Use of Fourier-transform infrared spectroscopy to determine immunoglobulin status in camelid and equine species

Abstract

Measurement of systemic immunoglobulin (Ig) concentrations in horses and camelids is important for early and accurate diagnosis of immunodeficiencies in order to provide proper medical intervention. As a consequence, there is a demand for up-to-date, economic, rapid and precise diagnostic assays for Igs. Accordingly, multiple methods for the evaluation of Ig concentrations have been evaluated in equine and camelid species, each with particular advantages and disadvantages. Fourier-transform infrared (FTIR) spectroscopy has recently emerged as a powerful diagnostic tool for the quantitative characterization of biological fluids in human and veterinary medicine. In particular, FTIR spectroscopy has proven to be an accurate, economical and reagent-free method for immunoglobulin G (IgG) quantitation in horses. Further research to investigate its potential application in the quantitation of other equine Ig isotypes and IgG subclasses is warranted. Additionally, as rapid quantitative assays for the measurement of camelid serum IgG are limited, further work to assess the use of FTIR spectroscopy in this area is desirable. The objectives of this thesis were: (1) to develop an FTIR-based assay for the measurement of IgG concentrations in alpaca serum and to compare its performance to that of the radial immunodiffusion (RID) assay, and (2) to develop FTIR-based assays for the measurement IgGa, IgGb, IgG(T), IgA, and IgM for equine plasma using ELISA assays as reference tests. The first objective of this thesis was achieved by performing RID IgG assays and collecting FTIR spectra for 175 alpaca serum samples. A FTIR-based assay was built using partial least squares regression to convert the spectroscopic data into quantitative IgG values which were compared to the RID results. Correlation coefficients and scatter plots indicated good to excellent levels of agreement between the assays. The results suggest that FTIR spectroscopy may be a useful method for IgG measurement in alpaca serum. For the second objective, IgGa, IgGb, IgG(T), IgA, and IgM concentrations were determined by ELISA assays and FTIR spectra were collected for 100 equine plasma samples. The spectra were randomly divided into training and prediction sets. The training set was used to build a calibration model for each Ig isotype or IgG subclass using partial least squares regression, while the prediction set was used to test the performance of the developed models. Pearson correlation coefficients and scatter plots displayed moderate to good agreement between FTIR and ELISA IgGb assay results but poor overall agreement for IgGa, IgG(T), IgA and IgM assay results. As well, significant differences were noted between the ELISA results of this study and the reviewed studies from the published literature. At present, a FTIR spectroscopic approach is an inaccurate technique for the measurement of Ig isotypes or IgG subclasses in equine plasma