An enzyme-linked fluoroimmunosensing system for Brucella melitensis antibody detection based on a novel substrate 4-hydroxycinnamic alcohol for HRP

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1 41 3 ( ) Vol.41 No Journal of Central South University (Science and Technology) Jun ( ) (4-hydroxycinnamic alcohol, HCA) (HRP) - - HRP H 2 O nm 467 nm HRP ph HRP-BrAb BSA 2.7~90 µg/l 2.7 µg/l 4.6% HRP HRP O656.2 A (2010) An enzyme-linked fluoroimmunosensing system for Brucella melitensis antibody detection based on a novel substrate 4-hydroxycinnamic alcohol for HRP ZHAN Xue-hui, ZHOU Sui-an, GONG Fu-chun, LI Fei, CAO Fen, LI Xia (School of Chemical and Biological Engineering, Changsha University of Science and Technology, Changsha , China) Abstract: A natural product, 4-hydroxycinnamic alcohol (HCA) was used as a substrate for HRP in enzyme-linked fluoroimmunoassay. In enzymatic reaction procedure, HRP-Brucella melitensis antibody conjugate (HRP-BrAb) catalyze the polymerization of HCA by H 2 O 2, and the HCA is partly converted to polymers, a fluorescent species. The fluorescence increase of the HRP-enzymatic product at emission of 467 nm (excitation at 315 nm) is proportional to the concentration of HRP-BrAb binding to the Brucella melitensis antigens, which were entrapped in cellulose-paraffin matrix. The linear range of determination for BrAb is µg/l with the relative standard deviation of 4.6%. The detection limit is 2.7 µg/l. HCA is stable in air and non-toxic to human health. The proposed method can be used for analysis of commercial formulation and plasma sample with satisfactory results. Key words: 4-hydroxycinnamic alcohol; HRP fluorogenic substrate; Brucella melitensis antibody; immunosensing system (HRP) ( ) (1972 ) zhanxueh@163.com

2 3 891 [1 5] HRP / HRP H 2 O 2 / / / HRP HRP [6] [7] [8] [9] (1) (2) H 2 O 2 (3) [10 11] HRP HRP ELISA HITACHI F4500 CSS (HRP EC , RZ 3) (4-hydroxylcinnamic alcohol HCA) Sigma (BrAg) (BrAb) 1.00 mmol/l 2.31 mg (Sigma ) 100 ml ph=6.8 B-R 1.3 (HRP-BrAb) 10 ml ph 6.8 (PBS) HRP(3.5 mg) 2 ml 1% 12 h 4 10 mmol/l PBS 0.15 mol/l NaCl (ph 7.2) 1 ml ph mol/l NaCl 0.1 mol/l Na 2 CO 3 BrAb(5 mg) HRP h 10 mmol/l ph 7.2 PBS Sephadose G-200 HRP-BrAb (0.48 g/l) mg BrAg 12 mg (BSA) 1.5 ml ph 7.0 PBS (4 ) 0.9 g 4 BrAg-BSA 1 1 ( ) ( 5 min) 6 mm (PVC) 1(a) mm (0.05 µm) 1.6 1(b) 1 A 5 ml 100 µl HRP-BrAb(0.48 mg/l) BrAb ( ) ( 0.1% BSA 1 PVC 2 PVC 3 PVC 4 5 (BrAg) (a) (b) 1 Fig.1 Configuration of biocomposite support body and schematic diagram of injection system coupling with fluometry

3 892 ( ) 41 ph= mmol/l Tris-HCl) 30 min 2 (0.1 mol/l Tris-HCl-KCl ph=7.5) mol/l HCA 315 nm 467 nm HCA mol/l H 2 O 2 HCA HCA H 2 O 2 HRP-BrAb-BrAg HRP H 2 O 2 HCA HCA HCA BrAb 2 BrAb HRP-BrAb BrAg HRP-BrAb BrAb BrAb( ) HCA BrAb HCA-HRP-H 2 O 2 HCA 312 nm 1 ( 299 nm) 3 HRP-BrAb H 2 O 2 HCA HCA HRP-BrAb H 2 O nm 1 ( 315 nm) HRP-BrAb 2.1 (4-hydroxycinnamic alcohol HCA) [12 13] HCA HRP BrAb HCA [14 16] 2 HCA HRP H 2 O 2 HCA 2 ( ) mol/l HCA mol/l H 2 O 2 ph=4.5 1 min HRP-BrAb /(mg L 1 ) ; ; ; ; ; HRP-BrAb H 2 O 2 HCA Fig.3 Fluorescence spectra of HCA solution catalyzed by HRP-BrAb (a) (b) 2 Fig.2 Mechanism of HRP-catalyzed reaction 2.2 BrAg HRP-BrAb BSA HRP-BrAb BrAb (Cellulose) (paraffin) 1

4 % BSA HRP-BrAb BSA BrAb HRP-BrAb HRP-BrAb / BSA Table 1 Effect of BSA on fluorescence intensity * 52 -BSA 11 -BrAg 287 -BrAg-BSA 273 * ph 5.5 HRP ph (ph=5.0) HRP BrAb HRP-BrAb ph ph ph BrAb BrAg HRP-BrAb ph HCA H 2 O 2 HCA H 2 O 2 HRP-BrAb HCA H 2 O 2 HRP-BrA 5 30 ml/h 2.3 ph HCA ph HRP-BrAb HRP-Ab-Ag ph ph HCA ph 6 ph ph 6 ph HCA ph HRP-BrAb HRP 4 HCA 315 nm 315 nm HRP-BrAb ph ph=5.5 ph 5 Fig.5 Effect of flow rate of substrate carrier solution on fluorescence nm 4 ph Fig.4 Effect of ph on activity of HRP HRP-BrAb 5 ml HRP-BrAb HRP-BrAb 100 µl HRP-BrAb(0.48 mg/l) 5 ml 100 µl HRP-BrAb min BrAb 7 BrAb 2.7~90 µg/l 2.7 µg/l (7.2 µg/l)

5 894 ( ) BrAb g/l h( 27 ) ( ) 3 3 ELISA BrAb HRP-BrAb 0.48 mg/l 6 HRP-BrAb Fig.6 Effect of amount of HRP-BrAb on fluorescence strength 3 BrAb Table 3 BrAb determination in rabbit serum samples µg/l 1 (31 d) 4.31± (41 d) 5.85± (53 d) 7.82± (268 d) 11.35± BrAb 0.48 µg/ml BrAb-HRP 0.1% BSA 0.1 mol/l Tris-HCl-1 mmol/l EDTA 4 (3.5%~6.1%) 7 BrAb Fig.7 Calibration curve for BrAb determination obtained by competitive immunoassay 55 mg/l BrAb 2 2 BrAb (1) BrAb (2) (3) (4) 2 Table 2 Reproducibility of immunosensing system / % 275, 280, 265, 277, 267, , 226, 230, 214, 240, [1] Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELSIA) quantitative assay of immunoglobulin G[J]. Immunochemistry, 1971, 8(5): [2] Carlo M D, Mascini M. Enzyme immunoassay with amperometric flow-injection analysis using horseradish peroxidase as a label. Application to the determination of

6 3 895 polychlorinated biphenyls[j]. Anal Chim Acta, 1996, 33(6): [3] Takatoshi S, Masanori Y, Hiroya I, Kiyoshi M. Flow injection analysis of amitrole by chemiluminescent immunosensor using alkaline phosphatase and adamantyl methoxyphosphoryloxy phenyldioxetane[j]. Anal Sci, 2001, 17(5): [4] Hossein S, Martin G S, Oliver H, et al. Development of enantioselective chemiluminescece flow- and sequential injection immunoassays for α-amino acids[j]. J Biochem Biophys Methods, 2002, 53(1): [5] Villarta R L, Guilbault G G, Suleiman A A. Flow injection analysis of glucose by fibre optic chemiluminescence measuremen[j]. Anal Lett, 1993, 26(4): [6] Lonergan G, Mew E, Schliephake K, et al. Phenolic substrates for fluorometric detection of laccase activity[j]. FEMS Microbiol Lett, 1997, 153(2): [7] Ci Y X, Chen L, Wei S. Fluorescence reaction of the system mimetic peroxidase [Mn-T(4-TAP)P]-homovanillic acidhydrogen peroxide. Spectrofluorimetric determination of H 2 O 2 [J]. Fresenius J Anal Chem, 1989, 33(4): [8] Gong F C, Zhou Z J, Shen G L, et al. Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3,5,5 -tetramethylbenzidine as substrate[j]. Talanta, 2002, 58(2): [9] Chen L, Chang W B. Studies on the structure of substrate for peroxidase[j]. Chemical Research in Chinese Universities, 1995, 16(5): [10] Tatyana V V, Ian J R. Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation[j]. Archives of Biochemistry and Biophysics, 2004, 43(1): [11] Michel P E, Rooij de N F, Koudelka-Hep, et al. Redox-cycling type electrochemiluminescence in aqueous medium. A new principle for the detection of proteins labeled with a ruthenium chelate[j]. J Electroanal Chem, 1999, 47(4): [12] Lee H S. Tyrosinase inhibitors of Pulsatilla cernua root-derived materials[j]. J Agri Food Chem, 2002, 50(6): [13] Zapata J M, Calderón A A, Muñoz R, et al. Oxidation of natural hydroxybenzoic acids by grapevine peroxidases: kinetic characteristics and substrate specificity[j]. Am J Enol Vitic, 1992, 43(1): [14] Beier R C, Young C R, Stanker L H. USDA agricultural research service, detection of bacteria from a cecal anaerobic competitive exclusion culture with an immunoassay electrochemiluminescence sensor[j]. Proc SPIE, 1991, 35(44): [15] Tuuminen T, Palomaki P, Rakkolainen A, et al. 3-phydroxyphenylpropionic acid-a sensitive fluorogenic substrate for automated fluorometric enzyme immunoassays[j]. Immunoassay, 1991, 12(1): [16] Limoges B, Degrand C, Brossier P, et al. Homogeneous electrochemical immunoassay using a persulfonated ionomermodified electrode as a detector for a cationic labeled hapten[j]. Anal Chem, 1993, 65(3): ( )

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