ИСТИНА

Determination of Levofloxacin (levorotatory stereoisomer of Ofloxacin) in milk by heterogeneous enzyme-linked immunoassay Shanin1 Ilya, Nguyen2 Thi Dieu Thuy, Eremin1 Sergei 1 – M. V. Lomonosov Moscow State University; 2 - Institute of Biotechnology Vietnamese Academy of Science and Technology. numenor-08@mail.ru Now medicine is unthinkable without synthetic antibacterial substances. In practice widely used sulfonamides, penicillins, cephalosporins, fluoroquinolones, etc. Also, drugs are often based on these substances for veterinary use, whereby they are found in foods (meat, milk, etc.). Currently fluoroquinolones have more than 20 substances and is constantly updated. Some substances in this group (enrofloxacin, ofloxacin, ciprofloxacin, etc.) are used in the medical treatment of cattle and small ruminants, poultry, fish and pigs. In Russia the amount of fluoroquinolones in milk according Regulation 2.3.2.1078-01 put the MRL 100 ng / ml. Furthermore, there are guidelines for milk, according to which only detect physiologically active substances. So, Ofloxacin - a mixture of two stereoisomers: levo - physiologically active and dextrorotatory - physiologically inactive. Therefore it is only necessary to determine Levofloxacin (levo stereoisomer). It should be noted that the specification of fluoroquinolone drugs have indications that their use is not recommended at the time the osteoarticular system, and the elderly. Therefore, it necessitates control of these antibiotics in food and in milk. Physico-chemical methods of analysis are quite long, expensive and unproductive in contrast to the immunochemical, which are based on highly specific antigen-antibody reaction. One of the most sensitive immunochemical method is an enzyme immunoassay. For determination of Levofloxacin we selected format of in-direct competitive heterogeneous enzyme-linked immunosorbent assay (ELISA). During the development of immunochemical techniques the one of the major steps is the obtaining of specific antibodies. Fluoroquinolones - low molecular weight substances (haptens), and additional step are needed - the synthesis of conjugates with bovine serum albumin (BSA) for amplification of immunogenicity of the antigen. Californian rabbits were used for production of polyclonal antibodies for Levofloxacin. Immunization is carried out subcutaneously in the 15-20 locations by freshly emulsion composition: 1-3 mg conjugate LEV-BSA, 1.0 mL Freunds adjuvant, 1.0 ml of water for injection. As coating antigens the conjugates LEV-OVA and GAR-OVA were synthesized for development homologous and heterologous ELISA format, respectively. The detection limits and range of detection of Levofloxacin for homologues ELISA were 0,01 and 0.01 to 10 ng / mL, respectively. The detection limits and range of detection of Levofloxacin for heterologous ELISA were 0,001 and 0.001 to 10 ng / mL, respectively. It means that heterologous ELISA using GAR-OVA as coating antigen and anti-LEV antibody could in 10 times improve the sensitivity for assay. The preparartion of antibody with high affinity and specifisity is difficulty and the search of new approaches is done for replacement of traditional antibodies to so named synthetic antibodies. One of the promising structures are molecular imprinting polymers (MIPs), a feature of which is the simplicity and stability of MIP-reagents and the ability to perform the assay in organic solvents. Acknowledgement The research was supported by grant of Russia (RFBR 13-03-93000) and Vietnam (VAST.HTQT.Nga.03/13-14) “Development of a new ELISA method using imprinted polymers as antibody for detection of drug residues in food”.