CN1077030A - An enzyme-linked immunoassay sensitizer and a step determination method - Google Patents
An enzyme-linked immunoassay sensitizer and a step determination method Download PDFInfo
- Publication number
- CN1077030A CN1077030A CN 92102313 CN92102313A CN1077030A CN 1077030 A CN1077030 A CN 1077030A CN 92102313 CN92102313 CN 92102313 CN 92102313 A CN92102313 A CN 92102313A CN 1077030 A CN1077030 A CN 1077030A
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- China
- Prior art keywords
- sensitizer
- enzyme
- antibody
- antigen
- linked immunoassay
- Prior art date
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Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000002965 ELISA Methods 0.000 title abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 23
- 108090000790 Enzymes Proteins 0.000 claims abstract description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000427 antigen Substances 0.000 claims abstract description 14
- 102000036639 antigens Human genes 0.000 claims abstract description 14
- 108091007433 antigens Proteins 0.000 claims abstract description 14
- 239000007791 liquid phase Substances 0.000 claims abstract description 11
- 229920001503 Glucan Polymers 0.000 claims abstract description 10
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 claims abstract description 10
- 108010010803 Gelatin Proteins 0.000 claims abstract description 8
- 229920000159 gelatin Polymers 0.000 claims abstract description 8
- 239000008273 gelatin Substances 0.000 claims abstract description 8
- 235000019322 gelatine Nutrition 0.000 claims abstract description 8
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 8
- 235000011187 glycerol Nutrition 0.000 claims abstract description 8
- 239000007790 solid phase Substances 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 5
- 239000000969 carrier Substances 0.000 claims abstract description 4
- 239000011159 matrix material Substances 0.000 claims abstract description 4
- 239000007787 solid Substances 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 230000001900 immune effect Effects 0.000 claims description 6
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 239000003547 immunosorbent Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000005686 Serum Globulins Human genes 0.000 description 1
- 108010045362 Serum Globulins Proteins 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an a kind of enzyme-linked immunoassay sensitizer and a step determination method.Enzyme-linked immunoassay with sensitizer mainly by glucosan, glycerine and gelatin are formulated by a certain percentage, during mensuration with liquid phase antigen, the liquid phase enzymic-labelled antibody, specific antibody and above-mentioned sensitizer are added in surface of solid phase carriers simultaneously, can disposable generation solid matrix antibody-antigen-enzymic-labelled antibody compound, because the present invention has adopted sensitizer, so quickened the enzyme linked immunoassay process, only can finish the inspection-free survey of enzyme with 20 minutes, and application of sample trace, detect quick, accurate, hurry up, letter is for the clinical medicine medical diagnosis on disease provides a kind of the fastest up-to-date accurate means and method.
Description
The invention belongs to the enzyme linked immunosorbent detection sensitizer, especially use this sensitizer to carry out the enzyme linked immunological single stage method and measure.
Existing enzyme linked immunosorbent detection adopts " two step method " more, and this technology was created by people such as Engvall Perlmann and Weemene in 1966, continued to use more than 20 year, was applied to clinical medical many fields after updating and improving.The experimental principle of enzyme linked immunological (Enzgme Immun Assag-EIA) technology is: specific antigen or antibody can be incorporated into the surface of solid phase carrier under certain condition, and keep its immunocompetence, antigen or antibody are connected the activity that formed bond still can keep its specific immune response and enzyme with enzyme, after bond and corresponding antibody or the antigen combination, can judge according to the color reaction that adds enzyme substrate solution and have or not corresponding immune response, and the proportional relation of the amount of corresponding antibodies or antigen in the depth of color reaction and the sample, course of reaction is seen Fig. 1.This is a kind of not only special but also responsive methods for the diagnosis of diseases, has safety, stable, easy characteristics such as observations, but existing determination techniques complex operation, and each mensuration need 3～5 hours, and susceptibility is undesirable, does not satisfy the requirement of timely diagnosis.
One step of the enzyme linked immunological determination method that the object of the present invention is to provide a kind of enzyme-linked immunoassay sensitizer and use this sensitizer to shorten minute, improves susceptibility and accuracy, simplifies the operation course.
Concrete technical scheme of the present invention is: adopt a kind of sensitizer in enzyme linked immunosorbent detection, this sensitizer is formulated by glucosan, glycerine and gelatin, wherein used glucosan can be glucosan 5000, the amount of allocating into is 53～32%, and the amount of allocating into of glycerine and gelatin is respectively 25～41% and 22～28%; One step of enzyme linked immunological determination method is to add liquid phase antigen, liquid phase enzymic-labelled antibody simultaneously at surface of solid phase carriers, specific antibody and above-mentioned sensitizer, sensitizer concentration is 0.5～4%, and between 35～39 ℃ incubation 10 minutes, the enzyme labeling thing and the trace antigen of washing surplus afterwards, disposable generation solid matrix antibody-antigen-enzymic-labelled antibody (Ab-Ag-Ab-E) compound, and then add chromogenic enzyme substrate.Its reaction mechanism is: this sensitizer uses liquid phase enzyme labelled antibody and liquid phase antigen preferentially to combine in course of reaction and generates the Ag-Ab-E compound:
Ag+Ab ()/() Ab+Ag K＝（AgAb）/（Ag）（Ab）
It is big that the equilibrium constant becomes, and along with the increase of molecular weight, under the effect of gravitation, quickened Ag-Ab-E and moved to solid phase surface, intermolecular attraction F=Q
+Q
-/ ad
2Molecule increases, the quantity of electric charge also increases, acting force also increases like this, quickened insolubilized antibody and liquid phase Ag-Ab-E reaction velocity, thereby entire reaction course is quickened, sensitizer is gone back the characteristics that possess hydrophilic property strengthens in above-mentioned course of reaction, because immunoglobulin (Ig) belongs to hydrophobicity euglobulin class, be little miscible fluid during high concentration behind the purifying, after with sensitizer provided by the invention, because variation has taken place for IgG spatial configuration of molecules and molecule peptide chain, soluble in water, and be clear solution, solubleness increases, thereby helps the carrying out that react, immune precipitation does not take place after adopting the present invention simultaneously, make enzyme labelled antibody have the characteristic of simple substance point, it is one to one with combining of specific antigen, or 1 antigen molecule is in conjunction with 2～10 enzymic-labelled antibodies, possess antibody excess immune response characteristics, so sensitivity is obviously improved.
The present invention compared with prior art has following advantage and good effect: owing to adopted sensitizer in the present invention, so quickened the enzyme linked immunoassay process, the mensuration process that two-step approach need can be finished in 3～5 hours shortens to only wants 20 minutes with regard to achievable single stage method, susceptibility has improved 5～10 times than two-step approach, and application of sample trace, detect quick, accurate, fast, simple, use this sensitizer and can produce the replacement new product of elisa diagnostic kit, for the clinical medicine medical diagnosis on disease provides a kind of renewal means and method more accurately.
Fig. 1 is DASP in the prior art " a sandwich enzyme-linked immune two-step approach synoptic diagram ";
Fig. 2 is DASP of the present invention " a sandwich enzyme-linked immune single stage method synoptic diagram ";
Fig. 3 is the antibody excess reaction.
Embodiment 1
Get 53% glucosan 5000, glycerine 25% makes them be dissolved in to steam and stay in the water, heats to 37 ℃, add 22% gelatin mix the back mixed solution.In this solution, add to rely root peroxidase labelled antibody, the enzyme dilution, stand-by.
Embodiment 2
Get 32% glucosan 5000 and 25% glycerine, be dissolved in the distilled water after, heat to 37 ℃, add 28% gelatin mix the back mixed liquor, following steps are with embodiment 1.
Embodiment 3
Add liquid phase antigen simultaneously at surface of solid phase carriers, the liquid phase enzymic-labelled antibody, specific antibody or antigen and concentration are 0.5% sensitizer, put 37 ℃ of incubation reaction 10 minutes, wash superfluous enzyme labeling thing and trace antigen, obtain solid matrix antibody-antigen-enzymic-labelled antibody (Ab-Ag-Ab-E) compound of disposable generation, add chromogenic enzyme substrate then, take out after 10 minutes, carry out analytical calculation with enzyme micro-plate reader and can obtain measurement result.
Claims (7)
1, a kind of sensitizer with single stage method mensuration enzyme linked immunological is characterized in that it is by glucosan, glycerine and gelatin preparation.
2, sensitizer according to claim 1 it is characterized in that used glucosan can be glucosan 5000, and the amount of allocating into is 53～32%.
3, sensitizer according to claim 1, the amount of allocating into that it is characterized in that used glycerine and gelatin is 25～40% and 22～28%.
4, a kind of one step of enzyme linked immunological determination method is characterized in that adding liquid phase antigen, liquid phase enzymic-labelled antibody, specific antibody and sensitizer simultaneously at surface of solid phase carriers disposable generation solid matrix antibody-antigen-enzymic-labelled antibody compound.
5, determination method according to claim 4 is characterized in that described sensitizer is by glucosan, glycerine, gelatin preparation.
6, determination method according to claim 4 is characterized in that temperature of reaction is 35 °～39 ℃.
7, according to claim 4 or 6 described determination methods, the concentration that it is characterized in that used sensitizer is 0.5～4%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92102313 CN1077030A (en) | 1992-04-03 | 1992-04-03 | An enzyme-linked immunoassay sensitizer and a step determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92102313 CN1077030A (en) | 1992-04-03 | 1992-04-03 | An enzyme-linked immunoassay sensitizer and a step determination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1077030A true CN1077030A (en) | 1993-10-06 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 92102313 Pending CN1077030A (en) | 1992-04-03 | 1992-04-03 | An enzyme-linked immunoassay sensitizer and a step determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1077030A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100338466C (en) * | 2000-08-16 | 2007-09-19 | 科学与工业研究委员会 | A quick test method for ELISA with microwave as medium |
| CN101501495B (en) * | 2005-07-05 | 2013-10-02 | 西托斯科莱顿股份有限公司 | Detection of Rho proteins |
-
1992
- 1992-04-03 CN CN 92102313 patent/CN1077030A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100338466C (en) * | 2000-08-16 | 2007-09-19 | 科学与工业研究委员会 | A quick test method for ELISA with microwave as medium |
| CN101501495B (en) * | 2005-07-05 | 2013-10-02 | 西托斯科莱顿股份有限公司 | Detection of Rho proteins |
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|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |