CN104181293B - Enzyme-linked immunosorbent assay method - Google Patents
Enzyme-linked immunosorbent assay method Download PDFInfo
- Publication number
- CN104181293B CN104181293B CN201410423837.7A CN201410423837A CN104181293B CN 104181293 B CN104181293 B CN 104181293B CN 201410423837 A CN201410423837 A CN 201410423837A CN 104181293 B CN104181293 B CN 104181293B
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- slide
- enzyme
- lid
- assay method
- linked immunosorbent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- Biotechnology (AREA)
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- Biochemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a kind of enzyme-linked immunosorbent assay method, use a kind of MBP enzyme linked immuno-adsorbent assay box, including the slide being provided with sulculus, slide back is provided with the base for supporting described slide, the crimping of slide front is provided with the silica gel plate of the aperture corresponding with sulculus, the top of silica gel plate is crimped with lid, and lid is provided with the through hole that aperture thereon with described silica gel plate is corresponding；Operating procedure is: the clone after washing is coated in slide sulculus, clone is fixed, air-dries；Slide is proceeded on base, cover silica gel plate and lid, add blood plasma to be detected, hatch at 37 DEG C, washing, add the anti-human IgA antibody of horseradish peroxidase-labeled, hatch at 37 DEG C, slide is taken out, washing, dyeing, microscopy observed result.The present invention uses MBP enzyme linked immuno-adsorbent assay box, and during having stopped to be loaded, the liquid between each sulculus is because flowing the cross pollution gone here and there and cause, and improves the accuracy of detection.
Description
Technical field
The present invention relates to enzyme linked immunological clinical application technique field, especially a kind of MBP enzyme linked immuno-adsorbent assay
Method.
Background technology
MBP enzyme linked immuno-adsorbent assay is antigen or the antibody of solubility to be attached on solid phase carrier, utilizes antigen-antibody to tie
Closing selectivity and carry out immunoreactive qualitative and quantitative detecting method, therefore, MBP enzyme linked immuno-adsorbent assay test is cured for clinic
Prevention and the diagnosis of learning disease have directive function.
Traditional enzyme-linked immunosorbent assay method is the slide using and being provided with multiple sulculus, and antigen-antibody reaction is at slide
Carry out in sulculus, but each sulculus is only capable of accommodating 40 microliters of liquid, only separate by the slight paint swelled between each sulculus, therefore,
The fluid sample easily cross pollution of adjacent two sulculuses, the accuracy of final impact detection.
Summary of the invention
It is an object of the invention to provide a kind of enzyme-linked immunosorbent assay method, it can solve fluid sample and easily intersect
The problem polluted.
In order to solve the problems referred to above, the technical solution used in the present invention is: it uses MBP enzyme linked immuno-adsorbent assay box to examine
Surveying, described MBP enzyme linked immuno-adsorbent assay box includes the slide which is provided with 24 sulculuses, and described slide back is provided with for supporting
The base of described slide, this base is grooved base, and described slide is 4, is disposed side by side in described bottom tub；Described slide
Front crimping is provided with the silica gel plate of the aperture corresponding with described sulculus, and the top of described silica gel plate is crimped with lid, this lid
It is provided with and the described silica gel plate through hole that aperture is corresponding on it；
Enzyme-linked immunosorbent assay method comprises the following steps:
A, the clone cultivated with phosphate buffer solution washing, be then spread evenly across glass by described clone
In the sulculus of sheet, it is placed in 4 DEG C of refrigerators, uses the acetone soln of precooling clone to be fixed, air-dry standby；
B, proceed to described slide, on the base of described MBP enzyme linked immuno-adsorbent assay box, cover silica gel plate successively
And lid, add blood plasma to be detected from the aperture of lid surface, be subsequently placed at 37 DEG C and hatch 30 minutes；
C, employing phosphate buffer solution wash 3 times, add horseradish peroxide from the aperture of lid surface
The anti-human IgA antibody of compound enzyme labeling, is subsequently placed at 37 DEG C and hatches 30 minutes；
D, by slide take out, use phosphate buffer solution wash 3 times, described slide is placed in benzidine nitrite ion, so
Rear addition hydrogenperoxide steam generator, dyes 5 minutes～10 minutes, microscopy observed result.
In technique scheme, more specifically technical scheme is it may also is that the addition of clone described in step A is 15
Microlitre～20 microlitres.
Further, in step B, blood plasma to be detected uses PBS to dilute 5 times～10 times；Described in step B
Dilution after the addition of blood plasma to be detected be 15 microlitres～20 microlitres.
Further, the addition of the anti-human IgA antibody of the horseradish peroxidase-labeled described in step C is 15 micro-
Rise～20 microlitres.
Further, lid is crimped on the upper surface of described base；The side of lid is provided with the gusset phase with described base
Embedding buckle.
Further, surface of glass slide one jiao is provided with a corner block for location, direction, and described bottom tub inner surface sets
There is multiple salient point for controlling slide locations.
Further, the material of lid is lucite.
Owing to have employed technique scheme, the present invention compared with prior art has the advantages that
1, the present invention uses MBP enzyme linked immuno-adsorbent assay box, the slide of MBP enzyme linked immuno-adsorbent assay box to be provided above one layer of band
Having the silica gel plate of multiple aperture, silica gel plate is arranged over one layer of lid with multiple through holes, silica gel plate aperture, the hole of lid through hole
Footpath is all identical with the sulculus of slide, and silica gel plate aperture, lid lead to the hole site all with the position one_to_one corresponding of slide sulculus, make
The liquid obtained in adjacent slide sulculus is preferably kept apart, and during having stopped to be loaded, the liquid between each sulculus is because of stream string
And the cross pollution caused, improve the accuracy of detection.
2, the present invention uses MBP enzyme linked immuno-adsorbent assay box, uses logical between the slide sulculus of MBP enzyme linked immuno-adsorbent assay box
96 pitchs of holes design so that sample-adding can use 8 roads or 12 road sample injectors, replace the mode that traditional craft is loaded, from
And improve sample-adding efficiency.
3, the present invention uses MBP enzyme linked immuno-adsorbent assay box, and MBP enzyme linked immuno-adsorbent assay box uses general 96
Pitch of holes designs so that the cleaning of MBP enzyme linked immuno-adsorbent assay box can wash trigger compatibility with general, thus subtracts
Lack manual operation.
4, the present invention is applicable to all clone antigen based on slide medium or the detection of antibody, in clinic
On, in addition to conventional Epstein-Barr virus detection of specific antibody, apply also for mycoplasma pneumoniae, CPN,
The detection of the Respiroviruses such as Respiratory Syncytial Virus(RSV), legionella pneumophilia, influenza virus.
Accompanying drawing explanation
Fig. 1 is the structural representation of the embodiment of the present invention.
Fig. 2 is the top view of the base of the present invention.
Fig. 3 is the structural representation of the slide of the present invention.
Fig. 4 is the structural representation of the silica gel plate of the present invention.
Fig. 5 is the structural representation of the lid of the present invention.
Detailed description of the invention
The invention will be further described for embodiment below in conjunction with the accompanying drawings:
Embodiment 1
A kind of enzyme-linked immunosorbent assay method, uses MBP enzyme linked immuno-adsorbent assay box as shown in Figure 1 to 4, such as Fig. 1
～shown in Fig. 4, this MBP enzyme linked immuno-adsorbent assay box includes the slide 2 which is provided with sulculus 2-1, slide 2 back is provided with for propping up
Supportting the base 1 of described slide 2, the crimping of slide 2 front is provided with and the silica gel plate 3 of described aperture 3-1 corresponding for sulculus 2-1, institute
The top stating silica gel plate 3 is crimped with lid 4, and lid 4 is provided with the through hole 4-corresponding with described silica gel plate 3 aperture thereon 3-1
2；Base 1 is grooved base, and slide 2 and silica gel plate 3 are arranged in described bottom tub, and lid 4 is crimped on the upper surface of base 1；
The side of lid 4 is provided with the buckle 4-1 mutually embedding with base 1 gusset, and the material of lid 4 is lucite；Slide 2 in the present embodiment
Quantity be 4,4 slides are disposed side by side in described bottom tub, and every slide 2 is provided with 24 sulculuses, every slide 2 surface
One jiao be equipped with one for direction location corner block, base 1 groove inner surface is provided with multiple for controlling described slide 2
The salient point put.Through hole 4-2 on lid 4 and the position of the aperture 3-1 of described silica gel plate and aperture all with the sulculus on slide 2
The position of 2-1 is corresponding with aperture, thus deepens the degree of depth of slide sulculus 2-1 so that the adjacent liquid in slide sulculus is relatively
Good keeps apart, it is to avoid the cross pollution of fluid sample during sample-adding.
Operating procedure is:
15 microlitres of cells systems are spread evenly across by the clone that A, employing phosphate buffer solution washing are cultivated
In the sulculus 2-1 of slide 2, it is placed in 4 DEG C of refrigerators, uses the acetone soln of precooling will be spread evenly across slide sulculus
Clone in 2-1 is fixed, and air-dries standby；
B, slide 2 is proceeded on the base 1 of MBP enzyme linked immuno-adsorbent assay box, cover silica gel plate 3 He successively
Lid 4, adds the blood plasma to be detected after 20 microlitre dilutions from the aperture of lid 4, is placed at 37 DEG C and hatches 30 points
Clock；
C, employing phosphate buffer solution wash 3 times, add the anti-of 20 microlitre horseradish peroxidase-labeled
People's IgA antibody, is placed at 37 DEG C and hatches 30 minutes；
D, slide 2 is taken out, use phosphate buffer solution to wash 3 times, described slide is placed in benzidine
In nitrite ion, it is subsequently adding hydrogenperoxide steam generator, dyes 10 minutes, microscopy observed result.
In the present embodiment, test plasma phosphate buffer solution dilutes 10 times.
Embodiment 2
In the present embodiment, the addition of clone is 20 microlitres, and test plasma phosphate buffer solution dilutes 5 times, to be detected
The addition of blood plasma is 15 microlitres, and the addition of the anti-human IgA antibody of peroxidase labelling is 15 microlitres；Other are all with real
Execute example 1 identical.
Operating procedure is:
20 microlitres of cells systems are spread evenly across by the clone that A, employing phosphate buffer solution washing are cultivated
In the sulculus 2-1 of slide 2, it is placed in 4 DEG C of refrigerators, uses the acetone soln of precooling will be spread evenly across slide sulculus
Clone in 2-1 is fixed, and air-dries standby；
B, slide 2 is proceeded on the base 1 of MBP enzyme linked immuno-adsorbent assay box, cover silica gel plate 3 He successively
Lid 4, adds the blood plasma to be detected after 15 microlitre dilutions from the aperture of lid 4, is placed at 37 DEG C and hatches 30 points
Clock；
C, employing phosphate buffer solution wash 3 times, add the anti-of 15 microlitre horseradish peroxidase-labeled
People's IgA antibody, is placed at 37 DEG C and hatches 30 minutes；
D, slide 2 is taken out, use phosphate buffer solution to wash 3 times, described slide is placed in benzidine
In nitrite ion, it is subsequently adding hydrogenperoxide steam generator, dyes 5 minutes, microscopy observed result.
Claims (8)
1. an enzyme-linked immunosorbent assay method, it is characterised in that: use MBP enzyme linked immuno-adsorbent assay box to detect, described enzyme
Connection immuno absorbence detection box includes the slide (2) which is provided with 24 sulculuses (2-1), and described slide (2) back is provided with for propping up
Supportting the base (1) of described slide (2), this base (1) is grooved base, and described slide (2) is 4, is disposed side by side on the described end
In seat slot；The crimping of described slide (2) front is provided with the silica gel plate (3) of the aperture (3-1) corresponding with described sulculus (2-1), institute
The top stating silica gel plate (3) is crimped with lid (4), this lid be provided with described silica gel plate (3) on it aperture (3-1) corresponding
Through hole (4-2)；
Enzyme-linked immunosorbent assay method comprises the following steps:
A, the clone cultivated with phosphate buffer solution washing, be then spread evenly across glass by described clone
In the sulculus of sheet (2), it is placed in 4 DEG C of refrigerators, uses the acetone soln of precooling clone to be fixed, air-dry standby；
B, proceed to described slide, on the base (1) of described MBP enzyme linked immuno-adsorbent assay box, cover successively
Silica gel plate (3) and lid (4), add blood plasma to be detected from the through hole (4-2) on lid (4) surface, be subsequently placed at 37 DEG C
Hatch 30 minutes；
C, employing phosphate buffer solution wash 3 times, add peppery from the through hole (4-2) on lid (4) surface
The anti-human IgA antibody of root peroxidase labelling, is subsequently placed at 37 DEG C and hatches 30 minutes；
D, slide (2) is taken out from MBP enzyme linked immuno-adsorbent assay box, use phosphate buffer solution washing 3
Secondary, described slide (2) is placed in benzidine nitrite ion, is subsequently adding hydrogenperoxide steam generator, dye 5 minutes～10 points
Clock, microscopy observed result.
Enzyme-linked immunosorbent assay method the most according to claim 1, it is characterised in that: clone described in step A
Addition is 15 microlitres～20 microlitres.
Enzyme-linked immunosorbent assay method the most according to claim 1 and 2, it is characterised in that: the blood to be detected in step B
Slurry uses PBS to dilute 5 times～10 times；The addition of the blood plasma to be detected after the dilution described in step B is 15
Microlitre～20 microlitres.
Enzyme-linked immunosorbent assay method the most according to claim 1 and 2, it is characterised in that: horseradish mistake described in step C
The addition of the anti-human IgA antibody of oxide enzyme labeling is 15 microlitres～20 microlitres.
Enzyme-linked immunosorbent assay method the most according to claim 3, it is characterised in that: horseradish peroxide described in step C
The addition of the anti-human IgA antibody of compound enzyme labeling is 15 microlitres～20 microlitres.
Enzyme-linked immunosorbent assay method the most according to claim 1, it is characterised in that: described lid (4) is crimped on institute
State the upper surface of base (1)；The side of described lid (4) is provided with the buckle (4-1) mutually embedding with the gusset of described base (1).
7. according to the enzyme-linked immunosorbent assay method described in claim 1 or 6, it is characterised in that: described slide (2) surface one
Angle is provided with a corner block for location, direction, and described base (1) groove inner surface is provided with multiple for controlling described slide (2)
The salient point of position.
Enzyme-linked immunosorbent assay method the most according to claim 1, it is characterised in that: the material of described lid (4) is
Lucite.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410423837.7A CN104181293B (en) | 2014-08-26 | 2014-08-26 | Enzyme-linked immunosorbent assay method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410423837.7A CN104181293B (en) | 2014-08-26 | 2014-08-26 | Enzyme-linked immunosorbent assay method |
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| Publication Number | Publication Date |
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| CN104181293A CN104181293A (en) | 2014-12-03 |
| CN104181293B true CN104181293B (en) | 2016-09-07 |
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| CN111308081A (en) * | 2020-03-10 | 2020-06-19 | 中国工程物理研究院职工医院 | Universal enzyme-linked immunoassay test board and preparation method thereof |
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| CN1053042C (en) * | 1985-08-22 | 2000-05-31 | 翰斯·Ｊ·沃尔夫 | Diagnosis method for the disease with EBV virus in vitro |
| CN1075208A (en) * | 1992-12-02 | 1993-08-11 | 张昌卿 | A kind of chromogenic reagent and manufacture method thereof that detects Epstein-Barr virus antibody |
| CN1186635C (en) * | 2002-03-29 | 2005-01-26 | 成都夸常科技有限公司 | Biological chip capable of being disassembled and assembled |
| CN1289904C (en) * | 2003-08-01 | 2006-12-13 | 博奥生物有限公司 | Micro array reaction unit and its use |
| CN2863809Y (en) * | 2005-12-15 | 2007-01-31 | 华东理工大学 | Biological chip reactor |
| CN102023205A (en) * | 2009-09-09 | 2011-04-20 | 上海裕隆生物科技有限公司 | Detachable film chip |
| CN202322854U (en) * | 2011-11-14 | 2012-07-11 | 江苏百奥特医疗仪器科技有限公司 | Silicon gel slice for culturing cells |
| CN202562928U (en) * | 2012-02-09 | 2012-11-28 | 许丹科 | Visualized protein chip device |
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