Increasing the specificity of colony hybridization when using heterologous probes.

Goat anti-mouse immunoglobulin horseradish peroxidase (HRP) conjugate was diluted in PBS containing 10% BSA. This conjugate has been successfully used from various manufacturers; however, specific lots need to be titrated for optimal performance. One particular preparation of HRPconjugated goat anti-mouse IgG, IgM and IgA (Catalog No. A 0412; Sigma Chemical, St. Louis, MO, USA) was diluted at 1/8000. One hundred microliters of the diluted conjugate were put into each well, and the binding was allowed to proceed for 45 min at room temperature on an orbital shaker. Plates were then washed 6 times with PBS/ 0.05% Tween-20. 3,3′,5,5′-Tetramethylbenzidine (TMB) peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was prepared according to the manufacturer’s instructions, and 100 μL were added to each well. The reaction was allowed to proceed about 30 min at room temperature, at which time the reaction was stopped by the addition of 100 μL 0.18 M H2SO4. The plate was read at 450 nm with a microplate reader. Sample antibody concentrations were estimated by comparing OD values to the OD values of the standard curve. An example of data from mice immunized with the SWM (110–121) peptide is shown in Figure 3. Inclusion of free unbiotinylated peptide has been shown to greatly diminish the signal generated in this assay (data not shown). The assay described in this report should be helpful to most researchers wanting to prepare anti-peptide antisera or to monitor anti-peptide antibody responses for other experiments. We have used the above protocol, with appropriate modifications, in a number of systems with consistent success. In addition, this protocol could be adapted to other systems, besides monitoring antipeptide antibodies, where a biotinylated substrate is obtainable.