Immunoglobulin G Measurement in Blood Plasma Using Infrared Spectroscopy

Abstract

A rapid, simple, and inexpensive method to measure the immunoglobulin G (IgG) concentrations in blood samples in human and veterinary medicine is highly desired. Infrared spectroscopy (coupled with chemometric manipulation of spectral data) has the advantages of simple sample preparation, rapid implementation of analysis, and low cost. Here a method that exploits infrared spectroscopy as the basis to measure IgG concentration in animal plasma samples is reported, with radial immunodiffusion (RID) used as the reference test method for partial least squares (PLS) calibration model development. Smoothed non-derivative and the second-order derivative spectra were used to develop calibration models. Various additional spectral preprocessing steps were evaluated to optimize the calibration models, and the possible benefits of using an internal standard (potassium thiocyanate [KSCN]) were investigated. Monte Carlo cross-validation was used to determine the optimal number of PLS factors, and an independent prediction set was used to test the predictive performances of provisional models. The effects of various preprocessing options (spectral smoothing, derivation, normalization, region selection, mean-centering, and standard deviation scaling) on quantification accuracy were investigated. The root mean squared error of prediction (RMSEP) for different combinations of spectra preprocessing steps was 394 ± 36 mg/dL for the non-derivative spectra and 427 ± 101 mg/dL for the second-order derivative spectra. Immunoglobulin G concentrations produced by the optimized PLS model for the non-derivative spectra (RMSEP = 352 mg/dL) were found to be stable with respect to different splits of the samples among the calibration, validation, and prediction sets. The precision of the Fourier transform infrared (FT-IR) method is found to be slightly superior to that of the RID method. The results of this work indicate that infrared spectroscopy is a promising technique for economically and rapidly determining the IgG concentrations of plasma and plasma-derived samples.

A rapid field test for the measurement of bovine serum immunoglobulin G using attenuated total reflectance infrared spectroscopy

“…As a result, the calibration model development was weighted towards low IgG concentrations, which are particularly more important for diagnosis of FTPI in farm animals [ 3 , 4 ]. Similar findings have been observed for transmission IR spectroscopy-based serum IgG assays for bovine serum [ 44 ] and IR-based assays for other species [ 45 , 46 ].…”

“…The ATR assay showed higher Pearson correlation and concordance coefficients than have been reported for previous transmission IR spectroscopy-based serum IgG assays for bovine [ 44 ], equine serum and plasma [ 10 , 45 ], and alpaca serum [ 46 ]. Agreement between the ATR and RID assays was poorer at high IgG concentrations than at low IgG concentrations (Fig.…”

Quantification of bovine immunoglobulin G using transmission and attenuated total reflectance infrared spectroscopy

“…4). The same results were reported during PLS model building for IgG concentrations of bovine, 14 equid, 21 camelid, 3 and canine samples. 40 The lower precision of the ATR-IR method is likely associated with the variability in sample dryness for each replicate, and to the low number of ATR-IR scans (32 scans), as compared to the TIR method (512 scans).…”

“…2). In comparison, these values are higher than those reported previously for counterpart TIR-based assays for equid IgG 21,32 and camelid IgG levels, 3 and for the ATR-IR method described for canine IgG concentrations. 40 Furthermore, the correlation coefficients between IgG concentrations predicted by both IR spectroscopic methods and the reference RID IgG assay were higher than those reported for refractometry, 9,28 similar to those of near-IR spectroscopy, 52 but lower than the value reported for automated turbidimetric immunoassay.…”

Spectroscopic properties of various blood antigens/antibodies

“…The spectrophotometric method of human IgG was performed in a wide concentration range [22]. A method for the mid-infrared spectral quantification of IgG has been developed in horse blood plasma [23]. The pre-processed SERS spectra is also examined in order to evaluate the spectral differences among ABO blood groups and the antigen-antibody interactions [24].…”