A sandwich ELISA for the detection of Wnt5a.
Wnt5a is a noncanonical member of the Wnt family of signaling molecules that has been linked to various physiological and pathological processes including cell differentiation, cell migration, cell growth, vascular remodeling, cancer and chronic inflammation. To understand the role of Wnt5a in these processes, it is necessary to determine the function and expression level of Wnt5a. In this study we developed a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Wnt5a. We found that a rabbit anti-human Wnt5a is a suitable capture antibody for establishing a sandwich ELISA. We used two systems to detect Wnt5a: (1) goat anti-mouse Wnt5a and horseradish peroxidase (HRP) conjugated F(ab')(2) donkey anti-goat IgG as detection and enzyme-linked antibodies respectively, or (2) biotinylated goat anti-mouse Wnt5a and HRP-streptavidin as detection antibody and enzyme-linked avidin respectively. A sandwich ELISA using either of these systems failed to detect recombinant mouse (rm)-Wnt5a diluted in Hank's balanced salt solution supplemented with Ca(2+) and Mg(2+) and 1% bovine serum albumin (HBBS+, 1% BSA). Addition of polyethylene glycol (PEG) to the HBBS+, buffer during the binding stage of rm-Wnt5a, afforded the detection of rm-Wnt5a. The use of PEG during both the binding of rm-Wnt5a and detection antibody stages of the assay yielded the maximum signal for rm-Wnt5a. The relationship between the ELISA signal and concentration of Wnt5a was linear with an R(2) of 0.9934. In summary, we have developed a specific and sensitive sandwich ELISA that detects rm-Wnt5a.
Kummitha
China Malakondaiah
CM
Chemical and Biomolecular Engineering, Ohio University, Athens, Ohio, USA.
Mayle
Kristine M
KM
Christman
Mark A
MA
Deosarkar
Sudhir P
SP
Schwartz
Anthony L
AL
McCall
Kelly D
KD
Kohn
Leonard D
LD
Malgor
Ramiro
R
Goetz
Douglas J
DJ
D000818:Animals; D000937:Antigen-Antibody Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018397:Equidae; D006041:Goats; D006801:Humans; D051379:Mice; D011485:Protein Binding; D011817:Rabbits; D012680:Sensitivity and Specificity; D051153:Wnt Proteins / Q000378:metabolism*; D000071818:Wnt-5a Protein
D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C438615:Wnt5a protein, mouse