Edited by Ma JY
Published online Oct 15, 2002. doi: 10.3748/wjg.v8.i5.863
Revised: March 1, 2002
Accepted: March 5, 2002
Published online: October 15, 2002
AIM: To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.
METHODS: The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning, 56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by Sfi I/Not I and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody.
RESULTS: The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.
CONCLUSION: We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.
- Citation: Zhong YW, Cheng J, Wang G, Shi SS, Li L, Zhang LX, Chen JM. Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry. World J Gastroenterol 2002; 8(5): 863-867
- URL: https://www.wjgnet.com/1007-9327/full/v8/i5/863.htm
- DOI: https://dx.doi.org/10.3748/wjg.v8.i5.863
Hepatitis C virus (HCV) has been identified as the major etiological agent of post-transfusion non-A non-B hepatitis[1-10], responsible for most cases of non-A non-B hepatitis. Hepatitis C is a disease of clinical importance because of its high infection rate in blood donors and its persistence as chronic infections which may lead to cirrhosis and hepatocellular carcinoma in the long term[11-26]. The variability of the HCV genome has difficulties in serological detection and vaccine design. Recent advance in phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may have potential therapeutic use. We now report the generation of phage display antibody using phage antibody library. From this library we obtained specific single-chain Fv antibody that recognizes the viral envelope protein E2, using HCV E2 protein as the immobilized antigen and proceeding immunohistochemistry.
Humanized scFv antibody phage library in which the variable region coding gene of VL and VH were amplified by polymerase chain reaction (PCR) with degenerate primers and connected with a glycine linker [(Gly4Ser)3] was widely used in the screen and identification of humanized scFv to various antigens[28-34]. The recombinant HCV E2 protein was purchased from Virostat Co, USA. Phage M13K07 was purchased from Pharmacia Co., Sweden. Other reagents used in this experiment are all domestic products of analytical grade.
The phage library was amplified in 37 °C. The host E. coli TG1 was infected with phage M13K07 and incubated at 37 °C for 12 h, the phage in the supernatant was harvested and concentrated by PEG. Culture plate (Nunc) was coated with recombinant HCV E2 protein at the concentration of 80 mg/L. The coating buffer was 0.05 mol/L NaHCO3, pH9.6. The plate was blocked with BSA at the concentration of 20 g/L for 2 h and the concentrated phages were added to the well of the plate, incubated at the room temperature for 90 min. The plates were washed 20 times with PBST and PBS buffer respectively. The bound phage was eluted by the 0.1 M of triethylamine, and neutralized with 1 M Tris buffer (pH7.4). Recovered phages were used to infect the host E. coli TG1 at the log phase growth and HCV E2 protein-binding phages were amplified. The procedure of absorption-elution-amplification was repeated 5 times.
After 5 rounds of biopanning, 56 phage clones were selected randomly. The clones grew in 400 μL 2 × TY-AMP-Glu at 37 °C overnight. The culture was transformed to another Eppendorf tube when its A600 reached 0.5. The culture was continued at 30 °C overnight after adding helper phage. ELISA for determining the supernatants was repeated at least two times. The cross-reaction of the supernatants to the BSA antigen was conducted. According to the ELISA results to the HCV E2 and BSA, one clone with high reaction to HCV E2 and low reaction to BSA was selected.
The plasmid DNA was prepared using Wizard plus minipreps DNA Purification System (Promega Co., USA) and sequenced using ABI automated DNA sequencing machine.
The selected HCV E2 scFv clone was subcloned as Sfi I/Not I fragments into the vector pCANTAB5E for expression as E-tagged soluble scFv. DNA digestion and electrophoresis confirmed the recombinant vector pCANTAB5E-E2-scFv. Competent E. coli XL1-Blue was transformed with pCANTAB5E-E2-scFv and transformed XL1-Blue was induced by IPTG for 20 h. The E. coli was harvested by centrifugation at 10000 rpm. The culture supernatant was rendered for ELISA test according to the standard procedure. In ELISA detection, Nunc plate was coated with 1 μg/well of recombinant HCV E2 antigen and blocked with 2% bovine serum albumin (BSA) at 37 °C for 2 h. The supernatants from induced and non-induced transformed E. coli were added and incubated at 37 °C for 2 h. The plate was washed with PBS buffer, and 100 μL of HRP/anti-E Tag 1:4000 ratio diluted in PBS buffer containing 1% BSA was added, and incubated at 37 °C for 1 h. The substrate solution was added and A450 value was measured.
Paraffin-embedded liver tissue slices were from patients with positive anti-HCV antibodies and HCV-RNA. After deactivating endogenous hyperoxidase, these slices were submersed in the methanol solution with 0.5% H2O2 in the room temperature. Fifty min later, they were washed with PBS buffer for 3 times, 5% BSA was added and slices were stored overnight at 4 °C. Self-made scFv primary anti- HCV E2 single-chain antibodies were diluted at 1:100 ratio and added on to the slice. They were kept in the 37 °C incubator for 1 h, then 4 °C refrigerator overnight. HRP-sheep anti-M13 antibodies (diluted to 1:200) solutions were dropped on to the tissue sections, incubated at 37 °C for 40 min. After washing 3 times with PBS buffer, DAB solutions (9 mg DAB, 13.5 mL Tris.Cl, 1.5 mL CoCl2, 15 μL 30% H2O2) were dropped on to the tissue sections at room temperature. After ten minutes, the slices were washed with PBS buffer for 3 times again, and 1% heamatin solution was used to stain the cell nucleus. Gradient ethanol was utilized to dehydrate and dimethylbenzene to clear the sections, then neutral resin to envelope them. The resultant slices were observed under microscope. The controls were set as follows: 1) PBS buffer instead of anti- HCV E2 scFv; 2) HBsAg, HBcAg double-positive liver tissue sections; and 3) Normal liver tissue sections.
Using HCV E2 protein as immobilized antigen, the humanized scFv phage library was biopanned. After 5 rounds of biopanning, 56 phage clones were selected randomly. ELISA and cross-reaction of these clones to BSA confirmed their specificity to HCV E2. Among the 56 phage clones, 16 showed good reactivity to the recombinant HCV E2 protein with high A value in the ELISA. In the cross-reaction screen, 6 among the 16 showed low cross-reaction with BSA. The combined results indicated that 1 of the 6 showed the highest reaction to HCV E2 protein and lowest reaction to BSA. One clone has been selected for further DNA digestion and sequence analysis. The DNA sequence digestion was made by Sfi I/Not I in Figure 1. Its nucleic acid sequence and deduced amino acid sequence about HCV-E2-scFv fragment are shown in Figure 2.
The expressed HCV-E2-scFv antibody from E. coli XL1-blue transformed by pCANTAB5E and induced by IPTG was confirmed by ELISA as shown in Figure 3. The recombinant HCV E2 antigen was taken as the positive control. The protein from induced and non-induced E. coli XL1-blue transformed by expression vector was positive. But the protein derived from the E. coli non-transformed by pCANTAB5E- HCV E2- scFv was negative. These results indicated that the soluble form of human HCV E2- scFv antibody has been successfully expressed in this procedure.
The different sections from liver tissues of healthy persons and patients with chronic hepatitis B or C were immunostained. The positive immunostaining was seen only in the liver tissue section of patients with chronic hepatitis C, but not in the liver tissue of normal person and patients with chronic hepatitis B as seen in (Figure 4A). The HCV E2 antigen was mainly located in the cytoplasm of the hepatocytes infected by HCV virus (Figure 4B).
HCV is the etiological agent responsible for most cases of non-A non-B hepatitis. It is a blood-related disease of clinical importance since a large number of cases developed chronic infections which may lead to cirrhosis and hepatocellular carcinoma. The variability of the HCV genome has posed serious problems in serological detection of HCV and vaccine design[27-38]. The possibility of using neutralizing antibodies in passive immunization is also hampered by the existence of genotypes and quasispecies[39-50]. To circumvent this, it is possible to envisage the use of cross reactive antibodies with a broad reactivity directed against functionally conserved surface domains common to various genotypes. There has been in vitro and in vivo evidence that HCV infection elicits a neutralizing antibody response in humans. The virus envelope protein is the primary target for the host immune system. Despite the high variability of the HCV envelope sequence, certain domains of biological importance, e.g. ligands required in viral attachment to host cell receptors, have to be preserved. Antibody specific for the envelope E2 protein has been shown to be cross-reactive HCV E2 and to be able to block viral attachment to cultured human fibroblast cells. However, attept to obtain human monoclonal anti -HCV antibodies is an urgency.
Phage library is a powerful tool for the selection of important and useful antibody specificities espeacially for getting humanized scFv[51-53]. It has many advantages. First, it is the only method to get specific antibody by passing the immunization step. It can mimic the maturation procedure of human antibody in vivo, so it is possible to get high affinity antibody from this selection. Second, the scFv with a low molecular weight, can make it potentially applicable in the clinical diagnosis and treatment of both infectious disease and cancer. Finally, it has no Fc fragment, so the backgroud is very low.
The phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may be potential for therapeutic and diagnostic use. In this report, we have obtained anti-HCV E2 scFv by affinity selection and purified recombinant hepatitis C virus E2 as coating antigen from synthetic phage display antibody library. After 5 rounds of selection, 56 clones were evaluated by enzyme-linked immunosorbent assay (ELISA). One phage clone was selected from 56 clones according to the highest A450 value in the ELISA identification and lowest cross-reaction to the bovine serum albumin (BSA). The coding fragments of scFv were sequenced. The affinity and specificity of scFv were evaluated by ELISA and immunohistochemistry. HCV E2-scFv DNA digestion and sequence data showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against HCV E2 antigen has a specific binding activity with hepatitis C virus E2 antigen. This study illustrated the feasibility of using antibody-engineering technology with the phage display library that may be useful for future therapeutic and detection purpose.
|1.||Assy N, Minuk G. A comparison between previous and present histologic assessments of chronic hepatitis C viral infections in humans. World J Gastroenterol. 1999;5:107-110. [PubMed] [Cited in This Article: ]|
|2.||Caselmann WH, Serwe M, Lehmann T, Ludwig J, Sproat BS, Engels JW. Design, delivery and efficacy testing of therapeutic nucleic acidsused to inhibit hepatitis C virus gene expression in vitro and in vivo. World J Gastroenterol. 2000;6:626-629. [PubMed] [Cited in This Article: ]|
|3.||Cheng JL, Liu BL, Zhang Y, Tong WB, Yan Z, Feng BF. Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction. World J Gastroenterol. 2001;7:370-375. [PubMed] [Cited in This Article: ]|
|4.||Dai YM, Shou ZP, Ni CR, Wang NJ, Zhang SP. Localization of HCV RNA and capsid protein in human hepatocellular carcinoma. World J Gastroenterol. 2000;6:136-137. [PubMed] [Cited in This Article: ]|
|5.||Deng ZL, Ma Y, Yuan L, Teng PK. The importance of hepatitis C as a risk factor for hepatocellular carcinoma in Guangxi. World J Gastroenterol. 2000;6:75. [Cited in This Article: ]|
|6.||Feng DY, Chen RX, Peng Y, Zheng H, Yan YH. Effect of HCV NS (3) protein on p53 protein expression in hep atocarcinogenesis. World J Gastroenterol. 1999;5:45-46. [PubMed] [Cited in This Article: ]|
|7.||Gao JE, Tao QM, Guo JP, Ji HP, Lang ZW, Ji Y, Feng BF. Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins. World J Gastroenterol. 1997;3:114-116. [Cited in This Article: ]|
|8.||Huang F, Zhao GZ, Li Y. HCV genotypes in hepatitis C patients and their clinical significances. World J Gastroenterol. 1999;5:547-549. [PubMed] [Cited in This Article: ]|
|9.||Li LF, Zhou Y, Xia S, Zhao LL, Wang ZX, Wang CQ. The epidemiologic feature of HCV prevalence in Fujian. World J Gastroenterol. 2000;6:80. [Cited in This Article: ]|
|10.||Maier KP. Iron, HCV and the liver. World J Gastroenterol. 1997;3:61-63. [Cited in This Article: ]|
|11.||Song ZQ, Hao F, Min F, Ma QY, Liu GD. Hepatitis C virus infection of human hepatoma cell line 7721 in vitro. World J Gastroenterol. 2001;7:685-689. [PubMed] [Cited in This Article: ]|
|12.||Sun DG, Liu CY, Meng ZD, Sun YD, Wang SC, Yang YQ, Liang ZL, Zhuang H. A prospective study of vertical transmission of hepatitis C virus. World J Gastroenterol. 1997;3:111-113. [Cited in This Article: ]|
|13.||Tang BZ, Zhuang L, You J, Zhang HB, Zhang L. Seven years follow up on trial of Interferon alpha in patients with HCV RNA positive chronic hepatitis C. World J Gastroenterol. 2000;6:68. [Cited in This Article: ]|
|14.||Zhao YY, Yang HY, Liu GX, Li ZQ, Liu L, He LL, Deng WJ. Hepatitis C virus infection in patients with primary liver cancer. Xin Xiaohuabingxue Zazhi. 1996;4:43-44. [Cited in This Article: ]|
|15.||Tang ZY, Qi JY, Shen HX, Yang DL, Hao LJ. Short- and long-term effect of interferon therapy in chronic hepatitis C. World J Gastroenterol. 1997;3:77. [Cited in This Article: ]|
|16.||Wietzkebetaraun P, Meier V, Braun F, Ramadori G. Combination of "low-dose" ribavirin and interferon alfa-2a therapy followed by interferon alfa-2a monotherapy in chronic HCV-infected non-responders and relapsers after interferon alfa-2a monotherapy. World J Gastroenterol. 2001;7:222-227. [PubMed] [Cited in This Article: ]|
|17.||Worman HJ, Lin F. Molecular biology of liver disorders: the hepatitis C virus and molecular targets for drug development. World J Gastroenterol. 2000;6:465-469. [PubMed] [Cited in This Article: ]|
|18.||Worman HJ, Lin F. Molecular biology of liver disorders: the hepatitis C virus and molecular targets for drug development. World J Gastroenterol. 2000;6:465-469. [PubMed] [Cited in This Article: ]|
|19.||Xiao LY, Yan XJ, Mi MR, Han FC, Hou Y. Preliminary study of a dot immunogold filtration assay for rapid detection of anti-HCV IgG. World J Gastroenterol. 1999;5:349-350. [PubMed] [Cited in This Article: ]|
|20.||Yan FM, Chen AS, Hao F, Zhao XP, Gu CH, Zhao LB, Yang DL, Hao LJ. Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C. World J Gastroenterol. 2000;6:805-811. [PubMed] [Cited in This Article: ]|
|21.||Yang JM, Wang RQ, Bu BG, Zhou ZC, Fang DC, Luo YH. Effect of HCV infection on expression of several cancer-associated gene products in HCC. World J Gastroenterol. 1999;5:25-27. [PubMed] [Cited in This Article: ]|
|22.||Yu SJ. A comparative study on proliferating activity between HBV related and HCV related small HCC. World J Gastroenterol. 1997;3:236-237. [Cited in This Article: ]|
|23.||Zhang LF, Peng WW, Yao JL, Tang YH. Immunohistochemical detection of HCV infection in patients with hepatocellular carcinoma and other liver diseases. World J Gastroenterol. 1998;4:64-65. [PubMed] [Cited in This Article: ]|
|24.||Zhang SL, Liang XS, Lin SM, Qiu PC. Relation between viremia level and liver disease in patients with chronic HCV infection. World J Gastroenterol. 1996;2:115-117. [Cited in This Article: ]|
|25.||Zhou P, Cai Q, Chen YC, Zhang MS, Guan J, Li XJ. Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C. World J Gastroenterol. 1997;3:108-110. [Cited in This Article: ]|
|26.||Zhu FL, Lu HY, Li Z, Qi ZT. Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E. coli. World J Gastroenterol. 1998;4:165-168. [PubMed] [Cited in This Article: ]|
|27.||Zhong Y, Cheng J, Shi S, Xia X, Wang G, Yang J, Chen J. [Screening and characterization of human phage antibody to hepatitis C core antigen]. Zhonghua Ganzangbing Zazhi. 2001;9:217-219. [PubMed] [Cited in This Article: ]|
|28.||Zhong YW, Chen J, Shi SS, Wang G, Dong J, Xia XB, Yang JZ, Chen JM. Screening and expression of human phage antibody to hepatitis virus C NS5A antigen. Chinese J Traditional Western Med. 2001;2:97-99. [Cited in This Article: ]|
|29.||Cheng J, Zhong Y, Shi S, Liu Y, Wang G, Dong J, Xia XB, Chen JM. [Screening and characterization of human phage antibody to hepatitis virus C NS5A antigen]. Zhonghua Shiyan He Linchuang Bingduxue Zazhi. 2001;15:216-218. [PubMed] [Cited in This Article: ]|
|30.||Zhong Y, Cheng J, Liu Y, Dong J, Yang J, Zhang L. Expression of human single-chain variable fragment antibody against non-structural protein 3 of hepatitis C virus antigen in E. coli. Zhonghua Ganzangbing Zazhi. 2000;8:171-173. [PubMed] [Cited in This Article: ]|
|31.||Wu HJ, Tsai RK. Ocular manifestations in children with developmental delay preliminary report. Kaohsiung J Med Sci. 2000;16:422-428. [PubMed] [Cited in This Article: ]|
|32.||Zhong Y, Wang S, Zhao J. [The preparation of human single chain Fv antibody specifically against hepatitis C virus NS3 antigen and its application in immunohistochemistry]. Zhonghua Shiyan He Linchuang Bingduxue Zazhi. 2001;15:186-188. [PubMed] [Cited in This Article: ]|
|33.||Zhong YW, Chen J, Liu Y, Dong J, Yang JZ, Zhang LX. Expression of soluble human single chain Fv antibody to hepatitis C NS 3 antigen in E. coli. Ganzhang Zazhi. 1999;4:71-73. [Cited in This Article: ]|
|34.||Zhong YW, Chen J, Liu Y, Dong J, Yang JZ, Zhang LX. Screening and characterization of human phage antibody with single-chain variable fragment specific to hepatitis C nonstructural 3 protein. Zhonghua Chuanranbing Zazhi. 2000;18:84-87. [Cited in This Article: ]|
|35.||He Y, Liu W, Zeng L. [The effect of interferon in combination with ribavirin on the plus and minus strands of hepatitis C virus RNA in patients with hepatitis]. Zhonghua Neike Zazhi. 1996;35:32-35. [PubMed] [Cited in This Article: ]|
|36.||Wei L, Wang Y, Chen HS, Tao QM. Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing. China Natl J New Gastroenterol. 1997;3:12-15. [Cited in This Article: ]|
|37.||Worman HJ, Feng L, Mamiya N. Molecular biology and the diagnosis and treatment of liver diseases. World J Gastroenterol. 1998;4:185-191. [PubMed] [Cited in This Article: ]|
|38.||Chen MY, Huang ZQ, Chen LZ, Gao YB, Peng RY, Wang DW. Detection of hepatitis C virus NS5 protein and genome in Chinese carcinoma of the extrahepatic bile duct and its significance. World J Gastroenterol. 2000;6:800-804. [PubMed] [Cited in This Article: ]|
|39.||Han FC, Hou Y, Yan XJ, Xiao LY, Guo YH. Dot immunogold filtration assay for rapid detection of anti-HAV IgM in Chinese. World J Gastroenterol. 2000;6:400-401. [PubMed] [Cited in This Article: ]|
|40.||Liu LH, Xiao WH, Liu WW. Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2. World J Gastroenterol. 2001;7:131-135. [PubMed] [Cited in This Article: ]|
|41.||Meier V, Mihm S, Braun Wietzke P, Ramadori G. HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV infection: does it really mean viral replication? World J Gastroenterol. 2001;7:228-234. [PubMed] [Cited in This Article: ]|
|42.||Si XH, Yang LJ. Extraction and purification of TGFbeta and its effect on the induction of apoptosis of hepatocytes. World J Gastroenterol. 2001;7:527-531. [PubMed] [Cited in This Article: ]|
|43.||Wang NS, Liao LT, Zhu YJ, Pan W, Fang F. Follow-up study of hepatitis C virus infection in uremic patients on maintenance hemodialysis for 30 mo. World J Gastroenterol. 2000;6:888-892. [PubMed] [Cited in This Article: ]|
|44.||Yan J, Dennin RH. A high frequency of GBV-C/HGV coinfection in hepatitis C patients in Germany. World J Gastroenterol. 2000;6:833-841. [PubMed] [Cited in This Article: ]|
|45.||Chen S, Wang YM, Li CM, Fang YF. Molecular epidemiology of HCV infection in intravenous drug abusers. Shijie Huaren Xiaohua Zazhi. 2001;9:526-528. [Cited in This Article: ]|
|46.||Ding HL, Cheng H, Fu ZZ, Deng QL, YanLAST_NAME& gt; Tang Yan L, Yan T. The relationship of lmp2 and DR3 genes with susceptibility to type I diabetes mellitus in south China Han population. World J Gastroenterol. 2000;6:111-114. [PubMed] [Cited in This Article: ]|
|47.||Huang F, Zhao GZ, Li Y. HCV genotypes in hepatitis C patients and their clinical significances. World J Gastroenterol. 1999;5:547-549. [PubMed] [Cited in This Article: ]|
|48.||Su YH, Zhu SN, Lu SL, Gu YH. HCV genotypes expression in hepatocellular carcinoma by reverse transcription in situ polymerase chain reaction. Shijie Huaren Xiaohua Zazhi. 2000;8:874-878. [Cited in This Article: ]|
|49.||Wang PZ, Zhou YX. Study on hepatitis C virus genotyping in Xi'an area. Shijie Huaren Xiaohua Zazhi. 1999;7:757-759. [Cited in This Article: ]|
|50.||Yan XB, Wu WY, Wei L. Clinical features of infection with different genotypes of hepatitis C virus. Huaren Xiaohua Zazhi. 1998;6:653-655. [Cited in This Article: ]|
|51.||Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991;222:581-597. [PubMed] [Cited in This Article: ]|
|52.||Hoogenboom HR, de Bruïne AP, Hufton SE, Hoet RM, Arends JW, Roovers RC. Antibody phage display technology and its applications. Immunotechnology. 1998;4:1-20. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 359] [Cited by in F6Publishing: 367] [Article Influence: 14.4] [Reference Citation Analysis (0)]|
|53.||Lamarre A, Talbot PJ. Characterization of phage-displayed recombinant anti-idiotypic antibody fragments against coronavirus-neutralizing monoclonal antibodies. Viral Immunol. 1997;10:175-182. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 6] [Cited by in F6Publishing: 6] [Article Influence: 0.2] [Reference Citation Analysis (0)]|