Use of automated capillary immunoassay for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins

Authors: Yeh, Hung-Yueh; Frye, Jonathan; Jackson, Charlene; Read, Quentin; Line, John; Hinton Jr, Arthur

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/31/2023
Publication Date: 6/5/2023
Citation: Yeh, H., Frye, J.G., Jackson, C.R., Read, Q.D., Line, J.E., Hinton Jr, A. 2023. Use of automated capillary immunoassay for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins. Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet.2023.106757.
DOI: https://doi.org/10.1016/j.mimet.2023.106757

Interpretive Summary: The immunoblot assay is a useful tool for specific protein identification. However, a standard protocol for this assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In this communication, we optimized this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. The results show a single band of each protein was detected in the gel like images by this system after purification. A good linear range of the protein concentrations were also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various antibody types against two recombinant Salmonella proteins from both non-immunized and immunized chicken sera. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken antibodies before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

Technical Abstract: The classic immunoblot technique is a useful tool for specific protein identification. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In this communication, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations were also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from both non-immunized and immunized chicken sera. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.