Enzyme-linked immuno sorbent assay detection method of Skeletonema costatum

An enzyme-linked immunosorbent reaction and detection method technology, which is applied in the direction of analysis by chemical reaction of materials, measurement devices, and material analysis by observing the influence of chemical indicators, which can solve the problem of difficulty in obtaining highly specific antibodies, etc. question

Inactive Publication Date: 2014-03-26
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the double-antibody sandwich ELISA method is mainly used in medical detection and microbiological detection research, and there is no report on the detection of algae cell PCNA protein antigen.
In addition, although the preparation process of algae cell antibody is simple, it is not easy to obtain highly specific antibody

Method used

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  • Enzyme-linked immuno sorbent assay detection method of Skeletonema costatum
  • Enzyme-linked immuno sorbent assay detection method of Skeletonema costatum
  • Enzyme-linked immuno sorbent assay detection method of Skeletonema costatum

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Experimental program
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Effect test

Embodiment 1

[0036]The Scutellaria costatum 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 a conventional f / 2 medium, and the culture conditions are as follows: the light / dark cycle is 12h / 12h, the light intensity is 4000Lx, and the culture temperature is 22°C-25°C.

[0037] The culture solution of Skeletalum costatum in the logarithmic growth phase was filtered with a 0.45um cellulose acetate membrane to obtain the filter membrane, and the volume was recorded. Put the obtained filter membrane into a 5ml centrifuge tube, add 2ml of SD buffer, and ultrasonically break for 2min to obtain the sample solution of Skeletalum costa, mix and emulsify with an equal volume of Freund's complete adjuvant (SIGMA company), and the degree of emulsification should be as complete as possible , the emulsion was used to immunize m...

Embodiment 2

[0039] In this embodiment, several strains of dominant phytoplankton common in Jiaozhou Bay were selected as reference algae, and they were: Chaetoceros curvisetus, Chaetoceros debilis, Chaetoceros gracilis, Skeletonema costatum, Gymnodinium mikimotoi, Gymnodinium sp, Thalassiosira nordenskioeldii, Pseudonitzschia pungens, Nitzschia crescent (Nitzschia closterium) and Navicula membraneacea were isolated from the natural seawater of Jiaozhou Bay, and pure cultures were obtained by repeatedly picking individual algal cells under a microscope. These algae were cultured and harvested as described in Example 1.

[0040] The specific identification of the anti-Skeletalum costa antiserum was carried out by double-antibody sandwich ELISA procedure. All algae were resuspended in 1ml PBS, counted under a microscope, and the same cell volume (3.2×10 6 ) was suspended in PBS (final volume 1ml). Each seaweed was diluted with PBS to form a concentration gradient, and treated as described...

Embodiment 3

[0047] 1. Cultivate and collect Skeletalum costatum by the method described in embodiment 1.

[0048] 2. Take out the prepared ELISA plate of Skeletalum costa, take an appropriate amount of microwell strips, fix them on the bracket, and equilibrate to room temperature (18-25°C) for later use. Add 0-32*10 6 75ul of Skeletalum costa gradient dilution solution of 1 cell, and 25ul of SD buffer of Skeletalum costatum, incubate at 37°C for 30 minutes, add 100ul of antibody diluent (1:2000), incubate at 37°C for 30 minutes and wash the plate , add enzyme-labeled secondary antibody solution (goat anti-rabbit IgG-HRP, diluted 5000 times), incubate at 37°C for 30 minutes, add enzyme reaction substrate TMB matrix solution after washing the plate, add 2mol / L sulfuric acid to terminate the reaction after incubation for 30 minutes, Read the absorbance at 450 nm for each well on the plate.

[0049] 3. Standard curve: Take the algae liquid of the standard sample of Skeletalum costa in gradie...

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Abstract

The invention discloses an enzyme-linked immuno sorbent assay (ELISA) detection method of Skeletonema costatum. The method comprises the steps of: (a) repeatedly picking individual algal cell to obtain pure culture under a microscope; (b) preparing an ELISA plate; (c) preparing and diluting test reagents: uniformly mixing concentrated washing liquid (if there are crystals, fully dissolve and mix before usage), and diluting the concentrated washing liquid with deionized water (or distilled water) according to the proportion of 1:1, and storing at 4 DEG C for standby; (d) using a double antibody sandwich ELISA method to complete an antibody test, adding a confining liquid and conducting enclosing treatment on removed micropore strips; and (e) detecting the OD value of an enzyme-labeled second antibody participated in the reaction after coloration by using a microplate reader at single wavelength of 450 nm or dual wavelength of 450 nm / 630 nm (use a blank aperture for zeroing when single wavelength determination is employed), recording the results and calculating the concentration. The detection method provided by the invention has the advantages of specificity, sensitivity, high efficiency, easy operation and low cost, and can analyze a large amount of samples at the same time; therefore, the method is an effective means for qualitative and quantitative analysis of target antigens.

Description

technical field [0001] The invention belongs to the technical field of using immunological methods to detect micro organisms in marine environment. Specifically, the present invention relates to an enzyme-linked immunological reaction detection method and a kit for Skeletalum costatum, and the use thereof for detecting Skeletalum costatum. Background technique [0002] Skeletonema costatum is a kind of phytoplankton that widely exists in the world, and it is also one of the common dominant species of dinoflagellates in my country's coastal waters. This species is a common red tide organism in my country's coastal waters. There have been reports of red tides many times, causing a large number of fish deaths, shellfish and human poisoning, and posing a huge threat to marine fisheries and human health. Therefore, it is of great significance to timely, accurately and quickly detect Sclerostetrasperms and keep abreast of its dynamic changes in the ocean. [0003] In taxonomy, S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/78
CPCG01N33/6803G01N33/543G01N2333/405
Inventor 李成峰米铁柱
Owner OCEAN UNIV OF CHINA
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