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Rabbit anti-Hematodinium sp serum and application thereof

An antiserum, whipworm technology, applied in the field of detection of Trichuris ovale, can solve the problems of inability to parasite form and description of existing forms, long time for antibody preparation, etc., and achieves short detection time, high sensitivity and simple operation. Effect

Inactive Publication Date: 2009-11-11
MARINE FISHERIES RES INST OF ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR detection technology can only be used for qualitative research, and cannot explain the shape and existence of the parasite. Therefore, it is particularly necessary to carry out research on other detection technologies for this parasite
Overseas, Field and Appleton conducted research on the detection method of indirect fluorescent antibody on a Hematodiniu sp. Antibody Qualitative Detection Research

Method used

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  • Rabbit anti-Hematodinium sp serum and application thereof
  • Rabbit anti-Hematodinium sp serum and application thereof
  • Rabbit anti-Hematodinium sp serum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-1

[0072] Take Portunus trituberculatus body fluids in the terminal stage of the disease in a culture pond in Taohua, Zhoushan, Zhejiang, centrifuge at 3000r / min for 8min, discard the supernatant, and wash the sediment obtained by centrifugation with 0.1M phosphate buffered saline (PBS) with a pH of 7.4 Three times, then fix with 0.5% formalin solution at 37°C for 1 hour, and then wash three times with 0.1M PBS with a pH of 7.4, and obtain 3.6 ×10 7 The solution of individual worms was repeatedly frozen and thawed 4 times to obtain a suspension of worms.

[0073] Then, New Zealand white rabbits (male, weighing about 2.0 kg) were immunized with the worm suspension obtained above. The immunization was divided into 4 times: for the first immunization, the emulsion was uniformly emulsified with Freund's complete adjuvant and the worm suspension in equal proportions, and the white rabbit was injected subcutaneously at 6 points on both sides of the spine, with a dose of 1 mL; the firs...

Embodiment 1-2

[0075] The rabbit antiserum obtained in Example 1-1 was further purified as follows:

[0076] Get the healthy Portunus crab hemolymph, fix it with 0.5% formalin solution for 1h, wash with 0.1M PBS containing 0.5% calf serum with a pH of 7.4, use this blood cell suspension and the rabbit obtained in Example 1-1 The antiserum was mixed and reacted at 4°C in the dark, and the mixture was mixed continuously during the reaction, and the reaction time was 24 hours. The obtained solution was centrifuged at 3000 r / min for 8 min to remove the precipitate, that is, hemolymph, and the supernatant was collected to obtain purified rabbit antiserum. The titer of the rabbit antiserum antibody after adsorption treatment was measured again by indirect ELISA method, and the titer was 2560. Add 20% glycerol by volume to the purified antiserum, and freeze it at -70°C for future use.

Embodiment 2-1

[0078]Take the body fluid of Portunus trituberculatus in the terminal stage of the disease in a culture pond in Taohua, Zhoushan, Zhejiang, centrifuge at 4000r / min for 10min, discard the supernatant, and wash the precipitate obtained by centrifugation with 0.1M phosphate buffered saline (PBS) with a pH of 7.4 Three times, then fix with 0.5% formalin solution at 37°C for 1 hour, and then wash three times with 0.1M PBS with a pH of 7.4, and obtain 3.6 ×10 8 The solution of individual worms was repeatedly frozen and thawed 4 times to obtain a suspension of worms.

[0079] Then, New Zealand white rabbits (male, weighing about 1.5 kg) were immunized with the worm suspension obtained above. The immunization was divided into 3 times: for the first immunization, the emulsion was uniformly emulsified with Freund's complete adjuvant and the worm suspension in equal proportions, and the white rabbit was injected subcutaneously at 6 points on both sides of the spine, with a dose of 1 mL;...

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Abstract

The invention discloses rabbit anti-Hematodinium sp serum, which is rabbit antiserum which is obtained by first using Hematodinium sp polypide-containing body fluid of an infected crab as an antigen, then processing the antigen to obtain an immunizing antigen which is a Hematodinium sp polypide suspension and finally vaccinating a white rabbit. The invention also discloses application of the rabbit antiserum to a method for indirect fluorescent antibody detection, which is implemented by dripping the rabbit antiserum into a sample to be detected for primary antibody incubation, dripping isosulfocyanic acid fluorescent labeled sheep and rabbit antibodies for secondary antibody incubation and performing microscopic examination after mounting processing. The rabbit antiserum for detecting Hematodinium sp of the invention has strong specificity, high sensitivity and short detection time, and can be used for diagnosing the potential infection and early infection with the Hematodinium sp. The detection method of the invention is simple in operation and performs qualitative and quantitative detection on whether sea food shellfish is infected with the Hematodinium sp.

Description

technical field [0001] The invention relates to the detection of parasites of marine crustaceans, in particular to the detection of vortex vortex. Background technique [0002] Hematodinium is an important pathogenic parasite that harms seawater crustaceans with strong pathogenicity. It can cause disease of many economically important crustaceans such as Norway lobster (N.norvegicus), blue crab (C.Sapidus), white snow crab (C.bairdi), pearl snow crab (C.opilo), and lead to mass mortality. The prevalence of this parasite has been reported in Australia, Scotland, Canada, and the eastern coast of the United States. Because the parasite causes high host mortality, it seriously endangers the production of crustaceans. [0003] Hematodinium sp. is the main parasitic protozoan that has endangered the breeding of Portunustrituberculatu and Scylla serratac in Zhejiang Province in recent years. Widespread and high mortality, bringing huge economic losses to the portunus and blue cra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/533G01N33/53
Inventor 许文军谢建军施慧张静
Owner MARINE FISHERIES RES INST OF ZHEJIANG
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