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Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit

A detection kit and a technology for Streptococcus nialis, which are applied in the field of specific serological detection of fish blight, can solve the problems of insufficient sensitivity, complex multi-antibody components, poor specificity, etc., and achieve strong specificity, high detection sensitivity, and titer. high effect

Active Publication Date: 2014-12-10
福建省淡水水产研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex components of tilapia immunoglobulin (IgM) rabbit polyantibody, there are problems of insufficient sensitivity and poor specificity in the detection of specific antibody in tilapia immune serum

Method used

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  • Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit
  • Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit
  • Tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Preparation of tilapia IgM monoclonal antibody cell line 2H5 / B5 with preservation number CCTCC NO:C201277

[0028] 1. Tilapia Serum Preparation

[0029] Blood was collected from the heart of healthy tilapia, centrifuged at 5000 r / min for 10 min, and the supernatant was obtained as tilapia serum.

[0030] 2. IgM separation and purification

[0031] After the tilapia serum was precipitated with 20% PEG 6000, it was subjected to HiPrep 16 / 10 Sephacryl S-200 HR gel filtration chromatography to collect the components of the first peak, refined by Hitrap IgM Purification, and analyzed by SDS-PAGE electrophoresis. After purification, relatively pure IgM was obtained, and SDS-PAGE showed 75kD and 25kD bands, as shown in the attached figure 1 .

[0032] 3. Immunization of Balb / c mice

[0033]Five 8-week-old Balb / c female mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.), emulsified purified tilapia IgM with an equal volume of Freund's complete adjuv...

Embodiment 2

[0044] Tilapia Streptococcus agalactiae IgM Antibody Capture ELISA Kit

[0045] 1. Composition of Tilapia Streptococcus agalactiae IgM Antibody Capture ELISA Kit

[0046] Coat the Tilapia IgM monoclonal antibody 2H5 / B5 with ELISA plate, SIP fusion protein, HRP enzyme-labeled SIP fusion protein rabbit polyclonal antibody, antibody diluent, washing solution, chromogenic solution, stop solution, positive control and negative The control group was assembled into an ELISA kit.

[0047] (1) ELISA plate coated with tilapia IgM monoclonal antibody protein

[0048] Tilapia IgM McAb was diluted with phosphate buffer PBS (pH7.2) to a concentration of 20 μg / mL, added to the reaction wells of a 96-well enzyme plate, 100 μL per well, and coated overnight at 4 °C; phosphate buffer PBS ( pH 7.2) + 0.1wt.% Tween-20 (PBST), wash three times for 2 min each time; add 120 μL of 5% bovine serum albumin prepared in PBS to each reaction well, and block at 37°C for 3 h; Wash with PBST, 2 min ...

Embodiment 3

[0092] Determination of Sensitivity and Specificity of Tilapia Streptococcus agalactiae IgM Antibody Capture ELISA Kit

[0093] 1. Sensitivity determination

[0094] 1000 ng of Tilapia Streptococcus agalactiae-specific IgM antibody samples were diluted into a series of gradients, and detected by using the Tilapia Streptococcus agalactiae IgM antibody capture ELISA detection kit to analyze its effect on Tilapia Streptococcus agalactiae Detection sensitivity of specific IgM antibodies.

[0095] The sensitivity detection results of the Tilapia Streptococcus agalactiae IgM antibody capture ELISA detection kit for the specific IgM antibody of Tilapia Streptococcus agalactiae are shown in Table 3, and the sensitivity is 16.12 ng.

[0096] Table 3. Sensitivity determination of kits for specific IgM antibodies against Streptococcus agalactiae in tilapia

[0097]

[0098] Note: "+" means positive; "-" means negative

[0099] 2. Specificity Determination

[0100] Streptoc...

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Abstract

The invention discloses a tilapia streptococcus agalactiae IgM antibody capture ELISA detection kit. An anti-tilapia IgM specific monoclonal antibody is secreted from an anti-tilapia IgM hybridoma cell strain 2H5 / B5 assigned the accession number CCTCC No:C201277. The kit includes an ELISA plate which is coated by the monoclonal antibody, an antigen tilapia streptococcus agalactiae SIP fusion protein, an IgG-HRP enzyme marker of an SIP fusion protein rabbit polyclonal antibody, an antibody diluent, a washing solution, a coloring reagent, a stop solution, a positive control and a negative control. The anti-tilapia IgM specific monoclonal antibody is high in titer and specificity. The kit is simple in operation, is high in sensitivity, is strong in specificity, can be used in detection of a tilapia streptococcus agalactiae IgM antibody and has a quite high application value.

Description

technical field [0001] The invention belongs to the technical field of specific serological detection of fish diseases, and in particular relates to a tilapia Streptococcus agalactiae IgM antibody capture ELISA detection kit and a special monoclonal antibody. Background technique [0002] Streptococcal disease is listed as three types of diseases in my country, and it is one of the important pathogenic bacteria of fish and one of the pathogenic bacteria of zoonosis. In recent years, tilapia streptococcosis has caused large-scale outbreaks in tilapia farming in Fujian, Guangdong, Guangxi, Hainan and other provinces, causing serious economic losses. Numerous studies have shown that Streptococcus agalactiae ( Streptococcus agalactiae ) is the causative agent of tilapia streptococcosis. [0003] Vaccine immunization is an effective method for the prevention and control of fish diseases. Immunoglobulin is an important immune effector molecule in the humoral immune response of f...

Claims

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Application Information

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IPC IPC(8): G01N33/96G01N33/577
CPCG01N33/56944G01N33/96
Inventor 吴斌樊海平张新艳郑磊钟全福
Owner 福建省淡水水产研究所
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