Blocking ELISA method for detecting cysticercosis pisiformis cyst fluid antibody of rabbit

A technique for cysticercosis lentiformis and cysticercosis, which is applied in the field of blocking ELISA to detect cystic fluid antibodies of cysticerci lentiformis in rabbits, can solve the problem that cysticercosis lentiformis has not been established, clinical detection cannot be used, and the positive coincidence rate is high. problems, to achieve the effect of increasing the income of raising rabbits, blocking the vicious biological cycle, and purifying the environment

Inactive Publication Date: 2016-03-09
HENAN INST OF SCI & TECH
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Problems solved by technology

The results showed that the cyst fluid of rabbit cysticercosis lentiformis was passed through the column of Sephadex G-200, and the second peak egg self-antigenity was the strongest, the specificity was the best, and the positive coincidence rate was the highest; Cysticercus lentiformis antigens in the cyst fluid; while the antigenicity of the scolex and cyst wall of cysticercosis lentiformis is small, so it cannot be used for the diagnosis of the disease
Since then, Wang Xiaoxia et al. (2009) established an Elisa method for laboratory diagnosis using the crude protein of Cysticercus lentiformis, but the detection rate is low and cannot be used for clinical detection
To date, no serological method for the clinical detection of cysticercosis lentiformis in rabbits has been established

Method used

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  • Blocking ELISA method for detecting cysticercosis pisiformis cyst fluid antibody of rabbit
  • Blocking ELISA method for detecting cysticercosis pisiformis cyst fluid antibody of rabbit
  • Blocking ELISA method for detecting cysticercosis pisiformis cyst fluid antibody of rabbit

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preparation example Construction

[0039] Preparation of hyperimmune serum (primary antibody):

[0040] (1) Immunized animals: 5 healthy female BALB / c mice aged 6 weeks.

[0041] (2) Immune antigen: cyst fluid protein with a molecular weight of 50-100kDa.

[0042] (3) Immune adjuvants: Freund's complete adjuvant and Freund's incomplete adjuvant.

[0043] (4) Immune site: subcutaneous injection at four points on the back.

[0044] (5) Immunization dose: The immune dose for each mouse for the first immunization is 50 μg. 200 μL.

[0045] (6) Immunization method:

[0046] S1. For the first immunization, dilute cyst fluid protein with sterilized PBS, then mix it with Freund's complete adjuvant at a volume ratio of 1:1, and immunize after fully emulsifying.

[0047] S2. The second immunization was carried out two weeks later, and the incomplete Freund's adjuvant was mixed with the diluted cyst fluid protein at a volume ratio of 1:1, and the immunization was carried out after fully emulsifying. After that, immun...

Embodiment 4

[0053] Establishment of a blocking ELISA method for detecting antibody in cystic fluid of rabbit cysticerci lentiformis:

[0054] (1) Determination of optimal antigen coating concentration and serum dilution

[0055] S1. Packing plate: wrap the 96-well PVC reaction plate in the order of row×column=12×8, and wrap the plate with the concentration of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 μg / mL in turn, and use the same concentration for every two rows Coating, 50 μL per well, overnight at 4°C, washed 3 times with PBST, with an interval of 5 minutes between each time;

[0056] S2. Blocking: Block with PBST blocking solution containing 5% skimmed milk powder, 200 μL per well, 37°C, block for 2-4 hours, wash with PBST 3 times, with an interval of 5 minutes;

[0057] S3. Primary antibody reaction: remove the first column, and add 50 μL of PBS to each well, then add 100 μL diluted mouse positive serum to the first well of odd-numbered rows, add 100 μL diluted mouse negative serum to the first...

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Abstract

A blocking ELISA method for detecting a cysticercosis pisiformis cyst fluid antibody of a rabbit is used for early detection and clinical diagnosis of cysticercosis pisiformis of the rabbit, solves the problem that at present, the cysticercosis pisiformis of the rabbit cannot be diagnosed by using a serological method, and is an effective detection method for specificity diagnosis and comprehensive prevention and control of the cysticercosis pisiformis of the rabbit. The blocking ELISA method includes the following three steps of purification of a cysticercosis pisiformis cyst fluid, antigenicity detection of purified protein and establishment of the blocking ELISA detection method. The rapid, sensitive and specific blocking ELISA method is used for detecting the specific cyst fluid antibody in the serum of the rabbit suffering from the cysticercosis pisiformis, is good in result stability, can be commercialized and automatic and can be also used for determination with naked eyes. Therefore, the blocking ELISA method is expected to become an important tool clinically for rapid diagnosis and immune detection of the cysticercosis pisiformis of the rabbit, will certainly play an important role in diagnosis and prevention and control of the cysticercosis pisiformis of the rabbit and has good application prospect.

Description

Technical field: [0001] The invention relates to animal immune detection, in particular to a blocking ELISA method for detecting cystic fluid antibodies of rabbit cysticerci lentiformis. Background technique: [0002] Cysticercus pisiformis in rabbits is a tapeworm saccharosis caused by the larvae of Taenia lentilus Cysticercus pisiformis parasitizing in the liver, omentum, mesentery and abdominal cavity of rabbits. The disease is distributed worldwide, and occurs in all parts of our country, and the condition is serious. According to reports, from Heilongjiang and Liaoning in the northeast, to Qinghai and Xinjiang in the northwest, from Sichuan, Chongqing and Guizhou in the southwest, to Fujian in the southeast and Guangxi in south China, to Henan in central China, Zhejiang and Shandong in east China, etc. Provinces, all have the occurrence of this disease, the incidence rate varies from 5% to 100%, the average infection rate reaches about 40%, and the mortality rate is be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6803
Inventor 潘耀谦潘博李瑞珍孙玉倩银梅唐海蓉张慧辉
Owner HENAN INST OF SCI & TECH
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