CN115684587A - Method for improving detection precision of fluorescence immunochromatographic test strip - Google Patents
Method for improving detection precision of fluorescence immunochromatographic test strip Download PDFInfo
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- CN115684587A CN115684587A CN202110828527.3A CN202110828527A CN115684587A CN 115684587 A CN115684587 A CN 115684587A CN 202110828527 A CN202110828527 A CN 202110828527A CN 115684587 A CN115684587 A CN 115684587A
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Abstract
The invention discloses a method for improving detection precision of a fluorescence immunochromatographic test strip, which comprises the steps of preparing a monoclonal antibody I for marking an antigen to be detected into a freeze-dried pellet, mixing a sample and the freeze-dried pellet for marking the monoclonal antibody I for the antigen to be detected uniformly in advance, and adding the mixture into a sample adding hole of the fluorescence immunochromatographic test strip for detection. The traditional mode of coating the marked antibody in the sample pad is changed into a mode of freeze-drying the marked antigen monoclonal antibody I to be detected into a small ball, the reaction sufficiency of the antigen and the antibody can be improved in a liquid medium when the sample and the marked antigen monoclonal antibody I to be detected are uniformly mixed, and the releasing property of a combination of the marked antibody and the antigen can be improved when the sample is added into a sample adding hole of a fluorescence immunochromatographic test strip after uniform mixing, so that the sensitivity and the precision of the reagent are improved.
Description
Technical Field
The invention relates to a method for improving detection precision of a fluorescence immunochromatographic test strip.
Background
The immunochromatography is a rapid diagnosis technology commonly used at home and abroad, and has the advantages of simple and rapid operation, convenient use, no time and place limitation, suitability for bedside detection and the like.
The current common fluorescence immunochromatographic test strip on the market comprises a sample pad, wherein the sample pad is coated with monoclonal antibodies I marked by antigens to be detected and an NC membrane (nitrocellulose membrane), and the NC membrane is coated with a quality control line and a test line. When a sample is added into the sample adding hole, when the antigen in the sample flows through the sample pad, the antigen and the monoclonal antibody I marked by the antigen to be detected on the sample pad and the monoclonal antibody II marked by the antigen to be detected on the NC membrane detection line form a double-antibody sandwich, the reaction time is short and insufficient, and the precision is poor.
Disclosure of Invention
The invention aims to provide a method for improving the detection precision of a fluorescence immunochromatographic test strip, which can improve the release property of a conjugate of a monoclonal antibody I for marking an antigen to be detected and the antigen to be detected, thereby improving the sensitivity and precision of a reagent.
The technical scheme adopted by the invention is as follows: a method for improving detection precision of a fluorescence immunochromatographic test strip comprises the steps of preparing a monoclonal antibody I for marking an antigen to be detected into a freeze-dried pellet, mixing a sample and the freeze-dried pellet for marking the monoclonal antibody I for marking the antigen to be detected uniformly in advance, and adding the mixture into a sample adding hole of the fluorescence immunochromatographic test strip for detection.
Preferably, the antigen-labeled monoclonal antibody I to be detected is placed in a quantitative sampling pipette or a sample needle, and a sample sucked by the quantitative sampling pipette or the sample needle and the antigen-labeled monoclonal antibody I to be detected are mixed uniformly in advance in the quantitative sampling pipette or the sample needle and then added into a sample adding hole of a fluorescence immunochromatographic test strip for detection. Preferably, fluorescence immunochromatography reagent strip includes the casing and is located the inside test paper strip of casing, reagent strip includes sample pad, NC membrane and the absorbent paper of overlap joint each other in proper order, it has antigen monoclonal antibody II to be measured to coat on the testing line of NC membrane, it has goat anti mouse IgG antibody or rabbit anti mouse IgG antibody to coat on the quality control line of NC membrane, it is corresponding with the position of sample pad to add the appearance hole and be located the casing.
Preferably, the monoclonal antibody I for marking the antigen to be detected is a monoclonal antibody I for marking cardiac troponin I, a monoclonal antibody I for marking an N-terminal pro-brain natriuretic peptide precursor, a monoclonal antibody I for marking procalcitonin or a monoclonal antibody I for marking glycated hemoglobin, and the monoclonal antibody II for the antigen to be detected is a monoclonal antibody II for the cardiac troponin I, a monoclonal antibody II for the N-terminal pro-brain natriuretic peptide precursor, a monoclonal antibody II for the procalcitonin or a monoclonal antibody II for glycated hemoglobin.
Preferably, the freeze-dried pellet preparation process: adding the labeled antibody into a freeze-drying protective agent according to the mass ratio of 0.01-3, uniformly mixing, dripping 3-30 ul of the mixture into liquid nitrogen, forming small balls after dripping the liquid nitrogen, collecting the small balls in the liquid nitrogen, putting the small balls into a freeze-drying machine for drying treatment, and obtaining the freeze-dried small balls after the drying is finished.
The invention has the following beneficial effects: the invention changes the traditional mode of coating the marked antibody in the sample pad into the mode of freeze-drying the marked antigen monoclonal antibody I to be detected into a small ball, can improve the reaction sufficiency of the antigen and the antibody when the sample and the marked antigen monoclonal antibody I to be detected are mixed uniformly in a liquid medium, and can improve the releasing property of a combination of the marked antibody and the antigen when the sample is added into a sample adding hole after being mixed uniformly, thereby improving the sensitivity and the precision of the reagent.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings required to be used in the embodiments will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive labor.
FIG. 1 is a schematic view of a quantitative sampling pipette, and FIG. 1 is a lyophilized pellet.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The technical solutions provided by the embodiments of the present invention are described in detail below with reference to the accompanying drawings.
A fluorescence immunochromatography detection kit comprises a quantitative sampling pipette and a fluorescence immunochromatography reagent strip, wherein the quantitative sampling pipette can be shown in figure 1 or in other forms.
Example 1 method for improving detection precision of cTnI fluorescence immunochromatographic test strip
1) Adding the marked cTnI monoclonal antibody I into a freeze-drying protective agent (3 wt% of mannitol, 5wt% of sucrose, 1wt% of glycine and 91wt% of water) according to a mass ratio of 1;
2) And (3) sucking 100ul of sample by a quantitative sampling pipette, mixing the sample with the cTnI monoclonal antibody I freeze-dried pellet in the quantitative sampling pipette or the sample needle uniformly in advance, and then dripping the sample into a sample adding hole of a fluorescence immunochromatographic test strip for detection.
The quantitative sampling pipette in this embodiment can be as shown in fig. 1, and can be in any other form as long as it can perform quantitative pipetting, in this embodiment, the labeled cTnI monoclonal antibody I lyophilized pellet 1 prepared in step 1) above is placed in a quantitative sampling pipette, and the number is 1; in addition, the quantitative sampling pipette liquid in the embodiment can be replaced by a sample needle (not shown in the embodiment) of a fluorescence immunoassay quantitative analyzer, the freeze-dried pellet is placed in the quantitative sample needle, and when in measurement, a sample automatically sucked by the sample needle is mixed with the freeze-dried pellet in the sample needle in advance, and then the sample is added into a sample adding hole of a fluorescence immunochromatographic test strip for detection.
In this implementation fluorescence immunochromatographic reagent strip, including the casing with be located the inside test paper strip of casing, the reagent strip is including sample pad, NC membrane and the paper that absorbs water that overlap joint each other in proper order, wrap 4.0mg/ml cTnI monoclonal antibody II with 0.7ul/cm on the testing line of NC membrane, wrap 2mg/ml goat anti mouse IgG antibody with 0.8ul/cm on the quality control line of NC membrane, it is corresponding with the position of sample pad to add the sample hole and be located the casing.
Comparative example 1
The kit adopts a conventional fluorescence immunochromatographic reagent strip which is the same as the fluorescence immunochromatographic reagent strip in the embodiment 1, and comprises a latex pad, wherein the latex pad is overlapped with a space between a sample pad and an NC membrane, 4.0mg/ml EU-labeled cTnI monoclonal antibody I is coated on the latex pad at 1.5ul/cm, and 100ul of a sample sucked by a quantitative sampling pipette is added into a sample hole of the fluorescence immunochromatographic test strip for detection.
6 cTnI samples (35 ng/ml, 11ng/ml, 5.5ng/ml, 1.4ng/ml, 0.3ng/ml and 0.05 ng/ml) with concentration levels are selected for repeated tests, 14 fluorescence immunochromatography reagent strips in the same batch are taken and are respectively detected by matched fluorescence immunoassay quantitative analysis, the total average value (X) and the intra-batch standard deviation (S batch) of the measured values are respectively calculated, and the intra-batch variation Coefficient (CV) is obtained according to the intra-batch standard deviation/total average value. The specific test results of example 1 and comparative example 1 are shown in table 1:
TABLE 1
The experimental result shows that the precision of the reagent can be well improved by adopting the freeze-drying process in the embodiment 1, and the CV can be controlled within 8 percent and is greatly improved compared with the conventional process.
hs-cTn proposed in the Chinese expert consensus for clinical application of detecting cardiac troponin by the high-sensitivity method should be capable of detecting cTn in more than 50% of surface healthy population, and the imprecision of detection (expressed as CV) of the 99 th percentile value at the upper limit of the reference range should be less than or equal to 10%. 500 cases of surface healthy population are detected, the 99 percent value is 0.038ng/ml, and the 50 percent value is 0.01ng/ml. Precision experiments were performed on levels near the 99 percentile, with experimental results shown in table 2:
TABLE 2
The experimental result shows that when the concentration is more than 0.03ng/ml, the CV can be controlled within 10 percent, the upper limit of the reference range is 0.04ng/ml and the sensitivity is 0.01ng/ml by combining the detection of 99 percent locus value and the imprecise detection
Embodiment 2 a method for improving detection precision of hemoglobin fluorescence immunochromatographic test strip
1) Adding the marked hemoglobin monoclonal antibody I into a freeze-drying protective agent (3 wt% of mannitol, 5wt% of sucrose, 1wt% of glycine and 91wt% of water) according to a ratio of 0.1;
2) And (3) sucking 10ul of sample by a quantitative sampling pipette, uniformly mixing the sample with the freeze-dried globule of the labeled hemoglobin monoclonal antibody I in the quantitative sampling pipette or the sample needle in advance, and then dropwise adding the sample into a sample adding hole of a fluorescence immunochromatographic test strip for detection.
The quantitative sampling pipette in this example can be as shown in fig. 1, and can be in any other form as long as it can perform quantitative pipetting, and in this example, the labeled hemoglobin monoclonal antibody I lyophilized pellet prepared in step 1) is placed in a quantitative sampling pipette, and the number is 1; in addition, the quantitative sampling pipette liquid in this embodiment can be replaced by a sample needle of a fluorescence immunoassay quantitative analyzer (not shown in this embodiment), the freeze-dried pellet is placed in the quantitative sample needle, and during measurement, a sample automatically sucked by the sample needle is uniformly mixed with the freeze-dried pellet in the sample needle in advance, and then the mixture is added into a sample hole of a fluorescence immunochromatographic test strip for detection.
The fluorescence immunochromatographic reagent strip comprises a shell and a test strip positioned in the shell, the reagent strip comprises a sample pad, an NC membrane and absorbent paper which are sequentially and mutually overlapped, 2.0mg/ml glycosylated hemoglobin monoclonal antibody II is coated on a detection line of the NC membrane by 1.2ul/cm, 2.0mg/ml goat anti-mouse IgG antibody is coated on a quality control line of the NC membrane by 1.2ul/cm, and a sample adding hole is positioned on the shell and corresponds to the position of the sample pad.
Comparative example 2
Adopt conventional fluorescence immunochromatography reagent strip and detection method, with embodiment 2 the fluorescence immunochromatography reagent strip is different, still include the latex pad, between latex pad overlap joint and sample pad and NC membrane, latex pad on with 1.2ul/cm cladding 5.0mg/ml mark hemoglobin monoclonal antibody I, quantitative sampling straw absorbs 10ul sample and after mixing in the diluent, take 100ul mixing liquid and drip to fluorescence immunochromatography test paper strip application of sample hole and detect.
Selecting 5 HbA1c samples (13.5%, 10.0%, 8.2%, 6.8%, 5.3%) to make repetitious test, taking 14 portions of fluorescence immunochromatographic reagent strips of same batch, using matched fluorescence immunoassay to make detection respectively, respectively calculating total average value of measured values (A, B, C) X ) And intra-batch standard deviation (intra-batch), and obtaining the intra-batch Coefficient of Variation (CV) according to the intra-batch standard deviation/total average value. The specific test results of example 2 and comparative example 2 are shown in table 3:
TABLE 3
Experimental results show that the precision of the reagent can be well improved by adopting the freeze-drying process in the embodiment 2, and the CV can be controlled within 3.6 percent, which is greatly improved compared with the conventional process.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for improving detection precision of a fluorescence immunochromatographic test strip is characterized in that a monoclonal antibody I for marking an antigen to be detected is prepared into a freeze-dried pellet, a sample and the freeze-dried pellet I for marking the monoclonal antibody I to be detected are mixed uniformly in advance, and then the mixture is added into a sample adding hole of the fluorescence immunochromatographic test strip for detection.
2. The method for improving the detection precision of the fluorescence immunochromatographic test strip according to claim 1, wherein the freeze-dried beads for labeling the antigen monoclonal antibody I to be detected are placed in a quantitative sampling pipette or a sample needle, and the sample sucked by the quantitative sampling pipette or the sample needle and the labeled antigen monoclonal antibody I to be detected are mixed uniformly in advance in the quantitative sampling pipette or the sample needle and then added into a sample hole of the fluorescence immunochromatographic test strip for detection.
3. The method for improving the detection precision of the fluorescence immunochromatographic test strip according to claim 1, wherein the fluorescence immunochromatographic reagent strip comprises a shell and the test strip positioned in the shell, the reagent strip comprises a sample pad, an NC membrane and absorbent paper which are sequentially overlapped with each other, a detection line of the NC membrane is coated with an antigen monoclonal antibody II to be detected, a quality control line of the NC membrane is coated with a goat anti-mouse IgG antibody or a rabbit anti-mouse IgG antibody, and the sample adding hole is positioned on the shell and corresponds to the position of the sample pad.
4. The method for improving the detection precision of the fluorescence immunochromatographic test strip according to claim 1, wherein the freeze-dried pellet preparation process comprises the following steps: adding the labeled antibody into a freeze-drying protective agent according to the mass ratio of 0.01-3, uniformly mixing, dripping 3-30 ul of the mixture into liquid nitrogen, forming small balls after dripping the liquid nitrogen, collecting the small balls in the liquid nitrogen, putting the small balls into a freeze-drying machine for drying treatment, and obtaining the freeze-dried small balls after the drying is finished.
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| CN117451992A (en) * | 2023-11-06 | 2024-01-26 | 中国人民解放军军事科学院军事医学研究院 | Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117451992A (en) * | 2023-11-06 | 2024-01-26 | 中国人民解放军军事科学院军事医学研究院 | Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof |
| CN117451992B (en) * | 2023-11-06 | 2024-07-02 | 中国人民解放军军事科学院军事医学研究院 | Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof |
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