CN109164265A - Vaccine antibody detection card and preparation method thereof - Google Patents
Vaccine antibody detection card and preparation method thereof Download PDFInfo
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- CN109164265A CN109164265A CN201811116305.3A CN201811116305A CN109164265A CN 109164265 A CN109164265 A CN 109164265A CN 201811116305 A CN201811116305 A CN 201811116305A CN 109164265 A CN109164265 A CN 109164265A
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- G—PHYSICS
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The present invention discloses a kind of vaccine antibody detection card and preparation method thereof.Vaccine antibody detection card of the invention includes Ka Gai, card body and reagent strip.Ka Gai is provided with sample pipetting volume hole, observation window and sample number into spectrum marked position, card body is equipped with the groove for placing reagent strip, test strips are provided with sample bed course, colloid layer gold, nitrocellulose filter and water absorption layer, colloid layer gold includes the colloid gold label object of combined vaccine specific antibody, the detection of antigenicity line T line of nitrocellulose filter setting coating target vaccine and the nature controlling line C line of coating sheep anti-mouse igg antibody.The sample dosage of vaccine antibody detection card of the invention is few, has the detection sensitivity improved, and easy to use, fast simple.
Description
Technical field
The invention belongs to immunological test technical fields, and in particular to colloidal gold immunity chromatography is for quickly detecting blood
The reagent strip and its detection card of middle vaccine antibody.
Background technique
Vaccine is the preventative biology system in order to prevent, control the generation of infectious disease, prevalence, for human body immunization campaign
Product.The biological products of immunization campaign include vaccine, vaccine and toxoid.It wherein, is vaccine made of germ；By virus,
It is vaccine made of Richettsia, conveyor screw, is referred to as vaccine.Vaccine be by pathogenic microorganism (such as bacterium, rickettsia,
Virus etc.) and its metabolite, by be manually attenuated, inactivate or using made of the methods of genetic engineering for keeping off infection
Active immunity preparation.After being inoculated with this pathogen for not having injury power, immune system will generate certain Protective substances,
Such as immune hormone, active physiological substances, distinct antibodies；When body is contacted again this pathogen, the siberian crabapple of body
System, which is activated promptly, generates specific antibody to protect body.
Because whether individual constitution difference or vaccine itself and other objective factors, produce in body after vaccine injection
Potent antibodies, there is presently no suitable convenient, simple, efficiently detection methods.Wherein vaccine causes body immune system institute
The antibody of generation can be detected with colloidal gold immunity chromatography.
Colloidal gold immunochromatographimethod technology is that one kind using colloidal gold as tracer label object applied to antigen-antibody is novel
Immunolabelling technique.Advantage:
1, easy to use quick, unlimited place, all reactions can be completed in 15 minutes；
2, at low cost, do not need special instrument and equipment；
3, have a wide range of application, be suitable for a variety of testing conditions；
4, multinomial detection can be carried out, if the more difficult acquisition of sample, multinomial detection can save sample, reduce cost；
5, marker is stablized, and it is more than year to store 2 years at 4 DEG C for label sample, no signal relaxation phenomenon；
6, colloidal gold sheet does not need that chromogenic agents are added, eliminates enzyme target carcinogenicity substrate and terminate liquid as red
The step of, it is harmless to the human body.
Currently, the method that the prior art is not carried out convenience, simple, quick and high sensitivity detection antibody.
Summary of the invention
One of the objects of the present invention is to provide a kind of detections of vaccine antibody to block comprising Ka Gai, card body and reagent strip, institute
It states Ka Gai and is provided with sample pipetting volume hole, observation window and sample number into spectrum marked position, the card body is equipped with the groove for placing reagent strip, institute
It states test strips and is provided with sample bed course, colloid layer gold, nitrocellulose filter and water absorption layer, the colloid layer gold includes combined vaccine
The colloid gold label object of specific antibody, the detection of antigenicity line T line of nitrocellulose filter setting coating target vaccine and
It is coated with the nature controlling line C line of sheep anti-mouse igg antibody.
Preferably, vaccine antibody detection card of the invention further comprises the first filter layer and the second filter layer, wherein institute
Stating the first filter layer has the first aperture for allowing antibody molecule to pass through but molecule aggregate cannot be allowed to pass through, the second filter layer
With allowing small molecule the second aperture for passing through but antibody molecule cannot being allowed to pass through, and first filter layer be set to it is described
Above sample bed course, second filter layer is set to below the colloid layer gold.
Preferably, in vaccine antibody of the invention detection card, the nitrocellulose filter is made of nanometer nitrocellulose.
Preferably, in vaccine antibody of the invention detection card, the colloid layer gold includes glass layer.
Preferably, in vaccine antibody of the invention detection card, the test strips further comprise supporting course, and in supporting course
The same side on along its length successively to be mutually connected the setting sample bed course, the colloid layer gold, described
Nitrocellulose filter and the water absorption layer.
Preferably, in vaccine antibody of the invention detection card, the sample pipetting volume hole corresponds to the position of the sample bed course
It installs, the position that the observation window corresponds to the nitrocellulose filter is arranged.
Preferably, in vaccine antibody of the invention detection card, the Ka Gai and the card body are close by engaging formation closure
Jacket.
The second object of the present invention is to provide a kind of preparation method of vaccine antibody detection card comprising following steps:
(1) preparation of colloidal gold, the colloid gold particle of size needed for being formed under reducing agent effect by gold chloride；
(2) label of colloidal gold, it is under conditions of being suitable for reacting that the colloidal gold solution and mouse that prepare in step (1) is anti-human
Immune globulin antibody mixing, obtains colloid gold label mouse human immunoglobulins' antibody；
(3) colloid gold label mouse human immunoglobulins' antibody is fixed on colloid layer gold by colloidal gold immobilization；
(4) it is coated with nitrocellulose filter, variant vaccine antigen is sprayed on the first position of nitrocellulose filter, is formed
Sheep anti-mouse igg antibody is sprayed on the second position of nitrocellulose filter by target vaccine antigen detection line, and it is anti-to form sheep anti-mouse igg
Then body nature controlling line makes nitrocellulose filter dry；
(5) preparation of test strips, the nitrocellulose filter obtained by the stickup of the middle position of PVC board, in the fixed nitric acid of PVC board
Water absorption layer is pasted in the side of cellulose membrane position, pastes colloid layer gold in the other side of the fixed nitrocellulose film location of PVC board
With sample bed course；
(6) gained test strips are placed in the groove of card body, Ka Gai and card body is fastened, obtain vaccine antibody detection card.
Preferably, in preparation method of the invention, blotting paper covers the top 0.5-1.5 milli at nitrocellulose filter edge
Rice, top 0.5-1.5 millimeters of the colloid layer gold covering nitrocellulose filter.
Preferably, in preparation method of the invention, the sample bed course, colloid layer gold and nitrocellulose filter are successively adjacent
Connection, and the first filter layer is set above the sample bed course, the second filter layer is set below the colloid layer gold.
Advantageous effects:
The present invention utilizes colloidal gold immunochromatographimethod technology, can specifically detect the specific antibody that different vaccines generate.
Since detection card is convenient, sample dosage is few, it is only necessary to and the detection of vaccine antibody can be realized in 50 μ L serum, blood plasma or whole blood, and
Easy to operate, detection speed is fast, unlimited place, is suitable for clinical and community and promotes.
Preferably, the present invention avoids impurity entrance by impurity such as the first filter layer filtering molecular group or large particles
Nitrocellulose filter, the influence so as to avoid these substances for detection.
It is further preferred that the present invention filters out the colloid gold particle and other small molecules of unbonded antibody by the second filter layer
Equal impurity, avoid these impurity and enter nitrocellulose filter.These impurity can fill cellulose nitrate in traditional method
The hole of plain film, to be unfavorable for the movement of antigen-antibody conjugate to be detected, and the colloidal gold of unbonded antibody
Grain influences the response intensity of detection line position.
It is further preferred that the present invention replaces traditional general nitrocellulose filter using nanometer nitrocellulose filter, pass through this
The technical solution of invention, nanometer nitrocellulose do not reduce detection sensitivity, still have the effect of excellent.This and this field
The difference of interior general understanding.
Detailed description of the invention
A kind of perspective view of Exemplary Vaccine antibody test card of Fig. 1 present invention.
A kind of figure of the reagent strip of Exemplary Vaccine antibody test card of Fig. 2 present invention.
The part-structure figure of the reagent strip of another Exemplary Vaccine antibody test card of Fig. 3 present invention.
Description of symbols:
Vaccine antibody detection card 100, card lid 110, card body 120, reagent strip 130, sample pipetting volume hole 111, observation window 112,
Sample bed course 121,221, colloid layer gold 122,222, nitrocellulose filter 123,223, water absorption layer 124, supporting course 125,225,
First filter layer 2211, the second filter layer 2221.
Specific embodiment
The existing various exemplary embodiment that the present invention will be described in detail, the detailed description are not considered as to limit of the invention
System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair
It is bright.In addition, for the numberical range in the present invention, it is thus understood that specifically disclose the range upper and lower bound and they it
Between each median.Median and any other statement value in any statement value or stated ranges or in the range
Lesser range is also included in the present invention each of between interior median.These small range of upper and lower bounds can be independent
Ground includes or excludes in range.
Unless otherwise stated, all technical and scientific terms used herein has the routine in field of the present invention
The normally understood identical meanings of technical staff.Although the present invention only describes preferred method and material, of the invention
Implement or also can be used and similar or equivalent any method and material described herein in testing.The institute mentioned in this specification
There is document to be incorporated by reference into, to disclosure and description method relevant to the document and/or material.It is incorporated to any
When document conflicts, it is subject to the content of this specification.Unless otherwise stated, " % " is the percentage based on weight.
Embodiment 1
The present embodiment is the preparation method of Exemplary Vaccine antibody test card comprising following steps:
(1) preparation of colloidal gold
The different type vaccine that need to be detected according to target can choose the colloid gold particle of suitable size, 15nm, 18nm-
The preparation of 20nm, 30nm or 50nm colloid gold particle: 0.01%HAuCl is taken4Aqueous solution 100ml, heating are boiled.It is fast as needed
1% citric acid three sodium water solution 4ml, 2.5ml, 1ml or 0.75ml is added in speed, continues to boil about 5min, occurs orange red.In this way
Manufactured colloid gold particle is then respectively 15nm, 18-20nm, 30nm and 50nm.Cooling is spare；
(2) label of colloidal gold
Colloid gold label mouse human immunoglobulins' antibody: taking the colloidal gold solution 50mL prepared in step (1), uses
0.1mol/L K2CO3It is adjusted to pH8.9-9.2, it is anti-that the mouse human immunoglobulin(HIg) that 4-6mL concentration is 0.8-1.2mg/mL is then added
Body stirs 2-5 minutes, and the PEG20000 aqueous solution that 4-6mL mass fraction is 0.8-1.2% is added, is centrifuged in 15000r/min
35-45 minutes, supernatant is sucked, precipitating is suspended in the aqueous solution that concentration is 0.4-0.6mg/mL PEG20000, with
0.5mg/mL Sodium azide anti-corrosion sets 3-5 DEG C of preservation；
(3) colloidal gold immobilization
Colloid gold label object solution even made from step (1) and (2) is mixed, solid phase obtains glass in colloid layer gold
Fiber gold-labelled pad；
(4) it is coated with nitrocellulose filter
4.1T line envelope antigen: variant vaccine antigen is diluted to 0.8-1.2mg/ with the PBS buffer solution of pH7.2-7.5
ML is sprayed on nitrocellulose filter with Membrane jetter with pump speed 16mm/s, film speed 320mm/s, forms target vaccine antigen detection line；
4.2C line is coated with sheep anti-mouse igg antibody: being diluted to sheep anti-mouse igg antibody with the PBS buffer solution of pH7.2-7.5
1.4-1.6mg/mL is sprayed on nitrocellulose made from above-mentioned steps 4.1 with Membrane jetter with pump speed 16mm/s, film speed 320mm/s
On film, sheep anti-mouse igg antibody nature controlling line is formed；
Nitrocellulose filter made from above-mentioned steps 4.2 is set drying 1.5-2 hours in 37 DEG C of baking ovens by 4.3；
(5) preparation of test strips
5.1 paste nitrocellulose filter prepared by above-mentioned steps 4.3 in the middle position of PVC board；
5.2, in the top of the fixed nitrocellulose film location of PVC board, paste blotting paper, and blotting paper covers nitrocellulose
Membrane edge is along 1.0 millimeters；
5.3 fix the lower section of nitrocellulose film location in PVC board, the colloid gold label object of gluing steps (3) preparation
Glass fibre gold-labelled pad and sample pad, glass fibre gold-labelled pad cover 1.0 millimeters of nitrocellulose filter,
After 5.4 flatten the product that above-mentioned steps 5.3 obtain, it is cut into the test strips of 5.0mm wide；
(7) test strips of above-mentioned preparation are placed in the groove of card body by card taking lid and card body, and Ka Gai and card body are fastened,
Obtain vaccine antibody detection card.
Embodiment 2
The present embodiment is a kind of Exemplary Vaccine antibody test card 100.As depicted in figs. 1 and 2, vaccine antibody detection card
100 include card lid 110, card body 120 and reagent strip 130.
Card lid 10 is provided with sample pipetting volume hole 111, observation window 112 and sample number into spectrum marked position (not shown), card body 120 and sets
There is the groove for placing reagent strip 130.Card lid 110 and card body 120 pass through engaging and form closing seam box.
Test strips 130 are provided with sample bed course 121, colloid layer gold 122, nitrocellulose filter 123 and water absorption layer 124, and
Sample bed course 121, colloid layer gold 122, nitrocellulose filter 123 and water absorption layer 124 are respectively arranged at as supporting course 125
The same side of PVC board, along the length direction of supporting course 125 successively to be mutually connected setting sample bed course 121, colloidal gold
Layer 122, nitrocellulose filter 123 and water absorption layer 124.Water absorption layer 124 is pasted above one end of nitrocellulose filter 123, and is inhaled
Water layer 124 covers 1.0 millimeters of the top at 123 edge of nitrocellulose filter, i.e. water absorption layer 124 is Chong Die with nitrocellulose filter 123
1.0 millimeter.Colloid layer gold 122 is pasted above the other end of nitrocellulose filter 123, and colloid layer gold 122 covers nitric acid fibre
Tie up 1.0 millimeters of the top at the edge of plain film 123, i.e. 1.0 millimeters of colloid layer gold 122 and 123 weight of nitrocellulose filter.Sample pad
About 1.0 millimeters Chong Die with the other end of colloid layer gold of layer.Colloid layer gold includes the colloid gold particle that diameter is 50nm.
Colloid layer gold 122 is glass layer, and it includes the colloid gold label objects of combined vaccine specific antibody.This implementation
Colloid gold label object is the antiantibody of vaccine specific antibody and the conjugate of colloid gold label object in example.Nitrocellulose filter
The detection of antigenicity line T line of 123 setting coating target vaccines and the nature controlling line C line of coating sheep anti-mouse igg antibody.
The position that the sample pipetting volume hole 111 of vaccine antibody detection card 100 corresponds to sample bed course 121 in the present embodiment is set
It sets, the position that observation window 112 corresponds to nitrocellulose filter 123 is arranged.
Embodiment 3
The present embodiment is another Exemplary Vaccine antibody test card.In the present embodiment, in addition to nitrocellulose filter is to receive
Rice nitrocellulose filter, colloid layer gold includes the colloid gold particle that diameter is 15nm, and is had outside structure shown in Fig. 3,
Remaining structure is same as Example 2.
As shown in figure 3, the test strips of the present embodiment are provided with sample bed course 221, colloid layer gold 222, nitrocellulose filter
223, and sample bed course 221, colloid layer gold 222, nitrocellulose filter 223 are successively arranged in parallel, and it is any between the two continuously
Upper and lower lap is also not present in gap.
In addition, being provided with the first filter layer 2211 in the top of sample bed course 221.It is arranged in the lower section of colloid layer gold 222
Second filter layer 2221.Fig. 3 A, which shows the second filter layer 2221, has size identical with colloid layer gold 222, and supporting course
225 have the groove with 2221 same size of the second filter layer, for fixing or installing the second filter layer 2221.Fig. 3 B is shown
Second filter layer 2221 has size identical with sample bed course 221 and 222 sum of the two of colloid layer gold, and the second filter layer
2221 are located at the lower section of sample bed course 221 and colloid layer gold 222.Fig. 3 C shows the second filtering of setting above supporting course 225
Layer 2221, and the size of the second filter layer 2221 is identical as the size of supporting course 225.
In the present embodiment, the average pore size of the first filter layer is 250-500nm, and the average pore size of the second filter layer 2221 is
50-70nm.Preferably, the second filter layer 2221 is made of the stronger material of water imbibition.It is highly preferred that colloid layer gold 222 and
Interface between two 2221 layers of filter layers is higher than the interface between supporting course 225 and nitrocellulose filter.
Since the second filter layer 2221 may filter that the colloid gold particle of the most of unbonded antibody of removal, it is anti-to be conducive to antigen
Body binding molecule moves in nitrocellulose filter, and the colloid of the unbonded antibody removed does not appear in detection line, from
And greatly improve the accuracy of detection.
Without departing substantially from the scope or spirit of the invention, the specific embodiment of description of the invention can be done more
Kind improvements and changes, this will be apparent to those skilled in the art.Other realities obtained by specification of the invention
Applying mode for technical personnel is apparent obtain.Present specification and embodiment are merely exemplary.
Claims (10)
1. a kind of vaccine antibody detection card, which is characterized in that including Ka Gai, card body and reagent strip, the Ka Gai is provided with sample
Well, observation window and sample number into spectrum marked position, the card body are equipped with the groove for placing reagent strip, and the test strips are provided with sample
Product bed course, colloid layer gold, nitrocellulose filter and water absorption layer, the colloid layer gold include the colloid of combined vaccine specific antibody
Golden marker, the detection of antigenicity line T line and coating sheep anti-mouse igg antibody of the nitrocellulose filter setting coating target vaccine
Nature controlling line C line.
2. vaccine antibody detection card according to claim 1, which is characterized in that further comprise the first filter layer and second
Filter layer, wherein first filter layer has the first hole for allowing antibody molecule to pass through but molecule aggregate cannot be allowed to pass through
Diameter, the second filter layer have the second aperture for allowing small molecule to pass through but antibody molecule cannot be allowed to pass through, and first mistake
Filtering layer is set to above the sample bed course, and at least part of second filter layer is set to below the colloid layer gold.
3. vaccine antibody detection card according to claim 2, which is characterized in that the nitrocellulose filter is by nanometer nitric acid
Cellulose is made.
4. vaccine antibody detection card according to claim 3, which is characterized in that the colloid layer gold includes glass fibre
Layer.
5. vaccine antibody detection card according to claim 4, which is characterized in that the test strips further comprise bearing
Layer, and along its length successively to be mutually connected the setting sample bed course, described on the same side of supporting course
Colloid layer gold, the nitrocellulose filter and the water absorption layer.
6. vaccine antibody detection card according to claim 5, which is characterized in that the sample pipetting volume hole corresponds to the sample
The position of product bed course is arranged, and the position that the observation window corresponds to the nitrocellulose filter is arranged.
7. vaccine antibody detection card according to claim 6, which is characterized in that the Ka Gai and the card body pass through engaging
Form closing seam box.
8. the preparation method of vaccine antibody detection card according to claim 1-7, which is characterized in that including following
Step:
(1) preparation of colloidal gold, the colloid gold particle of size needed for being formed under reducing agent effect by gold chloride；
(2) label of colloidal gold, it is under conditions of being suitable for reacting that the colloidal gold solution and mouse that prepare in step (1) is anti-human immune
Globulin antibody mixing, obtains colloid gold label mouse human immunoglobulins' antibody；
(3) colloid gold label mouse human immunoglobulins' antibody is fixed on colloid layer gold by colloidal gold immobilization；
(4) it is coated with nitrocellulose filter, variant vaccine antigen is sprayed on the first position of nitrocellulose filter, forms target
Sheep anti-mouse igg antibody is sprayed on the second position of nitrocellulose filter by vaccine antigen detection line T line, forms sheep anti-mouse igg antibody
Then nature controlling line C line makes nitrocellulose filter dry；
(5) preparation of test strips, the nitrocellulose filter obtained by the stickup of the middle position of PVC board, in the fixed cellulose nitrate of PVC board
Water absorption layer is pasted in the side of plain film location, pastes colloid layer gold and sample in the other side of the fixed nitrocellulose film location of PVC board
Product bed course；
(6) gained test strips are placed in the groove of card body, Ka Gai and card body is fastened, obtain vaccine antibody detection card.
9. preparation method according to claim 8, which is characterized in that the colloid layer gold covers the upper of nitrocellulose filter
It is 0.5-1.5 millimeters square, top 0.5-1.5 millimeters of water absorption layer covering nitrocellulose filter edge.
10. preparation method according to claim 8, which is characterized in that the sample bed course, colloid layer gold and nitrocellulose
Film successively adjacent connection, and between sample bed course and nitrocellulose filter and the colloid layer gold and the nitrocellulose filter
It is non-overlapping, and the first filter layer is set above the sample bed course, the second filter layer is set below the colloid layer gold.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811116305.3A CN109164265A (en) | 2018-09-25 | 2018-09-25 | Vaccine antibody detection card and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811116305.3A CN109164265A (en) | 2018-09-25 | 2018-09-25 | Vaccine antibody detection card and preparation method thereof |
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| CN109164265A true CN109164265A (en) | 2019-01-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115032404A (en) * | 2022-06-10 | 2022-09-09 | 杨富松 | Vaccine antibody detection card, detection system and detection method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115032404A (en) * | 2022-06-10 | 2022-09-09 | 杨富松 | Vaccine antibody detection card, detection system and detection method thereof |
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