CN103954753A - Quantitative determination method of immune chromatography test strip - Google Patents
Quantitative determination method of immune chromatography test strip Download PDFInfo
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- CN103954753A CN103954753A CN201410196384.9A CN201410196384A CN103954753A CN 103954753 A CN103954753 A CN 103954753A CN 201410196384 A CN201410196384 A CN 201410196384A CN 103954753 A CN103954753 A CN 103954753A
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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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Abstract
The invention discloses a quantitative determination method of an immune chromatography test strip. The quantitative determination method comprises the following steps: drawing a standard curve; dropwise adding a liquid to be detected to the immune chromatography test strip which comprises an internal reference line, at least one detection line and an excessive antigen detection line, detecting, and reading a detection signal capable of reflecting the intensity of antigen-antibody reaction to obtain a reading result; carrying out linear regression on the reading result of the nth detection line by utilizing the nth standard curve when the detection signals of the n-1 detection lines of at least one detection line exceed a threshold value according to the reading results, and calculating to obtain the concentration of the liquid to be detected. By adopting the mode, the quantitative detection method disclosed by the invention can solve the problem that the low-concentration measurement and the high-concentration measurement are simultaneously carried out, and can be used for the quantitative detection on biological macromolecules or micromolecules, such as serum markers, microbe antigens, virus particles and illicit drugs; the linear detection range of the test strip is enlarged, the detection accuracy is improved, and the accurate quantitative detection on a target detection object is realized.
Description
Technical field
The present invention relates to dry chemical detection field, particularly relate to a kind of quantitative detecting method of immuno-chromatographic test paper strip.
Background technology
Immunochromatography technique is the carrier that uses nitrocellulose filter etc. to support as reactant, utilizes the porous effect of film to react.This technology can be divided into two kinds of longitudinal diffusion and transverse dispersion.What be born in early days is the spot gold hybrid method of longitudinal diffusion, promotes the use of but fail to become main flow reagent because of its complicated operation eventually.Along with the development of membrane technology, lateral flow transverse dispersion test strips demonstrates good advantage, and is promoted greatly.In lateral flow immunochromatography technique, most widely used collaurum fast diagnose test paper is for the detection of very early pregnancy (HCG), because it is convenient, fast, specificity is high, be particularly suitable for care diagnostic, therefore its application is very extensive, becomes the mainstream product in lateral flow immunochromatography technique.
At present, the panimmunity chromatograph test strips such as collaurum on market, quantum dot, latex, fluorescence, can only complete qualitative detection or half-quantitative detection mostly, quantitatively detect very difficult.Main cause is in lateral flow immunochromatography process, and measured echo signal is a process increasing gradually, is a dynamic change.Traditional end-point method only can be used for qualitative or sxemiquantitative, cannot realize accurate quantification; Secondly,, when very big to the content scope of material to be checked face to face (such as three more than the order of magnitude), commercially available test strips is often helpless, especially, in the time that concentration of specimens is higher, even usually occurs false-negative result.Allow to detect higher concentration, in area with high mercury accuracy also extreme difference.
Therefore, how to increase the sensitivity of ELISA test strip, the linear detection range that simultaneously increases test strips is the bottleneck of puzzlement test strips development always.Higher sensitivity is often pursued by test strips manufacturer, but has ignored high sensitivity zone test strips linear detection range this present situation that narrows.When in the face of the higher sample to be tested of object concentration, must just can measure through diluting, otherwise will occur in a large number false negative result.And in the time of clinical practice, operating personnel also have to manually dilute, increase clinical examination staff's work complexity.Therefore,, in not affecting ELISA test strip limit, how increasing its linear detection range is the important embodiment of test strips performance.
Summary of the invention
The technical matters that the present invention mainly solves is to provide a kind of quantitative detecting method of immuno-chromatographic test paper strip, can carry out low concentration measurement and high concentration measures simultaneously, increase the linear detection range of test strips, improve the accuracy detecting, realize the accurate quantification of target detection thing is detected.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of quantitative detecting method of immuno-chromatographic test paper strip is provided, comprises that step is:
(1) preparation standard solution detecting with the immuno-chromatographic test paper strip that includes internal reference line, at least one detection line and excessive antigen detection line, the detection signal that obtains antigen-antibody reaction power on the described immuno-chromatographic test paper strip that can measure, obtains reading result;
(2) according to reading result described in step (1), in described at least one detection line, there is the detection signal of n-1 bar detection line to reach threshold value, carry out the drafting of typical curve by the measurement result of n article of detection line, obtain n bar taking standard solution concentration as horizontal ordinate, taking antigen-antibody reaction intensity on described immuno-chromatographic test paper strip as the typical curve of ordinate, wherein n is greater than 1 natural number;
(3) drop to be measured be added on immuno-chromatographic test paper strip and detect, obtaining the detection signal of antigen-antibody reaction power on the described immuno-chromatographic test paper strip that can measure, obtaining reading result;
(4) according to reading result described in step (3), in described at least one detection line, there is the detection signal of n-1 bar detection line to reach threshold value, n article of typical curve described in the reading result utilization of n article of detection line carried out to linear regression, and calculation obtains the concentration of described liquid to be measured.
In a preferred embodiment of the present invention, detection signal described in step (2) is for absorbing light signal, reflected light signal, fluorescence signal, magnetic field intensity signal, sound field intensity signal or electric field intensity signal.
In a preferred embodiment of the present invention, described reading result is the detection signal strength Area Ratio of described n article of detection line and described internal reference line.
In a preferred embodiment of the present invention, described reading result is described n article of detection line and the maximum detection signal strength ratio of described internal reference line.
In a preferred embodiment of the present invention, when the detection signal of described the above detection line of immuno-chromatographic test paper strip all reaches threshold value, and the detection signal of described excessive antigen detection line is 0 o'clock, the concentration of described liquid to be measured exceedes the linear detection range of described immuno-chromatographic test paper strip.
The invention has the beneficial effects as follows: the quantitative detecting method of immuno-chromatographic test paper strip of the present invention, the method can solve low concentration measurement and high concentration is measured the problem of simultaneously carrying out, increase the linear detection range of test strips, improve the accuracy detecting, realize the accurate quantification of target detection thing is detected, can be for biomacromolecule or micromolecular quantitative detections such as blood serum designated object, microbial antigen, virion, illegal drugs.
Brief description of the drawings
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing, wherein:
Fig. 1 uses double antibody sandwich method to work as antigen concentration lower than the lowest detection detection signal strength distribution plan of prescribing a time limit in liquid to be measured in quantitative detecting method of the present invention;
Fig. 2 uses double antibody sandwich method to work as antigen concentration detection signal strength distribution plan in sensing range time in liquid to be measured in quantitative detecting method of the present invention;
Fig. 3 uses double antibody sandwich method to work as antigen concentration higher than the highest detection detection signal strength distribution plan of prescribing a time limit in liquid to be measured in quantitative detecting method of the present invention;
Fig. 4 is the structural representation of immuno-chromatographic test paper strip in quantitative detecting method of the present invention;
Fig. 5 uses double antibody sandwich method to work as antigen concentration detection signal strength distribution plan when lower in liquid to be measured in quantitative detecting method of the present invention;
Fig. 6 is detection signal strength distribution plan while using double antibody sandwich method antigen concentration appropriate in quantitative detecting method of the present invention in liquid to be measured;
Fig. 7 is detection signal strength distribution plan while using double antibody sandwich method antigen concentration too high in quantitative detecting method of the present invention in liquid to be measured;
In accompanying drawing, the mark of each parts is as follows: 1, shell, 2, sample pad, 3, internal reference line, 4, the first detection line, 5, the second detection line, 6, excessive antigen detection line, 7, reaction film, 8, absorption pad.
Embodiment
To the technical scheme in the embodiment of the present invention be clearly and completely described below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making all other embodiment that obtain under creative work prerequisite, belong to the scope of protection of the invention.
In the present invention, the principle of quantitative detecting method is:
Adopt double antibody sandwich method, liquid to be measured detected to the three kinds of situations that there will be by test strips:
(1) refer to Fig. 1, when antigen concentration in liquid to be measured is prescribed a time limit lower than lowest detection, internal reference line generation antigen-antibody is in conjunction with obtaining a measurable detection signal, there is not antigen-antibody binding reaction in detection line, or determined antigen amount is too low, signal intensity is too low, can think that concentration is zero.
(2) refer to Fig. 2, in the time that in liquid to be measured, antigen concentration is in sensing range, now, along with the lifting of antigen concentration, the measurable signal intensity in detection line place increases gradually, can utilize relative signal intensity between detection line and internal reference line to calculate determined antigen concentration.
(3) refer to Fig. 3, prescribe a time limit higher than the highest detection of test strips design when antigen concentration in liquid to be measured, now detection line has been fixed whole antibody, there is no two anti-bindings of antibody and excessive antigen detection line, so locate not have measurable detection signal.
In the second situation, in the time that antigen concentration is in sensing range in liquid to be measured, refer to Fig. 4, on immuno-chromatographic test paper strip, measurable signal intensity profile can present following several situation:
(1) refer to Fig. 5, in the time that test substance concentration ratio is lower, the first detection line 4 gets final product the binary complex that complete capture antigen antibody forms, and is less than so locate to obtain one the detection signal of measuring threshold value.And not combined antibody can be with 6 to catch by excessive antigen detection, and there is larger detection signal.
(2) refer to Fig. 6, when testing concentration further increases, now the measuring-signal of the first detection line 4 has exceeded threshold value, unnecessary antigen-antibody binary complex is caught by the second detection line 5, and not combined antibody can be caught by excessive antigen detection line 6, also occur a signal, but the first situation diminishes relatively.
(3) refer to Fig. 7, when testing concentration further increases, now the fluorescence signal of the first detection line 4 and the second detection line 5 has all exceeded threshold, does not have unnecessary antibody to be caught by excessive antigen detection line 6.Now show that the excessive concentration of determinand has exceeded sensing range.
According to above-mentioned several situations, and consult Fig. 5-7, in the present invention, the concrete computation process of the one of the quantitative detecting method of immuno-chromatographic test paper strip is:
A: the drawing process of typical curve is:
(1) preparation standard solution detecting with immuno-chromatographic test paper strip, obtains the detection signal of antigen-antibody reaction power in the described test strips that can measure;
(2) internal reference line 3 being obtained to one can measuring-signal, is designated as A
c, can there are multiple A of being designated as in detection line signal
t1, A
t2a
tn, excessive antigen detection line signal is designated as A
m;
(3) if there is A in test strips
c, A
t1, A
mthree signals.Now utilize standard items concentration for horizontal ordinate, A
t1/ A
cvalue is ordinate, drawing standard curve S
1;
(4) if there is A in test strips
c, A
t1, A
t2a
tn, A
mfour or multiple detection signal, wherein A
t1signal exceedes threshold value.Now utilize standard items concentration for horizontal ordinate, A
t2/ A
cvalue is ordinate, drawing standard curve S
2; The like can obtain n bar typical curve S
1s
n.
B: sample quantitatively calculates idiographic flow and is:
(1) testing sample is detected with immuno-chromatographic test paper strip, obtain the detection signal of antigen-antibody reaction power in the described test strips that can measure;
(2) internal reference line 3 being obtained to one can measuring-signal, is designated as A
c, can there are multiple A of being designated as in detection line signal
t1, A
t2a
tn, excessive antigen detection line signal is designated as A
m;
(3) if there is A in test strips
c, A
t1, A
mthree signals.Now utilize typical curve S
1, use A
t1/ A
cvalue is carried out linear regression, obtains testing concentration;
(4) if there is A in test strips
c, A
t1, A
t2a
tn, A
mfour or multiple detection signal, wherein A
tn-1signal exceedes threshold value, and A
tnsignal does not exceed threshold value.Now utilize typical curve S
n, use A
tn/ A
cvalue is carried out linear regression, obtains testing concentration;
(5) if A in test strips
c, A
t1, A
t2a
tnsignal all exceedes threshold value, and does not occur A
msignal, points out testing sample concentration to exceed sensing range.
Embodiment mono-:
A kind of quantitative detecting method to c reactive protein is provided, comprises that step is:
1, the structural design of immuno-chromatographic test paper strip
On the reaction film of test strips, be fixed with four protein bands, be respectively one and be fixed with the internal reference line of 0.2 μ g rabbit igg, the Article 1 detection line that is fixed with 0.4 μ g CRP monoclonal antibody a, the Article 2 detection line that is fixed with 0.4 μ g CRP monoclonal antibody a, an excessive antigen detection line that is fixed with 0.5 μ g sheep anti-mouse igg.
2, detect damping fluid design
The mouse CRP monoclonal antibody of the fluorescently-labeled goat anti-rabbit igg of cy5 that contains 0.2 μ g in detection damping fluid, the cy5 mark of 1 μ g.
3, the quantitative detection algorithm of test strips
Get 10 normal concentration serum samples, add and detect in damping fluid, be added drop-wise in test strips and react after a period of time, coordinate special fluorescence to read instrument and can obtain following detection signal:
Obtaining two typical curves according to above-mentioned fluoroscopic examination result is respectively:
(1), in the time that testing concentration is 0-160mg/L, use A
t1/ A
ccarry out the drafting of typical curve, typical curve is Y=0.1803X+1.3707;
(2), in the time that testing concentration is 160-320mg/L, use A
t2/ A
ccarry out the drafting of typical curve, typical curve is Y=0.1365X-19.99.
Method of the present invention and enzyme-linked immunosorbent assay (ELISA) method is for the mensuration of serum CRP concentration, and in the time that serum-concentration is higher, if directly measure without dilution, contrast test result is as shown in the table:
When method of the present invention is quantitative for CRP test strip, its quantitative result is more accurate.Directly test for the serum sample of not diluted simultaneously, eliminated the false positive that high concentration reaction zone produces, a kind of CRP measuring method of whole process is provided.
Embodiment bis-:
A kind of quantitative detecting method to parathion-methyl fast detecting is provided, comprises that step is:
1, the structural design of test strips
On the reaction film of test strips, be fixed with four bands, be respectively one and be fixed with the internal reference line of 1 μ g rabbit igg, the Article 1 detection line that is fixed with 0.5 μ g parathion-methyl artificial antigen, the Article 2 detection line that is fixed with 0.5 μ g parathion-methyl artificial antigen, an excessive antigen detection line that is fixed with 0.5 μ g sheep anti-mouse igg.
2, detect damping fluid design
The mouse parathion-methyl monoclonal antibody of the fluorescently-labeled goat anti-rabbit igg of cy5 that contains 1 μ g in detection damping fluid, the cy5 mark of 1 μ g.
3, the quantitative detection algorithm of test strips
Get 7 normal concentration parathion-methyl solution, add and detect in damping fluid, be added drop-wise in test strips and react after a period of time, coordinate special fluorescence to read instrument and can obtain following detection signal:
| Standard items concentration (μ g/kg) | Internal reference line area (A C） | The first detection line area (A T1） | The second detection line area (A T2） | A T1/A C | A T2/A C |
| 100 | 2010 | 160 | 0.008 | ||
| 50 | 2048 | 1380 | 0.674 | ||
| 25 | 2032 | 2111 | 1.039 | ||
| 10 | 2031 | 3015 | 1.484 | ||
| 5 | 2015 | 4096 | 140 | 0.069 | |
| 1 | 2018 | 4096 | 3040 | 1.506 | |
| 0.5 | 2008 | 4096 | 3910 | 1.947 |
Obtaining two typical curves according to above-mentioned fluoroscopic examination result is respectively:
(1) in the time that testing concentration is 10-100 μ g/kg, Y=-0.0156X+1.5236;
(2) in the time that testing concentration is 0.5-10 μ g/kg, Y=-0.3951X+2.03.
Method of the present invention and ELISA method are measured for parathion-methyl, in the time that concentration of specimens is lower, directly measure without concentrated, and contrast test result is as shown in the table:
Method of the present invention, for directly testing without concentrated sample, can improve the linear detection range of test strips greatly.A kind of measuring method of the little molecular antigen of competition law ELISA test strip of wide-range is provided.
The method is by the collection to detection signal on immune chromatography test paper, and then use corresponding signal processing algorithm, low concentration can be realized and high concentration is measured simultaneously, increase the dynamic detection range of test strips, effectively prevent that false negative phenomenon from appearring in enriched sample while detection, and realize the accurate quantification of target detection thing is detected, can increase the accuracy of test strips quantitative result simultaneously.
The present invention combines test strips and late detection algorithm, in not changing the detection sensitivity of test strips, can greatly increase its sensing range, sample to be checked is without dilution direct-detection, reduce the interference that workload and dilution operation may bring, greatly improved detection efficiency.The advantage such as that described quantitative detecting method has is convenient and swift, be easy to carry about with one, reagent safety is nontoxic, environmental pollution is little, can be used for biomacromolecule or the micromolecular quantitative detections such as blood serum designated object, microbial antigen, virion, illegal drug, can be used for the quantitative detection of various clinical diagnosis biomarkers, specific biological molecule, pesticide residue, microorganism, water pollutant etc., be particularly suitable for external quick diagnosis and fast detecting field.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes description of the present invention to do; or be directly or indirectly used in other relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (5)
1. a quantitative detecting method for immuno-chromatographic test paper strip, is characterized in that, comprises that step is:
(1) preparation standard solution also detects with the immuno-chromatographic test paper strip that includes internal reference line, at least one detection line and excessive antigen detection line, the detection signal that obtains antigen-antibody reaction power on the described immuno-chromatographic test paper strip that can measure, obtains reading result;
(2) according to reading result described in step (1), in described at least one detection line, there is the detection signal of n-1 bar detection line to reach threshold value, carry out the drafting of typical curve by the measurement result of n article of detection line, obtain n bar taking standard solution concentration as horizontal ordinate, taking antigen-antibody reaction intensity on described immuno-chromatographic test paper strip as the typical curve of ordinate, wherein n is greater than 1 natural number;
(3) drop to be measured be added on immuno-chromatographic test paper strip and detect, obtaining the detection signal of antigen-antibody reaction power on the described immuno-chromatographic test paper strip that can measure, obtaining reading result;
(4) according to reading result described in step (3), in described at least one detection line, there is the detection signal of n-1 bar detection line to reach threshold value, n article of typical curve described in the reading result utilization of n article of detection line carried out to linear regression, and calculation obtains the concentration of described liquid to be measured.
2. quantitative detecting method according to claim 1, is characterized in that, detection signal described in step (2) is for absorbing light signal, reflected light signal, fluorescence signal, magnetic field intensity signal, sound field intensity signal or electric field intensity signal.
3. quantitative detecting method according to claim 1, is characterized in that, described reading result is the detection signal strength Area Ratio of described n article of detection line and described internal reference line.
4. quantitative detecting method according to claim 1, is characterized in that, described reading result is described n article of detection line and the maximum detection signal strength ratio of described internal reference line.
5. quantitative detecting method according to claim 1, it is characterized in that, when the detection signal of described the above detection line of immuno-chromatographic test paper strip all reaches threshold value, and the detection signal of described excessive antigen detection line is 0 o'clock, the concentration of described liquid to be measured exceedes the linear detection range of described immuno-chromatographic test paper strip.
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| CN105445449A (en) * | 2015-04-04 | 2016-03-30 | 吉林双正医疗科技有限公司 | Apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and making method thereof |
| CN105823880A (en) * | 2016-03-21 | 2016-08-03 | 中国科学院理化技术研究所 | Biochip utilizing hook effect to enlarge detection range and detection method thereof |
| CN107389625A (en) * | 2016-09-27 | 2017-11-24 | 上海艾瑞德生物科技有限公司 | Fluorescence immune chromatography test data processing method |
| CN107389626A (en) * | 2016-11-14 | 2017-11-24 | 上海艾瑞德生物科技有限公司 | Fluorescence immune chromatography test data processing method |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105445449A (en) * | 2015-04-04 | 2016-03-30 | 吉林双正医疗科技有限公司 | Apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and making method thereof |
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| CN107389625A (en) * | 2016-09-27 | 2017-11-24 | 上海艾瑞德生物科技有限公司 | Fluorescence immune chromatography test data processing method |
| CN107389625B (en) * | 2016-09-27 | 2020-02-18 | 上海艾瑞德生物科技有限公司 | Fluorescence immunochromatography test data processing method |
| CN107389626A (en) * | 2016-11-14 | 2017-11-24 | 上海艾瑞德生物科技有限公司 | Fluorescence immune chromatography test data processing method |
| CN107389626B (en) * | 2016-11-14 | 2020-01-17 | 上海艾瑞德生物科技有限公司 | Fluorescence immunochromatography test data processing method |
| 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 |
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Application publication date: 20140730 |