Preparation Of Lateral Flow Test Strip Based On Colloidal Selenium For Melamine Detection

Melamine (MEL) is a typical polar organic triazine compound, and it is usually used as an industrial chemical. It is released that several events of MEL being criminally added to milk, milk powder and other food products due to its high nitrogen content, thus leading to several poisonous powder events in China. Excess MEL intake can cause serious health problems, and there is an urgent need to develop a rapid, sensitive and cost effective detection approach for MEL detection, especially for onsite and in-home testing.Although many methods have been utilized to detect MEL, they need to be operated professionally with special equipment and costly, and can not be performed on site and in home. The only MEL detection device currently available for on-site test and in-home test is a one-step test strip based colloidal gold. But colloidal gold are expensive in raw material and the preparation process is relatively complicated. Considering the drawbacks of current methods for MEL detection, the objective of this study is to explore a lateral flow test strip based on colloidal selenium for MEL detection.Firstly, the artificial antigen of MEL combined with BSA was synthesised by carbodiimide, and proved by MS/MS spectrum. The ratio of MEL to BSA is 18-21:1 by calculating the 253.5 nm absorbance determination of MEL, BSA and BSA-MEL, artificial antigen purity is electrophoresis purity grade by SDS-PAGE measurement.Four hybridoma cell strains were obtained by cell fusion and sub-clone after limited dilution method, All the cell strains can secrete anti-MEL monoclonal antibody (McAb) with isotype of IgGl. After purified by Protein A affinity chromatography, the purity measured by SDS-PAGE is electrophoresis purity grade, the affinity constant of McAb is 3.58×109 L/mol and was regarded as the high affinity antibodies. Furthermore, there was no cross-reaction with homologs cyanuric acid, ammeline and ammelide.Selenium nanoparticles (Se NPs) were synthesized by reducing H2SeO3 with ascorbic acid, and the gum arabic (GA) powder as a stabilizing agent. In our experiment, Se NPs were evaluated under transmission electron microscope. Our results showed that the average diameter of spherical Se NPs was 57.97±6.84 nm, and there was no colloidal selenium particle agglomeration can be found. It is suggesting that GA can keep the colloidal selenium particles stably. Such Se NPs can be resuspended completely, thus meeting the needs of preparing colloidal selenium-protein probes.Based on color reaction and the gradient test, three factors affected assay performance were optimized. The optimal pH of gold-labeled McAb and selenium-labeled McAb, the optimal McAb amount and the optimal concentrations of coating on nitrocellulose membrane was 8.5,5 ug/mL,1.0 mg/mL, respecitively.Lateral flow test strip based on colloidal selenium and anti-MEL McAb was prepared for MEL detection. The maximum permitted concentration of MEL has been set at 2.5 mg/kg for both adult and animal feed, and 1.0 mg/kg for infant food by the World Health Organization. The limit of detection of the resultant test strip for MEL is 0.15 mg/kg in liquid milk,1.0 mg/kg in milk powder and 0.8 mg/kg in animal feed. No cross reactions with homologs cyanuric acid, ammeline and ammelide were found. In addition, the MEL test strip has excellent stability even after storage for one year at room temperature in desiccator. Our results demonstrated that the colloidal selenium MEL test strip can be conveniently, rapidly and sensitively used for MEL detection in adulterated milk products or animal food. Therefore, it could meet the needs for rapid detection of MEL.In contrast with colloidal gold, the colloidal selenium was easy to be prepared, and it is insensitive to electrolytes. Moreover, it is more economical in raw material costs.