Background

Human chromogranin A (CgA) consists of 457 amino acid with molecular weight ~ 49 kDa, and its gene is located in chromosome 14 of Homo sapiens. As a member of Granin protein family, CgA is mainly produced by endocrine cells or neuroendocrine cells and presents in the secretory dense core granules of neuroendocrine tissues [1, 2]. These CgA secrecting tissues include small intestine, large intestine, adrenal medulla and pancreatic islets [1, 2].

Further studies found that CgA was highly expressed in some tumors, and then determined as excellent marker in carcinoid tumor, pheochromocytomas, and paragangliomas [1, 3,4,5]. Compared with other neuroendocrine markers, CgA has the higher specificity for the detection of neuroendocrine tumors. Thus the CgA was widely used in immunohistochemistry (IHC) to diagnose a benign or malignant tumor, and determine the tumor grade. In this sense, it values to develop sensitive monoclonal antibodies (mAb) against CgA for IHC diagnosis.

Compared with the time-consuming and technique-requiring method to express CgA in a eukaryotic cell line, a method for CgA expression in prokaryotic system was developed. Hence, a cost-effective, feasible and reliable method was established to prepare an anti-CgA mAb by expressing CgA in E. coli. The prepared mAb against CgA from prokaryotic system can be used in Western Blot and IHC diagnosis based on its high specificity in this study.

Methods

Materials and chemicals

The anti-6 × His tag monoclonal antibody and HRP-labeled goat anti-mouse IgG were purchased from ZSGB-Bio Co., Ltd. (Bei**g, China). The cocktail of LK2H10 and PHE5 antibodies, and MaxVisionTM/HRP IHC kit were provided by Fuzhou Maixin Biotech Co., Ltd. (Fuzhou, China). SP12 antibody was from Thermo Fisher Scientific (Shanghai, China). Hypersensitivity ECL chemiluminescence detection kit was purchased from Wuhan Sanying Co., Ltd. (Wuhan, China). Myeloma cells SP2/0 were stored in our laboratory. Hypoxanthine, aminopterin and thymidine supplement (HAT), hypoxanthine and thymidine supplement (HT) and polyethylene glycol 1450 solution (PEG 1450) were purchased from Sigma-Aldrich (Shanghai, China). RPMI medium 1640 powder and fetal bovine serum (FBS) were purchased from Thermo Fisher (Shanghai, China). Balb/c mice were from Wushi Animal Laboratory (Shanghai, China). QuickAntibody-Mouse5W adjuvant (Quickadjuvant) was purchased from Bei**g Biodragon Immunotechnologies Co., Ltd. (Bei**g, China). All animal experiments obeyed the protocols approved by the Animal Ethics Committee of the Fujian Agriculture and Forestry University.

Cells cultivation

Murine myeloma cell line SP2/0 stored in our laboratory were cultured in RPMI-1640 medium with 10% FBS. Hybridoma cells were cultured in RPMI-1640 medium with HAT containing 20% FBS. After 10 d of cell fusion, hybridoma cells were cultured in RPMI-1640 medium with HT containing 20% FBS. A week later, the hybridoma cells were cultured in RPMI-1640 medium with 10% FBS.

Codon optimization and synthesis of CgA gene

The identification of CgA is P10645 from the Uniprot, according to the database. The coding part of CgA was selected to optimize codon for expression in E. coli BL21 (DE3) and evaluated by graphical codon usage analyser (http://gcua.schoedl.de) [6]. The optimized DNA of CgA was synthesized by Nan**g Genscript Biotech Co., Ltd. (Nan**g, China).

Expression and identification of the CgA fusion protein

The target gene was amplificated by PCR and digested by EcoRI/HindIII, then inserted into the plasmid pET-28a to transform E.coli BL21 (DE3) competent cells by calcium chloride transformation. Furthermore, the positive clone was induced by isopropyl-β-d-thiogalactoside (IPTG), and the target proteins were expressed and purified by affinity chromatography purification. Finally, the concentrations of CgA-His fusion protein were detected by BCA methods and Nanodrop (Thermofisher) [12]. Chromosome analysis was identified according to the publication in our lab [13]. The Hybridoma cells were stained by Giemsa solution, and the chromosome number was counted under the flurescence microscope.

Production of anti-CgA mAb

A Balb/c mouse was injected intraperitoneally with 0.5 mL paraffin oil. Seven days later, approximately 1 × 106 postive hybridoma cells were injected into the mouse abdominal cavity. After 1 week, the ascites fluid was collected by the needle and centrifuged at 12000 r/min for 20 min. The supernatant was absorbed and stored in − 20 °C fridge. According to the protocol of Protein G, the mAb was purified and analyzed by 10% SDS-PAGE [14]. The concentration of the purified mAb was determined by the BCA Protein Assay.

Affinity determination of mAb

The affinity determination of monoclonal antibody against CgA was carried out by the publication [2b). The cell line of 4E5 showed a good repeats in ELISA, Western blot and IHC, therefore 4E5 was chosen for the further experiments. The chromosome number of 4E5 cell line is 102 ± 6 (Fig. 2c), suggesting that the chromosome number of the hybridoma cell 4E5 was approximately equal to the total chromosome number of spleen cell (39 ± 1) and myeloma cell SP2/0 (66 ± 4). The results further demonstrated that the 4E5 cell line was successfully fused.

Fig. 2
figure 2

Screening of positive hybridoma clones. a The observation of hybridoma cells cultured in HAT medium. Hybridoma cells were observed after 6 d of fusion (× 100). b The isotype result of positive hybridoma cells. The isotype of monoclonal antibody was determined by isoty** (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM) kit, and the isotype of 4E5 is IgG2b. c Chromosome analysis of 4E5 hybridoma cell (10 × 100). The chromosome number of 4E5 cell line is 102 ± 6

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Purificaion of the anti-CgA mAb

To get enough CgA mAb, the 4E5 hybridoma cells were injected into the peritoneal cavity of pristine-primed Balb/c mice. Then the mice ascites fluids were collected and then purified with Protein G. As shown in Fig. 3a, two distinct bands appeared and miscellaneous band lacked after purification, consistent with the heavy chain and light chain of antibodies. The 50 kDa heavy chain and the 25 kDa light chain were observed in lane 3 and lane 4, indicating that 4E5 mAb was successfully purified by Protein G (Fig. 3a). The final concentration of the purified 4E5 mAb was 0.13 mg/mL determined with BCA protein assay, and the titer of anti-CgA mAb reached up to 5.12 × 105 with iELISA method (Fig. 3b). We also compared the titer of 4E5 mAb with that of LK2H10 and PHE5, a widely used anti-CgA mixture [17, 18]. Results showed that the titer of 4E5 is higher than that of anti-CgA mixture (Fig. 3b), demonstrating that the anti-CgA 4E5 mAb we purified was suitable for further characterization.

Fig. 3
figure 3

Purification of the anti-CgA mAb. a SDS-PAGE analysis of the purified mAb. Lane 1: Marker (Precision Plus Protein Dual color standards), lane 2: ascites fluid, lane 3–4: the duplicate purified mAb against CgA. b Titers of the purified 4E5 anti-CgA mAb and control antibody were determined by iELISA

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Characterization of the anti-CgA mAb

To assay the value of anti-CgA 4E5 mAb, the affinity and specificity of antibodies were determined by iELISA method [10, 11]. The results showed that the 4E5 mAb was sensitive in response to CgA-His, and not cross-reactive to random protein control, such as PDPN, PD1-L1, PGY, and BCL6. (Fig. 4a), indicating the 4E5 antibodies had high specificity for CgA. The specifity of 4E5 antibodies were also evaluated with Western Blot. As shown in Fig. 4b, the 4E5 mAb specifically recognized the target proteins CgA-His rather than control antigens. The affinity analysis of 4E5 mAb was also performed with iELISA, and the results showed that the affinity of 4E5 mAb was up to 9.23 × 109 L/mol (Fig. 4c). In contrast, the affinity of anti-CgA mixture (LK2H10 and PHE5) was similarly determined as 3.89 × 108 L/mol (Fig. 4d). These results indicated that the 4E5 mAb was more sensitive to CgA than LK2H10 and PHE5. Considering the mixture of LK2H10 and PHE5 antibodies was widely used in CgA detection [17, 18], thus the 4E5 mAb was value for further application for CgA detection or diagnosis. To determine the epitope of 4E5 antibody, we divided CgA gene into four fragments and expressed these gene fragments (Fig. 4e). Each fragment has about 110 amino acids. As shown in Fig. 4e, the second protein fragment (CgA111–220) was easily recognized by 4E5 antibody as CgA protein with WB, demonstrating the CgA111–220 fragment was the epitope of 4E5 antibody.

Fig. 4
figure 4

Characterization of the purified 4E5 mAb. a The specificity of the 4E5 mAb was determined by indirect ELISA (BCL6: B-cell lymphoma 6 protein, PD1-L1: Programmed cell death 1 ligand 1, PDPN: Podoplanin, PGY: P-glycoprotein). b The specificity of the 4E5 mAb was determined by Western Blot. Lane M: Marker, Lane 1–2: His-CgA protein duplicates, Lane 3: Random control (His-PD1-L1 human protein), Lane 4–6: The result of Western Blot. The proteins in Lane 4 and 5 are his-CgA protein duplicates, and Lane 6 was random control (His-PD1-L1). c Affinity of 4E5 mAb was analyzed by iELISA. Different concentrations (12.5, 25, 50, and 100 ng/mL) of coating antigen (CgA-His) were used to determine the affinity constant which is 9.23 × 109 L/mol. d Affinity of control antibodies (LK2H10 and PHE5) was analyzed by iELISA. Different concentrations (1, 1.5, and 2.5 μg/mL) of coating antigen (CgA-His) were used to determine the affinity constant which is 3.89 × 108 L/mol. e Epitope of 4E5 mAb was determined with western blot. Lane1–7: the result of SDS-PAGE analysis. Lane M: Marker, lane 1: the total protein of BL21 (DE3) with empty vector pET-28a, lane 2: the total protein of BL21 (DE3) with pET-28a-CgA fragment 1 (F1), lane 3: CgA fragment 2 (F2), lane 4: CgA fragment 3 (F3), lane 5: CgA fragment 4 (F4), lane 6: CgA. Lane 7–12: The result of Western Blot. Lane 7: the total protein of BL21 (DE3) with empty vector pET-28a, lane 8: CgA fragment 1 (F1), lane 9: CgA fragment 2 (F2), lane 10: CgA fragment 3 (F3), lane 11: CgA fragment 4 (F4), lane 12: CgA

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Tissue section detection for the mAb against CgA

Considering the CgA detection in clinic diagnosis, the 4E5 antibodies were tested with IHC assay. Different tissue samples were detected with Anti-CgA 4E5. As shown in Fig. 5, the 4E5 mAb is able to sensitively detect CgA equal to the positive control group (LK2H10 and PHE5 mixture and SP12). Thus 4E5 mAb can be used to specifically recognize CgA in the small cell lung carcinomas (SCLC), adrenal gland tissues, and Islet cell carcinoma (Fig. 5). The stained cells indicated tumor cells or CgA secreting cells were located in these tissues, which were valuable in clinical practice. In addition, the staining degree in 4E5 group was much deeper than the positive group, suggesting that the sensitivity of the 4E5 mAb was better than LK2H10 and PHE5 or SP12 antibodies. All these results showed the 4E5 mAb had good potential value to detect CgA secreting tumor cells on clinic.

Fig. 5
figure 5

The immunohistochemistry tests of mAb in difference tissues. The immunohistochemistry tests of 4E5 mAb was determined in small cell lung carcinomas (SCLC) (a) (× 200), adrenal gland tissues (b) (× 400), and Islet cell carcinoma (c) (× 200). The images of (d, e and f) showed the staining of LK2H10 and PHE5 mouse antibodies for corresponding tissues, as positive control. The images of (g, h and i) showed the staining of SP12 rabbit antibody for corresponding tissues, as positive control

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Discussion

In this study, we used a prokaryotic system to express CgA antigen. We firstly optimized codons of CgA gene for E. coli expression. Then we successfully cloned the CgA gene in prokaryotic expressing vector. Compared with CgA expression in eukaryotic system, CgA was easily and fast acquired by using E. coli host cell to construct the high level prokaryotic expressing system. Thus time and cost were saved by using the prokaryotic expression system. However, the CgA was not well expressed in E. coli with the eukaryote codon bias according to our attempt. Our codons optimization of CgA successfully resolved the problem of low CgA expression in prokaryotic system. Thus, an effective and reliable method has been established to prepare the anti-CgA mAb by expressing CgA in E. coli using the optimized codons.

Meanwhile, our results indicated that a valuable antibodies secrecting hybridoma for CgA was successfully screened with the antigen from prokaryotic system. As shown in Fig. 4a, the 4E5 mAb from hybridoma specifically reacted with CgA rather than other control antigens (PDPN, PD1-L1, PGY, BCL6) with iELISA method. The 4E5 mAb specifically for CgA was further confirmed with WB analysis (Fig. 5). Our results also showed that the affinity of 4E5 mAb was high up to 9.23 × 109 L/mol (Fig. 4c). This affinity is far beyond the baseline (107 L/mol) required for further applications [19], suggesting that the 4E5 mAb has good potential value in clinic application.

Moreover, although eukaryotic proteins usually underwent various post-translational modifications, CgA specific antibody applicable in IHC has been successfully generated with antigen from prokaryotic system in this study. We further characterized the epitope of the CgA specific antibody (Fig. 4e). To our knowledge, this epitope is little reported previously. This epitope should be valuable for the generation of CgA specific IHC antibodies. The 4E5 antibodies recognize this epitope, CgA111–220, will be useful for the detection of full-length CgA or long fragment of CgA, considering CgA110–220 rarely included in polypeptides from CgA [20]. Interestingly, CgA111–220 consist of many charged amino acids expresses 10–20 kDa lag in electrophoresis (Fig. 4e), leading to the lag effect of full-length CgA in electrophoresis compared with the theory molecular weight.

Our study also showed that the 4E5 mA is qualified for clinic application with IHC experiments. Nowadays, the cocktail of LK2H10 and PHE5 antibodies against CgA has been implemented in IHC diagnosis [17, 18, 21]. Thus it is used as the positive control in measuring the application of 4E5 mAb in clinic IHC experiments. We also used a CgA specific rabbit mAb, SP12, as positive control, which is also accepted as CgA detection [22, 23]. Our results showed the effect of 4E5 mAb can even better detect CgA in clinic samples than the cocktail of LK2H10 and PHE5 and SP12 antibody (Fig. 5), suggesting that it was valuable in further clinic application. The superiority of 4E5 mAb in CgA detection by immunohistochemistry may be mainly due to the high titer and affinity of 4E5 mAb compared with those of control group, which is observed in the example of comparison between 4E5 mAb and the LK2H10 and PHE5 antibodies (Figs. 3b, 4c, and d). As to the control antibodies, although there are other CgA-specific antibodies applicable in IHC, such as rabbit polyclonal anti-CgA antibody and EP38 rabbit mAb [24, 25], our positive controls (LK2H10&PHE5 and SP12) including mouse and rabbit monoclonal antibodies are still representative. On the other hand, although our results were shown just from lung carcinomas, adrenal gland and Islet cell carcinoma, it is reasonable that the 4E5 is also applicable in other related tissues.

Conclusions

In summary, we have successfully cloned and expressed CgA antigen with an effective and reliable method. This CgA antigen from prokaryotic system is obviously suitable for preparing a Chromogranin A mAb. Moreover, a high specific and affinity antibody named 4E5 for CgA has been screened in our study. The 4E5 mAb against CgA was valuable in IHC diagnosis. Our study provided an alternative method for the rapid preparation of CgA mAb for clinic application, considering the potentially complicated regulation and presence of CgA in various diseases.