[Establishment of sandwich ELISA for detecting Staphylococcal enterotoxin B in different matrices].

AIM: To establish sandwich ELISA for the detection of Staphylococcal enterotoxin B(SEB) and characterize the sensitivity and specificity of it in different materials. METHODS: The anti-SEB monoclonal antibodies (mAb) B4 and D6 were employed as capture antibody and detecting antibody respectively after being purified by Q sepharose fast flow chromatography and horseradish peroxidase(HRP) conjugation. RESULTS: The sensitivity of this assay reached 0.2 ng of SEB per mL of PBS with BSA and fetal bovine serum. It reached 0.39 ng of SEB per mL of 50 g/L skim milk, human urine and water. The concentration curve generated from SEB standard curve was linear with the range of 0.78-12.5 microg/L, (r(2)=0.99). This assay was highly reproducible and the coefficient of variation(CV) was less than 10%.No cross-reactivity between SEA and SECl was found. CONCLUSION: This assay offers a viable method with high sensitivity and specificity for detecting SEB and it may be used for SEB detection in clinical application and food samples.

[Establishment of chemiluminescence immunoassay for detecting Staphylococcal enterotoxin B and C1].

AIM: To establish chemiluminescence immunoassay (CLIA) for detecting staphylococcal enterotoxin B (SEB) and C1 (SEC1) and compare its sensitivity and stability with ELISA. METHODS: The anti-SEB and SEC1 monoclonal antibodies (mAb) were purified by Q Sepharose Fast Flow chromatographic column. The alkaline phosphatase (AP) conjugated mAbs FMMU-SEB.D6 and FMMU-SEC1.C4 were used as detecting antibodies and the FMMU-SEB.B4 and FMMU-SEC1.G8 mAbs were used as coating antibodies in both methods. Phenolphthalein monophosphate (PMP) and lumigen APS-5 were employed as substrates for AP in ELISA and CLIA, respectively. The light was detected and measured by the GENios analyzer (TECAN Group Ltd.). The sensitivities and detect ranges of CLIA and ELISA methods were compared. RESULTS: Compared with ELISA, CLIA was more sensitive (0.1 ng/mL vs 0.39 ng/mL) and timesaving. Furthermore, the liner range of CLIA was broader than that of ELISA (0.78-50 ng/mL vs 3.125-50 ng/mL). CONCLUSION: CLIA for detecting SEB and SEC1 are established successfully which may be useful in food monitoring, epidemiology survey and detecting SE contaminated samples in environment.

Identification of CD226 ligand on colo205 cell surface.

AIM: To confirm the existence of CD226 ligand and its distribution, which is a novel molecule cloned in 1996. METHODS: The mRNA was extracted from TPA activated Jurkat cells and used as a template for reverse-transcription. After PCR amplification, the fragment including CD226 extracellular region and the splice donor sequence "ACTTACCTGT" was obtained and cloned into fusion expression vector pIG. The recombinant vector pCD226/Ig was transfected in COS-7 cells by DEAE-Dextran method, the secreting fusion protein was identified by Sandwich ELISA, and was purified by anti-CD226 affinity chromatography. This fusion protein was used as a probe in the investigation of CD226 ligand by immunohistochemistry. Existence of CD226 ligand was further identified by adhesion experiment. RESULTS: Expression of a secreting fusion protein was identified by sandwich ELISA,indicating that both CD226 extracellular domain and IgGFc domain could be recognized respectively by anti-CD226 and anti-hIgFc mAb. About 130g CD226/Ig fusion protein could be obtained from 100mL COS-7 culture supernatants by anti-CD226 affinity chromatography purification. SDS-PAGE showed that this fusion protein has a molecular mass of 83ku. It was confirmed by immunohistochemistry that CD226 ligand expressed on the Colo205 cells, but not on Jurkat cell, U937 cell and mixed lymphocyte culture cells. In adhesive assay, resting Jurkat cells did not have significant adhesion to Colo205 cells. In contrast, activated Jurkat cells could bind to colon carcinoma Colo205 cells and this adhesive reaction could be blocked by CD226/Ig fusion protein or anti-CD226 mAb. Immunochemical experiment showed that Colo205 cells could be specifically stained by CD226/Ig, indicating that CD226 ligand exists on the surface of Colo205 cells. CONCLUSION: Existence of CD226 ligand on the surface of Colo205 cells was identified by immunohistochemistry and adhesion blocking experiment. In addition, the secreting CD226/Ig fusion protein prepared in this study will be a potential tool for further investigation of CD226 ligand.