Research Article: Quantitative immunoassay for mink immunoglobulin in serum and milk

Date Published: June 18, 2018

Publisher: BioMed Central

Author(s): Ronja Mathiesen, Mariann Chriél, Tina Struve, Peter Mikael Helweg Heegaard.

http://doi.org/10.1186/s13028-018-0391-7

Abstract

The significance of maternal immunoglobulin G (IgG) for the resistance against a number of infections affecting the health of young mink offspring is not known. Here, we present a validated immunoassay for quantification of mink IgG in serum and milk, using a commercially available polyclonal goat anti-ferret IgG antibody cross-reactive with mink IgG as both the catching and the detection antibody, in a sandwich format enzyme linked immunosorbent assay (ELISA). Using this ELISA, serum IgG concentrations was analyzed over time in both mothers and kits in order to establish a correlation between maternal IgG serum concentrations and those of the offspring.

Intra-assay coefficient of variation (CV) for a serum sample ranged from 2.15 to 5.97% depending on the dilution, while the inter-assay CV ranged from 5.17 to 17.78%. In addition, the range of milk intra-assay CV was 2.71–5.92%, while the range of the inter-assay CV was 4.20–16.03%. Calibrating the ELISA with purified mink IgG (an in-house preparation purified from mink serum) the lower limit of detection was found to be 5 ng/mL for serum and 1 ng/mL for milk. Both serum and milk showed high precision and good linearity over a two-log dilution range. When comparing the serum IgG concentrations of the mink kits a clear within litter effect was seen, while the mean serum IgG concentrations of litters differed significantly between some of the litters (P = 0.0013). Mean maternal serum IgG concentrations correlated positively with the IgG serum concentration of the corresponding offspring sampled over a 3 week period (R2 = 0.63).

A calibrated and reproducible sandwich ELISA for quantifying mink IgG concentrations in both milk and serum with high analytical sensitivity was developed and validated. The results in this study corroborate previous investigations supporting the usability of the ELISA, paving the way for investigations into the importance of maternal IgG in milk and in serum for the welfare and health of the offspring.

Partial Text

Loss of mink kits during the pre-weaning period is of major concern for welfare, management, and economy in the mink fur production. Mink kits are very vulnerable to pathogens at this stage as they are born with very low serum concentrations of maternal immunoglobulin G (IgG), received through trans-placental transfer from the mother, and must strengthen their immune system by taking up IgG from the mother’s colostrum and milk [1, 2]. Furthermore, mink kits do not initiate production of IgG until they are 7–8 weeks old, leaving them vulnerable for the first few weeks of life [1, 3]. IgG absorbed from colostrum and milk play a critical role in passive immunization of mink kits against pathogens (for an extensive review on IgG transfer from mother to offspring see [4]). In ferrets, maternal immunoglobulins transferred through the milk were found to protect offspring against influenza virus infection [5, 6] and maternal IgG is likely to play a similar important protective role against infection in mink. The purpose of this study was to develop and validate a quantitative enzyme-linked immunosorbent assay (ELISA) for quantifying total IgG (independent of antigenic specificity) in mink serum and milk. The assay allows the study of the dynamics of the exchange of immunoglobulin between mothers and their suckling kits and its significance for protecting against infectious disease to be studied in detail [7].

When studying the possible significance of IgG, with respect to protecting mink kits against various pathogens and diseases, it is important to have a reliable analytical method for analyzing the concentration of IgG in milk and blood. In the present study, the development and validation of a sandwich ELISA for quantifying IgG in mink serum and milk was described. We analyzed specificity, intra-and inter-assay variation, limit of detection, limit of quantification, and linearity of dilution. The specificity of the antibody used in the ELISA was elucidated by investigating its reactivity with full mink serum as well as mink milk by Western blotting. Except from a minor non-specific reaction with the albumin band in both serum and milk samples the antibody reacted exclusively with mink IgG light and heavy chains. The nonspecific binding to albumin may be an artefact related to the blotting procedure and did not present a problem during ELISA analysis as the ELISA background was low. The validation demonstrated that the assay is reproducible (low intra- and inter-assay variation), and sensitive with a lower limit of detection allowing minimal sample volumes (5 µL) to be used. Mean intra-assay CV for serum samples was 3.6 and 3.7% for milk samples and the mean inter-assay CV for serum samples was 11.0 and 9.6% for milk samples, which can be considered acceptable [10]. The lower limit of detection was 5 ng/mL and the limit of quantification was 11 ng/mL for serum samples, which is more than sufficient for measurement of mink serum concentrations of IgG as the lowest serum concentrations of IgG we analyzed in mink kits was 0.4 mg/mL and others have previously reported serum concentrations in the range of 0.1–0.73 mg/mL [1]. Furthermore, milk samples showed a lower limit of detection of 1 ng/mL and a limit of quantification of 3 ng/mL, which is also more than sufficient for milk samples as the lowest milk IgG concentration we analyzed from milk was 1600 µg/mL and others have reported milk IgG concentrations in the range of 1–6.3 mg/mL [1]. It is important for the precision of an ELISA to show good linearity of dilution, i.e. the calculated concentration of IgG in a given sample (within the linear range of the assay) should not be affected by the dilution at which the sample is tested [11]. As shown in Figs. 3 and 4, comparable values were obtained irrespectively of dilution for both serum samples (disregarding a few (3) non-systematically deviating samples) and milk samples indicating lack of interference by non-relevant matrix components. Additionally, an ideal ELISA assay must be robust and should not be affected by small changes in the procedure [11]. The assay was robust as measurements were unaffected by change of operator and as it performed well with analytes stored at − 20 °C for a longer period of time. Also, repeated freezing and thawing did not affect the readout. One limitation of the assay is that no calibrated standard mink IgG was available, thus precluding a definitive calibration of the assay. Here, a preliminary calibration of the purified mink IgG was achieved by spectrophotometry at 280 nm using the generally accepted mass extinction coefficient of 1.37 per 1 mg IgG/mL. Furthermore, in comparison with other studies regarding the concentration of IgG in adult female and kit serum and female milk, the results correspond well with our data [1, 12]. To our knowledge, no ELISA has previously been thoroughly validated for mink IgG quantification in serum and milk samples although a number of older studies report on IgG concentrations in mink blood and milk using non-validated methods. Porter et al. [2] used immunoelectrophoresis and a polyclonal rabbit anti-mink serum antibody to study differences in concentrations of gamma-globulins in maternal vs. offspring serum and in colostrum vs. adult serum [2] while Coe and Race [1] used single radial immunodiffusion and an in-house polyclonal antiserum against mink IgG to quantify mink IgG in serum samples. These two studies show conflicting results with regard to IgG concentrations in newborn kits; Porter et al. [2] indicate that there is no uptake of IgG from the mother’s placenta while Coe and Race [1] show that there is indeed uptake of IgG, allowing the newborn kit to have circulating IgG. The present study indicates trans-placental transfer of IgG, as serum IgG was detected in litter C, which was obtained by post mortem Caesarian section (Fig. 5). Other studies have also utilized ELISA for quantification of IgG in mink blood samples [13–15] and mink milk [12]. Previously described ELISAs [13, 14] employed an in-house absorbed rabbit anti-mink IgG serum, which is not commercially available. The assay was either not calibrated [13] or was using an undisclosed method for calibrating their mink IgG standard [14]. An indirect ELISA for quantification of hapten-carrier specific IgG from hapten-carrier conjugate immunized adult mink was also reported [15], however no attempt was made to quantify the naturally occurring total IgG population and no validation was described. Another study investigated the transfer of specific IgG from mother to offspring in the fetal stage by using mothers vaccinated with mink enteritis virus (MEV) and then analyzing the milk-derived MEV specific IgG in the kits’ serum, however the analytical method used in this study was not disclosed [12]. Thus, the litter specific serum MEV-specific IgG concentrations could in principle have been due to different efficiency of maternal vaccination from one mink female to the other. In the present study we clearly demonstrate that individual litters have a well-defined litter specific level of total circulating IgG (Fig. 5), which to some extent is associated with the concentration of total IgG in the maternal circulation (Fig. 6a). Differences in serum IgG concentration between litters (high vs. low IgG concentration, Fig. 5) could have consequences for the immune competence and ability to handle pathogens. Litters with low IgG concentrations might be more susceptible to disease, which could explain the observation that only some litters on a farm is affected by outbreaks of “pre-weaning diarrhea” [16]. Serum IgG concentrations in mink kits reached a plateau 8 days after parturition (Fig. 6b), while maternal IgG concentrations remained fairly constant. Looking back to the studies done by Porter et al. [2] and Core and Race [1], both studies also show that kits reach serum IgG concentrations that are similar to the adult female’s serum IgG concentrations 8 days after parturition [1, 2]. Our data thus corroborate the previous data, however with a much higher number of animals included. Furthermore, we analyzed milk samples collected from 19 adult females milked one time/week over a 3-week-period. The milk IgG concentrations did not correlate in any way with serum concentration in the same individual animal (data not shown). Results reported by Uttenthal et al. [12] indicate that the IgG concentrations in both milk and serum from adult females, sampled over a 4-week-period, did not change much over time, which is what we have also seen in our samples (Fig. 6b). The study by Fink et al. [17] shows that mink colostrum and milk have the same chemical composition indicating that the milk does not change much after the mink kits are born. To sum up, the IgG concentrations found in our collected mink blood and milk samples corroborates earlier studies and adds new data, which further validates our ELISA and establishes it as a reliable analytical tool for analyzing IgG concentrations in serum and milk of mink.

The validated sandwich ELISA presented here is a sensitive and reproducible calibrated assay suitable for analyzing total IgG concentrations in mink blood and milk. Serum IgG concentrations in mink kits are shown to be litter specific. In addition, a positive correlation between mean maternal serum IgG concentrations and mean kit serum IgG concentrations was established and it is confirmed that mink kit serum IgG concentrations reaches a plateau within 8 days after parturition. Our results corroborate and extent previous investigations, stressing the usability of the ELISA, paving the way for more investigations into the importance of maternal IgG for the welfare and health of the offspring.

 

Source:

http://doi.org/10.1186/s13028-018-0391-7