Immunogenicity of novel sulfadimethoxide conjugates

Sulfadimethoxine (SDM) is an antibiotic commonly added to animal feeds. Anti-SDM antibodies are useful for the detection of residual SDM in foods, feeds and biological fluids by ELISA. In this study, we show that SDM is immunogenic in rabbits when it is conjugated with soy 11S globulin or with βamylase. Rabbit anti-SDM antibodies obtained by immunization with SDM-11S and SDM-beta-amylase displayed low cross-reactivity against other sulfonamides, including sulfamethazine (SM2), sulfasulfonamides (SN), sulfadiazine (SD), sulfamethoxypyrazine (SMP), sulfalene (SMZ), sulfaquinoxaline (SQX). Thus, soy 11S globulin and β-amylase are suitable carriers for the induction of anti-SDM antibodies and have the advantage of being cheaper that BSA.


INTRODUCTION
Sulfadimethoxine (SDM) is an artificial antibiotic commonly used as feed additive added broadly to animal feeds.Because of its speed and high specificity, competitive indirect enzyme-linked immunosorbent assay (ELISA) is useful for the detection of residual sulfamido medicine in foods and feeds (Deborah et al., 1988).The development of an immunoassay requires the production of antibodies for the analyte.Sulfadimethoxine are small molecules and sulfadimethoxine derivatives, namely haptens, must being synthesized and coupled to a carrier to induce antibody production.Currently, the common hapten carriers used in ELISA are bovine serum albumin *Corresponding author.E-mail: jysu@scut.edu.cn.Tel: +86-02-87113252.Fax: +86-02-87113252.

Abbreviations:
SDM, Sulfadimethoxine; SN, sulfasulfonamides; SD, sulfadiazine; SM2, sulfamethazine; SMP, sulfamethoxypyrazine; SMZ, sulfalene; SQX, sulfaquinoxaline; DNP, 2,4-dinitrophenyl; BPO, benzylpenicilloyi; BaA, bacterial α-amylase; TAA,taka-amylase A; BSA, bovine serum albumin; HSA, human serum albumin; KLH, keyhole limpet hemocyanin (BSA), human serum albumin (HSA) and keyhole limpet hemocyanin (KLH).However, when these proteins are used as the immunogen carrier, the affinity of the corresponding antibodies is poor (Hochul and Jun, 2000).Additionally, KLH and HSA are also relatively expensive and not easily obtainable.The objective of this study was to find a suitable replacement for BSA, KLH and the like.The ideal carrier should have a moderate price, a comparatively stable structure and strong water-solubility.The carrier needs to be able to undergo cross-linking with hapten under organic solvent circumstances while retaining solubility.
Sulfadimethoxine conjugated with ovalbumin served as the coating antigen.In this study, sulfadimethoxine was conjugated with α-amylase, β-amylase, soy 11S globulins, lysozy, soy 7S globulins, poly-l-lysine and BSA.The immunogenicity of the conjugates was tested in rabbits and the specificity of anti-ADM antisera was test-ed by competitive ELISA.
Soy 11S globulin is an inhomogeneous protein whose molecular weight ranges from 340 to 375KDa (Deak et al., 2007;Peng et al., 1984;Wolf, 1997).Soy 7S globulin is a major soybean antigen protein which has the 43% ratio in the soybean protein.Soy 7S globulin is an  Hiroyuki et al., 1987;Wolf, 1997).Though researchers have long been focused on soy allergizationin immature animals,soy 11S globulins and soy 7S globulins have never been used as hapten carriers before α-Amylase contains 526 amino acids and has a molecular weight of 58.6 kDa.Bacterial α-amylase (BaA), Taka-amylase A (TAA) have been conjugated with 2,4-dinitrophenyl (DNP) and benzylpenicilloyi (BPO) groups and used as haptenic determinants (Hamaoka et al., 1971).Lymphoid cells obtained from mice primed with these hapten-protein conjugates were stimulated in vitro with various DNP-and BPO-proteins as the second antigen and transferred into X-irradiated recipients.β-Amylase is a glucoamylase that can be also called saccharifies enzyme.The difference between α-amylase and β-amylase is that β-amylase may cut-out α-1,4 glucoside keys from non-reducibility termination in starch, generate glucose one by one.It may also hydrolyze α-1,6 glucoside key of amylopectin bifurcation.β-Amylase exist in the mold, bacterium, yeast.While β-amylase is commonly used to saccharify amylum candy and other starchiness materials in fermentation industries, it has never been used as a hapten carrier (Masakazu et al., 2009;Hirata et al., 2004.)Poly-l-lysine is a homo-polymer composed of 25 to 35 L-lysines with acidamide bonds between alphaamidocyanogen and the epsilon-carboxyl.It has a molecular weight ranging from 500 to 4,500 kDa and favorably dissolves in water (Hiraki et al., 2003).It has been demonstrated that double-stranded RNA polyinosinic: polycytidylic acid can be used effectively as an endogenous interferon in rabbit and the rodent.Levy (1975) reported that poly-l-lysine dissolved in 0.5% cellulose can improve resistance to natural RNase in the human blood plasma by 5 to 10 times.Li Min (1999) used sodium alginate-poly-L-lysine-sodium alginate to capsulate cattle pheochromocyte and influxion rachnoidea spinalis, which reached more effective immune isolation.Fan (1984) used poly-L-lysine in enzyme linked immunosorbent assay (ELISA) to package T2 poison inoculated with rabbit T2 antibody and determined the antibody quantity based on specific antigen-antibody reaction.
Lysozyme, also known as muramidase, contains a single polypeptide chain of 129 amino acids.Its molecular weight is 14.4 KDa.The isoelectric point is 10.5 to 11.0.Lysozyme exists in the human being, animal, plants, microbe and aves albumen.

Preparation of soy 11S globulins and soy 7S globulins
Soy 11S globulins and soy 7S globulins were isolated according to a new method (Deak et al., 2007;Nicolas et al., 2007), which is a modification of the procedure of Nagano et al.

Preparation of coating antigen
Since sulfadimethoxine has reactive group for coupling reactions, it was conjugated with ovalbumin (OVA) as an ELISA solid phase through diazonium salt coupling (Chemistry of the Diazonium and Diazo Groups).

Preparation of immunogen
Since sulfadimethoxine has reactive group for coupling reactions, it was then conjugated with BSA as an immunogen, by the glutaraldehyde method (Lau et a1., 1981).Dosages in the preparation of immunogens by glutaraldehyde method are shown in Table 1.

Fluorescence spectra
Fluorescence measurements were performed on an F-4500 spectrophotometer (HITACHI, Japan) equipped with a 150 W xenon lamp and 5 nm slit width and 1.00 cm quartz cell at a scanning speed of 1200 nm min −1 , the temperature was controlled by digital aqueous thermostat (Shanghai, China).Sulfadimethoxine and sulfadimethoxine conjugates were dissolved in phosphate buffer (pH= 7.2, 0.05 M).The influence of the various component ratios on the formation of SDM conjugates has been considered (Chemistry of the Diazonium and Diazo Groups).

Antibody titration by indirect competitive ELISA
With the checkerboard procedure, the appropriate concentrations of coating antigen were prepared by serial dilutions from 1 to 4 µg/ml of SDM-OVA in the coating solution and primary sulfadimethoxine antibody (serially diluted purified sulfadimethoxine antiserum from 1:200 to1:256,000).The titration of sulfadimethoxine antibody was evaluated by indirect ELISA as described by Cazemier (2000).Sulfadimethoxine antibody titers are shown in Table 2.

Specificity of sulfadimethoxine antibody by indirect competition ELISA
To ascertain the specificity of the sulfadimethoxine antibody, the inhibition ratio of sulfadimethoxine with sulfadimethoxine antibody and the cross reactivity of the sulfadimethoxine antibody with other sulfonamides were calculated by a competitive indirect ELISA method.A competitive indirect ELISA was carried out by simultaneously incubating sulfadimethoxine with an appropriate dilution of rabbit sulfadimethoxine antiserum over a sulfadimethoxine ovalbumin solid phase (the appropriate concentration was 2 µg/ml) and then determining bound rabbit antibody with a goat anti-rabbit peroxidase conjugate (Muldoon et al., 2004).Plates were coated with an appropriate concentration of SDM-OVA in coating buffer by incubation at 4°C overnight.50 ul/well of sulfadimethoxine solution or sulfonamides [Sulfamethazine(SM2), sulfasulfonamides(SN), sulfadiazine(SD), sulfamethoxypyrazine(SMP), sulfalene(SMZ), sulfaquinoxaline (SQX)], followed by 50 ul/well of SDM antibody solution at twice the desired assay concentration was added.Inhibition standard curve Chen et al. 9941 was prepared by serial dilutions from 1 ng/ml to 4 ug/ml of sulfadimethoxine solution.Competitive immunological reaction was allowed to take place for 1 h and then plates were washed as before.Afterwards, a 1/1000 dilution of goat anti-rabbit IgG-HRP in dilution solution was added and the reaction lasted for 30 min.After washing, the retained peroxidase activity was determined as indicated earlier.The absorbance was immediately read at 492 nm with a reference wavelength at 630 nm (Korpimaki et al., 2004).
The resulting curves were fitted with a four parameter logistic equation to determine the IC50, which is defined as the concentration of inhibitor required to inhibit color development by 50% compared with control wells containing no competitor.The cross reaction rate is defined as the concentration of other sulfonamides inhibitor required to inhibit color development by 50% compared with control wells containing sulfadimethoxine.

Ultraviolet absorption spectrogram analysis
Ultraviolet absorption spectrogram of sulfadimethoxine, neotype carrier and sulfadimethoxine conjugates dissolved in phosphate buffer (pH= 7.2, 0.05 M) are shown in Figures 1 to 6.The coupling ratios of sulfadimethoxine with carriers were also determined (Thouvenot and Morfin, 1983) and shown in Table 1.The ultraviolet absorption intensity of sulfadimethoxine conjugates increase at the basic of carriers (soy 11S globulins, lysozy, soy 7S globulins and poly-l-lysine) and were between the intensity of carrier and sulfadimethoxine.This result suggests that sulfadimethoxine conjugates were prepared successfully.As shown in the figures, carriers of the higher molecule ratios when sulfadimethoxine were conjugates with carriers were soy 11S globulins, β-amylase, α-amylase, lysozy, soy 7S globulins and poly-l-lysine by turns.The coupling ratios of sulfadimethoxine with BSA/OVA were 14 and 12.

Fluorescence emission evidence of association process of sulfadimethoxine and carriers (11S, 7S)
Another argument in favor of the association of sulfadimethoxine with carriers comes from the appearance of the weak fluorescence of carriers by addition of SDM.The fluorescence spectra of sulfadimethoxine conjugates are shown in Figures 7 to 11 at different ratios.For example, in Figure 8, the characteristic fluorescence peak of 11S occurs at 350 nm.The blend of sulfadimethoxine to 11S not only results in the decrease of fluorescence intensity of 11S, but also leads to the red shift of maximum emission wavelength from approximately 350 to 370 nm.In Figure 9, the blend of sulfadimethoxine to 7S leads to the red shift of maximum emission wavelength from approximately 460 to 480 nm.It can be seen that the fluorescence intensities of the conjugates decrease as the ratio increases.In the mentioned systems, the intrinsic fluorescence of carriers (11S, 7S) are quenched.Apparently, sulfadimethoxine 9942 Afr.J. Biotechnol.have a quench for the fluorescence of carriers and there are interactions and energy-transfer happening among sulfadimethoxine and carriers.It effectively suggests that there is association process between sulfadimethoxine and carriers.It proved that sulfadimethoxine conjugate was prepared successfully.

Production of antiserum against sulfadimethoxine conjugate
An indirect ELISA was devised to monitor titers of sulfadimethoxine antiserum wherein rabbit antiserum was incubated over a micro titer plate solid phase coated with sulfadimethoxine ovalbumin (the appropriate concentration was 2 ug/ml).The total bound antibodies were subsequently detected with goat anti-rabbit peroxidase conjugate.The serum dilution visually distinct in color from preimmune serum control at the same dilution was arbitrarily designated as the titer.A summary of sulfadimethoxine antiserum titers against the seven immunogens over 76 days is shown in Table 2. ELISA titration of rabbit anti-sulfadimethoxine antiserum is shown in  SDM-β-amylase, SDM-11S, SDM-poly-l-lysine were all higher than 100,000.The antiserum which had higher titers was SDM-11S antiserum (102, 400, at days 53), SDM-β-amylase antiserum (100,000, at days 53), SDMα-amylase antiserum (80,000, at days 53), SDM-poly-llysine antiserum (80,000, at days 42).The titer of SDM-BSA antiserum was 102, 400 at days 42.

Specificity of sulfadimethoxine antibody
Standard curve of antibody against SDM immunogen (SDM-BSA, SDM-11S, SDM-β-amylase, SDM-poly-llysine) reacting with SDM is shown in Figure 13.The competition effect of sulfadimethoxine with sulfadimethoxine antibody presented linear correlation when the density of sulfadimethoxine ranged from 32 ng/ml to 10 µg/ml.
Typical competition curve and IC50 (concentrations which resulted in 50% inhibition) are shown in Table 3.The SDM-11S globin antibody had the IC50 of 56.5135 ng/ml; the SDM-β-amylase had the IC50 of 79.4328 ng/ml.The SDM-BSA antibody had the IC50 of 137.9461 ng/ml.9946 Afr.J. Biotechnol.Table 4 shows the cross reactivity of the sulfadimethoxine antibody with other sulfonamides (SMZ, SD, SMP, SQX, SM2 and SN).Cross-re-activities of the polyclonal antibody with other sulfonamides were all below 0.05% and reactivity was highest with sulfamerazine which had the most similar structure to sulfadimethoxine.

DISCUSSION
Though, procedures have been established for the production of sulfadimethoxine-BSA/OVA conjugates and antibodies to sulfadimethoxine-BSA/OVA in rabbits (Kamps et al., 1993) and these antibodies have been used in ELISAs for the detection of sulfadimethoxine in foods, feeds and biological fluids (Littlefield et al.,1990)  conjugated with the carriers by the glutaraldehyde method rather than by other methods such as diazonium salt coupling which has been proven unsuitable after several attempts (unpublished observations).Both direct and indirect competitive ELISAs have been used in sulfadimethoxine detection (Sherma, 1997).The antisera of SDM-11S, SDM-β-amylase, SDM-poly-l-lysine had high inhibition ratio to SDM.Carriers which had better immunogenicity were β-amylase, soy 11S globulins and poly-l-lysine.SDM-amylase was not further discussed in this study, as the data would appear in another study.
Compared with BSA, sulfadimethoxine-soy 11S globulin 9950 Afr.J. Biotechnol.and β-amylase conjugate had similar, if not better immunogenicity.The anti-sulfadimethoxine antisera obtained with conjugates based on soy 11S globulin and β-amylase had higher specificity and better affinity than antibodies obtained using BSA or poly-l-lysine as a carrier as evidenced by a lower IC50 in inhibition experiments.Soy 11S globulin could be isolated from defatted soybean seeds easily.Soybean seeds are cheap in China.β-Amylase of technical grade is cheaper in China than BSA and the required amount of β-amylase in the coupling reaction is low.Soy 11S globulin and β-amylase have stable structure and strong water-solubility.They both are capable of cross-linking with sulfadimethoxine under organic solvent circumstances and they confer immunogenicity to sulfadimethoxine in rabbits.
Our data showed that soy 11S globulin and β-amylase are suitable carriers for sulfadimethoxine in immunization experiments and represent a cheaper alternative to BSA as a carrier.

Figure 14 .
Figure 14.Blockade ELISA representative inhibition curve of SDM immunogen antiserum reacting with SDM.

Table 1 .
Dosages in the preparation of immunogens by glutaraldehyde method and the coupling ratios of sulfadimethoxine with the carriers.

Table 4 .
Cross reactivity of the polyclonal antibody with other sulfonamides.