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Test paper stripe for detecting botulinum toxin type, preparation and application thereof

A type A botulinum toxin and detection test paper technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of heavy workload, a large number of experimental animals, and long time consumption, and achieve the effect of fast and simple detection

Inactive Publication Date: 2009-07-29
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gold standard for the detection of botulinum toxin is the classic mouse lethal test, and its detection limit can reach 10-20pg, but this test also has many shortcomings, mainly in the long time-consuming, heavy workload, and the need for a large number of experimental animals, etc.
Many laboratories at home and abroad have done a lot of research on serological detection methods, such as enzyme-linked immunoassay (ELISA), radioimmunoassay, endopeptide chain enzyme-linked immunoassay, etc. There are more or less defects in special equipment and detection sensitivity and specificity contradictions, which limit the popularization and application of these methods. So far, no method can really replace the classic animal experiments.

Method used

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  • Test paper stripe for detecting botulinum toxin type, preparation and application thereof
  • Test paper stripe for detecting botulinum toxin type, preparation and application thereof
  • Test paper stripe for detecting botulinum toxin type, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1, Preparation of Botulinum Toxin Type A Polyclonal Antibody

[0035] (1) Preparation of anti-botulinum toxin type A polyclonal antibody

[0036] 200 μg of purified rBoNT / A protein was thoroughly mixed with an equal volume of Freund's complete adjuvant, and New Zealand purebred white rabbits were first immunized. Two weeks later, boost immunization once with 0.5 mg protein and an equal volume of Freund's incomplete adjuvant; after three weeks, boost immunization once again with 0.5 mg protein and an equal volume of Freund's incomplete adjuvant; thereafter, auricular vein every week Blood collection, titer determination by indirect ELISA. If further booster immunization is needed, the interval between each immunization is two weeks, and a large amount of blood is collected one week after the last immunization.

[0037]Place blood at 37°C for 1 hour after blood collection from the heart, carotid artery or inguinal vein, and overnight at 4°C to shrink the blood c...

Embodiment 2

[0041] Embodiment 2, preparation of botulinum toxin type A monoclonal antibody

[0042] (1) Preparation of anti-rBoNT / A monoclonal antibody

[0043] Type A hybridoma cells were all prepared by the Institute of Biological Sciences, Hebei Academy of Sciences. After the hybridoma cells were obtained, the cells were cultured first. The hybridoma cell lines identified as positive and with good cell growth status were screened for subclones. Two cell lines with high stability and titer were selected to prepare large quantities of monoclonal antibodies. The cell culture supernatant was used to identify the subtype of the monoclonal antibody, using the Mouse Monclonal Antibody Isotyping Kit from Roche Company, USA.

[0044] One week after Balb / c mice were sensitized by intraperitoneal injection of paraffin oil, the mice were injected with 0.5 ml of normal saline suspension containing about 1×106 hybridoma cells. After 1-2 weeks, when the abdomen of the mouse became obviously enlarg...

Embodiment 3

[0047] Embodiment 3, the detection test paper of type A botulinum toxin

[0048] Referring to accompanying drawing 1, reaction support is 6.2cm * 0.4cm PCV plate; Water absorption pad is the oil filter paper of 2cm * 0.4cm; The nitrocellulose membrane of 1.8cm * 0.4cm is coated with goat anti-rabbit IgG, type A botulinum toxin successively Polyclonal antibody (1mg / ml, diluted in PBS); containing 0.4cm×0.4cm colloidal gold-labeled botulinum toxin type A monoclonal antibody (16.8ug, diluted with 5mM PB), glass fiber membrane; gold-labeled antibody ( Its standard gold pH is 7.5, concentration is 0.2mg / ml) protective film is the glass fiber of 2.7cm * 0.4cm; That is to form the type A botulinum toxin antigen detection test paper.

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Abstract

The invention discloses detection test paper which comprises a reaction support, an absorbent pad, a nitric acid fiber membrane and a GAB gold-labelled antibody protective film, wherein the nitric acid fiber membrane is coated with an A type botulinum toxin polyclonal antibody band and a quality control goat-anti-rabbit IgG band, and the GAB gold-labelled antibody protective film comprises an A type botulinum toxin monoclonal antibody which is labled by colloidal gold. The invention also simultaneously discloses a method for preparing the test paper and application thereof. Based on a specimen and a piece of test paper, the invention can quickly and easily detect the A type botulinum toxin antigen in the specimen, can capture the A type botulinum toxin once, saves a great deal of manpower and material resources, is convenient, fast, and simple, and does not need special instruments and equipment nor professional training; the invention is clear and easily distinguished in result, is easy to be operated and promoted, is suitable for grass-roots units, batch on-site detection of unexpected events, and epidemiology investigation, and plays an auxiliary role to the infection diagnosis of the A type botulinum toxin.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a type A botulinum toxin detection test strip and a preparation method and application thereof. Background technique [0002] Botulinum neurotoxin (BoNT) is a highly toxic protein toxoid produced by anaerobic Clostridium botulinum, of which types A, B, and E can cause botulism in humans , my country reported that most of the patients caused by botulinum toxin were type A. The molecular weight of each type of botulinum toxin is about 150,000D; it consists of a heavy chain and a light chain linked by a disulfide bond, including a heavy chain (H, about 100kD) and a light chain (L, about 50kD), and the H chain includes the N-terminal (about 50 kD) and C-terminal (about 50 kD), the light chain is linked to the N-terminus of the heavy chain by a disulfide bond. Among them, botulinum toxin type A has the most in-depth research, and its application is also the most extens...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558G01N33/532
Inventor 王静王景林张晓龙杨宇孙晓红谢士嘉
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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