CN107656048A - A kind of immuno-chromatographic test paper strip and its application of half-quantitative detection antigen or antibody - Google Patents
A kind of immuno-chromatographic test paper strip and its application of half-quantitative detection antigen or antibody Download PDFInfo
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- CN107656048A CN107656048A CN201710831261.1A CN201710831261A CN107656048A CN 107656048 A CN107656048 A CN 107656048A CN 201710831261 A CN201710831261 A CN 201710831261A CN 107656048 A CN107656048 A CN 107656048A
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Classifications
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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
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
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- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Inorganic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention relates to immuno-chromatographic test paper strip and its application of a kind of the half-quantitative detection antigen or antibody of ion vitro immunization detection technique field.The test strips include：Antiadhesive stent；Paste sample application zone, label land, colour developing area and suction zones on antiadhesive stent successively with edge chromatography direction；In the colour developing area detection zone and quality control region are sequentially provided with along chromatography direction；The detection zone includes the detection line of more than two, the quality control region includes the nature controlling line of one or more, and in the detection line at least in two detection lines coated second homologue concentration or identical in quality, or at least in two detection lines coated second homologue can combine isoconcentration or wait the purpose thing to be detected of quality.When carrying out antigen or antibody test using test strips of the present invention, the error come due to color condition different band is reduced, improves testing result, and it is simple to operate, it is easy to use.
Description
Technical field
The invention belongs to ion vitro immunization detection technique field, and in particular to a kind of half-quantitative detection antigen or antibody are immunized
Chromatograph test strip and its application.
Background technology
With the continuous improvement of the various diseases incidence of disease, the popular need for easy, quick, high specificity detection technique
Ask increasing.
Immunochromatography technique is to be applied to internal antigen or antibody test using collaurum or fluorescence etc. as trace labelling thing
A kind of new detection technique.Due to the technology have cost it is relatively low it is easy to use, quick, be easy to basic unit use and scene make
With and all reactions advantage, the immunochromatography technique such as can complete in 15 minutes and be applied to human chorionic gonadotrophin
(HCG), alpha-fetoprotein (AFP), prostate specific antigen (PSA), bird flu, the plague, Human Hydatidosis and mycoplasma pneumoniae etc.
Clinical detection.Immunochromatography technique is substantially carried out qualitative detection or needs additional colorimetric card that progress is compared at present
Half-quantitative detection.And more accurate quantitative detecting method such as chemoluminescence method, process the methods of time-resolved fluorescence method are answered
Miscellaneous, cumbersome and needs carry out last result by the instrument of costliness and read.Thus developing one kind quickly can be in short-term
The interior detection method for directly reading scope residing for experiment Testing index just seems particularly urgent.Especially for prostate-specific
Antigen, C peptides, follicular stimulating hormone and lutropin etc. have the clinical detection index of term of reference, even if its testing result is same
For positive but concentration level difference, then meaning differs greatly, and represents and whether suffers from different disease or illnesses not
The same stage.Thus further determine that scope residing for its testing result is particularly important when testing result is positive.
The immunochromatography technique of application and development at present comprises only a detection line generally directed to same detected material, thus right
In the detection of the body index with certain term of reference, immunochromatography technique there is no the reality of direct rapid semi-quantitative detection application
Example.
The content of the invention
The present invention provides a kind of immune chromatography test paper of half-quantitative detection antigen or antibody in view of the shortcomings of the prior art
Bar, by observing the testing result of this test strip, can quickly determine testee's body index to be detected whether position
In with reference in normal range (NR) or ill different phase.
Therefore, first aspect present invention provides a kind of immuno-chromatographic test paper strip of half-quantitative detection antigen or antibody, its
Including：
Antiadhesive stent；With
Paste sample application zone, label land, colour developing area and suction zones on antiadhesive stent successively along chromatography direction；
In the colour developing area detection zone and quality control region are sequentially provided with along chromatography direction；
The detection zone includes the detection line of more than two, and the quality control region includes the nature controlling line of one or more.
Heretofore described term " more than two " represents to be more than or equal to two；Equally, term " one or more " represents big
In equal to one.
In some preferred embodiments of the present invention, the detection line is vertical with chromatography direction with nature controlling line.
In certain embodiments of the present invention, the label land include with label and can with it is to be detected
The first homologue that purpose thing combines；The second couple that can be combined with purpose thing to be detected is coated with the detection line of the detection zone
Answer thing；Quality Control thing corresponding with purpose thing to be detected is coated with the nature controlling line of the quality control region.
According to the present invention, in the detection line at least in two detection lines coated second homologue concentration or matter
Measure it is identical, or at least in two detection lines coated second homologue can combine isoconcentration or wait the purpose to be detected of quality
Thing.
In some preferred embodiments of the present invention, close to label land or quality control region in the detection line
At least in two detection lines coated second homologue concentration or content it is identical.
In certain embodiments of the present invention, the label is colloid gold particle, chemical illuminating reagent, chemical colour reaction
One kind in reagent, metallic, carbon nano-particle, latex particle, fluorescent material, rare earth ion, magnetic particle and quantum dot
It is or a variety of.
In some preferred embodiments of the present invention, the label is colloid gold particle.
In certain embodiments of the present invention, the purpose thing to be checked is antigen to be checked, and first homologue is
With the antibody a of the antigen binding to be checked, second homologue is the antibody b, the antibody a with the antigen binding to be checked
Antigenic determinant different on the antigen to be checked is identified with antibody b；Described Quality Control thing for containing antigen to be checked standard items or
The secondary antibody c combined with antibody a.
In other embodiments of the present invention, the purpose thing to be checked is antibody to be checked, and first homologue
For the secondary antibody d with the antibody binding to be checked, second homologue is the antigen with the antibody binding to be checked；The Quality Control
Thing is the standard items containing detection antibody or other Quality Control things that can reach corresponding Quality Control effect.
In certain embodiments of the present invention, the purpose thing to be checked is antibody to be checked, and first homologue is
With the antigen of the antibody binding to be checked, second homologue is the secondary antibody d with the antibody binding to be checked；The Quality Control thing
For the standard items containing detection antibody or other Quality Control things that can reach corresponding Quality Control effect.
In other embodiments of the present invention, the purpose thing to be checked is antibody to be checked, and first homologue
For the antigen e with the antibody binding to be checked, second homologue is the antigen f with the antibody binding to be checked, antigen e and
Antigen f is combined with the different loci of detection antibody, and the Quality Control thing can reach for the standard items containing detection antibody or others
To the Quality Control thing of corresponding Quality Control effect.
In certain embodiments of the present invention, the test strips are using double antibody sandwich method detection antigen.
In other embodiments of the present invention, the test strips use indirect method, prize law or dual-antigen sandwich method
Detect antibody.
In the present invention, detected detected sample can be blood sample, urine, saliva or excrement.
In the present invention, PVC board, the matrix material selection polymer PET of sample application zone can be selected in the matrix material of the antiadhesive stent
Or polymer PET, glass fibre element film or filter paper fibre can be selected in glass fibre, the matrix material of label land, develop the color area
Matrix material can be nitrocellulose filter, and the matrix material of suction zones can be the absorbent materials such as commercially available absorbent filter.
Second aspect of the present invention provides a kind of immunochromatographytest test kit, and it is included described in first aspect present invention
Test strips.
Third aspect present invention provides a kind of immunochromatography detection method, and it uses the examination described in first aspect present invention
Kit described in paper slip or second aspect of the present invention is detected.Specifically, testing sample is added drop-wise to sample application zone or will added
Sample area is immersed in testing sample, keeps flat and stands 5-20 minutes；Then by observing the band color of detection line and nature controlling line, judge
The concentration or content range of antigen or detected antibody are detected in testing sample.
Beneficial effects of the present invention are：The a plurality of of coating same substance is provided with the colour developing area of test strips of the present invention
Detection line (more than two), and at least in two detection lines the concentration of coated material or identical in quality, or at least two
In bar detection line coated material can combine isoconcentration or wait the purpose thing to be detected of quality.So chromatography side upwardly through
Antigen-antibody reaction (sandwich method, prize law or indirect method) carries out solidification colour developing to label, and then treats testing goal thing
Content carries out the detection of sxemiquantitative.The test strips can realize half-quantitative detection, and detection is quick, without any instrument and equipment, takes
Band is convenient, and testing cost is low, simple to operation, and high specificity and detection sensitivity are high.Carried out using test strips of the present invention
The method of antigen or antibody half-quantitative detection, the error come due to color condition different band is reduced, improves testing result, and
Operation inconvenience and use cost caused by unnecessary accurate detection can be farthest reduced, and it is simple to operate, use
It is convenient.
Brief description of the drawings
Illustrate the present invention below in conjunction with accompanying drawing.
Fig. 1 is the structural representation of the test strips employed in the embodiment of the present invention 1.The implication of reference is such as in figure
Under：1 sample application zone；2 label lands；3 colour developing areas；301 detection lines (T lines)；302 nature controlling lines (C lines)；4 suction zones；5 adhesions
Support.
Fig. 2 is that embodiment 1 is shown using what the test strips containing two detection lines were detected to Serum Alpha Fetoprotein content
Color schematic diagram.
Embodiment
To be readily appreciated that the present invention, the present invention is described in detail below in conjunction with accompanying drawing.
Test strips of the present invention can be detected using double antibody sandwich method to antigen, and can using indirect method,
Dual-antigen sandwich method or prize law detect to antibody.
Therefore, the immuno-chromatographic test paper strip of the half-quantitative detection antigen or antibody involved by first aspect present invention, it is wrapped
Include：
Antiadhesive stent；With
Paste sample application zone, label land, colour developing area and suction zones on antiadhesive stent successively along chromatography direction；
In the colour developing area detection zone and quality control region are sequentially provided with along chromatography direction；
The detection zone includes the detection line of more than two, and the quality control region includes the nature controlling line of one or more.
In some preferred embodiments of the present invention, the detection line is vertical with chromatography direction with nature controlling line.
In certain embodiments of the present invention, the test strips are that the test paper of antigen is detected using double antibody sandwich method
Bar.The label land of the test strips includes with label and can be with the antibody a of antigen binding to be checked；The detection zone
Detection line on be coated with identical material, specially can be with the antibody b of antigen binding to be checked, and the antibody a and antibody b know
Different antigenic determinant on antigen not to be checked；Be coated with the nature controlling line of the quality control region standard items containing antigen to be checked or with
The secondary antibody c that antibody a is combined.
In the detection line at least in two detection lines coated antibody b concentration or content it is identical, or at least
In two detection lines coated antibody b can combine the antigen to be checked of isoconcentration or equivalent.
When using above-mentioned ELISA test strip antigen, according to the term of reference of the concentration of tested antigen or content, antibody b quilts
It is divided into several pieces to be coated in detection line, and coated antibody b content is identical or at least two at least in two detection lines
Coated antibody b can combine or detect the antigen to be checked of equivalent at bar detection line.When measuring samples are added into sample application zone
Afterwards, measuring samples can be mobile to suction zones because of capillarity and reach label land by sample application zone.In label
Land, in sample antigen to be checked can specifically (the antibody a) of mark substance markers be tied with the antibody a with label
Close, form antibody-antigen conjugates.The antibody a of the antibody-antigen conjugates and excessive mark substance markers is under chromatography effect
Continue towards detection zone (T lines area) and quality control region (C lines area) migration.Because mark the antibody a of substance markers and detection zone coated
Antibody b identifies antigenic determinant different on same antigen, therefore the antigen to be checked for having combined the antibody a of mark substance markers arrives
After up to detection zone can with the immobilised antibody b in T lines area combine and produce develop the color band.At the same time, remaining Ag-Ab knot
Compound continue along chromatography direction reach, with another detection line fix antibody b combine and formed Article 2 develop the color band；It is surplus
Remaining antibody-antigen conjugates and the antibody a of excessive mark substance markers continue along the reach of chromatography direction until forming last
Bar colour developing band.If the concentration of antigen to be checked is higher in sample, then in the antigen that detection zone is combined with coated antibody b just
Can be more, and antigen has been combined with the antibody a for marking substance markers, then the colour developing formed by double-antibody sandwich in detection zone
The color of band also can be deeper.If the concentration of antigen to be checked is relatively low in sample, then is combined in detection zone with coated antibody b
Antigen will be fewer, also can be shallower in the colour developing band color that detection zone is formed by double-antibody sandwich.If treated in sample
Antigenic content difference is examined, then the shade that the band and every band that detection zone can develop the color are presented also can be different.Pass through
The coated antibody b of institute is dense on the T lines bands number of comparative analysis colour developing, the shade and each T line of each T lines band
Degree or content, testing staff are assured that the concentration or content range of antigen to be checked in sample.
In other embodiments of the present invention, the test strips are that the test strips of antibody are detected using indirect method.Should
The label land of test strips includes with label and can be with the secondary antibody d of antibody binding to be checked, the inspection of the detection zone
Identical material is coated with survey line, can be specially coated with the antigen of antibody binding to be checked on the nature controlling line of the quality control region
There are the standard items containing detection antibody or other Quality Control things that can reach corresponding Quality Control effect.
Or the test strips are that the test strips of antibody are detected using prize law.The label land bag of the test strips
Containing with label and identical material can be coated with the detection line of the detection zone with the antigen of antibody binding to be checked,
Specially the standard items containing detection antibody can be coated with the nature controlling line of the quality control region with the secondary antibody d of antibody binding to be checked
Or other Quality Control things that can reach corresponding Quality Control effect.
Or the test strips are that the test strips of antibody are detected using dual-antigen sandwich method.The label knot of the test strips
Area is closed to include with label and can be coated with the detection line of the detection zone identical with the antigen e of antibody binding to be checked
Material, specially can with the antigen f, the antigen e and the antigen f of antibody binding to be checked respectively can with it is described to be detected anti-
The different parts of body are combined, and the standard items containing detection antibody or others are coated with the nature controlling line of the quality control region to be reached
The Quality Control thing of corresponding Quality Control effect.
In the detection line at least in two detection lines coated antigen or secondary antibody concentration or content it is identical, or extremely
In rare two detection lines coated antigen or secondary antibody can combine the antibody to be checked of isoconcentration or equivalent.
Below by taking indirect method as an example, the process that antibody test is carried out using test strips of the present invention is described in detail.According to
The concentration of antibody to be checked or the term of reference of content, the antigen corresponding to antibody to be checked are divided into several pieces and are coated in detection line
On, and the content of at least two coated antigens of detection line is identical or coated antigen can be tied at least at two detection lines
Close or detect the antibody to be checked of equivalent.After measuring samples are added into sample application zone, measuring samples can be because capillarity be to water suction
Area is mobile and reaches label land by sample application zone.In label land, the determined antigen in sample is to be checked anti-
Body can specifically (the secondary antibody d) of mark substance markers be combined, and forms conjugate with the secondary antibody d with label.The conjugate
Detection zone (T lines area) is continued towards under chromatography effect and quality control region (C lines area) is moved with the secondary antibody d of the mark substance markers of excess
Move.In T lines area, meeting and T lines area are immobilised anti-after the antibody to be checked arrival detection zone for the secondary antibody d for having combined mark substance markers
Original combine and produce one develop the color band.At the same time, the secondary antibody d of remaining conjugate and mark substance markers continues to suction zones
It is mobile and with antigen binding fixed in detection zone Article 2 detection line and form Article 2 colour developing band；Remaining conjugate and
Excess marker, which obtains secondary antibody d, to be continued to move to suction zones until forming the last item colour developing band.If antibody to be checked in sample
Concentration is higher, then will be more in detection zone and the antibody of coated antigen binding, and antibody is with marking substance markers
Secondary antibody d is combined, then the color of the colour developing band formed by indirect method in detection zone also can be deeper.It is if to be checked anti-in sample
The concentration of body is relatively low, then will be fewer in detection zone and the antibody of coated antigen binding, by indirect method in detection zone shape
Into colour developing band color also can be shallower.If antibody content to be checked in sample is different, band that detection zone can develop the color and
The shade presented per band can be different.T lines bands number, each T lines band to be developed the color by comparative analysis
Shade and each T line on the coated antigen concentration of institute or content, testing staff be assured that to be checked in sample
The concentration or content range of antibody.
In certain embodiments of the present invention, the label is colloid gold particle, chemical illuminating reagent, chemical colour reaction
One kind in reagent, metallic, carbon nano-particle, latex particle, fluorescent material, rare earth ion, magnetic particle and quantum dot
It is or a variety of；Preferably, the label is colloid gold particle.
In the present invention, detected detected sample can be blood sample, urine, saliva or excrement.
Second aspect of the present invention is related to a kind of immunochromatographytest test kit, and it includes the examination described in first aspect present invention
Paper slip.
Third aspect present invention is related to a kind of immunochromatography detection method, and it uses the test paper described in first aspect present invention
Kit described in bar or second aspect of the present invention is detected.Specifically, testing sample is added drop-wise to sample application zone or will be loaded
Area is immersed in testing sample, and calmness places 5-20 minutes；Then by observing the band color of detection line and nature controlling line, judge to treat
The concentration or content range of antigen or detected antibody are detected in test sample product.
Embodiment
To make the present invention easier to understand, the present invention is further described below in conjunction with embodiment, these realities
Apply example only serve it is illustrative, it is not limited to application of the invention.If raw material or component nothing used in the present invention
Specified otherwise can be made by commercial sources or conventional method.
Embodiment 1：Using the content of double antibody sandwich method quick detection human serum alpha-fetoprotein antigen.
The preparation of 1.1 colloidal gold immunochromatographiassay assay reagent boxes
The structural representations of the test strips in immunochromatographytest test kit employed in the present embodiment as shown in figure 1,
Specially：
Chromatography direction in edge is end to end successively on the antiadhesive stent of the test strips and is stained with sample application zone, label combines
Area, colour developing area and suction zones.The test strips can make measuring samples successively by adding in the presence of suction zones by capillarity
Sample area, label land and colour developing area simultaneously eventually arrive at suction zones to complete to detect.Parallel arranged has successively in colour developing area
Two detection lines (T1 lines and T2 lines) and a nature controlling line (C lines), and two detection lines and nature controlling line are vertical with chromatography direction.
The anti-human alpha-fetoprotein IgG of mouse that the collaurum including 400ng/mL is marked in the matrix material of label land
Monoclonal antibody a (the first homologue), antibody a are by identifying that specific antigenic determinant is and to be checked with this on alpha-fetoprotein antigen
Antigen occurs antigen-antibody reaction and combined.
Develop the color area two detection lines on be coated with the anti-human alpha-fetoprotein IgG monoclonal antibody b of mouse (the second homologue), two
Coated antibody b concentration is identical in bar detection line.Antibody b by identify on alpha-fetoprotein antigen specific antigenic determinant and
Immune response occurs with the antigen to be checked and combines, the antigenic determinant of antibody b identifications and the antigenic determinant of antibody a identifications are not
Together.The standard items (Quality Control thing) of 10ng/mL alpha-fetoprotein antigen are coated with the bar nature controlling line of quality control region.
The alpha-fetoprotein concentration of reference is less than 20ng/mL in normal human's serum, i.e. reference upper level is 20ng/mL, therefore
And in two detection line T1s and T2 coated antibody b concentration be 10ng/mL.
The setting of above-mentioned first homologue, the second homologue and Quality Control thing follows following rule：The concentration of first homologue
At least above the concentration of the second homologue in all detection lines and the concentration sum of Quality Control thing, or the first homologue quality extremely
It is more than the quality of the second homologue and the quality sum of Quality Control thing in all detection lines less, to ensure that detection line and nature controlling line can
Normal colour developing.Detection line is set as two, and in two detection lines the concentration of coated second homologue or content it is identical and
The concentration of coated second homologue or the half that content is reference value in every detection line.By above-mentioned setting, can protect
Card has enough mark conjugates to pass through each detection line and nature controlling line successively in detection is applied, and can be by these
When line forms immobilization band, the content of the purpose thing to be checked combined on conjugate is marked, it is sufficient to the second couple in detection line
Answer thing to carry out immune response, and mark the content of the first homologue combined on conjugate, it is sufficient to the Quality Control thing on nature controlling line
Immune response is carried out, so could be in the detection by chromatographing situation, the redissolution situation for marking combination etc., judging semidefinite
Whether the quality of amount immuno-chromatographic test paper strip meets requirement, while detection line and nature controlling line are shown normally, could semidefinite
Amount ground judges the concentration range of purpose thing to be checked.
Such as in the present embodiment, it is according to the serum reference concentration of alpha-fetoprotein antigen, then coated at every detection line
The anti-human alpha-fetoprotein IgG monoclonal antibody b of mouse concentration is 10ng/mL；Coated alpha-fetoprotein standard items is dense at nature controlling line
Spend for 10ng/mL；The anti-human alpha-fetoprotein IgG monoclonal antibody a of mouse for the mark substance markers that label land includes concentration
For 400ng/mL.So in the present embodiment, the concentration sum of the second homologue and Quality Control thing is 10ng/mL+10ng/mL+
10ng/mL=30ng/mL.The concentration of first homologue should be greater than 30ng/mL, and the concentration of the first homologue is in the present embodiment
400ng/mL meets setting more than 30ng/mL.
The colloidal gold immunochromatographiassay assay reagent box of detection human serum alpha-fetoprotein antigenic content is prepared according to above-mentioned parameter.
Specific preparation process is as follows：
Step 1, the anti-human alpha-fetoprotein IgG monoclonal antibody a of mouse of colloid gold label is prepared
0.75mL-4mL 1wt% citric acid trisodium or lemon are rapidly added after the heating of 100mL chlorauric acid solutions is boiled
Sour three sodium water solutions, continue to boil about 5-30 minutes, cooling can obtain colloidal gold solution after colour stable.
The pH value for adjusting colloidal gold solution waits electricity to the anti-human alpha-fetoprotein IgG monoclonal antibody a of mouse (the first homologue)
Near point or meta-alkalescence (optimal pH), mouse anti-human igg list is slowly added into colloidal gold solution by most suitable labelled protein amount
Clonal antibody a.
With magnetic stirring apparatus it is well mixed after, add BSA or polyglycol solution, centrifuge 20-50 minutes, sediment is with containing
1%BSA PB liquid or the buffer solution redissolution containing polyethylene glycol, repeated centrifugation 2-3 times, it is original volume that final precipitation, which redissolves volume,
1/10, obtain the anti-human alpha-fetoprotein IgG monoclonal antibody a of mouse of colloid gold label.
Most suitable protein labeling method for determination of amount is：With 0.1mol/L K2CO3The pH value of colloidal gold solution is adjusted to
8.0-9.2, each colloidal gold solution of the content containing different proteins is sequentially added in some centrifuge tubes and is allowed into graded, mixed
Even standing 2-4 hours.Then 100 μ L 10% NaCl solution is added in every pipe, using the protein content of the constant pipe of color as minimum
Protein stabilized amount, it is most suitable protein labeling amount to add 10%-20% protein contents again on this basis.
Optimal pH is the isoelectric point or more slightly larger than isoelectric pH value of the first homologue, and its determination method is：By collaurum
If solution is added in main, often the addition of colloidal gold solution is 1mL in pipe.With 0.1mol/L K2CO3Adjust colloid in often pipe
The pH value of gold solution, is allowed into graded.The most suitable labelled amount albumen of equivalent is added in each Guan Zhongjun, mixes and stands 2 hours, add
Enter 100 μ L 10% NaCl solution, using the pH value of the constant pipe of color as optimal pH.
Step 2, the preparation of label land
The matrix material selection glass fibre membrane of label land.First using the treatment fluid processing glass fibers containing BSA
Tie up plain film, then by the anti-human alpha-fetoprotein IgG monoclonal antibody a solution even application of the mouse of 400ng/mL colloid gold label in
On glass fibre element film, label land is obtained.
Step 3, sample application zone is prepared
The matrix material of sample application zone is selected from polymer PET or glass fibre.Sample application zone is handled overnight through treatment fluid.
Step 4, colour developing area is prepared
The matrix material selection nitrocellulose filter in colour developing area, by the chromatography direction in use, the line point in colour developing area
For detection zone (T lines area) and quality control region (C lines area).Then the mouse that respectively coating concentration is 10ng/mL in T lines area detection line is anti-human
Alpha-fetoprotein IgG monoclonal antibody b, concentration 10ng/mL alpha-fetoprotein antigen standard is coated with C lines area nature controlling line, is obtained
Must be developed the color area.
Step 5, suction zones are prepared
The absorbent materials such as the matrix material selection filter paper of suction zones.Suction zones promote measuring samples successively by capillarity
By sample application zone, label land, colour developing area and eventually arrive at suction zones and chromatographed, to complete to detect.
Step 6, the assembling of detection kit
The sample application zone being prepared, label land, colour developing area and suction zones are pasted on antiadhesive stent successively
The immuno-chromatographic test paper strip of alpha-fetoprotein antigen in half-quantitative detection human serum is obtained, its structural representation as shown in figure 1, enter again
The assembling of one step just obtains immunochromatographytest test kit.
The detection of 1.2 human serum alpha-fetoprotein antigenic contents
When laboratory carries out conventional alpha-fetoprotein determination to crowd, alpha-fetoprotein is less than 20-25ng/ in normal human serum
mL.It is then normal less than the value, then for the positive during higher than the value, the possibility with liver cancer increases, and need to do more further
Detection.
In detection, a certain amount of blood serum sample to be checked is added dropwise on sample application zone, blood serum sample to be checked acts in chromatography
Under reach label land from sample application zone after, then alpha-fetoprotein passes through antigen-antibody reaction and the collaurum of label land
The antibody a of mark is combined, and obtains the antibody a of alpha-fetoprotein and colloid gold label compound.The compound and remaining colloid
The antibody a of gold mark is migrated under chromatography effect towards detection zone, and the detection of claret is combined to form with the antibody b at detection line
Band.The antibody a for the colloid gold label not being combined is migrated under chromatography effect towards quality control region, at nature controlling line and alpha-fetoprotein
Combine to form the nature controlling line of claret.What the antibody a of the colloid gold label combined at detection line and nature controlling line was more at most formed
Claret band color is deeper, and the wine that the antibody a of the colloid gold label combined at detection line and nature controlling line is formed more at least is red
Vitta band color is more shallow.The corresponding concentration range of purpose thing to be checked can be obtained by being compared by detection line with the color of nature controlling line,
Semi-quantitatively determine the concentration range of alpha-fetoprotein antigen in blood serum sample to be checked.Above-mentioned whole detection process, test strips are put down
Put standing 10 to 20 minutes, can just complete, it is fast and convenient and accurate.
Result judgement：If C lines do not develop the color, it is invalid to detect；If C lines develop the color, detection is effective.If testing result is as schemed
Shown in 2A, the colour developing of C lines, T1 lines and T2 lines all develop the color and color depth is identical and close with C lines, then it represents that first tire in serum to be checked
Protein level is higher than 20ng/mL, suffers from the increase of liver cancer possibility, it is necessary to which further checked.If testing result such as Fig. 2 B institutes
Show, the colour developing of C lines, T1 lines and T2 lines all develop the color but T1 line colors are deeper than T2 lines, then it represents that Serum Alpha Fetoprotein level to be checked is low
It is normal in 20ng/mL.If C lines develop the color, the only colour developing of T1 lines or T1 lines and T2 lines is not shown, then it represents that blood to be checked
Level of Alpha Fetoprotein is less than 20ng/mL in clear, is normal.
It should be noted that embodiment described above is only used for explaining the present invention, do not form to any of the present invention
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word wherein used is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to
And specific method, material and embodiment, it is not intended that the present invention is limited to wherein disclosed particular case, on the contrary, this hair
It is bright to can be extended to other all methods and applications with identical function.
Claims (10)
1. the immuno-chromatographic test paper strip of a kind of half-quantitative detection antigen or antibody, it includes：
Antiadhesive stent；With
Paste sample application zone, label land, colour developing area and suction zones on antiadhesive stent successively along chromatography direction；
In the colour developing area detection zone and quality control region are sequentially provided with along chromatography direction；
The label land includes with label and the first homologue that can be combined with purpose thing to be detected, the inspection
Survey in the detection line in area and be coated with the second homologue that can be combined with purpose thing to be detected, be coated with the nature controlling line of the quality control region
There is Quality Control thing corresponding with the purpose thing to be detected；
The detection zone includes the detection line of more than two, and the quality control region includes the nature controlling line of one or more；And the detection
In line at least in two detection lines coated second homologue concentration or identical in quality, or at least in two detection lines
Coated second homologue can combine isoconcentration or wait the purpose thing to be detected of quality.
2. test strips according to claim 1, it is characterised in that the label is colloid gold particle, chemiluminescence examination
Agent, chemochromic reagent, metallic, carbon nano-particle, latex particle, fluorescent material, rare earth ion, magnetic particle and quantum
One or more in point.
3. test strips according to claim 1 or 2, it is characterised in that the purpose thing to be checked is antigen to be checked, and described
First homologue is the antibody a with the antigen binding to be checked, and second homologue is anti-with the antigen binding to be checked
Body b, the antibody a and antibody b identify antigenic determinant different on the antigen to be checked；The Quality Control thing is containing antigen to be checked
Standard items or the secondary antibody c that can be combined with antibody a.
4. test strips according to claim 1 or 2, it is characterised in that the purpose thing to be detected is detection antibody, and
First homologue is secondary antibody d corresponding with the test antibodies, and second homologue is and the detection antibody pair
The antigen answered, the Quality Control thing are the standard items containing detection antibody or other Quality Control things that can reach corresponding Quality Control effect.
5. test strips according to claim 1 or 2, it is characterised in that：The purpose thing to be detected is detection antibody, and
First homologue is antigen corresponding with the test antibodies, and second homologue is corresponding with the detection antibody
Secondary antibody d, the Quality Control thing is the standard items containing detection antibody or other Quality Control thing that can reach corresponding Quality Control effect.
6. test strips according to claim 1 or 2, it is characterised in that the purpose thing to be detected is detection antibody, and
First homologue is antigen e corresponding with the detection antibody, and second homologue is and the detection antibody
Corresponding antigen f, the antigen e and the antigen f are combined with the different parts of the detection antibody, the Quality Control thing be containing
The standard items of detection antibody or other Quality Control things that can reach corresponding Quality Control effect.
7. test strips according to claim 1 or 2, it is characterised in that the test strips are detected using double antibody sandwich method
Antigen.
8. test strips according to claim 1 or 2, it is characterised in that the test strips are using indirect method, prize law or double
Antigen sandwich method detects antibody.
9. a kind of immunochromatographytest test kit, it includes the test strips any one of claim 1-8.
10. a kind of immunochromatography detection method, it uses the test strips or claim 9 any one of claim 1-8
Described kit is detected.
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| CN111033563A (en) * | 2019-11-27 | 2020-04-17 | 深圳加美生物有限公司 | Image analysis method and system for immunochromatography detection |
| CN111474351A (en) * | 2020-04-28 | 2020-07-31 | 上海泰辉生物科技有限公司 | Immunochromatographic test strip and kit for detecting coronavirus |
| CN112557647A (en) * | 2020-12-10 | 2021-03-26 | 宁波华仪宁创智能科技有限公司 | Detection method based on immunochromatography technology and application |
| CN114264819A (en) * | 2021-12-08 | 2022-04-01 | 上海理工大学 | Quantum dot nanosphere immunochromatography test strip for rapidly detecting new coronavirus |
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