Enzyme‐Linked Immunosorbent Assays (ELISA)

Abstract

This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.

Na+/H+ Exchanger NHE3 Activity and Trafficking Are Lipid Raft-dependent

“…The absorbance varied linearly with the amount of peroxidase bound. Serial dilution titration analyses were performed to determine the optimal concentration of reagents used in the ELISA as described (36). The optimal concentrations of primary and secondary antibodies as well as developing reagent (oPD) and cells were serially diluted and analyzed by crisscross matrix analysis.…”

Immunometric Antibody Sandwich Enzyme-Linked Immunosorbent Assay

“…Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) (Rockland Immunochemicals, #TMBE-1000) (for horseradish peroxidase [HRP]-based assays) Antibodies of known concentration for antigen-specific capture and detection These can be polyclonal/monoclonal, monoclonal/monoclonal, or monoclonal/polyclonal antibody combinations (Rabin et al 1992;Hornbeck et al 2001). Addition of a reporter-labeled detection antibody (which eliminates the need for a tertiary antibody) converts this assay into a direct sandwich ELISA (Fig.…”

Antigenicidad de piensos y materias primas proteicas en conejos

“…Se tomó como base un protocolo genérico de ELISA indirecto (Hornbeck et al, 1991), que se fue modificando, adaptando y ajustando en cada una de sus fases, que se describen a continuación.…”

“…De los protocolos ELISA disponibles, se eligió el ELISA indirecto por ser el más adecuado para los objetivos de la presente investigación (Hornbeck et al, 1991). Esta técnica ha sido utilizada en la investigación de las reacciones inmunitarias frente a proteínas dietarias en humanos (Husby, 2000), ratones (Christensen et al, 2004), perros (WillisMahn et al, 2014), terneros (Timmermans et al, 1992), lechones Lallès et al, 2004;Li et al, 1991a;Hankins et al, 1992;Lallès et al, 2004) y conejos (March, 2003).…”

“…Dada la heterogeneidad del contenido proteico de los hidrolizados, se consideró necesario estandarizarlo antes de realizar el proceso de tapizado de los pocillos, mediante las diluciones oportunas, ya que la concentración del antígeno utilizada para el tapizado puede afectar a los resultados (Hornbeck et al, 1991). De esta forma, los resultados del ELISA son independientes del nivel y la naturaleza de la proteína en los piensos o materias primas estudiadas.…”