Application of Serum ELAVL4 (HuD) Antigen Assay for Small Cell Lung Cancer Diagnosis

Materials and Methods

Materials. Enzymes for recombinant DNA technology such as Taq polymerase, T4 DNA ligase, BamHI and XhoI were purchased from New England Biolabs (NEB, Hitchin, Herts, UK). Polymerase chain reaction (PCR) amplification kit (including PCR buffer and dNTP mix) was also obtained from NEB. Plasmid mini kit I and PCR product recovery kit were purchased from Omega Bio-Tek (Norcross, GA, USA).

Patients. We collected serum samples from 47 SCLC patients who had undergone surgery at the Beijing Chest Hospital, Capital Medical University. Normal serum samples were from 29 healthy volunteers. The ages (mean±SD) of the SCLC Patients were 61.5±8.4 years. None of the patients had received chemotherapy or radiotherapy before surgery. Diagnosis was confirmed by conventional pathological morphology. Value of CEA, NSE and pro-GRP were tested by ELISA. Supernatants from serum samples were collected immediately after centrifugation (3,000 xg for 15 min) and stored at −80°C until required.

All patients provided written informed consent. All studies and procedures involving human serum samples were approved by the Beijing Chest Hospital Institutional Review Board. Patient samples were used in accordance with The Declaration of Helsinki.

Primer design and PCR amplification. The primers were designed according to the nucleotide sequence of ELAV4. In order to facilitate subsequent cloning, BamHI and XhoI restriction endonuclease sites were attached to the 5’-termini of the upstream and downstream primers, respectively: forward: 5’-CGGGATCCGAGCCTCAGGTGTCAAATG-3’; reverse: 5’-CCGCTCGAGGTCAGGACTTGTGGGCTTTGTTG-3’. The polymerase chain reaction was carried out using the human genomic DNA of HuD as a template. The PCR mixture was subjected to 35 cycles of denaturation (30 s, 95°C), annealing (30 s, 57°C), and extension (60 s, 72°C) using a DNA Thermal Cycler (Eppendorf). The PCR product was separated on a 1% agarose gel, purified using a PCR product recovery kit, and digested with BamHI and XhoI overnight.

Plasmid constructs, protein induced expression and purification. The PCR product was cloned into the BamHI and XhoI restriction-enzyme sites of the prokaryotic expression vector pET-28a (+). The recombinant plasmid was transformed into strain DH5α of Escherichia coli. Protein expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.1 mM final concentration) when the OD₆₀₀ of the lysogeny broth (LB) culture medium reached approximately 0.8. The cultures were allowed to grow at 37°C overnight. Bacterial cells were harvested by centrifugation at 5000 rpm for 15 min. Then, the cell pellets were resuspended in ice cold lysis buffer (MCAC-0, pH 7.9, 1mM phenylmethane sulfonyl fluoride (PMSF)) and homogenized by sonication on ice. The cell debris were removed completely by centrifugation of the lysate at 20,000 xg for 45 min at 4°C. All subsequent purification steps were performed at 4°C. The supernatant was added to a self-packaged Nickel-affinity column pre-equilibrated with lysis buffer. The contaminant protein was removed with MCAC-0 buffer and the target HuD protein was eluted with gradient concentration of 0-200 mM imidazole. The concentration of purified HuD protein was detected by BCA (Bicinchonininc Acid) method.

MALDI-TOF-TOF mass-spectrometric analysis. The purified protein was analyzed by SDS-PAGE and by MALDI-TOF-TOF mass spectrometry. 0.5 ml of digested sample was spotted onto an AnchorChip target (Bruker Daltonics, MA, USA). The sample droplet was dried at ambient temperature and coated with 0.5ml matrix solution. The mass spectrometer was calibrated externally using the monoisotopic (M+H) + ions of peptide-calibration standards (Bruker Daltonics, MA, USA). Mass data acquisitions were piloted using flexControl v.3.3 (Bruker Daltonics, MA, USA). The positive MALDI-TOF and MS/MS LIFT spectra of selected ions were collected and peak lists were generated using the SNAP peak-detection algorithm, with a signal-to-noise threshold higher than 6 and with top-hat baseline subtraction in flexAnalysis v.3.3 (Bruker Daltonics, MA, USA). The MS spectra and MS/MS spectra were combined in Biotools (Bruker Daltonics, MA, USA) and searched for Humanistic in the NCBI nonredundant database using the Mascot search engine (Matrix Science; London, England).

Competitive-inhibition enzyme linked immune-absorbent assay (ELISA). To optimize the reaction conditions of indirect competitive ELISA, such as determination of the optimal coating concentration of HuD and the best working concentration of monoclonal antibody. The HuD protein was fold diluted in phosphate-buffered saline (0.05 mol/l PBS, pH 9.6) to yield working solutions (20 ng/ml), anti-HuD was proportionally diluted, and HRP labeled goat anti-rabbit IgG was diluted 1:2,500. Square titration was performed for ELISA detection, according to OD₄₅₀ value equal to 1.0 was chosen as the ideal working concentration. The 96-well plates were coated with 20 ng/ml of HuD at 4°C overnight. After washing the plate three times with 300 μl of PBST (0.05% Tween 20), the plate was blocked with PBS/3% non-fat milk at 37°C for 1 h. Then different concentrations of HuD standard solution (marking standard curve) or the serum samples were mixed with the monoclonal antibody in each well. After incubation at 37°C for 1 h, the wells were washed with PBST (phosphate buffered saline with Tween) 3 times and then horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,500) was added and reacted for 1h at room temperature. After washing 3 times with PBST, 100 μl of 3,3’,5,5’-tetramethylbenzidine (Tiangen, Beijing, China) was added and incubated at 37°C for 15 min. The reaction was stopped with Stop Solution (2M H₂SO₄). All samples were examined in triplicate. The OD₄₅₀ values were measured and the inhibition curves were plotted. Standard curve and ROC curve analysis. Purified HuD protein as standard sample was serially diluted 2-fold in PBS (from 100 ng/ml) to yield an eight-point standard curve. The highest concentration of HuD was 100 ng/ml, followed by 50, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 ng/ml respectively. The standard samples were mixed with equal anti-HuD mAb. The logarithmic values of the dilution were calculated and plotted as on the horizontal axis. The values of B/B₀ were estimated and plotted as the vertical axis. The optical density at 450 nm of each fold dilution were regarded as B, and the optical density of the standards as B0. The absorbance was read at 450 nm for all test samples. All samples were tested in triplicate. The HuD concentration for test samples (all patients and normal controls) were extrapolated from a standard curve through Curve Expert 1.4. The concentration of HuD in serum was plotted for ROC curve.

Statistical analysis. Statistical analyses were carried out with SPSS 16.0. The cut-off values and the area under the curve were defined by ROC curve. Sensitivity and specificity values were obtained in SCLC patients. To compare the concentration of HuD between all patients and normal control groups independent sample T test was used. A p-value less than 0.05 was considered statistically significant. In addition, Pearson Chi-Square was used to compare the relationship between HuD level and existing biomarkers, including CEA, NSE and pro-GRP. A p-value less than 0.01 was considered statistically significant.