Preparation And Application Of The Monoclonal Antibody For L-hydroxyproline

China government has prohibited the addition of simply hydrolyzed animal proteinfrom solid leather waste into milk. Therefore, it is very important to monitor the possiblyadded solid leather waste in milk. L-hydroxyproline (HP) is a special amino acid inhydrolyzed animal protein. If HP is detected in a milk sample, the sample can bedetermined as the “leather-original-HP added milk”.In order to develop an effective method for detection of HP in milk, HP wasderivatized with p-hydroxybenzaldehyde, acetylsulphanilyl chloride and5-chlorine acidrespectively to synthesize three haptens: HPH, HP1and HP2. The hapten HPH wascoupled with bovine serum albumin and ovalbumin respectively by using ofN,N’-carbonyldiimidazo method to produce immunogen1and coating antigen1. Thehaptens HP1and HP2were also coupled with carrier protein to produce other twoimmunogens and other two coating antigens respectively by using of glutaraldehydemethod and mixed anhydride method. The hapten density in HPH-BSA was about14andwas about12in HPH-OVA. The hapten density in HP1-BSA was about22and was about15in HP1-OVA. The hapten density in HP2-BSA was13and was9in HP2-OVA. Thethree immunogens were all used to immunize Balb/c mice. During multipleimmunizations, serum titers of these mice were determined by indirect ELISA method toevaluate different coating antigen/antibody combinations. The results showed that theimmunogen HPH-BSA had better immunogenicity. Then the spleen cells from the micewith the highest titer were fused with SP2/0myeloma cells to produce hybridomas. Thepositive hybridoma cells were screened by using indirect competitive ELISA and thensubcloned by using the limited dilution method. Finally, three hybridoma cell linessecreting the specific monoclonal antibody for HPH were obtained. The ascites from themice were purified by acrylic acid-ammonium sulfate precipitation method to producethe monoclonal antibody. The antibody of HPH showed low crossreactivity to parentalL-hydroxyproline and showed negligible crossreactivity to D-hydroxyproline and otheramino acids. Milk sample was treated with concentrated sulphuric acid at hightemperature to release free L-hydroxyproline tha was then derivatized withp-hydroxybenzaldehyde for ELISA analysis. The limit of detection for HPH was0.08 μg/mL, and this value as L-hydroxyproline equivalent was0.04μg/mL. The recoveriesfrom L-hydroxyproline fortified blank milk were in the range of88.6%～102.5%withcoefficients of variation4.1%～9.7%. Therefore, the developped ELISA method wassuitable for monitoring the simply hydrolyzed animal protein from solid leather waste inmilk with L-hydroxyproline as the target analyte.