Antibody-sandwich ELISA analysis of a novel blood biomarker of CST4 in gastrointestinal cancers

Abstract

Background: Members of the cystatin family have increasingly been proven to be involved in several tumors, including gastric cancer (GC) and colorectal cancer (CRC). Cystatin S (CST4) was found to be upregulated at the gene expression level in GC cells, making it a potential novel biomarker for the early diagnosis of gastrointestinal cancer.

Materials and methods: Quantitative real-time polymerase chain reaction and Western blotting analysis were used to explore CST4 expression in gastrointestinal cancer tissues and cell lines. We purified CST4 recombinant protein and generated anti-CST4 monoclonal antibodies to develop an antibody-sandwich enzyme-linked immunosorbent assay (ELISA) analysis system for blood CST4 detection. The performance and clinical efficacy of the detection method were evaluated using a training set and validation set, respectively.

Results: According to the quantitative real-time polymerase chain reaction and Western blotting results, CST4-mRNA expression and protein expression were upregulated in gastrointestinal cancer tissues and cell lines. The ELISA detection system for CST4 showed significantly better sensitivities of 69.0% and 69.0% and specificities of 85.6% and 83.6% for GC and CRC, respectively, than other common clinical biomarkers, carcinoembryonic antigen, CA19-9, CA125, and CA72-4. Clinical verification experiments using GC and CRC validation sets also found distinguishable CST4 median concentrations (177.7 pg·mL^-1 and 174.2 pg·mL^-1 respectively) and high positive detection rates (72.3% and 88.4% respectively), further confirming the specificity and sensitivity of this method.

Conclusion: We validated the overexpression of CST4 in gastrointestinal cancer tissues and cell lines and developed an antibody-sandwich ELISA analysis system for blood CST4 detection, which exhibited high specificity and sensitivity. Novel blood biomarkers of CST4 have enormous potential in terms of clinical diagnostic value in GC and CRC.

Keywords: ELISA; biomarker; cystatin S; gastrointestinal cancer.

Figure 1

SDS-PAGE of recombinant CST4 protein. Abbreviations: CST4, cystatin S; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 2

CST4 linear regressions of OD and concentration in the linear range. Abbreviations: CST4, cystatin S; OD, optical density.

Figure 3

ROC of clinical serum samples of GC and CRC. Abbreviations: CRC, colorectal cancer; GC, gastric cancer; ROC, receiver-operating curve.

Figure 4

The respective concentrations of CST4 in serum samples of GC, CRC, GB, CB, Cs, Is, and Hs. Note: The quantitative statistical analysis was confined to results within the limit of detection (Mann–Whitney test, P<0.001). Abbreviations: CBD, colorectal benign diseases; CRC, colorectal cancer; Cs, other cancers; CST4, cystatin S; GBD, gastric benign diseases; GC, gastric cancer; Hs, healthy samples; Is, interference samples.

Figure 5

The positive coincidence rate contrast between CST4 and other biomarkers (CEA, CA19-9, CA125, CA72-4) which were detected in the same samples of GC and CRC. Note: The cut-off values of CST4 detection for GC and CRC tumors were set as 101 pg·mL⁻¹ with reference to the preceding ROC data (McNemar test, P<0.001). Abbreviations: CEA, carcinoembryonic antigen; CRC, colorectal cancer; CST4, cystatin S; GC, gastric cancer; ROC, receiver-operating curve.