In animal studies, antibody titers against antigens are often assayed to determine immune responses by treatments (1). Since the antibody titers highly depend on animal study protocol, especially immunization protocols. Therefore, determining sample dilution for our samples is always challenging in the antibody assays. For example, immunized mice require higher serum dilution which sometimes reach 1: 1,000,000 dilution or more, instead, control mice require lower serum dilution like 1:10 which may show undetectable.

Appropriate serum dilutions can be determined using several dilutions such as 1:100, 1:1000, 1:10,000 or more using small numbers of samples (one each from control and test groups) before assaying all samples.

In addition, antibodies for a standard curve must be prepared. Some studies just used OD values to evaluate immune response however, OD values may vary in assays because of temperature, pipetting, etc. The OD values can not be compared in between assays run on different days. If we have a standard, regardless of OD values, sample antibody levels can be calculated relatively correctly. This mean serum dilution can be determined in several trials using the standards.

If we are tired of using higher dilutions, we can ignore the antibody levels in the control group as not detected. Instead, an antibody assay can be optimized as lower assay sensitivity using a short incubation time, or a lower secondary antibody concentration. In the latter case, we may see saturated OD values at the higher standard range because of limited secondary antibody numbers compared with numbers of antibodies against an antigen. We can carefully titrate secondary antibody concentration to check the linear range of the s tandard curve.

1. A. V. Lin, Indirect ELISA. Methods Mol. Biol. 1318, 51–59 (2015).