Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay

LUO Kaiming; ZHAO Hu; GAO Xiangdong

doi:10.11665/j.issn.1000-5048.20150618

Journal of China Pharmaceutical University > 2015 > 46(6): 740-744. > DOI: 10.11665/j.issn.1000-5048.20150618

LUO Kaiming, ZHAO Hu, GAO Xiangdong. Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay[J]. Journal of China Pharmaceutical University, 2015, 46(6): 740-744. DOI: 10.11665/j.issn.1000-5048.20150618

Citation:

PDF (1331 KB)

Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay

More Information

Graphical Abstract

Abstract

Abstract

This study was to develop a quantitative immunoassay for hyaluronic acid(HA). We firstly prepared a monoclonal antibody(MAb)against it. HA, activated by 1-cyano-4-dimethyl-aminopyridinium tetrafluoroborate(CDAP), was coupled to bovine serum albumin(BSA). BALB/c mice were immunized with purified complete antigen BSA-HA and the monoclonal antibody was prepared by hybridoma technique. To characterize the specificity of the antibody, inhibition enzyme-linked immunosorbent assay(ELISA)was conducted and the cross reactivity along with affinity constant was determined. The results showed that the substitution degree of BSA-HA(m_BSA/w_HA)was about 9. Also, a hybridoma cell line named 8B6 was obtained by hybridoma technology. The indirect ELISA titer of the ascetic fluid was 1 ∶256 000; the isotype of MAb 8B6 was IgG1 and the affinity constant was K_a=6. 71×10⁹ mol^-1. To establish the indirect competitive ELISA method, polystyrene microwell plates were coated with 10. 0 ng/mL HA and blocked with 1. 0% BSA. The linear range of indirect competitive ELISA was between 10. 0 ng/mL to 10. 0 μg/mL which confirmed that the method was specific and sensitive.
- hyaluronic acid,
- monoclonal antibody,
- indirect competitive ELISA,
- immunoassay

FullText(HTML)

References (21)

References

[1]	Lapcík L Jr,Lapcík L,De Smedt S,et al.Hyaluronan:preparation,structure,properties,and applications[J].Chem Rev,1998,98(8):2663-2684.
[2]	Choi KY, Chung H, Min KH, et al. Self-assembled hyaluronic acid nanoparticles for active tumor targeting[J].Biomaterials,2010,31(1):106-114.
[3]	Mironov V,Kasyanov V,Shu XZ,et al.Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in an in situ crosslinkable hyaluronan-gelatin hydrogel[J].Biomaterials,2005,26(36):7628-7635.
[4]	Lee H, Lee K, Park TG. Hyaluronic acid-paclitaxel conjugate micelles:synthesis,characterization,and antitumor activity[J].Bioconjug Chem,2008,19(6):1319-1325.
[5]	Peng C, Ge H, He S, et al. Molecular cloning expression and identification of recombinant hyaluronan synthase from Pasteurella multocida[J].J China Pharm Univ(中国药科大学学报),2009,40(6):549-552.
[6]	Vasi AM,Popa MI,Butnaru M,et al.Chemical functionalization of hyaluronic acid for drug delivery applications[J].Mater Sci Eng C Mater Biol Appl,2014,38:177-185.
[7]	Kamat M,El-Boubbou K,Zhu DC,et al.Hyaluronic acid immobilized magnetic nanoparticles for active targeting and imaging of macrophages[J].Bioconjug Chem,2010,21(11):2128-2135.
[8]	Widner B,Behr R,Von Dollen S,et al.Hyaluronic acid production in Bacillus subtilis[J].Appl Environ Microbiol,2005,71(7):3747-3752.
[9]	Chien LJ,Lee CK.Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin[J].Biotechnol Prog,2007,23(5):1017-1022.
[10]	Jia Y,Zhu J,Chen X,et al.Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights[J].Bioresour Technol,2013,132:427-431.
[11]	Yu H,Stephanopoulos G.Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid[J].Metab Eng,2008,10(1):24-32.
[12]	Mao Z,Shin HD,Chen R.A recombinant E.coli bioprocess for hyaluronan synthesis[J].Appl Microbiol Biotechnol,2009,84(1):63-69.
[13]	Prasad SB,Jayaraman G,Ramachandran KB.Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactis[J].Appl Microbiol Biotechnol,2010,86(1):273-283.
[14]	Prasad SB,Ramachandran KB,Jayaraman G.Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis[J].Appl Microbiol Biotechnol,2012,94(6):1593-1607.
[15]	Oueslati N,Leblanc P,Harscoat-Schiavo C,et al.CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form[J].Carbohydr Polym,2014,112(21):102-108.
[16]	Jeong E,Shim WY,Kim JH.Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight[J].J Biotechnol,2014,185:28-36.
[17]	Luo HQ,Li NB,Liu SP.Resonance Rayleigh scattering study of interaction of hyaluronic acid with ethyl violet dye and its analytical application[J].Biosens Bioelectron,2006,21(7):1186-1194.
[18]	Lei X,Zhao J,Wang X,et al.Development of a hybridoma cell line secreting monoclonal antibody against S protein of a Chinese variant of PEDV[J].Monoclon Antib Immunodiagn Immunother,2015,34(1):12-16.
[19]	Namkoong H,Funatsu Y,Oishi K,et al.Comparison of the immunogenicity and safety of polysaccharide and protein-conjugated pneumococcal vaccines among the elderly aged 80 years or older in Japan:an open-labeled randomized study[J].Vaccine,2015,33(2):327-332.
[20]	Chu C,Schneerson R,Robbins JB,et al.Further studies on the immunogenicity of haemophilus influenzae type b and pneumococcal type 6A polysaccharide-protein conjugates[J].Infect Immun,1983,40(1):245-256.
[21]	Lees A,Nelson BL,Mond JJ.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents[J].Vaccine,1996,14(3):190-198.

Cited By

Get Citation

PDF

XML

Article views (1428) PDF downloads (2184)

Turn off MathJax

Article Contents

Abstract

References

Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay

Abstract

References

Catalog

Export File

Citation

Format

Content