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Inspection of englena

A detection method, the technology of Euglena dinoflagellate, which is applied in the direction of measuring device, color/spectral characteristic measurement, instrument, etc., can solve the problems that the research progress is not large, and the mature technology of qualitative and quantitative determination of algae has not been formed, so as to achieve high sensitivity, detection The output limit is low and the effect of reducing economic losses

Inactive Publication Date: 2005-02-23
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In short, there is not much progress in domestic and international research on this aspect, and a very effective mature technology for qualitative and quantitative determination of algae has not yet been formed.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The dinoflagellate used in the present invention is isolated from a natural seawater sample of Jiaozhou Bay, and a single algae cell is repeatedly picked under a microscope to obtain a pure algae species for cultivation, and the culture medium used is a conventional f / 2 medium. The algal culture solution in the logarithmic growth phase was fixed with formaldehyde (final concentration 1.5-2%), centrifuged at 10,000 rpm for 10 minutes to collect algal cells, and then washed twice with phosphate buffer (PBS, pH7.4). Then transfer the algae cells into Eppendorf tubes, shake off the residual water, resuspend with sterilized PBS, mix and emulsify with an equal volume of Freund's complete adjuvant, and immunize rabbits with the emulsion by subcutaneous injection and intramuscular injection, with a dose of about 10 7 cells / rabbit / time. Immunization was boosted after two weeks interval. The adjuvant used for boosting immunization was Freund's incomplete adjuvant. Other steps re...

Embodiment 2

[0022] to 10 3 ~10 10 Cells / milliliter dinoflagellate cell lysate was coated on the microtiter plate (100 microliters per well), blocked with 1%, 2% or 3% BSA solution after overnight at 4°C, 200 microliters per well, 37°C After incubation for 1 hour, 1.5 hours or 2 hours, wash the plate, add 0-10 8 Dilnodinella gradient dilution (50 microliters) of 1 cell and the sample to be tested (50 microliters), then add antibody dilution (1:10000), 50 microliters / well, and incubate at 37°C for 30, 45 or 60 minutes After washing the plate, add enzyme-labeled secondary antibody solution (horseradish peroxidase-labeled goat anti-rabbit IgG), 100 μl / well, incubate at 37°C for 30, 45 or 60 minutes, add enzyme reaction substrate after washing the plate TMB matrix solution, 100 μl / well, after incubation for 10 minutes, 2 mol / l sulfuric acid was added to terminate the reaction, and the absorbance value at 450 nm of each well on the plate was read with a microplate reader.

[0023] According ...

Embodiment 3

[0027] to 10 3 ~10 4 2 cells / ml of dinoflagellate cell lysate coated enzyme plate (50 microliters per hole), according to the competition ELISA step operation described in Example 2, the competition inhibition curve correlation equation obtained at last is: ln(A / (A 0 -A)=-1.4234*log (number of algae cells)+5.2919, the linear correlation coefficient is R 2 = 0.9654, the linear range of competitive inhibition was between 125,000 and 5,000,000 cells.

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Abstract

A method for detecting Euglena dinoflagellate includes preparing enzyme markplate, using enzyme marker to analyse light absorption value of each hole on the plate, converting obtained data to work out standard curve of quantitative detection for obtaining linear equation, carrying out quantitative determination of Euglena dinoflagellate in tested sample as per obtained equation.

Description

technical field [0001] The invention relates to the technical field of detection of marine microorganisms. In particular, it is a polyclonal antibody that can be used to detect Gymnodinosa and an immunological method for detecting Gymnodinosa based on the antibody. Background technique [0002] Gymnodinium sp. is a kind of phytoplankton that widely exists in the world, and it is also one of the common dominant species of dinoflagellate in my country's coastal waters. This species is a common red tide organism in the coastal waters of my country. There have been reports of red tides for many times, which will cause a large number of fish deaths, shellfish and human poisoning events, and pose a huge threat to marine fisheries and human health. It has been recorded in Jiaozhou Bay. Among more than 40 species of red tide organisms, Gymnodinosa is one of the main species of red tide algae. Therefore, it is of great significance to detect Gymnodinosa in a timely, accurate and rap...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N33/547G01N33/569
Inventor 李荣秀于志刚辛泽毓米铁柱甄毓
Owner SHANGHAI JIAO TONG UNIV
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