Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis

Abstract

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.

Generation and characterization of a monoclonal antibody against human BCL6 for immunohistochemical diagnosis

“…After 10 days, HAT in RPMI-1640 medium was replaced with HT. Then a week later, the hybridoma cells were cultured in RPMI-1640 medium with 10% FBS [10].…”

“…The coding region for BCL6 1-350 (GenPept, NP_001124317.1) was optimized for expression in E . coli BL21 (DE3) and evaluated by graphical codon usage analyser (http://gcua.schoedl.de) [10]. The optimized coding DNA for BCL6 1-350 was synthesized by Nanjing Genscript Biotech Co., Ltd (Nanjing, China).…”

“…Ten days later, positive hybridoma cells were determined by iELISA. The positive clones with high titer were chosen for further sub-cloning, until the positive percentage was up to 100% [10,11].…”

“…The anti-BCL6 mAb was purified with Protein G and analyzed with 10% SDS-PAGE. The concentration of the purified mAb was determined with BCA protein assay [10]. Titer determination of 1E6A4 mAb was determined with iELISA.…”

“…The affinity of antibody against BCL6 was determined with iELISA as described previously [10]. Various concentrations of BCL6 proteins (425.00, 106.25, and 26.56 ng/mL) were used as coating antigens.…”