Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof

An enzyme-linked immunosorbent reagent and immunoglobulin technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of difficult systematization and standardization of technology, unsuitable for on-site inspection and screening of a large number of samples, and cumbersome operation.

Inactive Publication Date: 2013-10-30
贵州谱尼测试技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty of systematization and standardization of the technology used in the above method, complex equipment and cumbersome operation, etc., it is not suitable for on-site inspection and screening of a large number of samples.

Method used

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  • Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof
  • Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof
  • Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of kit components

[0057] 1. Preparation of cloned antibody

[0058] a. Animal immunity

[0059] The immunogen was injected into Balb / c mice at a dose of 100 μg / mouse to produce polyclonal antibody serum.

[0060] b. Cell fusion and cloning

[0061] After the mouse serum determination result is high, the spleen cells are taken and fused with SP2 / 0 myeloma cells at a ratio of 9:1. The cell supernatant is determined by indirect competitive ELISA to screen positive wells. Use the limiting dilution method to clone the positive wells until a hybridoma cell line secreting monoclonal antibodies is obtained.

[0062] The monoclonal hybridoma cell line C-4-2CGMCC No.3110 of porcine immunoglobulin G was obtained after screening. The porcine immunoglobulin G monoclonal hybridoma cell line can produce an unlimited amount of porcine immunoglobulin G-specific antibodies, and the antibody is specific to porcine immunoglobulin G, with a sensitivity of 0.2 mg / L.

[0063] c....

Embodiment 2

[0076] Example 2 Establishment of an enzyme-linked immunoassay kit for detecting porcine immunoglobulin G

[0077] Set up an enzyme-linked immunoassay kit for the detection of porcine immunoglobulin G, which contains the following components:

[0078] (1) Enzyme-labeled plate coated with porcine immunoglobulin G coupled antigen;

[0079] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0080] (3) Porcine immunoglobulin G monoclonal antibody working solution;

[0081] (4) 6 bottles of porcine immunoglobulin G standard solution with concentrations of 0mg / L, 0.2mg / L, 0.6mg / L, 1.8mg / L, 5.4mg / L, 16.2mg / L;

[0082] (5) The substrate color-developing solution is composed of liquid A and B, the substrate color-developing liquid A is carbamide peroxide, and the substrate color-developing liquid B is tetramethylbenzidine;

[0083] (6) The stop solution is 2mol / L hydrochloric acid;

[0084] (7) The concentrated washing liquid is 0.5%-1.0% Tween 20 and 0.1-0.3‰ thimerosal prese...

Embodiment 3

[0086] Example 3 Detection of porcine immunoglobulin G in samples

[0087] 1. Sample pretreatment

[0088] Weigh 20 mg of the sample to be tested and dissolve it in 40 mL of deionized water to make the final solution concentration 0.5 mg / mL. After dissolving, shake and mix well, and then mix it with the diluted sample compound solution at a ratio of 1:49. spare. Take 50μl for analysis.

[0089] 2. Detect with the kit

[0090] Add 50μl of porcine immunoglobulin G standard solution or sample solution to the microwells of the ELISA plate coated with porcine immunoglobulin G antigen, then add 50μl of porcine immunoglobulin G monoclonal antibody working solution, and then add horseradish. Oxidase-labeled goat anti-mouse anti-antibody working solution 50μl, seal the plate with a cover mold, react in a 37°C incubator for 30 minutes, pour out the liquid in the well, add 250μl washing solution to each well, and pour out the liquid in the well after 30 seconds. Repeat the operation to wash t...

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Abstract

The invention provides an enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G, comprising an ELISA plate which is coated with coating antigen, an enzyme label, porcine immunoglobulin G specific antibody working liquid (being contained when the antigen is coated on the ELISA plate and the enzyme label is enzyme labeling antibody or antibody is coated on the ELISA plate and the enzyme label is enzyme labeling antigen), porcine immunoglobulin G standard product solution, substrate color development solution, stop solution, concentrated washing liquid and concentrated complex solution. The invention further discloses a method which applies the enzyme-linked immunosorbent kit for inspecting the porcine immunoglobulin G, and the method comprises the steps of firstly carrying out the pre-treatment on a sample, then using the kit for inspecting and finally analyzing the inspection result. The provided enzyme-linked immunosorbent kit can be used for inspecting the content of the porcine immunoglobulin G in porcine plasma protein powder and other samples, the operation is simple, the cost is low, and the enzyme-linked immunosorbent kit can be monitored on-site and is applicable in screening mass samples.

Description

Technical field [0001] The invention relates to an enzyme-linked immunoassay technique, in particular to an enzyme-linked immunoglobulin kit for detecting porcine immunoglobulin G, which is particularly suitable for the determination of porcine immunoglobulin G in porcine plasma protein powder. technical background [0002] As a new type of protein feed, plasma protein powder has the characteristics of comprehensive nutrition, easy digestion and absorption, good palatability, and rich in immunoglobulins. It is widely added in piglet feed. According to the source of blood raw materials and processing methods, there are mainly the following categories: porcine plasma protein powder (spray-dried plasma protein, SDPP), low ash porcine plasma protein powder (LAPP), sow plasma protein powder (SDSPP) and bovine plasma protein powder (SDBP), the effect is roughly the same, of which SDPP is more and the most commonly used, so in general, plasma protein powder refers to SDPP. [0003] A lar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577G01N33/535
Inventor 万宇平高东微王雅静马孝斌刘中勇蔡克周余厚美陈永红李明
Owner 贵州谱尼测试技术有限公司
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