BackgroundHuman chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker.ResultsThe synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG2b, and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 109 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively.ConclusionsA sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis.

Five strictly anaerobic strains, designated RG2T, RG3, RG10T, RF4T, and RG29, were isolated from paddy soils in China. Strains RG2T, RF4T, RG10T, RG3, and RG29 grew at temperatures ranging 5–42°C and pH ranging 5.5–8.5. Strains RG2T, RF4T, RG3, and RG29 could tolerate NaCl up to 0–0.7% (w/v) while strain RG10T could tolerate NaCl up to 0–0.8% (w/v). The isolated strains showed the highest 16S rRNA gene sequence similarities to the type strains of Geomonas terrae Red111T and Geomonas paludis Red736T. In phylogenetic (based on 16S rRNA gene sequence) and phylogenomic trees, strains clustered with the members of the genus Geomonas. Menaquinone-8 was the predominant quinone present in all strains. The major fatty acid profiles of all strains were C15:1 ω6c, C16:0, iso-C15:0, and Summed Feature 3. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the isolated strains and the closely related Geomonas species were lower than the cutoff value (ANI 95–96% and dDDH 70%) for prokaryotic species delineation. Based on physiological, biochemical, and chemotaxonomic properties, strains RG2T, RG10T, and RF4T could easily be differentiated with the members of the genus Geomonas. Additionally, all the isolated strains possessed nifHDK clusters and catalytic compartments of nitrogenase. Based on the above results, the isolated five strains represent three novel species of the genus Geomonas, for which the names Geomonas oryzisoli sp. nov., Geomonas subterranea sp. nov., and Geomonas nitrogeniifigens sp. nov. are proposed. The type strains are RG10T (= GDMCC1.2537T = KCTC 26318T), RG2T (= GDMCC1.2536T = KCTC 25317T), and RF4T (= GDMCC 1.2547T = KCTC 25316T).

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 108 L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14–263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.

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