Analysis of serum alpha-fetoprotein (AFP) and AFP-L3 levels by protein microarray

Abstract

Objectives This study aimed to examine a simple, effective, time-saving, and low-cost protein microarray method for detecting serum alpha-fetoprotein (AFP) and AFP-L3 levels. Methods Serum samples from patients with hepatocellular carcinoma (HCC) (n = 33) and control subjects (n = 39) were collected and evaluated for the presence of AFP using a novel protein microarray. Glycoprotein (including AFP-L3) was enriched from crude samples by a Hotgen Biotech glycosyl capture spin column and then detected by protein microarray. An electrochemiluminescence immunoassay (ECLIA) was used to validate the measured values. Results Neither AFP levels lower than 20 ng/mL in the HCC group nor AFP levels higher than 20 ng/mL in the control group were found when tested by the ECLIA and protein microarray. The kappa test showed good consistency in the diagnostic performance of measuring serum AFP levels and the percentage of AFP-L3 in total AFP by the ECLIA and protein microarray. Protein microarray had advantages of smaller sample size required, low cost, and convenience compared with the ECLIA. Conclusion The protein microarray assay that was developed in the present study shows potential as an economic and convenient technique for detecting AFP and AFP-L3 levels in serum samples from patients with HCC.

Keywords: AFP-L3; Lens culinaris agglutinin; Protein microarray; alpha-fetoprotein (AFP); electrochemiluminescence immunoassay; glycoprotein; hepatocellular carcinoma.

Figure 1.

Diagram of AFP antibody and BAS solution spotted on a protein microarray. AFP: alpha-fetoprotein; BSA: bovine serum albumin.

Figure 2.

(a) Image of a protein microarray for detecting AFP antigen standards. Different concentrations of AFP antigen standards were added to wells one to six (35.5, 17.8, 8.8, 4.4, 2.2, and 1.1 ng/mL). Serum samples from the control group were added to wells seven to nine and well 10 was used as a negative control. (b) Standard curve used in the protein microarray for determining AFP levels. AFP: alpha-fetoprotein.

Figure 3.

Image of a protein microarray for detecting serum samples before and after pretreatment of the Hotgen Biotech glycosyl capture spin column. Four pairs of serum samples were added to wells one and two, three and four, five and six, and seven and eight. Wells nine and 10 were used as a positive and negative control, respectively.