Monoclonal Antibody-Based Immunoassayfor Analysis of Octopamine in Housefly

Yujing Liu, Zhen Cao, Liang Zhang, Yongdan Li, Guiyu Tan, Baomin Wang, and Xiwu Gao1

Octopamine (OA) is one of the biogenic monoamines in the housefly, which acts as an important neurohormone inthe physiological process of this pest. In this study, a new hapten of OA was synthesized via aldol condensation.With the hapten, monoclonal antibodies (MAb) were generated and their characterizations were investigated. Anindirect competitive enzyme-linked immunosorbent assay (icELISA) based on MAb 3C11-E3 was established,which required simple sample pre-treatments and had low cross-reactivity with OA structural analogise. The halfmaximal inhibition concentration (IC50) and the detected range (IC20-IC80) of the icELISA were 128 ng/mL and12-1438 ng/mL, respectively. Average recoveries of OA ranged from 73 to 129% in the housefly.

Introduction

D iscovered in the salivary glands of octopus,

Octopamine (OA, 1-(p-hydroxyphenyl)-2-aminoetha-nol; Fig. 1) is one of the biogenic monoamines in the housefly(Musca domestica). It acts as a neuromodulator, neurotrans-mitter, and neurohormone, modulating almost every physi-ological process in the invertebrate’s peripheral organs, senseorgans, and central nervous system (CNS).(1) Although theOA is a strong polar molecule and presents at a low con-centration in the complex matrix, the distribution and contentof OA are of great physiologically significance to agriculturalinsects.

Nowadays several techniques have been used for OAanalysis in agricultural insects. High-performance liquidchromatography (HPLC) is one of the most frequently usedtechniques.(2–6) Other instrumental analysis methods, such asgas chromatography conjugated negative ion chemical ioni-zation mass spectrometry(7) and micellar electrokinetic cap-illary chromatography,(8) were also applied to detect OA ininsect tissues. In addition, a radioenzymic assay was used byDavenport and Wright to measure OA in moth.(9) Barron andcolleagues used a radioisotope assay to analyze the distri-bution and dynamics of OA in honeybees treated with 3H-octopamine.(10)

Although the methods mentioned above are highly sensitiveand accurate, they are expensive, time-consuming, and requirecomplicated sample preparation. Enzyme-linked immunosor-bent assay (ELISA) is one of the fastest, most sensitive, se-lective, and cost-effective analysis methods. It requires arelatively simple sample preparation. Both monoclonal anti-bodies (MAbs) and polyclonal antibodies (PAbs) against OAhave been reported for immunohistological studies in insect

tissues.(11–14) However, the MAbs and PAbs reported hadrelatively lower specificity to OA and have not been used forquantitative analysis. In the present study, a new MAb (3C11-E3) against OA was produced with a newly synthesized hap-ten. An indirect competitive ELISA (icELISA) based on3C11-E3 was developed and optimized for the analysis of OAin the housefly.

Materials and Methods

Hapten synthesis and conjugate preparation

The hapten OA-CBA (4-((2-hydroxy-2-(4-hydroxyphenyl)ethylamino)methyl)benzoic acid) was synthesized via aldolcondensation(15) (Fig. 1). Briefly, OA (200 mg) and NaOH(42 mg) dissolved in 1 mL methanol were added to a solutionof 4-CBA (158 mg) and NaOH (42 mg) dissolved in 5 mLmethanol. The mixture was incubated at 60�C for 4 h withstirring to obtain a yellow color. Thin-layer chromatographywas used to monitor the reaction progress. After an ice bath,NaBH4 (50 mg) was added to the reaction product and themixture was stirred for another 30 min. While the yellow colorfaded, concentrated HCl was added drop-wise to remove theunreacted NaBH4 and the solution was adjusted to pH 5.0.Acidification of the solution resulted in precipitation of a whitesolid, which was subsequently centrifuged, washed withmethanol, and recrystallized. The crystal (OA-CBA) wasdissolved in DMSO and analyzed with 1H-nuclear magneticresonance (1H-NMR) spectra. The chemical shift was relativeto 1H-NMR (300MHz, DMSO): 2.89 (m, CH2, 2H); 3.16 (s,OH, 1H); 4.25 (s, CH2, 2H); 4.91 (m, CH, 1H); 6.00 (s, OH,1H); 6.73 (d, aromatic-H, 2H); 7.12 (d, aromatic-H, 2H); 7.67(d, aromatic-H, 2H); 7.95 (d, aromatic-H, 2H); 9.48 (m, NH;COOH, 2H).

College of Agronomy and Biotechnology, China Agricultural University, Beijing, China.

MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPYVolume 33, Number 4, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/mab.2014.0005

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OA-CBA was conjugated with BSA and OVA (Fig. 1) viathe active ester method.(16) To a solution of hapten OA-CBA(10 mg) and NHS (6 mg) dissolved in 1 mL DMF, 8.6 mgDCC were added. The mixture was gently stirred at roomtemperature for 1 h and then centrifuged at 10,000 g for10 min. An aliquot of 250mL and 255 mL of the supernatantwas slowly added into BSA solution (30 mg in 2.0 mL car-bonate buffer) and OVA solution (20 mg in 2.0 mL carbonatebuffer) in 1 h, respectively. After the two solutions werestirred overnight at 4�C, the conjugate products OA-CBA–BSA and OA-CBA-OVA were dialyzed and then diluted to1 mg/mL.

Method of immunization

The protocol of immunization was similar to that previ-ously described.(17) Five female BALB/c mice, 6 weeks old,were initially injected with 100mg of OA-CBA-BSA emul-sified with an equal volume of complete Freund’s adjuvant.Two subsequent injections were carried out at 2-week in-tervals using Freund’s incomplete adjuvant. One week afterthe third injection, mice were eye-bled and sera were testedvia ELISAs. The best-performing mouse was selected forhybridoma production and boosted with 100 mg of OA-CBA-BSA 3 days before fusion.

Antibody preparation

MAbs were prepared by fusing mouse spleen cells andmurine SP2/0 myeloma cells according to the procedurepreviously described.(18) The hybridoma supernatants weretested by ELISAs for positive clones. The resulting clonewas expanded in mice for ascites production and the MAbwas purified by ammonium sulphate precipitation. The

immunoglobulin isotype was determined with a mouseantibody isotyping kit. The titer was determined by indi-rect ELISA.

Establishment of icELISA

A 96-well microtiter plate was coated with 100 mL/well ofOA-CBA-OVA in carbonate buffer at 37�C for 3 h. Afterbeing washed with 200mL/well of PBST three times, 50 mL/well of analytes or standard in PBSTG were pipetted, fol-lowed by the addition of 50mL MAb solution diluted inPBSTG. The plate was incubated at 37�C for 0.5 h and thenwashed with PBST as above. 100mL of the goat anti-mouseIgG-peroxidase conjugate in PBSTG were added to each wellfollowed by incubation at 37�C for 0.5 h. After being washedagain, 100mL/well of OPD substrate solution were added.The reaction was stopped by adding 50mL of 2 M H2SO4.Absorbance was read at 492 nm. All data were analyzed byOrigin 8.0 software.

Cross-reactivity studies

The cross-reactivity (CR) of the established icELISA assaywas determined against several OA analogise, includingadrenaline, 5-hydroxytryptamine, dopamine, norepinephrine,and tyrosine. CR was calculated as shown in Equation (1):

CR (%)¼ IC50(OA)

IC50(Tested chemical)· 100 (1)

Spiking tests

Laboratory-maintained houseflies were used for recoverystudies. An amount of 500 mg adult housefly sample was

FIG. 1. Reactions schemes used to synthesize the OA derivatives and conjugates. OA-CBA was used as the hapten; OA-CBA-BSA and OA-CBA-OVA were used as immunogen and coating antigen, respectively.

276 LIU ET AL.

ground, and then divided into aliquots of 20 mg. OA stocksolution was diluted to 20, 40, 80, 160, and 320mg/mL inPBS, and then 10 mL of the standard solutions were added tothe aliquots of ground sample, respectively, followed bygentle vortexing. A housefly sample without OA added wasused as blank controls. After being dissolved in 1.0 mL ofPBS, each sample was extracted at 4�C for 0.5 h, and sub-sequently centrifuged at 8000 rpm for 10 min. Each super-natant was detected with icELISA in triplicate.

icELISA analysis of housefly samples

Adult houseflies (resistant and sensitive strains to Imida-cloprid, Malathion, Propoxur, Spinosad, and Beta-cyperme-thrin) were weighed and ground. The following extractionand detection of OA was conducted as the spiking tests.

Results and Discussion

Hapten synthesis and hapten-protein conjugation

OA is a small (MW 153) and simple molecule (Fig. 1),making it difficult to obtain specific and high affinity anti-body as hapten. It presents in insects at a relatively low level,which represents a challenge for OA hapten design to obtainantibodies to develop a sensitive ELISA. Although we triedto synthesize hapten with glutaradehyde as previously de-scribed,(12) the antibodies produced have high affinity againstglutaradehyde-OA, but low affinity against free OA. Thishapten synthesization method only provoked antibody suit-able for immunocytochemistry study, but not for ELISA todetect free OA. In the present study, hapten OA-CBA wassynthesized via aldol condensation. It contained a terminalcarboxyl acid for conjugation with carrier proteins. Then weconjugated OA-CBA with BSA and OVA via the active estermethod as immunogen and coating antigen.

Monoclonal antibody generation and characterization

After the third immunization, blood was obtained frommice to determine titers and inhibition against OA. Themouse producing antisera of the best inhibition was then usedto collect spleen cells for hybridoma cell production. Fourclones secreting MAbs against OA were obtained after lim-iting dilution. One clone, designed as MAb 3C11-E3, withthe best inhibition with OA was expanded for ascites pro-duction. The titer of the ascites was 1:16,000. MAb 3C11-E3is an IgG1 isotype.

icELISA development and evaluation

Optimum concentrations of the coating antigen (0.25mg/mL), purified MAb (50 ng/mL), and the IgG-HRP conjugate(1 mg/mL) for icELISA were screened by checkerboard ti-tration. Figure 2 shows a standard curve of OA by the icE-LISA under the optimized conditions. The IC50 value of theicELISA was 128 ng/mL and calibration range (20–80% in-hibition) was from 11 to 1438 ng/mL of OA. The specificity ofMAb was evaluated with OA analogues (Table 1). The resultsshowed high cross-reactivity with adrenaline (440%), whereasthere was little with dopamine (0.4%) and norepinephrine(0.9%), and no cross-reactivity with 5-hydroxytryptamine andL-tyrosine. Although the cross-reactivity with adrenaline washigh, adrenaline has not been found in invertebrates. So the

high cross-reactivity would not affect the analysis of OA inhouseflies. Compared with the reported MAb(11,13) (63% toepinephrine, 21% to tyramine, 8% to norepinephrine, dopa-mine and serotonin showed 0% inhibition), 3C11-E3 showedlower cross-reactivity with structural analogues, but polishedwith non-conjugated OA.

The housefly samples were extracted at 0.5, 1.0, 2.0, and4.0 h, respectively, to detected OA content for optimal re-coveries. The icELISA data show that there was no differencein recoveries with an extraction time between 0.5 h and 4 h.So in the following analysis, we used the extraction time of0.5 h. Table 2 shows that average recoveries of OA fortifiedin houseflies ranged from 73 to 129%. In addition, we forti-fied OA in German cockroach with the same concentrations;the average recoveries ranged from 82 to 104% by icELISA.

icELISA analysis of housefly samples

We used the established icELISA to determine OA contentin houseflies of resistant or sensitive strains to Malathion,Propoxur, Imidacloprid, Spinosad, and Beta-cypermethrin,all of which are widely used insecticides, the latter threeacting as insect neurotoxins. The result (Fig. 3) showed thatOA was more in the sensitive strain of houseflies to Imida-cloprid, Spinosad, and Beta-cypermethrin compared to re-sistant strain houseflies. But there was no difference in thetwo strains of houseflies to Malathion and Propoxur. In otherwords, the resistant strain houseflies may strengthen resist-ibility partly by reducing the content of OA. According to aprevious report, OA could induce the increase of cyclicadenosine monophosphate (cAMP) content in insects, andthen the cAMP would act as a second messenger to furtherregulate physiological responses.(19) The lower content ofOA in a resistant strain housefly would control the cAMPlevel to make the housefly less sensitive to the treatment ofsome neurotoxins. We used the female and male housefliesfor further studies, respectively. The results indicated that thecontents of OA in the resistant strain (2.20 ng/mg in femalehousefly, 3.18 ng/mg in male housefly) were still lower thanthat in the sensitive strain (4.89 ng/mg in female housefly,4.72 ng/mg in male housefly) after being treated with

FIG. 2. Typical icELISA standard curve of inhibition byOA based on MAb 3C11-E3.

MAb-BASED IMMUNOASSAY FOR OCTOPAMINE IN THE HOUSEFLY 277

Spinosad. We also assayed the OA in the head and body ofmale and female houseflies, respectively. The conclusion wasconsistent with Roeder’s research(1); more OA was present inhousefly heads (13.58 ng/mg in female housefly, 15.86 ng/mgin male housefly) than in their bodies (5.50 ng/mg in femalehousefly, 5.16 ng/mg in male housefly).

Conclusions

To our knowledge, this is the first report describing an OAhapten synthesization method via aldol condensation. TheMAb produced by OA-CBA-BSA had higher affinity to OA

than that previously published and was successfully used todevelop an icELISA for the analysis of OA in the housefly.The IC50 value of icELISA was 128 ng/mL. The workingrange (20–80% inhibition) was from 11 to 1438 ng/mL. TheMAb 3C11-E3 had weak cross-reactivity with other meta-bolic products in insects. Spiking tests of OA in housefly

Table 1. Cross-reactivity of Octopamine and Analogues

Analytes Structures IC50 (ng/mL) CR (%)

Octopamine 128 100

Adrenaline 28.9 440

Norepinephrine 14,300 0.9

Dopamine 33,400 0.4

5-Hydroxytryptamine >1,000,000 -

L-Tyrosine >1,000,000 -

CR, cross-reactivity.

Table 2. Recoveries of Octopamine Spiked

in Housefly and German Cockroach

Samples Determined by icELISA

Average recoveries (%) – SDa

Concentrationsspiked (ng/mL) Housefly German cockroach

0.0 0.0 0.0100 85 – 11 -200 73 – 15 104 – 7400 96 – 5 104 – 10800 129 – 20 93 – 51600 104 – 6 82 – 9

aData were averages of three determinations.FIG. 3. Contents of OA in resistant and sensitive strainhouseflies.

278 LIU ET AL.

samples showed that the developed icELISA was a quick,reliable, and sensitive method.

Acknowledgments

The authors would like to thank Professor Qingxiao Li(Department of Molecular Biosciences and Bioengineering,University of Hawaii) for valuable suggestions.

Author Disclosure Statement

The authors have no financial interests to disclose.

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Address correspondence to:Guiyu Tan

College of Agronomy and BiotechnologyChina Agricultural UniversityNo.2 Yuanminyuan West Road

Haidian DistrictBeijing 100193

China

E-mail: tanguiyu@126.com

Received: January 7, 2014Accepted: April 22, 2014

MAb-BASED IMMUNOASSAY FOR OCTOPAMINE IN THE HOUSEFLY 279