Indirect enzyme-linked immunosorbent test method for quantitative detecting red tide hinge algae

An enzyme-linked immunosorbent assay and H. akashiwo technology, applied in the field of quantitative detection of H. akashiwo, can solve the problems of reduced practical application value of antibodies, increased detection process workload, and failure to obtain high specificity, etc., to achieve simple quantitative detection , reduce economic loss, good species-specific effect

Inactive Publication Date: 2005-03-02
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This competitive enzyme-linked immunosorbent assay has high detection sensitivity, but this detection method needs to use more algae cell lysis solution to coat the enzyme plate in advance, so a large number of continuous cul...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Preparation of antibodies for detecting Heterocurvium akashiwo

[0017] The Heterocurvium akashiwo species used in the present invention is provided by the Faculty of Life Science and Technology, Ocean University of China. Pure cultures are obtained by repeatedly picking individual algal cells under a microscope. The medium used is conventional f / 2 medium, and the culture conditions are: light / dark cycle of 14h / 10h, light intensity of 3000Lx, and culture temperature of 18°C-22°C.

[0018] After the Heterocurvium akashiwo culture solution in the logarithmic growth phase was fixed with formaldehyde (final concentration 0.5-1%), the algal cells were collected by centrifugation at 10,000 r / min for 10 minutes, and then washed once with distilled water and twice with phosphate buffered saline solution. The algae cells were collected by centrifugation in each step, and the centrifugation conditions were 10000r / min, 10min. Transfer the algae cells into a 1.5ml cent...

Embodiment 2

[0019] Example 2. Identification of antiserum specificity against Heterocurvium akashiwo

[0020] In this embodiment, several strains of common phytoplankton in my country's coastal waters are selected as reference algae, and they are: Chaetoceros curvisetus, Chaetoceros debilis, Chaetoceros gracilis, Chaetoceros gracilis, and Chaetoceros gracilis. Chaetoceros minutissimus, Skeletonema costatum, Thalassiosira nordenskioldi, Navicula membranacea, Pseudo-nitzschia pungens, Crescent rhomboid Nitzschia closterium, Gymnodinium mikimotoi, Gymnodinium sp., Gymnodinium sanguineum, Alexandrium tamarens, Prorocentrum minimum ), Prorocentrum micans, and Prorocentrum donghaiense are all isolated from natural seawater, and pure cultures are obtained by repeatedly picking individual algal cells under a microscope. These algae were cultured and harvested as described in Example 1.

[0021] According to the indirect enzyme-linked immunosorbent assay step described in the claims, the specific i...

Embodiment 3

[0023] Example 3. Quantitative determination of Heterocurvium akashiwo

[0024] 1. Cultivate and collect Heterocurvium akashiwo by the method described in Example 1.

[0025] 2. Coat the microtiter plate with H. akashiwo cell standard solution containing 0, 38, 192, 960, 4800, 24000, 120000, 600000 cells / ml and the sample solution to be tested, 100 microliters per well, 37°C After incubation for 20 minutes, block with 10% skimmed milk powder solution, incubate at 37°C for 20 minutes, wash the plate, add anti-H. Secondary antibody solution (goat anti-rabbit IgG-horseradish peroxidase, diluted 12000 times), incubate at 37°C for 15 minutes, wash the plate, add enzyme reaction substrate TMB matrix solution, incubate for 10 minutes, add 2mol / L sulfuric acid to stop the reaction , read the absorbance at 450 nm of each well on the plate.

[0026] 3. Standard curve: take the common logarithmic value of the number of algae cells in the standard sample solution of H. 0 ×100, is the v...

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Abstract

The invention discloses a method for measuring indirect enzyme linked immunosorbent assay of red current yiwan alga, which belongs to sea biology technology field. The invention is based on the incrimination of antibody of yiwan alga, at first, the standard sample of yiwan alga and the samples needed to be measured are enclosed by enzyme labeling board, they are sealed with degreased milk powder solution after being hatched in 37deg.C, adds in idiosyncratic red current resisting yiwan alga and serum resisting diluted solution after the board is cleaned, then carries on board cleaning after being hatched in 37deg.C, then adds in enzyme labeling solution, then they are hatched in 37deg.C, adds in enzyme reacting material after the board is cleaned, then the reaction is stopped after the board is cleaned; then uses enzyme labeler to measure the light absorption valve of each aperture on the measuring board, through data conversion, acquires the standard curve of the measured yiwan alga, the linear formula can be acquired with calculation. The invention is quick, convenient, and it overcomes the deficiency in red current yiwan alga measurement.

Description

technical field [0001] The invention relates to a method for quantitatively detecting Heterocurvium akashiwo, in particular to an indirect enzyme-linked immunosorbent assay method for quantitatively detecting Heterocurvium akashiwo. Used in the field of marine biotechnology. Background technique [0002] Heterosigma akashiwo is a common harmful algae widely distributed in the coastal waters of the world. threaten. Therefore, it is of great significance to detect H. akashiwo timely, accurately and quickly, and keep abreast of its dynamic changes in the ocean. Taxonomically, Heterocurvium akashiwo belongs to the genus Heterocurvium of the phylum Xanthophyta. The traditional identification method, that is, to distinguish Heterocurvium akashiwo from the morphology, mainly uses optical microscope to directly observe and count. There are certain difficulties in the operation, and the process is cumbersome, time-consuming, and laborious. When the sample volume is large, the work...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/52G01N33/535G01N33/547
Inventor 李荣秀于志刚亓海刚辛泽毓米铁柱
Owner SHANGHAI JIAO TONG UNIV
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