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Development of Immunosensor-based Technique for Diagnosis of Sub-clinical Mastitis in Dairy Cows

Author: DingShouQiang
Tutor: TanXun
School: Zhejiang University
Course: Basic Veterinary Science
Keywords: Dairy cow Recessive mastitis Haptoglobin Immunosensor
CLC: S858.23
Type: Master's thesis
Year: 2010
Downloads: 58
Quote: 1
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Abstract

The cow mastitis serious harm to cows healthy one of the major diseases, including two types of clinical mastitis and recessive mastitis. Clinical mastitis has obvious clinical symptoms, and the diagnosis is relatively easy; recessive mastitis lack of clinical symptoms, and requires the help of the laboratory means to diagnose. Over the years, the clinical diagnosis of recessive mastitis method is mainly dependent on milk somatic cells (somatic cell) count, directly or indirectly, or microbial culture of milk. In recent years, through the detection of milk in the acute phase proteins (acute phase protein, APP) to establish the diagnosis of Subclinical mastitis attention gradually. The haptoglobin (haptoglobin, Hp) is a major APP cattle the substance synthesized by the liver, in normal milk content is very low, so that can not be checked out. In the the cows mastitis occurrence of the process, due to the increased barrier permeability of blood - milk, Hp by blood - milk barrier into the milk. Therefore, the increased content of milk Hp is considered to be one of the hallmarks of cows mastitis. Although commercially available ELISA test kit for the detection of Hp, but the detection process takes a long time, the degree of automation is low and does not meet the requirements of the on-site detection. Immunosensor detection technology developed in recent years, a new type of detection technology, the basic detection principle with the ELISA method similar, but the antigen - antibody reaction signal is amplified by the electrochemical sensing device and output, in order to achieve rapid, sensitive and automated detection. The purpose of this study is to construct the immune sensors detect milk Hp content to provide new technology and methods for the diagnosis of Subclinical mastitis. The content of this study is divided into three parts. The first part of the combination of the current type of globin antibody immune sensor developed in Prussian Blue / o-phenylenediamine / gold nanoparticles modified electrode assembly cattle. The electrochemical three-electrode system as the basic device, a gold electrode as working electrode, a platinum wire electrode as the counter electrode, Ag / AgCl electrode as the reference electrode, by an electrodeposition method to Prussian blue (PB) is deposited on the surface of the gold electrode, the its power-polymerized o-phenylenediamine (o-PDA) to prevent PB leakage, and in turn the nano-gold particles and anti-bovine Hp antibody (concentration of 5.0 mg / L) assembled onto the surface of the gold electrode prepared immunosensor. The optimal incubation time of the sensor working electrode Hp antigen solution for 15min, the the scan base solution optimum pH value of 6-7. Optimum test conditions, the sensor detection sensitivity in 5-10mg / L of Hp. The second part of L-cysteine ??/ gold nanoparticles modified electrode fixed combination globin antibody immune sensor. As the working electrode to the gold electrodes in the three-electrode system, with layers of self-assembly techniques for the L-cysteine, nano-gold particles and anti-bovine HP (concentration of 5.0 mg / L) antibodies are sequentially assembled on the working electrode, the system an immune sensor. Immune sensor made of the Hp linear detection range for 15-100mg / L, the lowest detection limit of 0.63mg / L. Using the sensor and the ELISA test kit to detect the recessive mastitis suffering Hp content in the milk sample, the results show that the test results between the two methods was no significant difference (P gt; 0.05). The third part, immune sensor diagnostics with common recessive mastitis diagnosis method comparison. Selected 20 clinically healthy cows in a dairy farm in the Outskirts of Hangzhou as subjects. Randomly selected from each cow before and after the two mammary collecting milk samples, milk pathogens culture, HMT method, SCC method and constructed in this study L-cysteine ??/ gold nanoparticles / Hp antibody sensor diagnostic recessive mastitis . The bacteriological testing positive, HMT test positive (rating scale of 1-3), the SCC ≥ 50 × 104 / ml and Hp ≥ 3.9mg / L for recessive mastitis diagnostic criteria. The various methods of diagnosis using the chi-square test significant difference analysis, and the Kappa test diagnostic rate analysis. The chi-square test results show that the sensor diagnostic methods derived recessive mastitis positive and negative diagnosis results with the SCC method diagnostic results significantly different (P lt; 0.05), but with the of milk pathogens culture and HMT Act diagnostic results the difference was not significant (P gt; 0.05). These results of the Kappa test results also show that the sensor detection method with SCC Law diagnosis results in line with the lower rate (Kappa lt; 0.40), but with the diagnostic results obtained by the the bacteriological detection method, and HMT detection method consistent with (Kappa gt; 0.40). constructed in this study L-cysteine ??/ gold nanoparticles / Hp immunosensor for the diagnosis of Subclinical mastitis. In summary, this study were used Prussian blue / / gold nanoparticles modified electrode and L-cysteine ??/ gold nanoparticles modified electrode fixed Hp antibody prepared o-phenylenediamine become immune sensors detect Hp antigens, including L-cysteinyl The leucine / gold nanoparticles / anti-Hp immunosensor for the diagnosis of Subclinical mastitis.

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