CN104007102A - Universal solid-phase coated microplate for plate-shaped chemical luminescent immunodiagnosis reagent and preparing method thereof - Google Patents
Universal solid-phase coated microplate for plate-shaped chemical luminescent immunodiagnosis reagent and preparing method thereof Download PDFInfo
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- CN104007102A CN104007102A CN201310055070.2A CN201310055070A CN104007102A CN 104007102 A CN104007102 A CN 104007102A CN 201310055070 A CN201310055070 A CN 201310055070A CN 104007102 A CN104007102 A CN 104007102A
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Abstract
The invention discloses a preparing method of a universal solid-phase coated microplate for a plate-shaped chemical luminescent immunodiagnosis reagent. According to the method, different detection reagents can be used for the same universal coated microplate, the universal coated microplate can be combined with streptavidin and fluorescein, then biotin or fluorescein is marked on coated antibodies of different detection items, and the coated antibodies are indirectly fixed to the universal coated microplate. Coated microplates in the prior need to be produced independently along with different items, or the mark success rate of biotin is low, or fluorescein is low in binding rate, and respective defects exist. After the method is adopted, the mark success rate and the binding rate can be improved to the largest extent, the coated microplates are universal, and production and application convenience is improved.
Description
Technical field
The invention belongs to immune diagnostic technique field, be applied to medical inspection mechanism.
Background technology
The board-like chemiluminescence immunoassay diagnostic kit using both at home and abroad at present generally has two kinds, and a kind of is by the different coated different coated antibodies of Detection of content thing, and then makes different solid-phase coating plates.Every kind of solid-phase coating plate can only correspondence be applied to a kind of test item.Human body can have by the index of immunodiagnosis hundreds of.Therefore wanting all to produce corresponding diagnostic reagent just need to produce hundreds of solid-phase coating plates.This is for producing and applying and all brought complicacy.Another kind method improves: by originally, for directly preparing fluorescein or biotin on the coated antibody mark that is coated with plate, then anti-fluorescein antibody or streptavidin were coated on solid phase plate.Because anti-fluorescein antibody can so indirectly be simplified to the different solid-phase coating plates of original different test items the coated plate of unified anti-fluorescein antibody or the coated plate of streptavidin by specific combined with fluorescent element (or the specific combination biotin of streptavidin meeting).But introduced again new problem here.Because the diversity of the space structure of antibody causes biotin labeling coated antibody success ratio to only have 50% left and right, thus often there will be specificity amino sites on the variable region that antibody is combined with antigentic specificity by biotin in conjunction with losing the ability of being combined with antigen.And fluorescein labelled antibody although to be disulfide-bonded on antibody do not affect the specificity that antibody is combined with antigen, general success ratio is greater than 90%.But the reaction rate of fluorescein antibody and fluorescein only have ten thousand of streptavidin and biotin association reaction speed/.
Summary of the invention
For the defect existing in prior art, the invention discloses the preparation method of the general solid-phase coating plate of a kind of board-like chemiluminescence immunoassay diagnostic reagent, the method can make the general coated plate of same for different detection reagent, greatly reduces the complicacy of production and strengthens the simplicity of applying.
The present invention synthesizes a kind of new albumen in order to prepare a kind of general coated plate, and this general coated plate can be combined with streptavidin and fluorescein.Then biotin or fluorescein on mark on the coated antibody of different test items, be indirectly fixed on coated antibody on general coated plate.Adopt the coated plate before this invention to need to produce separately along with project difference, or biotin labeling success ratio is low, or fluorescein association rate is slow, has respectively shortcoming separately.After employing the present invention, can improve to greatest extent and be marked as power and association rate, can also general coated plate, raising production and application simplicity.
The preparation method of the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent of the present invention comprises the steps:
Step 1: get the streptavidin of 0.5～1.5mg molecular weight 66KDa and the anti-fluorescein antibody of 2.0～2.8mg molecular weight 160KDa is dissolved in the carbonic acid buffer of 0.05～0.15ml, 0.05M, pH9.6, add stirrer standby;
Preferably getting the streptavidin of 1mg molecule and the anti-fluorescein antibody of 2.4mg is dissolved in the carbonic acid buffer of 0.1ml;
Step 2: get glutaraldehyde 0.05～0.15ml of 70% and become 0.5～1.5% concentration standby with 70 times of distilled water dilutings;
Preferably getting glutaraldehyde 0.1ml, to be diluted to 1% concentration standby;
Step 3: add freshly prepared 1% glutaraldehyde solution 0.05～0.15ml in the solution making in step 1, stirring at room 1 hour.Take out reactant liquor and add bag filter, the carbonic acid buffer dialysed overnight to 0.05M, pH9.6, during change liquid 3 times; Requiring bag filter to see through diameter is 160KDa; Add equivalent glycerine to be stored in-15～25 ℃ the reactant liquor after dialysis;
Preferably add glutaraldehyde solution 0.1ml; Preferably add equivalent glycerine to be stored in-20 ℃ the reactant liquor after dialysis;
Step 4: the carbonic acid buffer of newly synthetic coating protein 0.05M, pH9.6 for glycerite is diluted to 1: 1000, and every hole adds 50～150 μ l dilute solutions, 3～5 ℃ of standing over night in 96 hole Chemiluminescent plates; By plank dabber, add solution 150～250 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 0.5～1.5 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing, obtain the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent.
Preferably in Chemiluminescent plate, every hole adds 100ul dilute solution, 4 ℃ of standing over night; Preferably add the solution 200 μ l that contain 1% bovine serum albumin(BSA), 37 ℃ of cappings 1 hour.
The coated plate making by method of the present invention, can, so that no matter manufacturing enterprise produces the board-like chemiluminescence detection reagent of which kind of type, can be used general coated plate.Be conducive to expand the scale of production, reduce semi-manufacture type and batch, minimizing quality inspection step, can improve product stability and quality.
For user, the solid-phase coating plate of all test items is general to be reduced and detects a plurality of projects simultaneously and cause misoperation.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below by specific embodiment, preparation method of the present invention is further described.
Embodiment 1:
Get 1mg streptavidin (molecular weight 66KDa) and 2.4mg anti-fluorescein antibody (molecular weight 160KDa) is dissolved in the carbonic acid buffer of 0.1ml0.05M pH9.6, add stirrer standby.
Getting 70% glutaraldehyde 0.1ml becomes 1% concentration standby with 70 times of distilled water dilutings.
In the solution of streptavidin and the mixing of anti-fluorescein, add freshly prepared 1% glutaraldehyde solution 0.1ml, stirring at room 1 hour.Take out reactant liquor and add bag filter, the carbonic acid buffer dialysed overnight to 0.05M pH9.6, during change liquid 3 times.Requiring bag filter to see through diameter is 160KDa.Add equivalent glycerine to be stored in-20 ℃ the reactant liquor after dialysis..
Newly synthetic coating protein glycerite is diluted to 1: 1000 with the carbonic acid buffer of 0.05M pH9.6.In 96 hole Chemiluminescent plates, every hole adds 100 μ l, 4 ℃ of standing over night, and by plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour.Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing.
Get the carbonic acid buffer that 0.1mg anti-alpha-fetoprotein AFP coated antibody is placed in 0.1ml0.05M pH9.6, add 4 μ g esterification biotins and 4 μ g fluoresceins, stirring at room 1 hour.The carbonic acid buffer dialysed overnight of reactant liquor to 0.05MpH9.6, liquid is changed 3 times in centre.Requiring bag filter to see through diameter is 40KDa.Add equivalent glycerine to be stored in-20 ℃ the reactant liquor after dialysis.
Biotin that previous step is prepared into is 1000 times of label diluted for fluorescein-labelled thing, then add another strain antibody horseradish peroxidase-labeled thing of a certain amount of anti-AFP thing working fluid that serves as a mark.
After adding 50 μ l standard serums or test serum and 100 μ l label working fluids in general coated plate, mix, be placed in 37 ℃ of constant temperature oven incubations 1 hour.Take out after plank washs 5 times and add luminous substrate, detect luminous signal, and then Criterion curve is obtained AFP concentration in test serum.
In above process, in label working fluid, in fact have 2 kinds of AntiAFP antibodies, on a strain antibody mark biotin and fluorescein, be combined and be fixed on Chemiluminescent plate with streptavidin or anti-fluorescein antibody immediately after general coated plate contact.On another strain AntiAFP antibody, connecting horseradish peroxidase, tracer when this is detection.These two antibody form after by incubation: general coated plate-biotin (or fluorescein) is changed the compound of antibody-AntiAFP antibody-AFP antigen-another strain AntiAFP antibody horseradish peroxidase-labeled thing.
Be more than that to take alpha-fetoprotein (AFP write a Chinese character in simplified form in English) detection method be example, similarly method can be applied in carcinomebryonic antigen, on prostate specific antigen etc. test item, as long as wherein a strain antibody is as above processed.
Embodiment 2:
Step 1: get the streptavidin of 1mg molecular weight 66KDa and the anti-fluorescein antibody of 2.4mg molecular weight 160KDa is dissolved in the carbonic acid buffer of 0.1ml, 0.05M, pH9.6, add stirrer standby;
Step 2: get 70% glutaraldehyde 0.1ml and become 1% concentration standby with 70 times of distilled water dilutings;
Step 3: add freshly prepared 1% glutaraldehyde solution 0.1ml in the solution making in step 1, stirring at room 1 hour.Take out reactant liquor and add bag filter, the carbonic acid buffer dialysed overnight to 0.05M pH9.6, during change liquid 3 times; Requiring bag filter to see through diameter is 160KDa; Add equivalent glycerine to be stored in-20 ℃ the reactant liquor after dialysis;
Step 4: newly synthetic coating protein glycerite is diluted to 1: 1000 with the carbonic acid buffer of 0.05M pH9.6; In 96 hole Chemiluminescent plates, every hole adds 100 μ l, 4 ℃ of standing over night; By plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing, obtain the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent.
The beneficial effect that the present invention realizes be combine that two-forty that streptavidin is combined with biotin and fluorescein be combined with anti-fluorescein antibody do not lose specific advantage.Before preparing solid-phase coating plate, in advance by streptavidin and anti-fluorescein antibody coupling in 1: 1, synthetic new albumen.The site that this albumen has simultaneously with biotin and fluorescein are combined.This albumen is coated with and on Chemiluminescent plate, is prepared into general Chemiluminescent plate.Then by biotin and fluorescein on the coated antibody difference mark of original disparity items, when some antibody labeling biotin causes inactivation, just only use fluorescein-labelled.Process so later all in theory test items and can adopt general solid-phase coating plate.Produce like this or apply and all greatly simplified flow process, reduced the probability of makeing mistakes.
More than described the preferred embodiments of the present invention, but it will be understood by those skilled in the art that and do not departing under the prerequisite of design philosophy of the present invention, its various modification or combination are all included in the protection domain of claim of the present invention.
Claims (10)
1. a preparation method for the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent, is characterized in that, described method comprises the steps:
Step 1: get the streptavidin of 0.5～1.5mg molecular weight 66KDa and the anti-fluorescein antibody of 2.0～2.8mg molecular weight 160KDa is dissolved in the carbonic acid buffer of 0.05～0.15ml, 0.05M, pH9.6, add stirrer standby;
Step 2: get glutaraldehyde 0.05～0.15ml of 70% and become 0.5～1.5% concentration standby with 70 times of distilled water dilutings;
Step 3: add freshly prepared 1% glutaraldehyde solution 0.05～0.15ml in the solution making in step 1, stirring at room 1 hour.Take out reactant liquor and add bag filter, the carbonic acid buffer dialysed overnight to 0.05M, pH9.6, during change liquid 3 times; Requiring bag filter to see through diameter is 160KDa; Add equivalent glycerine to be stored in-15～25 ℃ the reactant liquor after dialysis;
Step 4: the carbonic acid buffer of newly synthetic coating protein 0.05M, pH9.6 for glycerite is diluted to 1: 1000, and every hole adds 50～150 μ l dilute solutions, 3～5 ℃ of standing over night in 96 hole Chemiluminescent plates; By plank dabber, add solution 150～250 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 0.5～1.5 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing, obtain the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent.
2. preparation method as claimed in claim 1, is characterized in that, in step 1, gets the streptavidin of 1mg molecule and the anti-fluorescein antibody of 2.4mg is dissolved in the carbonic acid buffer of 0.1ml.
3. preparation method as claimed in claim 1, is characterized in that, in step 2, getting glutaraldehyde 0.1ml, to be diluted to 1% concentration standby.
4. preparation method as claimed in claim 1, is characterized in that, in step 3, adds glutaraldehyde solution 0.1ml.
5. preparation method as claimed in claim 1, is characterized in that, in step 3, adds equivalent glycerine to be stored in-20 ℃ the reactant liquor after dialysis.
6. preparation method as claimed in claim 1, is characterized in that, in step 4, in Chemiluminescent plate, every hole adds 100 μ l dilute solutions, 4 ℃ of standing over night.
7. preparation method as claimed in claim 1, is characterized in that, in step 4, adds the solution 200 μ l that contain 1% bovine serum albumin(BSA), 37 ℃ of cappings 1 hour.
8. a preparation method for the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent, is characterized in that, described method comprises the steps:
Step 1: get the streptavidin of 1mg molecular weight 66KDa and the anti-fluorescein antibody of 2.4mg molecular weight 160KDa is dissolved in the carbonic acid buffer of 0.1ml, 0.05M, pH9.6, add stirrer standby;
Step 2: get 70% glutaraldehyde 0.1ml and become 1% concentration standby with 70 times of distilled water dilutings;
Step 3: add freshly prepared 1% glutaraldehyde solution 0.1ml in the solution making in step 1, stirring at room 1 hour.Take out reactant liquor and add bag filter, the carbonic acid buffer dialysed overnight to 0.05M pH9.6, during change liquid 3 times; Requiring bag filter to see through diameter is 160KDa; Add equivalent glycerine to be stored in-20 ℃ the reactant liquor after dialysis; .
Step 4: newly synthetic coating protein glycerite is diluted to 1:1000 with the carbonic acid buffer of 0.05M pH9.6; In 96 hole Chemiluminescent plates, every hole adds 100 μ l, 4 ℃ of standing over night; By plank dabber, add the solution 200 μ l that contain 1% bovine serum albumin(BSA) next day, 37 ℃ of cappings 1 hour; Plank is patted dry again, be placed in dry house dehumidifier and dry final vacuum packing, obtain the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent.
9. the general solid-phase coating plate of board-like chemiluminescence immunoassay diagnostic reagent, is characterized in that, according to the method preparation described in claim 1 or 8.
10. a board-like chemiluminescence immunoassay diagnostic kit, is characterized in that, comprises coated plate claimed in claim 9.
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Patent Citations (8)
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| DE10343442A1 (en) * | 2003-09-19 | 2005-04-14 | Fachhochschule Flensburg | Quantitative detection of biological contaminants in complex cell mixtures, useful for testing cultures in food processing, by flow cytometry, using fluorescent antibody and magnetic particles |
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Application publication date: 20140827 |