliters of freshly prepared substrate solution to each well.
Monitoring the reaction as it develops over the next few minutes at room temperature. When the reaction is robust enough to analyze place the plate in a microplate reader and click settings to set the wavelength to 405 nanometers. Click ok then click template to assign the appropriate blank, standard, and unknown wells according to the 96 well plate set up.
Then when the most concentrated standard reaches an optical density of about one, 1.5, two, and 2.5 click read to read the plate and save the resulting data file after each analysis. In the absence of immunization, an increase in basal serum levels of IgM, IgG2a, IgG2b, IgG3, and IgA is observed in B cell specific TNF receptor associated factor three or B-TRAF3 knockout mice, compared to gender and age matched B-TRAF3 sufficient litter mate control animals. Seven days after immunization with TNP-polysaccharide or TNP-KLH as just demonstrated analysis of the thymus-independent Ig isotype responses to immunization reveals significantly higher thymus-independent TNP-specific IgG3 levels as well as elevated thymus-dependent TNP-specific IgG2b levels in myeloid cell-specific TRAF3 knockout mice compared to litter mate controls.
Further, evaluation of thymus-dependent mouse primary and memory responses to TNP-KLH immunization indicates the presence of partially decreased thymus-dependent IgM primary responses and defective IgG1 primary and memory responses in T cell specific TRAF3 knockout mice. While attempting this procedure, it's important to remember that ELISA have detection limits that are usually defined by the linear range of the standard curves and therefore multiple dilutions of your sample might be required. Following this procedure, other complementary methods like flow cytometry or L spot can be performed to gain a comprehensive understanding of antibody responses in a given experimental setting.
