Journal of Proteomics & Bioinformatics
Open Access
ISSN: 0974-276X
+44 1223 790975
ISSN: 0974-276X
+44 1223 790975
Charles Yuan Jie Liu, Ben Yu and Lucy Yin
Accepted Abstracts: J Proteomics Bioinform
Immunohistochemistry (IHC) in human hepatic tissue is often obstructed by the presence of nonspecific antibody interactions, the cause of high background staining, resulting in difficulties in the interpretation of data. Cognizant of previous encounters with high levels of background in human hepatic tissue when using polyclonal antibodies, we systemically investigated the potential causes by testing the impact of different antibody variants and assay conditions. Firstly, we performed IHC on hepatic tissue with immunized and nonimmunized polyclonal antibodies, demonstrating nonimmunized antibodies to produce insignificant background while immunized antibodies pervaded the samples with nonspecific staining. Following, we coincubated the primary antibody with different concentrations of protein blocks, acid/base solutions, crystalline ionic compounds, and combinations of these individual components in order to assess their potency in removing background staining. Optimization of these assay conditions revealed that background could be significantly reduced by the addition of sodium chloride to primary antibody solution and/or protein block to sodium azide-stabilized Tris-HCl buffer, while little beneficial effect was observed from coincubating the primary antibody with nonserum protein block/goat serum or hydrochloric acid/sodium hydroxide or a higher concentration of peroxidase block. The experimental data suggest that the liver specific background is associated with the variable light chain region of polyclonal IgGs rather than the nonimmunized heavy chain region. Our results also suggest that hepatic background in IHC is mainly attributed to strong ionic interactions between the light chains of polyclonal antibodies and polar molecules specifically prevalent in hepatic tissue, which can be reduced by increasing the ionic strength of the antibody-antigen reaction microenvironment. Thus, our findings can enhance the specificity of hepatic tissue immunoassays and has potential to benefit novel forms of targeted immunotherapies for liver diseases.
Charles Yuan Jie Liu completed his Bachelor degree in Chemistry from University of Toronto and later received his Masters degree in Translational Science from University of Manchester. During his Masters education, he received a joint scholarship from Cancer UK/AstraZeneca to work in the oncology discovery unit. Following his education, he continued his work with the pharmaceutical company and is now mainly focusing on Asian diseases.