Development of high-performance magnetic chemiluminescence enzyme immunoassay for alpha-fetoprotein (AFP) in human serum.
BACKGROUND
A high-performance chemiluminescence enzyme immunoassay (CLEIA) for alpha-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma (HCC), was constructed by using magnetic particles (MPs) as both the immobilization matrix and separation tools.


METHODS
A double sandwiched immunocomplex was formed through the reaction among anti-fluorescein isothiocyanate (FITC) antibody coated MPs, FITC-labeled anti-AFP antibody, AFP antigen, and alkaline phosphatase (ALP)-labeled anti-AFP antibody. The subsequent chemiluminescence reaction of ALP with 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD) gave light intensity that was directly proportional to the amount of analyte present in the samples. The effects of several physicochemical parameters, including the concentration of FITC-labeled anti-AFP antibody, the dilution ratio of ALP-labeled anti-AFP antibody, the volume of MPs and substrate, the immunoreaction time and other relevant variables upon the immunoassay were studied and optimized. RIA and microplate CLEIA were used as comparison methods.


RESULTS
The proposed method had a sensitivity of 3.0 ng/ml, low cross reactivities, and an assay time of 1 h. The linear range was 0-1200 ng/ml through using MPs and is useful for samples with extremely high AFP concentrations without dilution while avoiding the hook effect. The intra- and inter-assay precision was <3% and <5%. The present method has been successfully applied to the detection of AFP human serum with recoveries from 90 to 108%, and showed a good correlation with the commercially available AFP RIA kit.


CONCLUSIONS
This proposed assay provided apparent advantages over microplate CLEIA and RIA, and facilitated the development of high-throughput screening and automated operation systems in the clinical practice.
Wang
Xu
X
The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing, 100084, China. wang-x@mail.tsinghua.edu.cn
Zhang
Qian-Yun
QY
Li
Zhen-Jia
ZJ
Ying
Xi-Tang
XT
Lin
Jin-Ming
JM
D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D049449:Luminescence; D008280:Magnetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000509:alpha-Fetoproteins / Q000032:analysis*
D000509:alpha-Fetoproteins