Synthetic Antigen Controls for Immunohistochemistry.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

Synthetic Antigen Gels as Practical Controls for Standardized and Quantitative Immunohistochemistry.

Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX-XXX, 2019).

Antibody response to the surface envelope of caprine arthritis-encephalitis lentivirus: disease status is predicted by SU antibody isotype.

This study evaluated the hypothesis that the disease status of Saanen goats infected with caprine arthritis-encephalitis lentivirus (CAEV) is associated with the focus of immune responses to viral antigens, particularly the surface envelope glycoprotein (SU). Specifically, we have proposed that Th2 responses promote progressive immune-mediated arthritis, whereas Th1 responses restrict virus replication and development of clinical disease. The present study determined the isotype of SU antibodies associated with progressor and long-term nonprogressor (LTNP) status. We show that chronically infected goats that develop clinical arthritis have predominantly IgG1 antibodies to SU during both preclinical and clinical stages of disease, whereas SU antibodies of LTNP goats are relatively biased toward IgG2. Additional studies determined the isotype of SU antibodies induced initially by CAEV infection. These experiments show that initial IgG1-dominated responses to SU are associated with subsequent development of preclinical inflammatory joint lesions, whereas lack of joint pathology is associated with an IgG2 bias of initial responses to SU. Our results using the CAEV model suggest that isotype bias of SU antibodies is a reliable indicator of clinical disease caused by lentiviruses. Isotype analysis may be a useful method to screen candidate lentiviral vaccines intended to prevent disease progression.