Advanced Materials Research Vols. 581-582 (2012) pp 77-80 © (2012) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.581-582.77
Effect of carrier surface modification on the resultant antibody to folic acid Qiushi Tang1, a, Hongtao Lei1, b *, Longbin Huang1, c, Yuanming Sun1, d, Jinyi Yang1, e 1
Guangdong Provincial Key Laboratory of Food Quality and Safety / Key Risk Assessment Laboratory of Agricultural Product Preservation, Ministry of Agriculture, Guangzhou 510642, China a
[email protected], b*
[email protected],
[email protected],
[email protected], e
[email protected]
Keywords: Hapten; Carrier protein; Surface modification; Antibody
Abstract. For the generation of antibodies against small hapten molecules, the hapten is crosslinked with some carrier protein to make it immunogenic. However, it was rarely systematically studied about the effect of modified carrier protein on the obtained antibody nature. In this study, folic acid as the model hapten was coupled to natural and surface-modified bovine serum albumin (BSA) using modifying agent amidocaproic acid (ACA) and hexamethylene diamine (HDA). The three immunogens of FA-BSA, FA-ACA-BSA, FA-HDA-BSA were confirmed by SDS-PAGE and ultraviolet spectrum (UV). The Balb/C mice were immunized with the artificial antigen to obtain three specific antibody with indirect competitive enzyme-linked immunosorbent assay (icELISA) method to compare the performance of antibodies obtained based on different imunogen. The results indicate that the conjugates were successfully synthesized and the coupling ratio from the high to the low was HDA-BSA > ACA-BSA > BSA, while the titer of antibody was ACA-BSA > BSA >HDA-BSA and the IC50 was HDA-BSA < ACA-BSA < BSA. This suggests that sensitivity of antibody was improved by cationizing the carrier protein. Introduction In recent years, immunoassay technology for the low-molecular-weight analyte is a key research topic for the safety of food and agricultural products [1, 2]. However, it is still a challenge to obtain the high quality antibody for immunoassay of low molecular because these compounds have no immunogenesis and have to be conjugated to macromolecular carriers such as proteins before immunization. The immunogen design is an important factor in the development of immunoassay. It is reported that the spacer arm between hapten and carrier protein, coupling ratio and surface charge of the carrier protein, etc, affect the sensitivity and specificity of the antibody [3, 4]. But a comparative and systematic study of three effects on the antibody performance has rarely reported. The aim of the present research is to develop kinds of polyclonal antibodies against folic acid based on studying the modification effect of carrier protein to the antibody nature. The hapten folic acid was coupled to natural and modified BSA [5]. The performance of the obtained antibodies using different carriers was compared with an enzyme immunoassay. Experiments Materials. Ultraviolet spectra were recorded on a UV-2550 Shimadzu spectrophotometer (Kyoto, Japan). Polystyrene ELISA plates were from Guangzhou Jiete Biotech Co. and treated with a Multiskan MK2 microplate washer (Thermo Labsystems). Absorbances were read with a Multiskan MK3 microplate reader (Thermo Labsystems). 6K-15 High speed refrigerated centrifuge (SigmaSatorius, Germany). General reagents and organic solvents were of analytical grade unless specified otherwise. Folic acid (99.0%) was obtained from Shanghai Kaiyang Biotechnology Co., Ltd (Shanghai, China). Aminocaproic acid (ACA), 1, 6-Hexanediamine (HDA), Dimethylformamide (DMF), BSA and All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 58.254.92.167-21/09/12,17:07:22)
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Ovalbumin (OVA) were purchased from Shanghai Boao Biotechnology Co., Ltd (Shanghai, China). HRP-conjugated goat anti-mouse IgG was obtained from Boster Biotech Co., Ltd. (Wuhan, China). 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), complete and incomplete Freund’s adjuvants were purchased from Sigma (St. Louis, MO, USA). Carrier modification with ACA and HDA (Fig. 1). 30 mg (0.23 mmoL) ACA and 35 mg (0.18 mmoL) EDC was added slowly into a BSA solution (150 mg in 3 mL deionized water) with continuous stirring. The mixture was preceded for 4 h at room temperature and then dialyzed against PBS (0.01 mol/L, pH 7.4) to remove the free ACA, and then get the immmunogen ACABSA. HDA was coupled to modify BSA with the same method as ACA-BSA. Preparation of conjugates. Immunogenic conjugates and coating antigen FA-OVA were prepared by coupling folic acid to BSA, modified BSA and OVA according the carbodiimide method (Fig. 1) [5]. The obtained conjugates were identified by SDS-PAGE method [6] and ultraviolet spectrophotometry [7]. Production of antibody. Balb/C female mice aged 6-8 weeks (supplied by the Guangdong Medical Laboratory Animal Center) were immunized subcutaneously with a 1:1 (v/v, 200 µL) mixture of an immunogen (100 µg) in PBS and Freund’s complete adjuvant. Booster injections were given intraperitoneally with the same amount of immunogen emulsified with incomplete Freund’s adjuvant at 2 week intervals after the initial injection. After one week from the fourth injection, the mice were tail-bled and the antisera were tested by icELISA. An indirect competitive immunoassay for antibody. The concentrations of antibodies and coating antigens had been optimized by checkerboard titration and competitive curves were then obtained by plotting the normalized signal B/B0 against the logarithm of analyte concentration, where B0 is the signal without analyte and B is the signal of each concentration of analyte [8]. A four-parameter logistic equation (OriginPro 7.5 for Windows) was used to fit the sigmoidal curve. 50% inhibition value was defined as IC50; 10% inhibition value was defined as LOD; 20% -80% inhibition values were defined as detectable range. Results and discussion Synthesis of antigen. Coupling ratio and spacer arm were important factors affecting the sensitivity and specificity of an antibody, usually the spacer arm with 3-6 carbons was ragarded to be suitable for antibody production with high sensitivity and specificity [3, 4]. In this study, ACA and HDA were introduced to connect carrier protein and hapten, respectively. ACA's role is only as a connecting arm, while HDA's role is not only a connecting arm but also make the surface cationized. The concentration of FA-BSA, FA-ACA-BSA, FA-HDA-BSA and FA-OVA were 6 mg/mL, 4 mg/mL, 2 mg/mL, and 2 mg/mL through BCA method [9].
Fig. 1 Surface modification and folic acid coupling of the carrier BSA
Advanced Materials Research Vols. 581-582
Identification of antigen. SDS-PAGE method[6] was used to confirm the coupling (Fig. 2). Electrophoretic bands of ACA-BSA and HDA-BSA did not demonstrate much difference compared to that of BSA. However, significant differences can be found in the migration distance among FA-BSA, FA-ACA-BSA, FA-HDA-BSA, FA-OVA compared to that of BSA, this suggested that the conjugations are successful. The coupling ratio of FA-BSA, FA-ACA-BSA and FAHDA-BSA were calculated to be 19.1, 20.3 and 23.5 based on the literature method [7], respectively.
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Fig. 2 SDS-PAGE profile of folic acid hapten and immunogen. M: maker; 1: BSA; 2: ACABSA; 3: HDA-BSA; 4: FA-BSA; 5: FA-ACABSA; 6: FA-HDA-BSA; 7: OVA; 8: FA-OVA
Antibody Characterization. The antibody to FA-HDA-BSA and FA-ACA-BSA showed lower IC50 than the antibody to FA-BSA (Table 1). This induced that the spacer arm between the carrier protein and hapten can usually result to higher sensitivity. It could also be found that the sensitivity of FA-ACA-BSA to antibody was highest than that of other antibodies. Comparing to BSA and ACA-BSA surface, the HDA-BSA has more free amino groups and the primary amine that positively charged block the BSA surface which negatively charged with carboxyl groups. Therefore, it is probably that the HDA raised the charge of the carrier protein surface to be positive. As a result, the highly positively charged protein significantly enhanced antigen immunogenicity[10, 11] . This suggests that sensitivity of antibody was improved by cationizing the carrier protein. Conclusions The work we present in this paper represents the effect of modified BSA to the antibody. Folic acid was taken as the analyte that coupled with BSA and two modified BSA to study the effect of carrier surface modification on performance of obtained antibody against hapten. The results showed that the conjugates FA-BSA, FA-ACA-BSA and FA-HDA-BSA were successfully synthesized and the titer of antibody were 64000, 128000 and 32000 while the IC50 were 0.5 µg/mL, 0.4 µg/mL and 0.1 µg/mL, respectively. It indicates that high quality of antibody can be obtained by the carrier protein which was cationized with a spacer arm. Table 1 The comparison of the antibody affinity Immunogen
Titer
FA-BSA FA-HDA-BSA FA-ACA-BSA
64000 0.5 32000 0.1 128000 0.4
0.2 0.02 0.08
Detectable range (µg/mL) 0.3~1.2 0.03~0.64 0.1~1.2
FA-BSA FA-HDA-BSA FA-ACA-BSA
80
B/B 0(100%)
IC50 IC10 (µg/mL) (µg/mL)
100
60
40
20
0 0.01
0.1
1
10
Concentration(µg/mL)
Fig. 3 Standard curves of folic acid antibody Acknowlegements The work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201003008-08).
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References [1] M. Oplatowska and C.T. Elliott: Analyst Vol. 136 (2011), p. 2403 [2] Y.Z. Wang, H. Yang, B. Wang and A.P. Deng: Journal of the Science of Food and Agriculture Vol. 91 (2011), p. 1836 [3] H. Lee, G. Shan, K.C. Ahn, E. Park, T. Watanabe, S.J. Gee and B.D. Hammock: Journal of Agricultural and Food Chemistry Vol. 52 (2004), p. 1039 [4] K.V. Singh, J. Kaur, G.C. Varshney, M. Raje and C.R. Suri: Bioconjugate Chemistry Vol. 15 (2004), p. 168 [5] G. Wu, S. Wang, X. Wang, X. Li, X. Deng, Z. Shen and T. Xi: Peptides Vol. 32 (2011), p. 1484 [6] M. Guo and S.Q. Pang: Asian Journal of Chemistry Vol. 21 (2009), p. 5621 [7] Y. Zhou, Y.S. Li, F.G. Pan, Z.S. Liu and Z. Wang: Food Chemistry Vol. 112 (2009), p. 582 [8] J.P. Wang, G.C. Yu, W. Sheng, M. Shi, B.X. Guo and S. Wang: Journal of Agricultural and Food Chemistry Vol. 59 (2011), p. 2997 [9] E.V. Piletska, S.S. Piletsky, M.J. Whitcombe, I. Chianella and S.A. Piletsky: Analytical Chemistry Vol. 84 (2012), p. 2038 [10] R.J. Apple, P.L. Domen, A. Muckerheide and J.G. Michael: Journal of Immunology Vol. 140 (1988), p. 3290 [11] A. Muckerheide, R.J. Apple, A.J. Pesce and J.G. Michael: Journal of Immunology Vol. 138 (1987), p. 833
