Chemiluminescence Immunoassay (CLIA) for Candida Antigen

摘要

The project demonstrated that the combination of chemiluminescent (CL) acridinium esters and monoclonal antibodies to Candida cell-wall mannan is capable of generating a simple non-isotopic immunoassay for Candida mannan in serum. The sensitivity of the indirect two-site chemiluminescence assay was approximately 10 ng/ml and that of the competitive binding chemiluminescence assay was approximately 100 ng/ml with a detection time of only 2 seconds. The aims of the project were to identify and use a pair of antibodies against mannan to generate a sensitive and simple non-isotopic chemiluminescence immunoassay for mannan. However, only one monoclonal antibody with very low affinity for mannan and the IgM form to Candida mannan was available. The direct chemiluminescence labeling of this anti-mannan IgM with the CL acridinium ester resulted in the loss of its immunoreactivity, which complicates the problem further. These difficulties were circumvented by the availability of high affinity polyclonal anti-sera from human patients with systemic candidiasis for use as the capture antibody in the sandwich assay. The difficulties of labelling the anti-mannan IgM were overcome by attempting to use CL labelled succinyl Concavalin A (sCon A) or a CL-labelled secondary antibody to the anti-mannan IgM. The CL-labelled sCon A did not perform well as the label in the sandwich assay due to the high blanks caused by the binding of sCon A to the anti-mannan IgG coated tubes. This was not surprising as concavalin A has high affinity for carbohydrates and IgG has at least 20-30% carbohydrate.