CN111896743A - Fluorescence immunochromatography test strip and preparation method and application thereof - Google Patents
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G01N2333/4737—C-reactive protein
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The invention discloses a fluorescence immunochromatographic test strip and a preparation method and application thereof, and the fluorescence immunochromatographic test strip comprises a sample pad, a fluorescence pad, a cellulose nitrate membrane and a water absorption pad which are sequentially overlapped, wherein the cellulose nitrate membrane is coated with a detection line T and a quality control line C, the cellulose nitrate membrane is further coated with a detection line T2 positioned between the detection line T and the quality control line C, the types of antibodies in the detection line T and the detection line T2 are the same, and the concentration of the antibodies in the detection line T and the detection line T2 is 1: 1.2-3. According to the invention, the detection line T2 is added, so that unreacted microsphere-antibody-antigen complex to be detected in the detection line T is captured by the antibody in T2, and the antibody concentrations in T and T2 are regulated, so that the (T peak + T2 peak) signal/C peak signal pull-off gradient is larger when the concentration of a sample to be detected is calculated, and the upper end of a calibration curve model is in an ascending trend, thereby obtaining the fluorescence immunochromatographic test strip with wider linear range and higher high end measurement value precision.
Description
Technical Field
The invention belongs to the technical field of immunochromatography detection and analysis, and particularly relates to a fluorescent immunochromatography test strip and a preparation method and application thereof.
Background
The fluorescence immunochromatography technology is a novel membrane detection technology based on antigen-antibody specific immunoreaction. The technology takes strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) as a stationary phase, a test solution as a mobile phase, a fluorescence labeling antibody or antigen fixed on a fluorescence pad, and an analyte moves on the chromatography strip through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with a plurality of antigenic determinants, a sandwich type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase, and then is combined with a coating antibody to form a sandwich type double-antibody sandwich when reaching a detection line. The fluorescence chromatography test strip has the characteristics of convenient and quick use, low cost and wide application range, can adapt to various detection conditions, and is widely used for clinical detection, such as the detection of C-reactive protein, serum amyloid A, glycated albumin, myoglobin, microalbumin, procalcitonin and other substances.
For example, patent CN102023211B discloses an immunochromatographic test strip for quantitative detection of C-reactive protein in the whole course, which is formed by sequentially overlapping and adhering a sample pad, a label pad, a coating film, and absorbent paper to a bottom plate, wherein the label pad is coated with a CRP monoclonal antibody labeled with fluorescent latex microparticles and rabbit IgG labeled with fluorescent latex microparticles; the coating film comprises a detection area and a quality control area, and the detection area is coated with another CRP monoclonal antibody which is at different epitope with the CRP monoclonal antibody marked by the fluorescent latex particles. Patent CN109085358A discloses a Serum Amyloid A (SAA) determination kit (immunochromatography) and a manufacturing method thereof, the kit comprises an immunochromatography test strip and a plastic shell, the immunochromatographic test strip is a test strip consisting of a base plate, a sample pad, a reagent pad, a nitrocellulose membrane and an absorption pad, the sample pad, the reagent pad, the nitrocellulose membrane and the absorption pad are fixed on the bottom plate in a staggered way in sequence, the reagent pad is coated with color fluorescent microspheres marked by mouse anti-human SAA monoclonal antibodies, the detection line on the nitrocellulose membrane is coated with rabbit anti-human SAA polyclonal antibodies, the quality control line is coated with goat anti-mouse polyclonal antibodies, the monoclonal antibody is a purified and mixed monoclonal antibody, is derived from monoclonal antibody cell strains aiming at 2-6 different SAA epitope, and can quickly obtain a qualitative result and later obtain a quantitative result by adopting a colored fluorescent microsphere. However, the existing immunochromatographic test strip is provided with only one detection line, when the sample contains more antigens, the T line signal is saturated and difficult to rise, and the linear upper end of the T/C test signal tends to be flat, so that the problems that the linear range of the detection line is too narrow, and the detection value of the high end of the test is inaccurate can occur.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the fluorescence immunochromatographic test strip, and the fluorescence immunochromatographic test strip has the advantages that the detection line T2 is additionally arranged between the detection line T and the quality control line C, so that the unreacted microsphere-antibody-antigen complex to be detected in the T is captured by the antibody in the T2, and the concentration of the antibody coated in the T and the T2 is adjusted, so that the fluorescence immunochromatographic test strip with a wider linear range and higher high-end measuring value precision is obtained.
In order to achieve the purpose, the invention adopts the technical scheme that:
a fluorescence immunochromatographic test strip comprises a sample pad, a fluorescence pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, wherein the nitrocellulose membrane is coated with a detection line T and a quality control line C, the nitrocellulose membrane is further coated with a detection line T2 positioned between the detection line T and the quality control line C, the types of antibodies in the detection line T and the detection line T2 are the same, and the concentrations of the antibodies in the detection line T and the detection line T2 are 1: 1.2-3.
The principle of the technical scheme is as follows: the fluorescence immunoassay analyzer acquires T, C line peak diagrams by scanning, obtains reaction intensity by calculating peak height or peak area, and reduces the difference between test strips by using T/C value because the reaction intensity of T line may be obviously changed due to the difference of chromatographic process and the C line has the same influence with T because the complexity of the sample to be tested and the immunoreaction process are influenced by the fluidity of the sample, the matrix of the sample, the capture difference of protein coating, the uniformity of nitrocellulose membrane, the left and right difference of capillary traction and the like in the chromatographic process. When only one detection T line is coated on the nitrocellulose membrane but the antigen in a sample is more, the signal of the T line is saturated and difficult to rise, so that the linear upper end of the T/C of the test signal tends to be flat; according to the invention, through setting double detection lines and adjusting the concentration ratio of antibodies in the two detection lines, the T coating concentration of the detection line is slightly lower, the T2 coating concentration is high, and when more antigens are in a sample, the unreacted microsphere-antibody-antigen-to-be-detected complex of the coated antibody at the T position can be captured by the antibody at the T2 position, so that two T peaks can be scanned. The gradient of the (T peak + T2 peak) signal/C peak signal is larger than that of the T peak signal/C peak signal in a single T line mode, namely the linear range is expanded; meanwhile, the upper end of the calibration curve model is in an ascending trend, so that when a high-concentration sample is measured and is brought into the calibration curve for calculation, the obtained concentration variation coefficient is small, and the fluorescence immunochromatographic test strip is wider in linear range and higher in high-end measurement value precision.
Preferably, the concentration of antibody in the detection line T and the detection line T2 is 1: 1.5.
Preferably, the fluorescent pad is provided with a fluorescent microsphere labeled antibody I, the detection line T and the detection line T2 are coated with an antibody II, both the antibody I and the antibody II can be coupled with an antigen to be detected, and the quality control line C is coated with an antibody III which can be coupled with the antibody I.
Preferably, the antibody III in the quality control line C is a goat anti-mouse IgG antibody.
Preferably, the sample pad is a glass fiber film or a polyester fiber film; the fluorescent pad is a glass fiber film;
the invention also provides a preparation method of the fluorescence immunochromatographic test strip, which comprises the following steps: processing a sample pad, preparing a fluorescent pad, preparing a nitrocellulose membrane, and assembling a fluorescence test immunochromatography paper strip.
The invention also provides a detection kit, and the detection kit comprises the fluorescence immunochromatographic test strip.
The invention also provides application of the fluorescence immunochromatographic test strip and/or the detection kit in clinical detection.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the detection line T2 is additionally arranged between the detection line T and the quality control line C, so that the unreacted microsphere-antibody-antigen complex to be detected in the T is captured by the antibody in the T2, and the concentration of the antibody coated in the T and the T2 is adjusted, therefore, when a T, C line crest map is scanned to calculate the concentration of a sample to be detected, the gradient of a (T peak + T2 peak) signal/C peak signal is larger, and the upper end of a calibration curve model is in an ascending trend, so that the fluorescence immunochromatographic test strip with a wider linear range and higher high-end precision is obtained.
Drawings
FIG. 1 is a calibration curve for groups 1-1 and groups 1-7 in example 1 of the present invention;
FIG. 2 is a calibration curve for groups 2-1 and 2-7 in example 2 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
Example 1 optimization of Serum Amyloid A (SAA) kit
The embodiment mainly optimizes the test strip in the SAA fluorescence immunochromatographic kit, and the preparation process of the immunochromatographic fluorescence test strip is as follows:
1. preparation of sample pad
Processing the glass fiber membrane by using Tris-HCl with the pH value of 8.0, containing 2 percent of trehalose, 0.5 percent of BSA and 0.2 percent of Tween 20, and drying in an oven at the temperature of 45 ℃ for 8 hours.
2. Preparation of the fluorescent pad:
1) preparing fluorescent solution microspheres: fluorescent microspheres (available from Bangs Laboratories, Inc.) were diluted to 0.1% (W/W) with 0.05M MESpH6.0;
2) activation of microspheres: dissolving NHS with purified water to 10mg/ml, quickly adding the solution into a fluorescent microsphere solution, uniformly mixing by vortex, wherein the addition amount of the NHS solution is 10 mu g/ml, dissolving EDC with purified water to 10mg/ml, quickly adding the solution into the fluorescent microsphere solution, uniformly mixing by vortex, wherein the addition amount of the EDC solution is 20 mu g/ml, and placing the solution in a shaking table at 37 ℃ to shake for 30 min.
3) High speed centrifugation at 15000rpm for 20min to remove supernatant, diluting the precipitate with 0.02M PBS, pH7.4 crosslinking reaction solution to 1ml, and ultrasonic dispersing for 2 min.
4) The SAA monoclonal antibody 1 (purchased from Wuhan Huamei bioengineering Co., Ltd.) is diluted to 1mg/mL, added into the fluorescent microsphere solution with the final antibody concentration of 100 mu g/mL, vortexed and mixed evenly, and placed in a shaking table at 37 ℃ for 2 h.
5) Adding 100 mul of sealant into the fluorescent microsphere solution, and uniformly mixing by vortex; the blocking agent is 10% BSA solution, and is placed in a shaker at 37 ℃ to shake for 1 h.
6) Centrifuging at 15000rpm for 20min, removing supernatant, precipitating with Tris-HCl redissolution with pH of 8.0 to 1mL, and ultrasonically homogenizing.
7) And (3) spraying the fluorescent microsphere solution on a glass fiber membrane, and drying in an oven at 45 ℃ for 8 h.
3. Preparation of nitrocellulose membrane:
1) quality control line C: coating by diluting goat anti-mouse IgG antibody (purchased from Wuhan Huamei bioengineering Co., Ltd.) to 1.5mg/mL with 0.01M PBS pH7.4 containing 1% trehalose, with streaking parameter of 1 μ L/cm and speed of 80 mm/s;
2) and (3) detection line: SAA monoclonal antibody 2 (purchased from Wuhan Huamei bioengineering, Co., Ltd.) was diluted to different concentrations with 0.01M PBS pH7.4 containing 1% trehalose, and coated separately for two test lines, wherein the antibody concentration of test line T was 1.5mg/mL, the parameters of the assay were 1. mu.L/cm, the speed was 80mm/s, and the antibody concentrations at test line T and test line T2 are shown in the following table:
3) after scribing, putting the nitrocellulose membrane into a baking oven at 45 ℃ for drying for 8h
4. Assembling: assembling the sample pad, the fluorescence pad, the nitrocellulose membrane and the water absorption pad into a cutting strip and mounting the cutting strip to obtain the Serum Amyloid A (SAA) fluorescence immunochromatographic test strip.
And (3) linear evaluation: SAA positive samples with different concentrations are prepared, the 7 groups of test strips are tested, and the fluorescence signal intensities of T, T2 and C lines are respectively collected by a fluorescence immunoassay analyzer. Because the complexity of the sample to be detected and the immunoreaction process are influenced by the sample fluidity in the chromatography process, the sample matrix, the protein coating capture difference, the uniformity of an NC membrane and the difference of the left and right capillary traction, the reaction strength of the detection line can be obviously changed due to the difference in the chromatography process, and the quality control line has the same influence with the detection line, so that the detection result can be corrected by the quality control line, and the difference between the test strips can be reduced. (T + T2)/C value or T/C value was calculated for different SAA positive samples, respectively. The results are shown in Table 1, and corresponding calibration curves are plotted based on the data of sets 1-1 and 1-7 in Table 1, as shown in FIG. 1.
And (3) precision evaluation: in order to detect the measurement precision of 7 groups of test strips on high-concentration SAA positive samples, the 7 groups of test strips are used for measuring the SAA samples with the concentration of 110mg/L respectively, the measurement is repeated for 10 times, and the Coefficient of Variation (CV) of each group of test strips is calculated, and the results are shown in Table 2.
TABLE 1SAA sample T/C Signal ratio
| SAA concentration (mg/L) | Group 1-1 | Groups 1 to 2 | Groups 1 to 3 | Groups 1 to 4 | Groups 1 to 5 | Groups 1 to 6 | Groups 1 to 7 |
| 3 | 0.2183 | 0.1171 | 0.1345 | 0.0830 | 0.0741 | 0.0912 | 0.066 |
| 12.5 | 0.6781 | 0.5582 | 0.6133 | 0.3077 | 0.2894 | 0.4762 | 0.3677 |
| 25 | 1.0849 | 0.9249 | 0.9921 | 0.7340 | 0.7042 | 0.8413 | 0.6498 |
| 50 | 1.7273 | 1.4344 | 1.6427 | 1.4346 | 1.4133 | 1.5447 | 1.2391 |
| 100 | 2.8234 | 2.3601 | 2.5783 | 1.9387 | 1.8592 | 2.1134 | 1.7985 |
| 200 | 5.1216 | 4.3978 | 4.6679 | 2.7447 | 2.6875 | 3.8751 | 2.5489 |
| Linear phaseCoefficient of correlation (r)2) | 0.9971 | 0.9966 | 0.9952 | 0.9559 | 0.9564 | 0.9896 | 0.9651 |
TABLE 2 precision measurement data
| Group of | Coefficient of Variation (CV) |
| 1-1 | 7.52％ |
| 1-2 | 8.81％ |
| 1-3 | 9.27％ |
| 1-4 | 16.55％ |
| 1-5 | 17.24％ |
| 1-6 | 13.48％ |
| 1-7 | 18.73％ |
Combining the results of table 1, table 2 and fig. 1, when there is no detection line T2 (groups 1-7), as the concentration of SAA sample increases, saturation is reached at the T line, resulting in a lower T/C value and a flatter upper end of the calibration curve model and a lower linear correlation value, i.e. a narrow linear range and a poor linear correlation, and for a high concentration sample, the coefficient of variation of the measured value is greater than 10%, i.e. the precision of the measured value of high concentration is poor. When the concentration of the antibody in the detection line T is equal to or higher than that of the antibody in the T2 (groups 1-4 and 1-5), the antigen captured in the T line is too high, the antigen captured in the T2 is less or not, and the measurement result is inaccurate, and the (T + T2)/C value and the linear correlation coefficient are low, so that the precision is poor when a high-concentration sample is measured; similarly, too high concentration of the antibody at T2 (groups 1-6) also affects the captured antigen at the T line, and further affects the (T + T2)/C value, the linear correlation coefficient and the precision of the measurement value of the high concentration sample, and only when the concentration of the antibody at the detection line T and the detection line T2 is 1:1.2-3 (groups 1-1, 1-2 and 1-3), the (T + T2)/C value measured in the high concentration SAA sample is high, i.e., the linear range is widened, and the linear correlation coefficients are both greater than 0.99, so that the linear correlation is good. And because the upper end of the corresponding calibration curve is in an ascending trend, for a high-concentration sample, when a test result is brought into the calibration curve equation to calculate the concentration of the sample, the obtained concentration variation coefficients are small (all are less than 10%), namely the precision of a high-end measured value is higher.
Example 2 optimization of C-reactive protein (CRP) kits
The preparation method of the present embodiment and the immunochromatographic fluorescent test strip is substantially the same as that of embodiment 1, and the differences are as follows: SAA monoclonal antibody 1 was replaced with CRP monoclonal antibody 1 (purchased from warburg bioengineering, inc.), SAA monoclonal antibody 2 was replaced with CRP monoclonal antibody 2 (purchased from warburg bioengineering, inc., wuhan), wherein the concentration of CRP monoclonal antibody 2 of test line T was 1.5mg/mL, and the antibody concentrations in test line T and test line T2 were as shown in the following table:
| group of | T: concentration ratio of T2 antibody |
| 2-1 | 1：1.5 |
| 2-2 | 1：1.2 |
| 2-3 | 1：3 |
| 2-4 | 1：1 |
| 2-5 | 1：0.5 |
| 2-6 | 1：4 |
| 2-7 | No T2 |
The linear and precision evaluations were carried out according to the method described in example 1 and the corresponding calibration curves were plotted based on the data of the sets 2-1 and 2-7, the results being shown in Table 3, Table 4 and FIG. 2
TABLE 3CRP sample T/C Signal ratio
| CRP concentration (mg/L) | Group 2-1 | Group 2-2 | |
|
|
|
|
| 1 | 0.1319 | 0.1105 | 0.1235 | 0.0732 | 0.0695 | 0.0754 | 0.0528 |
| 10 | 0.3540 | 0.3251 | 0.3467 | 0.2424 | 0.2087 | 0.2561 | 0.2685 |
| 25 | 0.7377 | 0.6876 | 0.7189 | 0.5397 | 0.4763 | 0.5581 | 0.5737 |
| 50 | 1.2579 | 1.1497 | 1.2355 | 1.0241 | 0.9537 | 1.0892 | 0.9472 |
| 100 | 2.1559 | 1.9849 | 2.0763 | 1.4690 | 1.3569 | 1.6143 | 1.3729 |
| 200 | 3.8131 | 3.3413 | 3.5413 | 2.3082 | 2.1043 | 2.6849 | 1.6781 |
| Linear coefficient of correlation (r)2) | 0.9975 | 0.9951 | 0.9955 | 0.9812 | 0.9790 | 0.9887 | 0.9313 |
TABLE 4 precision measurement data
Similar to the results of example 1, combining the results of table 3, table 4 and fig. 2, it was found that only when the antibody concentration at the detection line T and the detection line T2 was 1:1.2-3 (groups 2-1, 2-2 and 2-3), the (T + T2)/C value measured in the high concentration SAA sample was high, i.e., the linear range was wide, and the linear correlation coefficients were all greater than 0.99, the linear correlation was good, and since the upper end of the calibration curve corresponding thereto showed an upward trend, for the high concentration sample, when the test results were taken into the calibration curve equation to calculate the sample concentration, the concentration variation coefficients were small (all less than 10%), i.e., the high-end measurement value was more precise.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The fluorescence immunochromatographic test strip comprises a sample pad, a fluorescence pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, wherein the nitrocellulose membrane is coated with a detection line T and a quality control line C, and the fluorescence immunochromatographic test strip is characterized in that the nitrocellulose membrane is further coated with a detection line T2 positioned between the detection line T and the quality control line C, wherein the types of antibodies in the detection line T and the detection line T2 are the same, and the concentrations of the antibodies in the detection line T and the detection line T2 are 1: 1.2-3.
2. The fluorescence immunochromatographic test strip according to claim 1, wherein the concentration of the antibodies in the detection line T and the detection line T2 is 1: 1.5.
3. The strip of claim 1, wherein the fluorescent pad has a first antibody labeled with fluorescent microspheres, the detection line T and the detection line T2 are coated with a second antibody, both the first antibody and the second antibody can be coupled to an antigen to be detected, and the quality control line C is coated with a third antibody coupled to the first antibody.
4. The fluorescence immunochromatographic test strip according to claim 3, wherein the antibody III in the quality control line C is a goat anti-mouse IgG antibody.
5. The fluorescence immunochromatographic test strip according to claim 1, wherein the sample pad is a glass fiber membrane or a polyester fiber membrane; the fluorescent pad is a glass fiber film.
6. The method for preparing the fluorescent immunochromatographic test strip of any one of claims 1 to 5, which comprises the following steps: processing a sample pad, preparing a fluorescent pad, preparing a nitrocellulose membrane, and assembling a fluorescence test immunochromatography paper strip.
7. A detection kit, characterized in that the detection kit comprises the fluorescence immunochromatographic test strip of any one of claims 1 to 5.
8. The use of the fluorescence immunochromatographic test strip of any one of claims 1 to 5 and/or the detection kit of claim 7 in clinical tests.
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