Preparation Of Monoclonal Antibody Against Melamine And Its Application In ELISA

In September 2008, an incident about food safety occurred in China. Baby milk food produced by more companies were contaminated with melamine,which brought a great harm to some people,especially the group of babies. Just the incident, it caught the wide attention for people to the toxicity of melamine after exposure to melamine-contamined milk.Melamine, with a 1,3,5-triazine skeleton, is an organic compound and a trimer of cyanamide, which is widely used in the production of plastics, dyes, fertilizers, and fabrics. Melamine is combined with formaldehyde to produce melamine resin which is a very durable thermosetting plastic used in Formica. If mixed with resins, melamine has fire retardant properties due to its release of nitrogen gas when burned, and has several other industrial uses. Because of melamine's molecule has very high nitrogen content, it can cause false assay results for protein content of food if melamine was added. So melamine was sometimes illegally added to food products in order to increase the apparent protein content of the food . Ingestion of melamine may lead to reproductive damage, bladde, chronic kidney inflammation, nephrolithiasis and even bladder carcinoma.Before 2007, pet food been recalled, melamine had not routinely been monitored in food, so no melamine tests have been conducted. This could be due to the previously low toxicity of melamine assumed, and the relatively expensive methods of detection. Traditional tests of protein levels such as the Kjeldahl and Dumas were performed to measure the nitrogen content, so the results may be misled by adding nitrogen-rich compounds such as melamine. The existing methods for melamine determination such as liquid chromatography– mass spectrometry (LC/MS) and electrospray ionization methods coupled with mass spectrometry were complicated sample preparation, expensive instruments needed and time consuming. Therefore, to develop a kind of highly efficient and rapid method to detect melamine in food, not only may be applied to market monitor whether the melamine added or not to the food, but also be used for import and export detection, which is quite important.The complete antigen of the melamine were prepared and the McAb against melamine was obtained. An indirect competitive inhibition enzyme-linked immunosorbent assay was developed , and then optimized. It will provide foundations for further studying of the test kits for detecting melamine-contamined food. Melamine, as a kind of hapten, the molecular weight is only 126.12.Glutaraldehyde method was used to make the conjugates of melamine with the carrier protein BSA and OVA. Coupling ratios of the melamine to BSA and OVA were 12:1 and 20:1 respectively.Melamine-BSA was used to immunize 8-week BALB/c female mouse by foot pad immunization method. The serum titer had reached 1:4000 higher before cell fusion. One hybridoma cell line named 3H8 secreting McAb against melamine was selected. The characteristics of the McAb secreted by 3H8 were studied:the titer of ascite was 1: 512000, the subclass and the affinity constant were IgG2b and 2.59×108 M-1.The McAb in culture supernatant secreted by 3H8 was used to develop the ELISA . An ic-ELISA method was developed for the quantitative detection of melamine and the optimized condition as follow: The melamine-OVA was added to microtiter plates at a concentration of 1μg/mL and incubated overnight at 4℃, then blocked it with 100μL/well 0.1M NH4Cl and incubated 1h at 37℃; the culture supernatant (1:200) and varying concentrations of standard melamine 50μL/well respectively were add and incubated 30 min at 37℃; then 100μL/well goat anti-mouse IgG-HRP (1:4000) was added and incubated 30 min at 37℃; 100μL/well OPD was chosen to be enzyme reaction substrate; The stopping solution (2M H2SO4) was added after incubated 15 min in the dark at 37℃. Absorbance values at 490 nm were determined by an enzyme immunoassay reader. A linear dose-response standard curve was prepared by plotting log[melamine] versus inhibiting rate. The regression equation of standard curve was y= -24.531x + 37.862. The correlation coefficient, the limit detection and a linear range were R2 = 0.9937, 0.12 ng/mL and 0.12 ng/mL to 8 ng/mL respectively. Finally, additive reclamation test had been done for food such as milk and eggs which were added to melamine by ic-ELISA developed. The average recovery rate of melamine from milk, egg white and egg yolk were 80.45%, 77.5% and 76.03% respectively. Experiment results showed that the McAb against melamine prepared by this research can be used for detecting melamine-contamined food. It will provide foundations for the development of melamine test kit and colloidal gold test strip.