26th Annual Meeting and Symposium of the
Desert Tortoise Council, March 16-18, 2001
Abstracts

Refinement of the Mycoplasma agassizii ELISA: From Ratio to Titer

Lori D. Wendland, D.V.M.¹, Paul A. Klein, Ph.D.², Laurie A. Zacher¹, and Mary B. Brown, Ph.D.^1
1Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610
²Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida 32610

Current diagnostic methods available for upper respiratory tract disease (URTD) include culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) serology. Of these, the detection of specific antibody is the method of choice for determination of exposure to Mycoplasma agassizii. The ELISA for the detection of antibodies to M. agassizii in desert and gopher tortoises was developed in 1992. Since that time, we have analyzed over 10,000 samples from at least 32 different chelonian species. With the extensive database now available to us, we have further refined the existing ELISA.

The results are currently being reported as an enzyme immunoassay (EIA) ratio (the optical density at 405 nm [OD405] of the sample/OD405 of the negative control). Although this method of reporting is not uncommon for many ELISA assays, it is not as familiar for many veterinarians and wildlife disease researchers who are more comfortable with the concept of titers. In most situations, either a fourfold increase in titer over time or a single high titer result is considered clinically significant. The large number of samples tested over the past few years has allowed us to get an accurate picture of the distribution of antibody levels in gopher and desert tortoises. This has allowed us to refine the test, to include more stringent quality control measures, and to report results as a titer that will make the assay more consistent with other serologic tests.

The OD405 of sera from over 1,000 gopher and desert tortoises was plotted to identify natural points of inflection within the data to evaluate current positive and negative cutoff points. A subset of these sera was used to determine end point titer and construct a standard curve. A linear relationship between end point titer and the absorbance value for these samples was established. From this graph, the equation of the line can be utilized to calculate sample titer. This also provides an internal standard for quality assurance between and within assays. The relationship between titer and OD405 was validated using sera from known positive and negative control tortoises from prior transmission studies.

This refinement does not invalidate previous results obtained with the EIA ratio method. The titer system does, however, have numerous benefits. Under the new protocol, samples are assessed at a higher dilution that decreases the chance of false-positive results due to nonspecific cross-reactions. A smaller volume of serum is needed to run the analysis, which conserves serum for other diagnostic tests. The standard curve provides a mechanism for more closely monitoring inter- and intra- assay variance for improved quality assurance. Further, it provides a more clinically relevant system for monitoring antibody changes in individual animals over time.