Hapten synthesis and antibody production for the development of a melamine immunoassay

Lei, Hongtao; Shen, Yu-Dong; Song, Lingguang; Yang, Jin-Yi; Chevallier, Olivier P.; Haughey, Simon A.; Wang, Hong; Sun, Yuan; Elliott, Christopher T.

doi:10.1016/j.aca.2010.03.007

Cited by 79 publications

(56 citation statements)

References 19 publications

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“…23 The approach of Yin et al was to derivatize melamine itself using tert-butyl bromoacetate followed by acid hydrolysis to create a hapten that was also coupled to protein via the carbodiimide reaction. 24 In this study, a polyclonal antibody to melamine was produced using a structural mimic, 6-hydrazino-1,3,5-triazine-2,4-diamine (HTD; see Figure 1) which negated the need for hapten derivatization.…”

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Development of an Optical Biosensor Based Immunoassay to Screen Infant Formula Milk Samples for Adulteration with Melamine

et al. 2011

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The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 μg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.

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Development of an Optical Biosensor Based Immunoassay to Screen Infant Formula Milk Samples for Adulteration with Melamine

et al. 2011

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“…The mixture was stirred at 4°C for 3 h. The orange conjugate solution was centrifuged at 10 000 g for 5 min and the supernatant passed through a Sephadex G-25 column to remove unconjugated DBAP. DBCP was conjugated to BSA or OVA to generate artificial antigen DBCP-BSA or DBCP-OVA by the active ester method according to the method of Lei et al [13]. All the hapten-protein conjugates were characterized by UV-vis spectrometry.…”

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An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

et al. 2011

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BackgroundDibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed.Methodology/Principal FindingsA monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC50 = 106 ng/mL), the direct hapten coated format (IC50 = 14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples.Conclusions/SignificanceThe stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples.

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“…The continuing need for food safety and increasing need for protection against adulteration of food products is putting increased demands on food analysts to develop rapid and novel test methods [28][29][30]. Thus, genomic, proteomic, metabolomic and other foodomic methods will have an important role in the food industry and in public health in ensuring the production of safe and high-quality food in coming years [8,17,31,32].…”

Section: What About the "Developed" Western World?mentioning

confidence: 99%

Food Authenticity and Safety in China: What about the Western World?

Josić¹,

Giacometti²

2014

Peptidomics

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Hapten synthesis and antibody production for the development of a melamine immunoassay

Cited by 79 publications

References 19 publications

Development of an Optical Biosensor Based Immunoassay to Screen Infant Formula Milk Samples for Adulteration with Melamine

Development of an Optical Biosensor Based Immunoassay to Screen Infant Formula Milk Samples for Adulteration with Melamine

An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

Food Authenticity and Safety in China: What about the Western World?

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