Sensitive and Selective Immunofluorescence Assay for CA15-3 Detection Using Fluorescein Derivative A10254.

Abstract

Carbohydrate antigen 15-3 (CA15-3), a specific breast cancer-related biomarker in serum, can provide direct information to monitor a patient's postoperative status and can predict recurrence and metastasis of breast cancer. The conventional Enzyme-Linked Immunosorbent Assay (ELISA) is an effective approach for CA15-3 detection, but there is a high limit of detection which is more than 1 U mL-1. Therefore, a convenient, highly sensitive and specific CA15-3 detection method is needed for earlier prognoses of breast cancer. In this work, we aim to develop a novel sensitive and selective immunofluorescence assay for the highly effective detection of CA15-3. We pre-coat the antibody I onto the solid phase to capture antigen CA15-3. The A10254- labeled antibody II identified by electrophoresis and fluorescence spectra recognized the antigen CA15-3 and formed the sandwich format. The fluorescence signal of A10254 directly relating to the amount of antigen could be detected and quantified by the microplate reader excited at 440nm. The obtained method for CA15-3 detection showed lower limits of detection (0.11 and 0.18 U mL-1) in a linear range of 0.25-14 U mL-1 in the buffer and 2% serum, respectively. This assay exhibited a good specificity and reproducibility in 2% serum with lower relative standard deviations in different tested concentrations. The developed immunofluorescence assay enhanced the performance of the conventional CA15-3 ELISA kit. Moreover, the acceptable accuracy and good reproducibility in diluted serum indicated the assay has great potential for the diagnosis of breast cancer in clinical practice.