CN1892224A - Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison - Google Patents
Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison Download PDFInfo
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- CN1892224A CN1892224A CN 200510081658 CN200510081658A CN1892224A CN 1892224 A CN1892224 A CN 1892224A CN 200510081658 CN200510081658 CN 200510081658 CN 200510081658 A CN200510081658 A CN 200510081658A CN 1892224 A CN1892224 A CN 1892224A
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- losing
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- shellfish poison
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Abstract
An enzyme-liked immunodetection reagent box can rapid sensitively detecting red tide toxins Amnesic Shellfish Poisoning (ASP) in environmental sample. It utilizes synthetic high polymer ratio antigen DA-KLH, through many times immunity BLAB/c mouse, cell fusion, screening, induced ascites and protein column purification to obtain monoclonal antibody of idiocratic anti domoic acid (main constituent of Amnesic Shellfish Poisoning), selecting 1 : 1000 coat proportion and 1 : 8000 antibody dilution, developing out indirection competition enzyme-liked immunity analysing Amnesic Shellfish Poisoning (ASP) reagent box with sensitivity to 10 ng/ml.
Description
Affiliated technical field
The present invention relates to a kind of monoclonal antibody of anti-losing the memory of property shellfish poison of utilizing and combine with losing the memory of property shellfish poison antigen, and the quick and convenient detection kit of the losing the memory of property shellfish poison content in the analyzing and testing environmental sample.
Background technology
The analytical approach of losing the memory of property shellfish poison has biological mouse method, high performance liquid chromatography and immuno-chemical method at present.Biological mouse method is that the extract with biological tissues such as algae or shellfishes is injected in the mouse peritoneal, and according to mouse diing time, the reference standard table checks in the content of losing the memory of property shellfish poison, and this method error is big, poor reproducibility, time-consuming trouble; High performance liquid chromatography is that extract with biological tissues such as algae or shellfishes is after pre-treatment, carrying out liquid chromatography separates, in conjunction with ultraviolet or mass detector, contrast through value with standard items, calculate the content of losing the memory of property shellfish poison, this method is accurate, ripe, and the different isomeride of energy Analysis and Identification, but shortcoming is the pre-treatment trouble, the instrument costliness, need special analytical technology personnel, be not suitable for the analysis of scene and batch samples; Immuno-chemical method is to make specific antibody (polyclonal antibody or monoclonal antibody) by immune animal, utilize the association reaction of antibody to losing the memory of property shellfish poison, make detection kit according to enzyme-linked immune analytic method, the analyzing and testing that is used to losing the memory of property shellfish poison, this method is easy, quick, can analyze batch samples simultaneously, external finished product kit on sale, but cost an arm and a leg.
Summary of the invention
For the scene that solves losing the memory of property shellfish poison, fast, the problem analysis of batch samples, present technique invention provides a kind of kit of losing the memory of property of indirect competitive enzyme-linked immunosorbent analysis shellfish poison of homemade, the monoclonal antibody development that utilizes anti-losing the memory of property shellfish poison, price than the cheap 5-10 of similar import reagent box doubly, be suitable for the scene, fast, the detection of batch samples.
The present technique invention solves the technical scheme that its technical matters adopted: use the water-soluble carbodiimide method, the principal ingredient domoic acid (DA) of losing the memory of property shellfish poison is coupled on the hemocyanin (KLH), add the adjuvant immunity BALB/c mouse, getting immune mouse spleen cell and SP2/0 myeloma cell merges, the cell line of the screening anti-domoic acid monoclone antibody of stably excreting (MAb-DA), enlarged culture, induce ascites in the mouse body, Protein G post purifying, make the monoclonal antibody of anti-domoic acid, wrap by 96 hole ELISA Plate with synthetic domoic acid-ovalbumin (DA-OVA), add DA standard items or sample, add Mab-DA again, envelope antigen DA and the antibody of DA competition freely Mab-DA, DA and antibody complex are by flush away freely, the antibody Mab-DA that combines with envelope antigen DA closes with two resistive connections of enzyme labeling again, develop the color through substrate, cessation reaction, measure the light absorption value (OD) in each hole with microplate reader, the OD value is big more, DA content is few more freely in the sample, the equation that reference standards returns, the content of calculation sample DA.
The present technique beneficial effect of the invention is: the positive cell that utilization obtains can make the monoclonal antibody of anti-domoic acid in a large number, with low cost, the low price of the kit of being developed, and the sensitivity of this kit can reach 10ng/mL.
Embodiment
Method with excessive carbodiimide and DA and KLH reaction, make the DA-KLH conjugate of high molecule ratio, repeatedly immunity, obtain the good splenocyte of immunity, after the fusion, should detect as early as possible, screen the strong cell line of secretory antibody ability to obtain good antibody, select suitable bag by ratio (1: 1000) and antibody dilution (1: 8000), to obtain best detection sensitivity (10ng/mL).
Claims (1)
- A kind of kit of fast simple losing the memory of property of analyzing and testing red-tide toxin shellfish poison, the removable 96 hole ELISA Plate of having wrapped detected antigen are arranged in packing box, concentrate washing lotion, specific detection antibody, ELIAS secondary antibody, losing the memory of property shellfish poison standard items, dilution, developer, stop buffer, the standard items that it is characterized in that sample to be analyzed that by specification makes and serial dilution are added in different Kong Zhongyu respectively and detect antibody responses, through hatching, washing, order and ELIAS secondary antibody, colour developing liquid and stop buffer reaction, on microplate reader, record the light absorption value in each hole, the light absorption value regretional analysis of standard items is obtained equation, calculate the content of toxins in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510081658 CN1892224A (en) | 2005-07-06 | 2005-07-06 | Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510081658 CN1892224A (en) | 2005-07-06 | 2005-07-06 | Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1892224A true CN1892224A (en) | 2007-01-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510081658 Pending CN1892224A (en) | 2005-07-06 | 2005-07-06 | Enzyme-linked immuno-detection reagent case for red-tide toxin memony-losing shellfish poison |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1892224A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949923A (en) * | 2010-09-16 | 2011-01-19 | 上海交通大学 | Enzyme-linked immunization kit of domoic acid and detection method thereof |
| CN102539746A (en) * | 2011-12-26 | 2012-07-04 | 上海交通大学 | Reagent kit for fast detecting domoic acid and application method thereof |
| CN102680693A (en) * | 2012-05-18 | 2012-09-19 | 上海交通大学 | Domoic acid collaurum immunochromatographic test strip and preparation method thereof |
-
2005
- 2005-07-06 CN CN 200510081658 patent/CN1892224A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949923A (en) * | 2010-09-16 | 2011-01-19 | 上海交通大学 | Enzyme-linked immunization kit of domoic acid and detection method thereof |
| CN102539746A (en) * | 2011-12-26 | 2012-07-04 | 上海交通大学 | Reagent kit for fast detecting domoic acid and application method thereof |
| CN102680693A (en) * | 2012-05-18 | 2012-09-19 | 上海交通大学 | Domoic acid collaurum immunochromatographic test strip and preparation method thereof |
| CN102680693B (en) * | 2012-05-18 | 2015-10-14 | 上海交通大学 | The preparation method of domoic acid colloidal gold immuno-chromatography test paper strip |
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