Bioconjugate Chem. 1999, 10, 583−588
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Molecular Modeling of Hapten Structure and Relevance to Broad Specificity Immunoassay of Sulfonamide Antibiotics C. A. Spinks,* G. M. Wyatt, H. A. Lee, and M. R. A. Morgan Department of Biochemistry, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, U.K. Received May 26, 1998; Revised Manuscript Received February 22, 1999
Molecular modeling of hapten structure was used to predict and influence, through appropriate synthetic work, the outcome of an immunization program. Examination of the structures of sulfonamide antibiotics led to the development of a hypothesis and the consequent synthesis of a sulfacetamideprotein immunogen aimed at the generation of broad specificity anti-sulfonamide antibodies. The antisera generated, alongside anti-sulfachlorpyradizine antisera generated at the same time, were characterized for cross-reactions against a range of sulfonamide drugs, and were found to exhibit good but not the desired broad specificity. Discussion is presented as to the reasons for the failure of the hypothesis. Further hypotheses are developed and speculation is made as to the future of molecular modeling in immunochemical research.
INTRODUCTION
Compounds of molecular masses less than about 100010000 Da are usually not intrinsically immunogenic. To generate antibodies to such compounds, it is required that they are covalently linked to a carrier of high molecular weight (almost always a protein) that is itself capable of stimulating the immune system. Of the many antibodies so produced, some are capable of recognizing the hapten molecule alone. On this basis, immunoassays have been established for many low molecular weight analytes, including mycotoxins (Wilkinson et al., 1992), pesticides (Van Emon et al., 1989) and vitamins (Finglas and Morgan, 1994). These reviews give an indication of the diversity and convenience of immunoassays for small molecules. It has long been recognized that the manner of the linking of hapten to carrier protein for immunogen synthesis is critical in determining the nature of the antibody response; thus, antibody specificity is maximal for those sites distal to the point of conjugation (Landsteiner, 1945). With this knowledge, it has been possible to generate antibodies of very high specificities, capable of distinguishing very closely related structures. For example, high specificity has been particularly important in circumstances where close structural relationships do not reflect similar bioactivities. Wie and Hammock (1984) devised novel routes of synthesis in order to obtain maximum specificity for benzoylphenylurea insecticides. However, it is sometimes also necessary to analyze a group of compounds of similar structure. This aim can often be achieved by judicious synthesis of the haptenprotein immunogen to expose maximally common features while minimizing the presentation of structural differences to the immune system. An example of this approach would be the immunoassay developed for quantification of the potato glycoalkaloids (Morgan et al., 1983). A further possibility is provided by monoclonal antibody technology, whereby low-frequency antibodies from the polyclonal population can be selected via the cloning and screening process. Accordingly, Ward et al. * To whom correspondence should be addressed.
(1990) have described a surprisingly diverse population of anti-aflatoxin antibodies obtained through use of one immunogen. In past years, the development of high specificity antibodies has been a paramount consideration in the acceptance of immunoassay technology. However, the importance of broad specificity analysis is increasing, particularly in circumstances where a high proportion of samples will be negative, since economic and regulatory factors dictate that more resources are required for positive samples. Given their robustness, simplicity, high sample through-put, and low cost per sample, the immunoassays should be as well-suited to application in screening assays as they are in specific determinations. The limiting factor is the development of suitable antibodies. The sulfonamide antibiotic drugs are analytes for which there is considerable demand for a broad specificity assay, detecting all members of the group and allowing the high proportion of negative samples to be rejected. The sulfonamides (used in pigs, cattle, and poultry, for example) seem to be particularly resistant to attempts to produce broad specificity antibodies. This is rather surprising since at first glance the general structure of the sulfonamides (Figure 1) with the comparatively large common aromatic ring moiety at one end of the molecule looks eminently suitable for broad specificity antibody generation. Indeed, Sheth and Sporns (1991) have described the production of antibodies using a sulfathiazole derivative linked through the variable R-group and exposing the common ring. The resultant antibodies were not as broadly specific as would be liked. Other groups of antibiotic drugs have proved easier to deal with (Kitagawa et al., 1983; Stanker et al., 1993; Brandon et al., 1994), but sulfonamide-protein immunogens have invariably generated antibodies of high specificity (Singh, et al., 1989; McCaughey, et al., 1990; Sheth and Sporns, 1990; Hoffmeister, et al., 1991; Martlbauer, et al., 1992; Garden and Sporns, 1994; Thomson and Sporns, 1995). Molecular modeling of hapten structure has recently been used with some success in developing insight as to the reasons for the cross-reaction characteristics of
10.1021/bc980054m CCC: $18.00 © 1999 American Chemical Society Published on Web 05/11/1999
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Figure 1. Sulfonamide drugs.
particular antibodies. There are clear advantages to looking at three-dimensional representations of structure, even without the further possibilities of investigating hydrophobic and electrostatic properties. Carlin et al. (1994) modeled the diuretic furosemide and related compounds and were able to explain the observed crossreactions of a monoclonal anti-furosemide antibody. Elissalde et al. (1995) modeled the mycotoxin fumonisin and revealed unexpected folding within the structure with high significance for anyone interested in immunochemical studies on this compound. Such studies and others have been carried out after the antibodies have been produced. In the present report, we describe the results of efforts to use molecular modeling in a predictive manner in order to enhance the possibility of developing a broad specificity anti-sulfonamide antibody. MATERIALS AND METHODS
Chemicals. All starting materials for hapten synthesis and conjugation were obtained from Sigma Chemical Co. (Dorset, U.K.). All reagents were of the highest quality available. Molecular Modeling. Modeling studies were carried out on a Silicon Graphics Workstation (Mountain View, CA) running QUANTA software (Molecular Simulations Inc., Burlington, MA). Twenty-two different sulfonamides were modeled. Minimum energy conformations of the compounds were calculated using the CHARMm force field. (CHARMm uses empirical energy functions to describe the forces on atoms in molecules, calculating
conformational energies, local minima, barriers to rotation, energy surfaces, and time-dependent dynamic behavior). Conformational searches were carried out by rotating all rotatable bonds through 360° in 30° increments and the lowest energy conformation computed. Electrostatic potentials were mapped across the surface of the structures by tumbling a positively charged particle over the surface of the molecule and calculating the energy for repulsion. Accordingly, high-energy regions correspond to a negative charge distribution within the molecule. Synthesis of Drug-Protein Conjugates for Immunization. (i) Sulfacetamide Immunogen (SAMBTG). Sulfacetamide (SAM) was reduced by sodium borohydride in a manner similar to the method described for triadimefon by Newsome (1986). SAM (400 mg) was dissolved in ethanol (20 mL) and mixed with sodium borohydride (700 mg in 12 mL ethanol). Frothing was observed, and the mixture was left overnight, after which concentrated HCl (4 mL) was added and the ethanol removed by rotary evaporation. The SAM derivative (110 mg) was dissolved in pyridine (5 mL), to which succinic anhydride (500 mg) was added and left stirring gently for 3 days at room temperature. Pyridine was removed by rotary evaporation, and the residue dissolved in dichloromethane (6 mL). After washing with 1 M HCl (2 mL) followed by water (4 mL), the sample was again dried and the hemisuccinate product (52 mg) dissolved in dioxan (3 mL). The mixed anhydride method for conjugation to protein was then carried out (Erlanger et al., 1959) where isobutylchloroformate (4 µL)
Molecular Modeling of Hapten Structure
and tributylamine (8 µL) was added to the SAM hemisuccinate solution and stirred for 30 min, ensuring that the temperature did not rise above 5 °C. In the meantime, bovine thyroglobulin (BTG; 45 mg) was dissolved in water (2 mL). Dioxan (2 mL) was added and mixed with half of the solution containing the activated SAM hemisuccinate, maintaining the pH at 9.0 with 1 M NaOH. After exhaustive dialysis against water, the conjugate was lyophilized. (ii) Sulfachlorpyridazine (SCP) Immunogen (SCP-BTG). On the basis of the method described by Schlaeppi et al. (1989), SCP was derivatized by replacing the terminal chlorine atom with a hydroxyl group. SCP (0.5 g) was dissolved in 6 M HCl (10 mL) and stirred at room temperature for 4 h. The mixture was dried by rotary evaporation to give a brown syrup, half of which was dissolved in pyridine (4 mL). 2-Fluoro-1-methylpyridinium p-toluenesulfonate (FMP; 100 mg) was added and then stirred gently at room temperature for 1 h, after which BTG (86 mg) in carbonate buffer (pH 9.6; 10 mL) was added. After exhaustive dialysis against water, the conjugate was lyophilized. Synthesis of Drug-Protein Conjugates for Coating Microtitration Plates. (i) SCP-BSA Conjugate. As with the immunogen, SCP was derivatized to produce a hydroxyl group. Succinic anhydride (500 mg) in pyridine (5 mL) was added to the derivative and gently stirred for 3 days at room temperature. The pyridine was removed by rotary evaporation, and half of the product was taken and added to 0.2 M phosphate buffer (pH 7.3; 1 mL) containing EDC (100 mg). After 30 min, BSA (75 mg) dissolved in 0.2 M phosphate buffer (pH 7.3; 2 mL) was added, and gently stirred for 1 h. After dialysis against water, the conjugate was lyophilized and stored at -20 °C. (ii) SAM-BSA Conjugate. The SAM derivative (190 mg), used for the immunogen, was dissolved in pyridine (5 mL). Pyridine is necessary both as a solvent and scavenger. FMP (200 mg) was added to the solution and left for 30 min at room temperature. The solution turned dark red in color. Bovine serum albumin (BSA; 140 mg) was dissolved in 0.2 M carbonate buffer (pH 9.6; 5 mL), and this solution was added to the SAM derivative to cause fizzing and a change to a yellow/brown color. The whole mixture was stirred gently overnight, and then dialyzed exhaustively against water and lyophilized. (iii) Diazo Conjugates. Several drug-protein conjugates were synthesized by the diazotization method and used as solid phases of the ELISA. This method links the drug to the protein via the para-amino benzene group of the common moiety. Essentially, the drug was dissolved in 1 M HCl (10 mL) with 0.1 M NaNO2 added dropwise until a positive starch test was obtained. The solution was added to 0.1 M sodium borate buffer (10 mL) containing the protein carrier. The pH of the total mixture was maintained at pH 9.0 with 1 M NaOH and left gently stirring for 1 h at room temperature. The conjugation mixture was subsequently dialyzed against water and lyophilized. Amounts of drug and protein, respectively, used in diazo conjugate synthesis were for sulfachlorpyridazine, 23 mg and 100 mg of BSA; sulfaquinoxaline, 44 mg and 100 mg of keyhole limpet haemocyanin (KLH); sulfamethazine, 25 mg and 75 mg of KLH; sulfanilamide, 18 mg and 80 mg of BSA; 4-aminodibenzene-sulfonamide, 25 mg and 50 mg of KLH; 4-aminophenyl-4-methyl benzenesulfonamide, 25 mg and 50 mg of KLH. (iv) Sulfabenzamide (SBA) Conjugate. Sulfabenzamide (SBA, 0.38 g) was dissolved in ethanol (15 mL)
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and mixed with sodium borohydride (0.2 g) in ethanol (15 mL). The mixture effervesced and a white precipitate formed. The ethanol was removed by rotary evaporation and the solid refluxed for 5 h with succinic anhydride (570 mg) in pyridine (10 mL). FMP (225 mg) was added, and the mixture left at room temperature for 30 min. KLH (75 mg) in carbonate buffer (pH 9.6, 15 mL) was added and left overnight at room temperature. The conjugation mixture was exhaustively dialyzed against water and then lyophilized. Immunization of Rabbits. For each immunogen, two New Zealand White rabbits were immunized with 100 µg/animal of SCP-BTG or SAM-BTG conjugate emulsified in 1 mL of complete Freund’s adjuvant and sterile physiological saline, 1:1 (v:v). The animals were immunized subcutaneously in multiple sites on the back. Subsequent monthly immunizations were carried out as above, except that incomplete Freund’s adjuvant was used. Blood (10 mL) was taken from the marginal ear vein of each animal 10 and 14 days after immunization. The blood was collected in heparinized tubes and centrifuged at 2500 rpm for 10 min. The supernatant was collected and stored frozen, with aliquots at 4 °C for use in ELISA. Determination of Antibody Response. In 0.05 M sodium bicarbonate/carbonate buffer, pH 9.6, 96-well microtitration plates were coated with 1 µg mL-1 of the appropriate drug-protein conjugate. All wells were filled with 300 µL of the coating solution and incubated overnight at 2 °C, at which point they were washed 3 times with water using a Denley Wellwash 5000 microtitration plate washer (Denley Instruments Limited, Sussex, England). Plates were stored dry at room temperature until required for use. A Beckman BioMek 1000 robotic pipetting workstation and side loader was programmed using BioMek software version 2.11 to carry out the liquid handling stages of the ELISAs. Each rabbit antiserum was diluted 1:1000 (v:v) in PBST and 200 µL added in triplicate to coated plates. A 10-fold serial dilution was carried out four times and the plates were incubated at 37 °C for 2 h. After washing and aspirating the plates five times with PBST, anti-rabbit IgG-HRP conjugate diluted 1:1000 (v:v; 200 µL) in PBST was added to each well and incubated for 1 h at 37 °C. After a further washing with PBST, to each well was added 200 µL of tetramethylbenzidine (TMB)based substrate (Vetoquinol, Bicester, U.K.). After 10 min, the reaction was stopped using 50 µL/well of 2 M H2SO4. The absorbance of each well was measured at 450 nm using a Titertek Multiskan MCC plate reader. Competitive ELISA. Since it was necessary to screen the antibodies raised against as many of the drugs as possible, the Beckman BioMek 1000 robotic system was programmed to perform all the liquid handling stages of the competitive ELISAs. The basic format of the assay involved the addition (in triplicate) of 100 mL of each standard at each concentration in PBST to drug-protein coated plate, followed by 100 mL of antiserum diluted in PBST to a concentration previously determined by ELISA. The concentrations of drug standards used were 1 mg mL-1, 100 ng mL-1, 1 ng mL-1, and zero. The plates were incubated for 2 h at 37 °C, washed five times with PBST, and then 200 mL/well of anti-rabbit IgG-HRP diluted 1:1000 (v:v) in PBST was added to all wells. After a further incubation of 2 h at 37 °C the plates were washed and TMB substrate added (200 mL/well). After 10 min. the reaction was stopped with 2 M H2SO4 and absorbances of each well determined at 450 nm.
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Figure 2. Van der Waals representation of the models of sulfacetamide and sulfachlorpyridazine. Table 1. Spatial Information Obtained from Molecular Modeling drug
angle of bend (deg)
distance between C1 and R-group (Å)
SCP SMP SEZ SAM SDM SID SDZ SME SD SMZ SMX STZ SQ SAN ADBS SP
88.8 89.17 97.1 86.6 90.64 97.09 92.88 90.34 95.93 86.01 103.7 90.49 88.98 107.8 95.21 133.1
5.706 5.761 6.502 5.847 6.394 6.348 6.504 6.12 6.651 5.26 6.028 6.067 6.325 5.325 9.088 9.396
RESULTS AND DISCUSSION
The initial modeling studies carried out after performing a search rotating all torsions and energy minimizing for a number of different sulfonamides (Figure 2 shows only SCP and SAM) revealed that the drugs each had a characteristic “bend” around the tetrahedral -SO2grouping. The bend varied considerably, being of least angle in SMZ (with the two halves of the drug almost folded back on each other) and of greatest angle in SP (where the drug is almost planar). The angles recorded are given in Table 1. Given the nature of the general structure of the sulfonamides, the differences are clearly due to the influence of the R-group alone. Our initial hypothesis was based on the idea that maximal exposure of the common moiety was compromised in those drugs where the bend was of smallest angle. Consequently, recognition of the common moiety would be maximal in those drugs where the bend was of greatest angle. We synthesized two immunogens, the SAM-BTG and SCP-BTG conjugates, with the former being a more planar structure, the latter having a greater bend. Reasonable titers were obtained with both immunogens, and subsequent characterization was carried out by determining the ability of the antibodies to recognize different drug conjugates immobilized by passive adsorption to the wells of microtitration plates. Primary antiserum dilution was always 1:1,000 (v:v) for both anti-
Table 2. Summary of Antisera Binding to Different Hapten Conjugatesa immunogen based on: coating conjugate based on: sulfachlorpyridazine (R-group linkage) sulfamethazine (R-group linkage) sulfacetamide acetyl sulfanilyl chloride sulfabenzamide sulfachlorpyridazine (common group linkage) 4-aminodibenzenesulfonamide 4-aminophenyl-4-methyl benzenesulfonamide sulfamethazine (common group linkage) sulfaquinoxaline sulfanilamide sulfanillic acid sulfasalazine
sulfachlorpyridazine
sulfacetamide
+++
++++
+++
++
+++ ++
++++ +++
++ ++++
+++ -
-
nt
+++
+++
++++
++++
+++ +++ ++
++++ +++ -
a Absorbance values obtained in ELISA of antiserum diluted 1:1000 v:v (++++ ) >2.0; +++ ) 1.5-2.0; ++ ) 1.0-1.5; - )
