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Shigella boydii enzyme-linked immunosorbent assay kit

An enzyme-linked immunosorbent reagent, Salmonella technology, applied in the field of biotechnology and immunology, can solve the problems of cumbersome operation, long detection cycle, screening, etc.

Active Publication Date: 2014-05-28
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of Salmonella choleraesuis mainly relies on the biochemical identification stipulated in the national standard. The disadvantage is that the operation is cumbersome, the detection cycle is long, and it cannot adapt to the screening of a large number of samples.
Immunological detection is the fastest, accurate and stable rapid detection method that has appeared in recent years, but there is no rapid detection product that can be used in detection practice at home and abroad. This patent uses Salmonella choleraesuis as the detection target, and the technology claimed Involving rapid testing products for Salmonella choleraesuis

Method used

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  • Shigella boydii enzyme-linked immunosorbent assay kit
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  • Shigella boydii enzyme-linked immunosorbent assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Preparation of anti-Salmonella choleraesuis monoclonal antibodies Mab05-F10 and Mab05-D10

[0059] 1. Preparation of immunogen and positive standard

[0060] Salmonella choleraesuis (CICC21493) was inoculated on a Sorbitol MacConkey (SMAC) plate, cultured at 37°C for 24h, picked a single colony in buffered peptone water (BPW), cultured at 37°C, 150r / min shaking for 17h, counted, added 0.3% Formaldehyde solution was inactivated at room temperature for 1 day. Adjust the concentration of Salmonella choleraesuis (CICC21493) to 5×10 with normal saline 9 CFU / ml was used as immunogen; the concentration was adjusted to 10 with normal saline 8 cfu / ml was used as a positive control standard, and buffered peptone water (BPW) was used as a negative control standard.

[0061] 2. Preparation of monoclonal antibodies

[0062] 1) Experimental animals: Three 8-week-old, weighing about 20g female Balb / c mice were selected as experimental animals.

[0063] 2) Immunization ...

Embodiment 2

[0087] Example 2. Characterization of monoclonal antibodies Mab05-F10 and Mab05-D10

[0088] 1. Monoclonal Antibody Subclass Identification

[0089] 1. Antigen coating: Coat goat anti-mouse secondary antibody IgG+A+M with 0.01M PBS, 50 μl per well, coat overnight at 4°C, discard the liquid in the well the next day, and wash the plate 3 times.

[0090] 2. Blocking: add 200 μl of 1% BSA to each well, and block overnight at 4°C. Pat the board dry the next day without washing it.

[0091] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0092] 4. After washing the plate 4 times, add specific binding rabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, and incubate at 37°C for 1 hour.

[0093] 5. After washing the plate 4 times, add diluted horseradish peroxidase-labeled anti-rabbit secondary antibody IgG (H+L) to each well, and incubate at 37°C for 30 minutes.

[0094] 6. After washing t...

Embodiment 3

[0107] Example 3. The composition, preparation and application of the ELISA kit for detecting Salmonella choleraesuis

[0108] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0109] (1) Antibody pre-coated ELISA plate: Dilute with 0.02M acetate buffer (pH2.0) solution and coat 96-well ELISA plate with anti-Salmonella choleraesuis monoclonal antibody Mab05-F10, 100 μl per well. Incubate overnight at 4°C, block and wash according to conventional ELISA methods.

[0110] (2) Salmonella choleraesuis positive control standard and negative control standard.

[0111] (3) Horseradish peroxidase-labeled anti-Salmonella choleraesuis monoclonal antibody Mab05-D10.

[0112] (4) Enzyme-labeled antibody diluent: 0.01M PBS, pH7.6.

[0113] (5) 10× concentrated lotion: 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% sodium azide, pH 7.4, just dilute the concentrated lotion 10 times before use.

[0114] (6) Chromogenic solution A, and chromogenic ...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting shigella boydii. The kit contains two strains of monoclonal antibodies which can be specifically combined in shigella boydii, one strain is a monoclonal antibody Mab05-F10 specialized as a capture antibody, and the other strain is a monoclonal antibody Mab05-D10 as a detection antibody. A large number of tests confirm that the kit can be specifically and efficiently detect shigella boydii, moreover, has no cross reaction with other 77 kinds of common pathogenic bacteria, and is a pathogenic bacteria detection product with excellent performance.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology, and in particular relates to an enzyme-linked immunoassay kit for detecting Salmonella choleraesuis. Background technique [0002] Salmonella choleraesuis (Shigella boydii) belongs to Shigella group C and is an important food-borne pathogen that can enter the human body through contaminated meat, eggs, fish and other animal products, melon seeds, etc. , causing fever, abdominal pain, tenesmus, and sometimes manifested as systemic poisoning symptoms as toxic dysentery, which becomes chronic if the treatment is not complete. [0003] At present, the detection of Salmonella choleraesuis mainly relies on the biochemical identification stipulated in the national standard. The disadvantage is that the operation is cumbersome, the detection cycle is long, and it cannot adapt to the screening of a large number of samples. Immunological detection is the fastest, accurate and stable rapid dete...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/56916G01N33/581G01N2333/255
Inventor 刘箐张超
Owner UNIV OF SHANGHAI FOR SCI & TECH
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