The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (K_d), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC₅₀ approaches the K_d value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to K_d. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.

The detection and quantification of protein–protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion-protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

Biofilms enable the persistence of pathogens in food processing environments. In order to inactivate these microorganisms, sanitizing agents are needed that are effective against pathogens entrapped in biofilms which are more difficult to inactivate than planktonic cells that are displaced and found on equipment surfaces. We examined conditions to develop, analyze, and enumerate robust biofilms of 3 different foodborne pathogens assisted by fluorescence adherence assay and enzymatic detachment. We compared 3 different isomeric forms of fluorescent substrates that are readily taken up by bacterial cells based on carboxy-fluorescein diacetate (5-CFDA, 5,6-CFDA, 5,6-CFDA,SE). Biofilm-forming strains of L. monocytogenes, E.coli O157:H7, and Salmonella serovars were detected using the chosen substrate in a microplate fluorescence assay define previously for use with Listeria. Adherence levels were determined by differences in relative fluorescence units (RFU). Multiple hydrolytic enzymes were examined for each representative pathogen for the most suitable enzyme for detachment and enumeration to confirm data obtained by fluorescence assay. Cultures were grown overnight in microplates, incubated, washed and replenished with fresh sterile growth medium; this cycle was repeated 7 times before used as ‘biofilms’. All treatments were performed in triplicate and compared by one-way analysis of variance (ANOVA) to determine significant differences (p < 0.05). Analysis of 7-day biofilms by SEM indicated possible extracellular polysaccharide involvement with Salmonella and E. coli.