Sequential Determination of Two Proteins by Temperature-Triggered Homogeneous Chemiluminescent Immunoassay
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- 作者：Yang Zhou ; Yuhao Zhang ; Choiwan Lau ; Jianzhong Lu
- 刊名：Analytical Chemistry
- 出版年：2006
- 出版时间：August 15, 2006
- 年：2006
- 卷：78
- 期：16
- 页码：5920 - 5924
- 全文大小：159K
- 年卷期：v.78,no.16(August 15, 2006)
- ISSN：1520-6882
文摘
A novel protocol for performing a sequential dual-proteinimmunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to currentmultilabel-based detection techniques, a single HRP labelis employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magneticbeads as bimolecular immobilizing carriers to separatedifferent targets by taking advantage of thermal response,as demonstrated by sequential detection of human IgGand IgA. PNIP is known to aggregate and precipitate outof water when the temperature is raised above the lowercritical solution temperature (LCST) of 31 
C; thus, it canbe separated from supernatant by centrifugation. Besides,magnetic beads can be separated from PNIP by magneticforce as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed byprimary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in thesample, and HRP-labeled second antibodies. Moreover,highly sensitive CL detection of HRP was applied, and thedetection limits of IgG and IgA were as low as 2.0 and1.5 ng/mL, respectively. Within the calibrated amount,the protocol had excellent precision within 11% for eachtarget and was comparable in performance to commercialsingle-analyte ELISAs. Furthermore, the proposed methodhas been sucessfully applied to the determination of dualanalyte in real samples without cross-reaction, and a goodcorrelation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serumsamples.
C; thus, it canbe separated from supernatant by centrifugation. Besides,magnetic beads can be separated from PNIP by magneticforce as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed byprimary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in thesample, and HRP-labeled second antibodies. Moreover,highly sensitive CL detection of HRP was applied, and thedetection limits of IgG and IgA were as low as 2.0 and1.5 ng/mL, respectively. Within the calibrated amount,the protocol had excellent precision within 11% for eachtarget and was comparable in performance to commercialsingle-analyte ELISAs. Furthermore, the proposed methodhas been sucessfully applied to the determination of dualanalyte in real samples without cross-reaction, and a goodcorrelation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serumsamples.