Development Of Immunosensor-based Technique For Diagnosis Of Sub-clinical Mastitis In Dairy Cows

Bovine mastitis represents the most prevalent disease affecting dairy cattle. The infection is usually classified into clinical and sub-clinical forms. Cows with clinical signs of mastitis are easily recognized, In the contrast, sub-clinical mastitis is difficult to detect due to the absence of any visible indications, and is commonly diagnosed by laboratory analysis.Sub-clinical diagnose always depends on counting the somatic cells (SCC) or culturing pathogenic bacteria in milk. In recent years, the significance of some acute phase proteins (APP) for detection of sub-clinical mastitis was highlighted. Haptoglobin (Hp) is one of the APP, which is synthetized mainly in the liver, and can hardly be detected in normal milk. However, Hp in milk is elevated in response to mammary infecton due to increased permeability of blood-breast barrier. Therefore, increase in milk Hp is thought to be the sensitive indictor of mastitis. Though commercial ELISA kit for Hp detection has been available,, it did not meet the demand for site diagnosis because of time-consuming or much lower automation. Immunosensor was a newly introduced method to detect antigen in samples. Although its detecting pricinple similar to that of ELISA technology, it can amlify and automatically output the antigen-antibody reaction signal by electrochemistry system, resulting in more quick and sensitive detection. The objective of the present study was to develop an immunosensor for analysis of Hp in milk.The experiment contents included three parts. Research of amperometric immunosensor based on prussian blue/o-PDA/gold nanoparticle modified gold electrode for the detection of Hp.The electrochemistry three electrodes system was used as the basic instrument. The gold electrode was defined as the work electrode, the Pt electrode as the relative electrode, and the Ag/AgCl electrode as the counter electrode. The work electrode was assembled in sequence by electrodeposit of PB, voltalization of O-PDA and coat of Hp. PBS (pH=7.0) was used as the scan base solution. The oxidation peak current was acquired by cyclic voltammetry (CV) scan. Optimization for the working conditions of the immunosensor showed that the time for incubation of the working electrode in antigen solution was 15 min, and the pH value of the scan base solution was between 6 and 7. The immunosensor's sensitivity for detection of Hp antigen was between 5 and 10 mg/LThe second part:Research of immunosensor based on L-cys/gold nanoparticles modified glod electrode for the detection of Hp.The gold electrode modified L-cys, glod nanoparticles, anti-Hp body by self-assemble method in three electrode system was the work electrode. The anti-Hp body concentration was 5.0mg/L. Immunosensor detection range for Hp was 15-100mg/L, and lowest detection limit was 0.63mg/L. Results from ELISA and Immunosensor showed that there're no significant differences (P＞0.05).The third part:Comparation of immunosensor and tradional diagonosis methods. 20 cows from a dairy farm in the suburb of Hangzhou city were randomly selected. Bacterial culture, Hangzhou mastitis test (HMT), SCC and the L-cys/GNP/Hp antibody immunosensor were used to diagnose sub-clinical mastitis. Bacterial positive, HMT positive (with score between 1 and 3), SCC≥50×104/L and Hp≥3.9mg/L in milk were set as the according criteria for diagnose of sub-clinical mastitis. The results were analysed using chi-square test and Kappa for significant difference and coincidence, respertivly. The chi-square test showed that the diagnostic results of immunosensor significantly differed from those of the SCC results, but there was no significant difference between the results of immunosensor, bacterial detection and the HMT method. The Kappa test showed high diagnostic coincidence between immunosensor, bacterial culture and HMT method, although there was low diagnostic coincidence between immunosensor and SCC methods. It was concluded that the developed immunosensor in this study can be used to diagnose bovine sub-clinical.Overall, the study of immunosensor was based on prussian blue/o-PDA/gold nanoparticle and L-cys/gold nanoparticles modified gold electrode for the detection of Hp respectively. And the L-cys/gold nanoparticles modified modified gold electrode immunosensor can be used for dairy cow sub-clinical diagnosis.