Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay for Determination of Dealkylated Hydroxytriazines

Abstract

The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm(-)(1)) and basic pH values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.

Development of a Competitive ELISA for the Evaluation of Sunflower Pollen in Honey Samples

“…Immunochemical techniques such as the proposed ELISA have the advantages of being sensitive, specific, and often provide the necessary limit of detection to analyze directly low concentrations of the target protein. In addition, these techniques have advantages over many current classical methods in speed and cost analysis ( − ).…”

Aproximaciones inmunoanalíticas para el control de xenobióticos y biotoxinas en alimentos

“…Así pues, si bien en todo método inmunoanalítico el anticuerpo constituye un reactivo clave, en el caso de que la sustancia a detectar sea un compuesto de bajo peso molecular, la síntesis de haptenos se considera una etapa crítica por sus enormes implicaciones sobre la afinidad y especificidad de los anticuerpos generados. Es más, la introducción del grupo funcional en la posición deseada con frecuencia solo es posible mediante estrategias de síntesis total que conllevan un esfuerzo experimental considerable y una sólida formación en química orgánica sintética (Sanvicens, Pichon, Hennion y Marco, 2003). Incluso disponiendo de experiencia en esta temática, y pese a los avances que se han producido en los últimos años en técnicas de modelado molecular que posibilitan un diseño más racional de las estructuras más adecuadas con vistas a generar anticuerpos con las características deseadas, sigue siendo difícil predecir cuál de entre todas las alternativas viables será la más idónea.…”

Preliminary Production of Anti- Glufosinate Monoclonal Antibodies

“…Standard curve for computation was generated by using standard substance concentration for X-axis, OD660 for Y-axis; (2) Protein level in the antigen: The Folin-phenol reagent method (Lowry method) was applied for determination; (3) synthetic antigen ultraviolet absorption spectrum scan: OVA, standard Glufosinate, synthetic antigen were measured in UV-spectra and visible-region absorbance on a spectrophotometer; (4) synthetic antigen 31 P NMR scan: synthetic antigen and standard Glufosinate were analyzed on a DRX-400 spectrometer; (5) animal immunize and detection [7]: ① immunize: immunogen (OVA-Glufosinate) 0.5mL and adjuvant were mixed in the same volume for the emulsifier, and the resulted solution was then used to immunize female BALB/c mice (weight 18g ～ 22g). After the third immunization about 7d ～ 10d, blood serum antibody was determined; ② blood serum titer detection [8][9][10]: The serum was assembled, and was then diluted in a serial multiple, at last dilution associated with the envelope antigen. The more that multiple of the antibody serum was diluted, the less antigen and antibody were combined; meanwhile the thinner the enzyme color reaction is, conversely the thicker the color reaction should be.…”