Protein microarray with horseradish peroxidase chemiluminescence for quantification of serum α-fetoprotein

Abstract

Objectives

To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of α-fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC).

Methods

A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA).

Results

Serum AFP concentrations determined via protein microarray were positively correlated (r = 0.973) with those determined via ELISA in patients with HCC (n = 60) and healthy control subjects (n = 30). Protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. Protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974; 95% CI 0.000, 1.000).

Conclusion

Protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. ¹ It occurs predominantly in Asia and Africa, although its incidence is steadily increasing throughout the rest of the world. ² Early stage HCC is asymptomatic but advanced disease has a poor prognosis, therefore early detection is critical. HCC serum tumour markers include α-fetoprotein (AFP), AFP-L3, des-γ-carboxyprothrombin (DCP) and golgi protein 73 (GP73), with AFP being the most widely used marker.^3,4

Enzyme-linked immunosorbent assay (ELISA) is successfully used in the detection of AFP,^5,6 but requires costly commercial kits. An alternative methodology is protein microarray with horseradish peroxidase (HRP)-linked chemiluminescence. The HRP chemiluminescent reaction is based on the catalyzed oxidation of luminol by peroxide, producing light. ⁷ HRP is currently used in numerous diverse industrial and medical applications, such as waste-water treatment, ⁸ chemical synthesis, coupled enzyme assays, biosensors, diagnostic kits and immunoassays.^9,10

This study reports the development and application of protein microarrays with HRP chemiluminescence to detect AFP in serum from patients with HCC and healthy control subjects.

Participants and methods

Study population

The study recruited patients with HCC who underwent liver resection surgery at the Department of Hepatobiliary Surgery, Beijing You’an Hospital, Beijing, China between 1 January 2014 and 31 July 2014. Patients were required to be positive for serum hepatitis B surface antigen and free from chronic hepatitis C infection. HCC diagnosis was histologically confirmed after surgical resection. Healthy control subjects were recruited from volunteers with no reported gastrointestinal or hepatobiliary disease who were undergoing routine health screening at the outpatient department of Beijing You’an Hospital, Beijing, China.

The study protocol was approved by the ethics committee of Beijing You’an Hospital. Written informed consent was obtained from all participants prior to enrolment in the study.

Blood collection

Whole blood (1 ml) was collected from each subject. Blood was allowed to clot, centrifuged at 3000 g for 10 min at 4℃, and the resulting serum was stored at −70℃. Prior to use, serum was diluted 1 : 4 with 0.05% PBS-Tween 20 (pH 7.5) and stored at 4℃ for a maximum of 7 days.

Microarray preparation

Reaction wells were created by attaching slips of hydrophobic paper (10 holes per slip) to aldehyde-coated glass slides. The prey antibody (2 mg/ml mouse antihuman AFP monoclonal antibody with 30% glycerol to prevent evaporation; Fapon Biotech Company Inc., Shenzhen, China) was spotted onto aldehyde-coated slides in quadruplicate using a microarray spotter (Cartesian Technologies, Irvine, CA, USA; microarrays 4 × 2 spots; spot size 700 µm; spot-to-spot distance 1000 µm). Each well contained four anti-AFP antibody and four negative control (5% bovine serum albumen [BSA]) spots. Following spotting, the slides were incubated for 24 h at 4℃ to fully immobilize proteins. Slides were then blocked with 20 μl blocking buffer (10% normal goat serum with 0.1% sodium azide) for 2 h at 37℃, washed four times with 0.05% PBS-Tween 20 (pH 7.5) at room temperature (5 s each wash), air dried at room temperature and stored at 4℃ until use.

AFP quantification

Rabbit antimouse polyclonal antibody (Abcam; Cambridge, UK) was labelled with biotin using a biotin (type A) conjugation kit (Abcam), according to the manufacturer’s instructions. Serum samples were added to the microarray reaction wells, hybridized for 30 min at 37℃, then washed four times with 0.05% PBS-Tween 20 (pH 7.5) at room temperature (5 s each wash). The biotin-labelled rabbit antimouse polyclonal antibody (1 : 50 dilution) was added to the wells and hybridized for 30 min at 37℃. Slides were washed twice with 0.05% PBS-Tween 20 (pH 7.5) at room temperature (5 s each wash), then incubated with HRP-labelled streptavidin (1 : 50 dilution) for 30 min at 37℃. Slides were washed four times with 0.05% PBS-Tween 20 (pH 7.5) at room temperature (5 s each wash), then incubated with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) for 5 s. Slides were scanned using a chemiluminescence imaging scanner (Academy of Military Medical Sciences, Beijing, China).

Levels of AFP in study samples were quantified by reference to a standard curve, established using serial AFP dilutions (10 ng/ml, 5 ng/ml, 2.5 ng/ml and 1.25 ng/ml) and 0.05% PBS-Tween 20 as negative control (Figure 1). Reliability and stability of the assay were verified by repeat quantification (10 assays) of the AFP standard curve to calculate within-run and between-run variation (Table 1). The cut-off value for optimal diagnostic performance for serum AFP quantification has been shown to be 20 ng/ml (sensitivity 41–65%, specificity 80–94%).^11,12 Therefore, the present study used a cut-off value of 20 ng/ml.

Figure 1.

(a) Photograph of protein microarray assay for α-fetoprotein (AFP). Wells 1–4: 10, 5, 2.5 and 1.25 ng/ml AFP, respectively; well 5: blank; well 6: serum from healthy control subject; wells 7–10: serum from patients with hepatocellular carcinoma. (b) Standard curve for assay. Individual data points are the mean of each well (n = 4).

Table 1.

Within- and between-run variations for protein microarray for detection of α-fetoprotein (AFP) in human serum.

AFP standard ng/ml	Within-run variation	Between-run variation
10	243 ± 29 (12)	245 ± 34 (14)
5	137 ± 15 (11)	136 ± 18 (13)
2.5	66 ± 9 (14)	69 ± 10 (14)
1.25	36 ± 4 (11)	33 ± 4 (12)
0	9 ± 2 (22)	11 ± 2 (18)

Data presented as mean ± SD (coefficient of variation) of 10 experiments.

No statistically significant differences (P > 0.05; Wilcoxon’s rank sum test).

ELISA

Serum AFP was quantified using ELISA (AFP Human ELISA kit; Abcam), according to the manufacturer’s instructions.

Statistical analyses

Data were presented as mean ± SD (coefficient of variation) or median (95% confidence interval [CI]). Between-group comparisons of AFP concentrations were made using Mann–Whitney U-test for both protein microarray and ELISA. Wilcoxon’s rank sum test was used to compare between-test differences in AFP concentration. Spearman’s correlation coefficient was used to determine the associations between serum AFP concentrations as determined by protein microarray or ELISA. Receiver operating characteristic (ROC) analysis was performed to determine the diagnostic performance of protein microarray and ELISA. Statistical analyses were conducted using SPSS® version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. All tests were two-tailed, and P-values <0.05 were considered statistically significant.

Results

The study included blood samples from 60 patients with HCC (39 male/21 female; mean age 58.0 ± 5.2 years; age range 48–67 years) and 30 healthy control subjects (17 male/13 female; mean age 54.2 ± 4.5 years; age range 46–64 years). There were no significant between-group differences in age or sex distribution.

Data regarding serum AFP concentrations detected via ELISA and protein microarray are shown in Figure 2. Serum AFP concentrations were significantly higher in patients than in controls when quantified via ELISA (2.86 ng/ml [95% CI2.33, 3.39 ng/ml] versus 32.58 ng/ml [95% CI 29.28, 35.88 ng/ml], P < 0.001) and protein microarray (3.53 ng/ml [95% CI 3.09, 3.97 ng/ml] versus 33.17 ng/ml [95% CI 29.99, 36.35 ng/ml], P < 0.001). There were no significant between-test differences in AFP concentrations in patients or controls. When the cut-off value was set to 20 ng/ml, protein microarray had 80% sensitivity and 100% specificity for HCC diagnosis, and ELISA had 83.3% sensitivity and 100% specificity (Table 2).

Figure 2.

Box whisker plots of α-fetoprotein (AFP) concentrations in patients with hepatocellular carcinoma (HCC; n = 60) and healthy control subjects (n = 30), quantified using protein microarray or enzyme-linked immunosorbent assay (ELISA). Extremities of the boxes represent 25th and 75th percentiles, error bars represent minimum and maximum outliers, asterisks represent extreme outliers. The colour version of this figure is available at: http://imr.sagepub.com.

Table 2.

Sensitivity and specificity of serum α-fetoprotein (AFP), quantified using protein microarray or enzyme-linked immunosorbent assay (ELISA) for the diagnosis of hepatocellular carcinoma (HCC), using a cut-off value of 20 ng/ml AFP.

	HCC group n = 60		Control group n = 30
Assay	AFP ≥ 20 ng/ml	AFP < 20 ng/ml	AFP ≥ 20 ng/ml	AFP < 20 ng/ml
Protein microarray	48 (80)**	12 (20)**	0	30 (100)
ELISA	50 (83.3)**	10 (16.7)**	0	30 (100)

Data presented as n (%) of patients.

**

P < 0.01 vs control group (within AFP category and assay; Mann–Whitney U-test).

There was a statistically significant positive correlation between AFP concentrations quantified via ELISA and those quantified via protein microarray (r = 0.973, P < 0.001; Figure 3). The ROC curve for diagnosis of HCC is shown in Figure 4. Serum AFP quantified via protein microarray had a similar diagnostic performance to ELISA in distinguishing patients with HCC from healthy controls (area under ROC curve: 0.974 [95% CI 0.000, 1.000] for protein microarray, and 0.966 [95% CI 0.932, 0.999] for ELISA).

Figure 3.

Scatter plot of serum α-fetoprotein (AFP) concentrations in patients with hepatocellular carcinoma and healthy control subjects (n = 90), quantified using protein microarray or enzyme-linked immunosorbent assay (ELISA).

Figure 4.

Receiver operating characteristic curve of serum α-fetoprotein (AFP) quantification using protein microarray or ELISA for diagnosis of hepatocellular carcinoma in patients with hepatocellular carcinoma and healthy control subjects (n = 90).

Discussion

Microarray technology is a powerful tool for high throughput assays of protein expression, protein–protein interaction and enzyme activity.^13,14 It has become an effective diagnostic tool, since many disease-related proteins are detectable in serum.^15,16 The array is incubated with purified proteins, serum or cell extract (“bait”); a labelled second antibody then recognizes the bait. Antibody microarrays have extensive applications, including evaluation of tumour progression and the detection of toxins and bacteria.^17–19

The current study applied HRP and chemiluminescence to protein microarray technology for the quantitative detection of AFP in human serum. Our small study found this method to have excellent specificity for the diagnosis of HCC, generating no false positives in healthy control subjects. The sensitivity of diagnosis via protein microarray was similar to that of ELISA (the gold standard for AFP detection) in the present study. Protein microarray has several advantages over ELISA, including rapidity, simplicity, and low cost. The protein microarray was performed in ∼1.5–2 h in the present study (excluding the time taken to manufacture the microarray), whereas the ELISA kit used in this experiment required >3.5h. Protein microarray assays are relatively simple to perform and do not require highly skilled technicians. In addition, the small size of the microarray reduces the quantity of antibodies, antigens and other materials required compared with ELISA. The amounts of serum required for the protein microarray are greatly reduced: only 10 µl of original or diluted serum is needed, whereas 50 µl of serum is needed for ELISA. The quantities of antibodies for the protein chip are also greatly reduced: only 0.25μl of mouse antihuman AFP monoclonal antibody is needed for spotting on each protein microarray, and 10μl of diluted biotin-labelled rabbit antihuman AFP polyclonal antibody is needed for detection. Conversely,50μl of mouse antihuman AFP monoclonal antibody and 50μl of diluted biotin-labelled rabbit antihuman AFP polyclonal antibody are needed for ELISA.

There is room for improvement in protein microarray technology. Detection with fluorescent labelled antibodies provides a great dynamic range for quantification and signal amplification. ²⁰ Moreover, further improving the uniformity of spots and reducing background noise would allow even more precise protein quantification.

Serum AFP has limited use as a single biomarker for the early detection of HCC, as it may not be sufficient to distinguish HCC from chronic hepatitis and liver cirrhosis. ²¹ Protein microarrays designed to detect multiple tumour markers would greatly increase the utility of this technology for HCC diagnosis.

In conclusion, protein microarray is a potential alternative to ELISA for detecting AFP in serum samples, in patients with HCC. It is low cost, easy to perform, and suitable for clinical diagnosis and public health use.