Excess soluble antigen used to block antibody binding to target cells.

Excess unlabeled antibody used to block binding of labeled version of the same antibody.Targeted knock out cell lines (or cells from transgenic animals) where the antigen expression has been specifically eliminated.Primary cells or mock transfected cell line known to not express the antigen of interest.Cell lines transfected to express the antigen of interest (fusion tags such as GFP or HA can give an independent way to confirm expression when no previously validated antibody is available for direct comparison).Primary cells known to express the antigen of interest (sometimes only after stimulation).There are several different controls that can be used to validate the specificity of your antibodies. Controls to Validate Antibody Specificity: Western blotting are raised against linear epitopes that may not be present/exposed in living cells whereas common flow cytometry sample processing steps such as enzymatic digestion and fixation/permeabilization can alter the native epitopes (or the antibodies and fluorophores themselves). Note also that many antibodies used in e.g. Typical problems in flow cytometry include unspecific binding and specific cross-reactivity with off targets (resulting in false positives) as well as lack of reactivity with the stated target antigen (resulting in false negatives). Unfortunately, majority of commercially available antibodies are poorly validated and it is up to the end user to make sure that the antibodies work as expected. Nearly 50% of the antibodies submitted to the Human Leucocyte Differentiation Antigen Workshops fail to function as intended!

Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.Accurate flow cytometry analysis depends on the specificity of the antibodies used. Requiring only 1.25 uL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance.

Controls for antibody cross reactivity