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 dete

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
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Example Embodiment

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

[0035] (1) Preparation of polyclonal antibodies against botulinum toxin type A

[0036] Mix 200μg of purified rBoNT / A protein with an equal volume of Freund's complete adjuvant to immunize New Zealand purebred white rabbits for the first time. Two weeks later, a booster immunization with 0.5 mg protein and an equal volume of Freund’s incomplete adjuvant; three weeks later, another booster immunization with 0.5 mg protein and an equal volume of Freund’s incomplete adjuvant; every week thereafter, the ear vein Blood was collected and the titer was determined 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]After collecting blood from the heart or carotid artery or groin vein, place it at 37°C for 1 hour and overnight at 4°C to shrink the blood clot. Car...

Example Embodiment

[0041] Example 2. Preparation of monoclonal antibodies to botulinum toxin type A

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

[0043] Type A hybridoma cells are prepared by the Institute of Biological Sciences, Hebei Academy of Sciences. After obtaining the hybridoma cells, first carry out cell culture. After the cells are cultured, the indirect ELISA method is used to determine whether the cell culture supernatant is bound to the natural toxin. Choose two The hybridoma cell lines that are identified as positive and have good cell growth are screened for subclones. Then two cell lines with higher stability and titer were selected to prepare a large amount of monoclonal antibodies. Use the cell culture supernatant to identify the subtype of monoclonal antibodies, using the Mouse Monclonal Antibody Isotyping Kit from Roche, USA.

[0044] One week after Balb / c mice were sensitized by abdominal injection of paraffin oil, the mice were injected with 0.5 ml of normal sa...

Example Embodiment

[0047] Example 3 Test paper for detection of botulinum toxin type A

[0048] Refer to Figure 1, the reaction support is a 6.2cm×0.4cm PCV plate; the absorbent pad is a 2cm×0.4cm oil filter paper; a 1.8cm×0.4cm nitrocellulose membrane is successively coated with goat anti-rabbit IgG, type A botulinum toxin Polyclonal antibody (1mg / ml, diluent is PBS); Containing 0.4cm×0.4cm colloidal gold-labeled type A botulinum toxin monoclonal antibody (16.8ug, diluted with 5mM PB), glass fiber membrane; gold-labeled antibody ( The standard gold PH is 7.5, the concentration is 0.2mg / ml) The protective film is 2.7cm×0.4cm glass fiber; that is, a type A botulinum toxin antigen test paper is formed.

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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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