hapten–antibody recognition studies in competitive immunoassay of α-zearalanol analogs by...

Received: 27 August 2010, Revised: 16 November 2010, Accepted: 16 November 2010, Published online in Wiley Online Library: 2011

Hapten–antibody recognition studies incompetitive immunoassay of a-zearalanolanalogs by computational chemistry andPearson Correlation analysisZhanhui Wanga,b, Pengjie Luoc, Linli Chenga,b, Suxia Zhanga,b

and Jianzhong Shena,b*

Themolecular recognition of hapten–antibody is a fundamental event in competitive immunoassay, which guaranteesthe sensitivity and specificity of immunoassay for the detection of haptens. The aim of this study is to investigate thecorrelation between binding ability of one monoclonal antibody, 1H9B4, recognizing and the molecular aspects ofa-zearalanol analogs. The mouse-derived monoclonal antibody was produced by using a-zearalanol conjugated tobovine serum albumin as an immunogen. The antibody recognition abilities, expressed as IC50 values, weredetermined by a competitive ELISA. All of the hapten molecules were optimized by Density Function Theory(DFT) at B3LYP/ 6-31G* level and the conformation and electrostatic molecular isosurface were employed to explainthe molecular recognition between a-zearalanol analogs and antibody 1H9B4. Pearson Correlation analysis betweenmolecular descriptors and IC50 values was qualitatively undertaken and the results showed that one moleculardescriptor, surface of the haptenmolecule, clearly demonstrated linear relationship with antibody recognition ability,where the relationship coefficient was 0.88 and the correlation was significant at p< 0.05 level. The study shows thatcomputational chemistry and Pearson Correlation analysis can be used as tool to help the immunochemistries betterunderstand the processing of antibody recognition of hapten molecules in competitive immunoassay. Copyright �2011 John Wiley & Sons, Ltd.

Keywords: hapten–antibody recognition; a-zearalanol; DFT; molecular descriptors; Pearson Correlation analysis

INTRODUCTION

Competitive format of immunoassay based on highly specificrecognition of hapten–antibody has been widely used to detectthe presence of small molecular weight contaminant, calledhapten, in many matrices because of its some advantages overtraditional instrumental methods, such as easy-to-use, highsensitivity and specificity (Morozova et al., 2005; Knopp 2006;Blasco et al., 2007). However, in most of the cases, antibodyelicited by one hapten–carrier conjugate can cross-react withother molecules if these molecules share an identical or verysimilar epitope with the hapten used (Xu et al., 2006; Mercaderet al., 2008). Actually, individual molecule always belongs toone class of compounds, for instance, sulfonamides family,thereby, the antibodies, particularly in the case of polyclonalantibodies, produced against one molecule often couldrecognize structurally similar analogs.For immunochemist, there is a challenging problem to

well understand the cause of the antibody’s promiscuity fromonly looking at two-dimensional structural formulas of thecompounds studied (Sanvicens et al., 2003). There is considerableinterest in understanding the structural basis of antibody–haptenrecognition at the molecular level. Although some papers havereported the recognition mechanism and models of antibody–hapten based on the X-ray graphs of antibody with or withouthapten, it is neither possible nor practical to crystallize eachinteresting antibody for immunochemist because of high cost

and much labor (Burmester et al., 2001; Kusharyoto et al., 2002;Valjakka et al., 2002). A method that can provide usefulinformation about the topological and electrostatic propertiesof hapten can be sufficient in elucidating the diversity of antibodybinding hapten and then may aid to design optimal hapten,thereby, producing improved antibody with the desired

(wileyonlinelibrary.com) DOI:10.1002/jmr.1121

Research Article

* Correspondence to: J. Shen, China Agricultural University, Beijing 100193,People’s Republic of China.E-mail: [email protected]

a Z. Wang, L. Cheng, S. Zhang, J. Shen

College of Veterinary Medicine, China Agricultural University, Beijing 100094,

People’s Republic of China

b Z. Wang, L. Cheng, S. Zhang, J. Shen

National Reference Laboratories for Veterinary Drug Residue, District Haidian,

Beijing 100094, People’s Republic of China

c P. Luo

National Institute for Nutrition and Food Safety, Chinese Center for Disease

Control and Prevention, Beijing 100050, People’s Republic of China

Abbreviations: ELISA, enzyme-linked immunosorbent assay; DFT, Density Func-

tion Theory BSA, bovine serum albumin; OVA, ovalbumin; DMF, dimethyl for-

mamide; HOMO, high occupied molecular orbital hydrogen; LUMO, low

unoccupied molecular orbital; IC50, 50% inhibition of control activity; MAbs,

monoclonal antibodies; S, molecule surface; V, Volum; Log P, lipophobicity index;

m, Dipole moment; H, heat of hydration; R, Refraction; P, molecular polarizability;

MW, molecular mass; D, distance.

J. Mol. Recognit. 2011; 24: 815–823 Copyright � 2011 John Wiley & Sons, Ltd.

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affinity and specificity (Beier and Stanker, 2001). In an effortto determine structural and electronic aspects of hapteneffecting on antibody–hapten recognition, molecular modelingtechniques have been previously employed and have beenproved a useful tool (Holtzapple et al., 1996; Galve et al., 2000;Spinks 2000). In these reports, molecules were mostly optimizedbyMM2 force field; moreover only conformation and electrostaticsurface of hapten molecules were presented. Apparently,the information for distinguishing similar molecules from eachother is limited. To more accurately understand the recognitionof antibody to hapten, the parametered information shouldbe considered and included.a-zearalanol is one of the non-steroidal estrogenic growth

promoters with a phenolic resorcyclic acid lactone structure,banned by the EU since 1985 (Zollner and Mayer-Helm, 2006).In most cases, a-zearalanol is presented with the structurallyrelated mycotoxins, b-zearalanol, a-zearalenol, b-zearalenol,and zearalenone, which exist in a metabolic relationship witha-zearalanol and its metabolites. Thesemycotoxins are frequentlyproduced by Fusarium spp. fungi in maize, oat, barley, wheat,and sorghum and may contaminate feed. Due to concernsabout long-term effect on humans, the Joint FAO/WHO ExpertCommittee on Food Additives has set the maximum residuelevels in liver at 10mg/kg and in muscle of 2mg/kg. Sensitiveanalysis methods for screening and confirmation of a-zearalanoland its metabolites including antibody-based techniques havebeen developed (Bennekom et al., 2005). We have producedone monoclonal antibody to a-zearalanol, named 1H9B4 andcompetitive ELISA was developed to detect a-zearalanol in beefsample. The specificity determination analysis demonstrated thatthe antibody 1H9B4 could recognize other five structurally similaranalogs with diverse affinity (Wang et al., 2004).In this paper, a-zearalanol and its five structural analogs

were optimized by precise Density Function Theory and thelowest conformation and electrostatic potential on van der Waalssurface of each molecule were calculated and visualized. Theparametered information of molecules is expressed by moleculardescriptors calculated. The information of haptenmolecules wereused to correlate recognition ability of antibody with molecularaspects of hapten molecules.

EXPERIMENTAL

Chemicals

a-zearalanol, b-zearalanol, a-zearalenol, b-zearalenol, zearala-none, zearalenone, diethylstilbestrol, bovine serum albumin(BSA), ovalbumin (OVA), isobutyl chloroformate, and tributyla-mine were purchased from Sigma Chemical Co. (St. Louis, MO).All other chemicals and solvents were of analytical grade or betterfrom Beijing Chemical Reagent Co. (Beijing, P.R.C.). Deionizedwater was prepared using a Milli-Q water purification system(Millipore, Bedford, MA). Polystyrene microtiter plates werepurchased from Beijing Wanger Bio. Tec. Co. (Beijing, P.R.C.).The ELISA plate reader was from TECAN U.S.A. Inc. (Durham, NC.).

Production of monoclonal antibody

The hapten used to link carrier protein was synthesized asdescribed previously (Wang et al., 2004). The immunogenwas prepared as follows: the hapten was dissolved in 5ml ofdimethyl formamide (DMF) with ultrasound, and the solution

was cooled with cold ethanol. Then 30ml of triethylamine and40ml of isobutyl chloroformate were added. The resulting mixedanhydride solution was stirred at room temperature for 20minbefore 3ml of 10mg/ml BSA in carbonate buffer (pH 9.6) wasadded drop-wise and continuously stirred at room temperaturefor 6 h. The hapten–BSA conjugates were dialyzed againstPBS (pH 7.4) at 48C for 72 h. The coating antigen was prepared bythe same method instead of BSA but OVA. The procedures forgenerating the immune response in mice and producingmonoclonal antibodies were similar to those described by Wanget al. The antibody screened by the one satisfied hybridomanamed 1H9B4 was used to develop competitive ELISA for thedetection of a-zearalanol and analogs.

Antibody recognition ability dereminated bycompetitive ELISA

The competitive ELISA was carried out as follows: polystyrene96-well micro-titration plates were coated with the coatingantigen (100ml/well) and incubated at 378C for 2 h, and thenat 48C overnight. The plates were washed three times withthe washing buffer, and then blocked with the blocking bufferat 378C for 1 h. Antibodies supernatants (50ml/well), variousconcentrations of hapten molecules (50ml/well) and goatanti-mouse IgG-HRP (1:5000 in PBS, 100ml/well) were addedto the pre-coated micro-plates incubated at 378C for 1 h. Afterincubation, the substrate solution (100ml/well) was added andincubated at 378C for 30min before the enzymatic reaction wasstopped by adding 2MH2SO4 (100ml/well). The absorbance (A) ofeach well was measured at 490 nm.The cross-reactivity values were calculated according to the

following equation:

CR ¼ IC50ðZERÞIC50ðAnalogsÞ � 100%

Computational method

Determination of minimum energy conformation of haptenmolecules

The structures of a-zearalanol and analogs were constructed andthen converted to three-dimensional models in Chemoffice Ultra11.0 software (Cambridge soft, Corporation, MA, USA). Geometryoptimizations of all molecules were performed using theGaussian03 interface in Chemoffice Ultra 11.0. To save compu-tational time, initial geometry optimizations were carried out withMMFF94 force field under the maximum number of iterationof 500 and minimum RMS gradient of 0.01 kcal/mol A. Based onthe optimized structures of hapten molecules by MMFF94,the lowest energy conformations were extensively searchedwith density function theory (DFT) at the B3LYP / 6-31 G* leveland, their fundamental vibrations were also calculated usingthe same method to check if they were true minima. To obtain ahigh-quality graph, the lowest energy conformation of haptenmolecules was visualized by Diamond3.0 (Crystal Impact, Bonn,Germany).

Determination of electronic property

In order to obtain the electrostatic potential on van der Waalssurface of a-zearalanol and other analogs, the resultant fileof final calculated structures was submitted to the CAChe Pro7.5

wileyonlinelibrary.com/journal/jmr Copyright � 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 815–823

Z. WANG ET AL.

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(CAChe Group, Fujitsu Systems Business of America, CA)which converts the electrostatic potential results into three-dimensional coordinates (Beier and Stanker, 1997). These arethen viewed in the CAChe Visualizer. The van der Waals surfaceis approximated by plotting an electron isodensity surface at0.002 e/A3. The electron density isosurface, colored by electro-static potential, is generated for optimized geometry of haptenmolecules by DFT at B3LYP/ 6-31G* level. The wavefunction ofmolecules have been computed by DFT.

Molecular descriptors

The steric descriptors, electronic descriptors, hydrophobicdescriptors, and geometry parameters, including moleculesurface (S), lipophobicity index (Log P), molecular polarizability,molecular mass, hydration energy, high occupied molecularorbital (HOMO) energy, low unoccupiedmolecular orbital (LUMO)energy, etc., of these hapten molecules were calculated byHyperchemProfessional 8.0 package (Hyperube Inc, Gainesville, FL).

Table 1. ELISA-determinated antibody recognition ability and structure of molecules tested

Haptens Structure IC50 (ng/ml) Cross-reactivities

a-zearalanol 3.0 100

b-zearalanol 8.0 36

a-zearalenol 5.5 53

b-zearalenol 433.3 0.67

Zearalanone 3.9 74

Zearalenone 5.4 54

Diethylstilbestrol >3000 <0.1

J. Mol. Recognit. 2011; 24: 815–823 Copyright � 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr

HAPTEN–ANTIBODY RECOGNITION STUDIES

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Correlation analysis

Pearson Correlation in SPSS software was used to find relation-ship between molecular descriptors of a-zearalanol analogs andrecognition ability of monoclonal antibody (IC50 values).

RESULTS AND DISCUSSION

Molecular recognition ability determined by ELISA

Competitive ELISA was used for the analysis of a-zearalanol,b-zearalanol, a-zearalenol, b-zearalenol, zearalanone, zearalenone,and diethylstilbestrol. The recognition abilities, expressed by IC50

values, of the antibody 1H9B4 toward these haptenmolecules areshown in Table 1. It can be seen that the antibody showedsignificantly varied recognition abilities to a-zearalanol,b-zearalanol, a-zearalenol, b-zearalenol, zearalanone, and zear-alenone although these molecules are structurally similar butdiethylstilbestrol. It is not surprising that the antibody 1H9B4cannot recognize diethylstilbestrol molecules (IC50 values>3000 ng/ml) since the chemical structure of diethylstilbestrolis significantly different from that of a-zearalanol by viewing themolecular scheme shown in Table 1. The antibody proved tobe most sensitive to a-zearalanol that was used as haptencoupled to BSA for producing monoclonal antibody. The IC50value required for a-zearalanol is only 3.0 ng/ml, while it showed

Figure 1. Models of the minimum energy conformations of the a-zearalanol and other analogs. For concision, the hydrogen atoms are hided. The

elements are represented in the following manner: red, oxygen; off-white, carbon. The double bonds are represented by dot line.


Z. WANG ET AL.

818

similar recognition ability to zearalanone (IC50¼ 3.9 ng/ml).The structural difference between a-zearalanol and zearalanonelies only in the substitute group at position 7 (Table 1). The similarrecognition ability of antibody 1H9B4 to these two compoundsindicated that the replacement of the hydroxyl group by theketone group at position 7 has limited effect on the recognitionof antibody to antigen. The possible reason may be the fact thatthe hydroxyl group on a-zearalanol had been modified andlinked to carrier protein. The moiety very close to protein cannotbe recognized by animal immune system. The structure ofa-zearalanol and zearalanone is identical except the substitutegroup at position 7, so it is easy to understand the similarantibody recognition ability. For b-zearalanol and zearalenone,1.8-fold and approximately 2.7-fold lower recognitionabilities were observed, respectively, as compared to that ofa-zearalanol. The concentrations that cause 50% inhibition were5.4 and 8.0 ng/ml, respectively. The presence of double bond atposition 11–12 is believed to contribute to the relatively lower

recognition ability of antibody 1H9B4 to zearalenone based onthe experimental results. The influence of double bond onantibody–hapten recognition was once again proved byinspecting the IC50 value of antibody to a-zearalenol. Thecomparison of antibody recognition ability to zearalenone(5.4 ng/ml) and a-zearalenol (5.5 ng/ml) also supports the aboveconclusion, suggesting that the diversity of group at position 7,in this case where the hydroxyl group replaces the ketone group,is not detrimental to antibody recognition ability to some extent.As noted previously, an approximately 2.7-fold lower recognitionability to b-zearalanol compared to that of a-zearalanol wasobserved. Apparently, the chiral characteristic of b-zearalanol toa-zearalanol resulted in the decrease in antibody recognitionability. The joint effect of changing the chirality at position7 and replacing single bond by double bond at positions 11–12can be evaluated by comparing the IC50 values for b-zearalenol(433.3 ng/ml) to that of a-zearalanol (3.0 ng/ml) exhibited hugedeleterious to molecular recognition.

Figure 2. The electron density isosurface colored by electrostatic potential is mapped on the van der Waals surface of the minimum energy

conformations of the a-zearalanol and other analogs, which approximated by plotting an electron isodensity surface at 0.002 e/A3. Red coloring indicatesthe most positive areas, blue the most negative areas, and white indicates relatively neutral areas.



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It was known that the recognition of antibody with haptenis dependent on the molecular shape, defined by the geometry,and on low-energy interaction such as hydrogen bonding,hydrophobic interaction, and electrostatic (Galve et al., 2000).Inspection of the two-dimensional structures of a-zearalanol andanalogs could not provide future information to explain the ELISAdata and is difficult to truly state which force mostly contributesthe antibody recognition with these compounds tested. Furtherstudies using computational chemistry and Pearson Correlationanalysis were undertaken.

Steric complementary

The global lowest energy conformations of a-zearalanol andother five structurally similar analogs were calculated by the DFTmethod at the B3LYP/ 6-31 G* level and are shown in Figure 1. Tobe concise, all hydrogen atoms of hapten molecules are hiddenand only the backbone of each hapten molecule is viewed. In therecognition process of antibody to hapten, the first event thatoccurs should be that hapten moves into the pocket of antibodysurrounded by functional groups of peptide residues; therefore,the conformation of hapten molecules is clearly important forantibody–hapten recognition. It can be seen from Figure 2 thatthe conformation of all hapten molecules is located in the samemanner. The common moiety of all haptens but dihydroxyben-zene is in the same plane, so this part of hapten molecules couldbe ignored when evaluating the effect of conformationcomplementary on recognition of antibody–hapten. The diversityof conformation exactly lies in the macrocycle moiety as dictatedin Table 1 from position 1 to position 12. The only differenceidentified while comparing the conformation of a-zearalanol tothat of b-zearalanol is the orientation of O18. The O18 atom ofa-zearalanol bends up about 608 from the plane of dihydrox-ybenzene while that of b-zearalanol bends up about 1208, whichmay responsible for the 2.7-fold decrease in antibody recognitionability. The importance of orientation of O18 can also be observedin other three structurally similar hapten analogs but zearala-

none. Although without considering bond value of O18, thesimilar orientation of that atom in a-zearalanol and zearalanoneexerted the similar antibody recognition ability. The alignment ofcarbon atom on the macrocycle of molecules showed limitedinfluence on hapten–antibody recognition since the similaralignment of carbon atoms has no corresponding similarantibody recognition ability such as the pair of a-zearalanoland b-zearalanol, and pair of b-zearalenol and zearalenone.Except for dihydroxybenzene moiety, the O1 and O3 on eachhapten molecules also keep the similar orientation that wasspeculated to be responsible for hapten–antibody recognitionin diversity extent. However, the antibody recognition tob-zearalenol is found to hardly be similar to the conformationbetween b-zearalenol and zearalenone, except for the orientationof O18. Though not sufficient to complete a stable hapte-n-antibody complex, the complementary shapes of the haptenand the antibody, supported by the conformation of diethyl-stilbestrol in the study, are necessary factors for a hapten and anantibody to interact.

Electrostatic potential on van der Waals surface

The electrostatic potential calculations displayed on van derWaals surfaces of global lowest energy conformation for haptenmolecules are shown in Figure 2 (in the same orientation as forFigure 1). In Figure 2, the van der Waals surface gives theinformation about shape and volume of hapten molecules,whereas, the electrostatic potential describes the potentialenergy of a proton placed at a point near the molecule. The mostpositive potential energy is represented by red areas on themolecules, and these areas are repulsive to a proton. The mostnegative potential energy is represented by blue areas on themolecules, and these areas are attractive to a proton. The valueof electrostatic potential above 0.01 e but below 0.03 e isindicated by yellow color and counter-value in the range of0.01–0.03 e is indicated by white-blue. The value of color

Table 2. Physical and chemical indexes of a-zearalanol and analogs

Haptens V S R Log P P m H MW D18–14 D18–16

a-zearalanol 937.37 601.85 90.38 1.16 34.31 2.9499 52.51 322.40 11.18 9.37b-zearalanol 925.51 597.57 90.38 1.16 34.31 0.9922 54.12 322.40 9.56 7.99a-zearalenol 903.64 583.54 91.50 0.90 34.12 3.2033 52.68 320.39 7.99 6.47b-zearalenol 916.33 644.55 91.50 0.90 34.12 4.208 50.39 320.39 9.35 7.21Zearalanone 909.49 593.31 89.35 1.37 33.76 3.4748 113.07 320.39 10.05 9.33Zearalenone 905.71 603.74 90.47 1.10 33.57 5.6308 111.13 318.37 8.10 6.44

Table 3. Quantum chemical indexes of a-zearalanol and analogs (e)

Haptens QO1 QO3 QO14 QO16 QO18 QC11 QC12 QC17 EHOMO ELUMO DE

a-zearalanol �0.4326 �0.4929 �0.6257 �0.6153 �0.6187 �0.2388 �0.3347 �0.4067 �12.297 �1.706 10.591b-zearalanol �0.4346 �0.4910 �0.6262 �0.6155 �0.6129 �0.2374 �0.3352 �0.4071 �11.988 �0.887 11.101a-zearalenol �0.4753 �0.4830 �0.6256 �0.6132 �0.6588 �0.0885 �0.1590 �0.3931 �12.252 �2.388 9.864b-zearalenol �0.4275 �0.4938 �0.6267 �0.6147 �0.6223 �0.0709 �0.1646 �0.4037 �12.190 �2.148 10.042Zearalanone �0.4088 �0.5095 �0.6254 �0.6117 �0.4218 �0.2544 �0.3338 �0.4040 �11.722 �1.808 9.914Zearalenone �0.4201 �0.5000 �0.6295 �0.6155 �0.4277 �0.0919 �0.1465 �0.4120 �11.882 �2.539 9.343


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boundary is also shown in Figure 2. The conformation in Figure 1can provide information concerning the underlying chemicalstructure of molecules; the electrostatic potential on van derWaals surface in Figure 2 shows how the haptenmolecules wouldinteract electronically and shapely with the paratope of antibody.When hapten molecule pocketing into the paratope of

antibody, the antibody would preferentially bind to thedihydroxybenzene moiety of molecules duo to the hydroxylgroup on position 7 is used to conjugate to the carrier protein, itsaffinity depending on how strong the non-covalent interactionare between the hapten molecules and the antibody. It is clearfrom Figure 2 that the left part of each of a-zearalanolcompounds has a strong area of positive potential (shown inred) that is associated with the carbon atoms on benzene ring. Asall a-zearalanol compounds contain the dihydroxybenzenemoiety, the similar contribution to antibody recognition couldbe achieved, resulting in masking the contribution of this partsince the recognition ability data were calculated by using acompetitive ELISA. We speculated that the dihydroxybenzenemoiety had a crucial effect on the interaction betweena-zearalanol compounds and antibody and came to zero levelon the process of competition compared to other leaving moietyof hapten molecules. The presumption can be supported bycomparing the antibody recognition ability for a-zearalanolcompounds to that for diethylstilbestrol that lacking dihydrox-ybenzene but having hydroxybenzene in spite of stericcomplementary were not roundly considered. Upon a simpleinspection of the electrostatic potential of each a-zearalanolcompound (except for the common moiety), most of the valueswere found to range between 0.3 to�0.3 e, corresponding to theyellow, white and white- blue color on molecular shape. Theresults imply that the electrostatic factor may play a limited role inthe antibody recognition to hapten in a competitive system.Although there are red color, representing strong positive area,on zearalanone (face to the author) and zearalenone (on theright), the antibody affinity observed from experiment does notseem to support that the strong electronic effect exists aroundthe area.The three-dimensional molecular modeling of hapten, includ-

ing the global lowest energy conformation and electrostaticpotential displayed on the above conformation, can only providea plausible explanation for the variation in antibody affinity tostructurally similar compounds. However, in most of the cases,the orientations of conformations of molecules are difficult towell align in the same direction, while, the color representing thevalue of electron is hard to distinguish from each other withnaked eye. Comparison of difference of conformation andelectrostatic potential of structurally similar compounds by onlynaked eye is hard to exactly correlate antibody recognitionto molecular characteristics of hapten molecules. In the processof judgment, subjectivity will appear in the scientist’s mind. Forthe better insight into the event of antibody interaction withhapten molecules, the quantitative parameters defining theunique structure of molecule are expected.

Molecular aspect of hapten molecular analysis

Molecular descriptors

It is important to consider appropriate molecular descriptors forthe recognition of antibody to hapten molecule. In this research,three groups of descriptors are generated to provide as completedescription of each molecule as possible.

Table

4.PearsonCorrelationan

alysisformoleculardescriptors

andan

tibodyrecognitionab

ility

(exp

ressed

byIC

50values)

VH

LogP

RMW

Sm

DE

QO1

QO3

QO14

QO16

QO18

QC11

QC12

QC17

D18–14

D18–16

Pearson

Correlation

�0.002

�0.350

�0.545

0.547

�0.107

0.886*

0.252

�0.077

0.120

0.071

�0.060

�0.124

�0.288

0.518

0.408

0.057

�.0014

�0.223

Sig.

(2-tailed)

0.997

0.496

0.263

0.261

0.840

0.019

0.630

0.885

0.821

0.894

0.909

0.814

0.581

0.293

0.422

0.915

0.980

0.671

*Correlationissignificantat

0.05level(2-tailed).



821

As mentioned above, the conformation complementary isfirstly to be considered when antibody binding to hapten.After the geometry of hapten and the binding site of antibody fitwell, other forces can subsequently occur. Geometry parametersare kind of steric ones and have great relationship with themolecular conformations. In the study, three geometryparameters, molecular volume (V), molecular surface (S), andrefractivity (R), are calculated and shown in Table 2. The distanceof atoms can also represent the conformation characteristics ofhapten molecule; thereby, the length of hapten moleculesdescribed by the two atoms at the extremity, D18–14 and D18–16,was given.The recognition between hapten molecule and antibody

mainly happens on the frontier molecular orbital and that nearby.Transition states may form during interaction between thelowest unoccupied molecular orbital energy (LUMO, electronacceptor) and the highest occupied molecular orbital energy(HOMO, electron donor) of the reacting compounds. Therefore,it is significant to study the characters of frontier molecularorbital to confirm the active position of hapten molecules.Also, molecular charge is another important factor to affectthe molecular property. In this research, several electronicparameters were calculated, including EHOMO, ELUMO, energydifference (DE) between HOMO and LUMO. Additionally, thecharge of atoms possible directly contacting with ammineacid residue on the paratope of antibody were calculated suchas oxygen atom at positions 1, 3, 14, 16, 18 and carbon atom atpositions 11, 12, 17 (shown in Table 3).The binding affinity of antibody to hapten has intimate

connection to hydrophobicity of hapten molecules. The hydro-phobicity is also vital character for recognition between antibodyand hapten such as Log P, dipole moment (m), polarizability, etc.All the molecular descriptors selected, might affect the

recognition of antibody to hapten we think, are shown inTables 2 and 3.

Correlation analysis

The correlation between molecular descriptors and antibodyaffinity was calculated by Pearson Correlation Matrix and is

shown in Table 4. As can be seen in Table 4, the parametersrelative to antibody affinity (expressed by IC50 values) include LogP, R, S, and QO11. The Log P has negative relationship withantibody affinity, while R, S and QO11 have positive relationshipwith antibody affinity. Among these parameters, S has the keyrelationship with antibody affinity since the coefficient is 0.8and is significant at 0.05 level (2-tailed). The experimentalobservation agreed that a hapten has to exhibit correct shapecomplementary to the residues exposed towards the bindingpocket of a target antibody. The results also demonstrated thatthe hapten recognition in competitive immunoassay could bequalitatively enucleated by computational chemistry techniquesand correlation analysis.

CONCLUSIONS

One monoclonal antibody toward a-zearalanol was producedand the recognition ability of the antibody to six structurallysimilar analogs was characterized by using competitive ELISA.The geometry optimization, frequency for a-zearalanol analogswere calculated. The optimized structure, electrostatic potentialon van der Waals surface as well as various molecular descriptorswere obtained and used to correlate with antibody recognition toa-zearalanol analogs. The results indicate that the conformation,hydrophobicity, and electrostatic of hapten have joint influenceon the recognition of antibody and hapten and the conformationof hapten is the most important factor in the competitiveimmunoassay. The method used in the paper is simple butunpublished and can assist in understanding the recognition ofantibody to hapten molecule in a competitive system.

Acknowledgements

This work is supported by the grants from National NaturalScience Foundation of China (No.30901086), State Key Programof National Natural Science of China (No. 30830082), and theProgram for Cheung Kong Scholars and Innovative ResearchTeam in University of China (No. IRT0866).

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hapten–antibody recognition studies in competitive immunoassay of α-zearalanol analogs by...

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