阅读说明：本技术 一种无钩状效应的免疫层析试纸条及制备方法 (Immunochromatography test strip without hook effect and preparation method thereof ) 是由 董涛 何国桢 杨朝初 图莉尔·伦宁达 阿若·布兰斯塔德 于 2021-08-12 设计创作，主要内容包括：本发明公开了一种无钩状效应的免疫层析试纸条及制备方法,该免疫层析试纸条包括底板,所述底板的上方设置有硝化纤维膜、样品垫和吸收垫,所述硝化纤维膜位于所述样品垫和所述吸收垫之间,所述硝化纤维膜上方设置有过渡垫、测试线、控制线,所述测试线设置在所述过渡垫和所述控制线之间,所述过渡垫的上方设置有共轭垫,所述共轭垫的上方设置有标记抗体。在无需额外人工操作的情况下,低成本地解决了传统三明治法LFIA试纸条中出现的钩状效应,有效消除了测试结果中的假阴性,破除现有LFIA试纸条应用限制；新型免疫侧流层析试纸条针对分析物展现出极高的检测特异性。(The immunochromatographic test strip comprises a bottom plate, wherein a nitrocellulose membrane, a sample pad and an absorption pad are arranged above the bottom plate, the nitrocellulose membrane is positioned between the sample pad and the absorption pad, a transition pad, a test line and a control line are arranged above the nitrocellulose membrane, the test line is arranged between the transition pad and the control line, a conjugate pad is arranged above the transition pad, and a marker antibody is arranged above the conjugate pad. Under the condition of no need of additional manual operation, the hook effect in the LFIA test strip of the traditional three-Ming-and-technology method is solved at low cost, the false negative in the test result is effectively eliminated, and the application limit of the traditional LFIA test strip is broken; the novel immune lateral flow chromatography test strip shows extremely high detection specificity aiming at the analyte.)

1. The utility model provides an immunity chromatography test paper strip of no hook-shaped effect, includes bottom plate (1), its characterized in that: the upper portion of bottom plate (1) is provided with nitrocellulose membrane (2), sample pad (3) and absorption pad (4), nitrocellulose membrane (2) are located sample pad (3) with between absorption pad (4), nitrocellulose membrane (2) top is provided with transition pad (5), test wire, control line, the test wire sets up transition pad (5) with between the control line, the top of transition pad (5) is provided with conjugate pad (6), the top of conjugate pad (6) is provided with the mark antibody.

2. The immunochromatographic strip without a hook effect according to claim 1, characterized in that: the transition pad (5) is a film processed by a chemical method or a film processed by a physical method and with gradient distribution of porosity.

3. A preparation method of an immunochromatographic test strip without a hook effect is characterized in that: the method comprises the following steps:

the method comprises the following steps: fixing a test line and a control line on the nitrocellulose membrane (2), and drying for 2 hours at 36 ℃ or until the nitrocellulose membrane (2) is completely dried;

step two: blocking the nitrocellulose membrane (2) with a 0.01MPBS buffer solution containing 2% w/v BSA and 0.02% w./v.tween-20 at room temperature, drying at 36 ℃ for 2h or until the nitrocellulose membrane (2) is completely dried;

step three: adding sucrose in a supersaturated amount into deionized water, and stirring at room temperature for 3 to 4 days to obtain a saturated aqueous solution of sucrose; removing sucrose precipitate in the solution, and diluting the saturated sucrose solution to a desired concentration by using deionized water;

step four: cutting the nitrocellulose membrane (2), and soaking the nitrocellulose membrane (2) in a sucrose solution from the side of the nitrocellulose membrane (2) until the nitrocellulose membrane (2) is completely soaked in the sucrose solution; no air bubbles can remain on the nitrocellulose membrane (2); completely drying the nitrocellulose membrane (2);

step five: immobilizing the labeled antibody which is coupled in advance on the conjugate pad (6), and completely drying at 36 ℃;

step six: assembling an LFIA test strip: firstly, a nitrocellulose membrane (2) is pasted on a bottom plate (1), then a transition pad (5), an absorption pad (4) and a sample pad (3) are pasted in sequence, and a conjugate pad (6) is pasted on the transition pad (5).

4. The method for preparing an immunochromatographic test strip without a hook effect according to claim 1, which is characterized in that: and in the second step, the pH value of the 0.01MPBS buffer solution is 7.2.

5. The method for preparing an immunochromatographic test strip without a hook effect according to claim 1, which is characterized in that: the labeled antibody is a fluorescence labeled antibody or a nanogold labeled antibody.

Technical Field

The invention belongs to the technical field of in-vitro detection, and particularly relates to an immunochromatographic test strip without a hook effect and a preparation method thereof.

Background

The Lateral Flow Immunochromatography (LFIA) technology based on the sandwich method has been widely developed in the field of in vitro detection due to the advantages of low cost, quick effect, simple and convenient operation and the like, and can be used for qualitative or quantitative analysis. The LFIA technology of qualitative analysis is mostly based on a colorimetric method, utilizes the surface plasmon resonance phenomenon of gold nanoparticles for detection, and is suitable for application scenes such as pregnancy, drug abuse, infectious disease detection and the like. The LFIA technique of quantitative analysis is similar to the colorimetric method of qualitative LFIA, and the color depth is utilized to realize quantitative detection; in recent years, a fluorescence method using quantum dots or fluorescent microspheres as a label has also been developed. Quantitative LFIAs are useful in the early diagnosis of major diseases such as heart disease, chronic obstructive pulmonary disease, cancer, etc. On the other hand, all current immune lateral flow chromatography test strips based on the sandwich method have the hook effect when the concentration of the analyte to be tested is too high, and the test result shows false negative no matter qualitative or quantitative.

The hook effect greatly limits the commercial application of LFIA and there is no ideal solution. The current solutions are: 1. diluting the sample; 2. rinsing; 3. increasing the concentration of the labeled antibody; 4. adding a third test line or applying a plurality of test zones; 5. and (4) dynamic measurement. The dilution sample and rinse method introduces an additional manual operation step in the LFIA test. The three methods of increasing the concentration of the labeled antibody, adding a third test zone and applying multiple test zones can significantly increase the manufacturing cost of the LFIA test strip. The dynamic measurement method is a method of making false negative, but still limits the detection range in quantification. Therefore, none of these methods completely solves the problem caused by the hook effect.

Disclosure of Invention

The invention provides an immunochromatographic test strip without hook effect and a preparation method thereof for solving the problems.

The technical scheme adopted by the invention is as follows:

the utility model provides an immunochromatographic test strip of no hook form effect, includes the bottom plate, the top of bottom plate is provided with nitrocellulose membrane, sample pad and absorption pad, the nitrocellulose membrane is located the sample pad with absorb between the pad, nitrocellulose membrane top is provided with transition pad, test wire, control line, the test wire sets up the transition pad with between the control line, the top of transition pad is provided with conjugate pad, keeps apart nitrocellulose membrane and conjugate pad's direct contact through transition pad, plays the effect that delays conjugate pad release mark antibody, conjugate pad's top is provided with the mark antibody.

Wherein, the transition pad is a film processed by a chemical method or a film processed by a physical method and has gradient distribution of porosity; adjusting the permeability and infiltration speed of a solution sample to be detected in the transition pad by a chemical method or a physical method, so that the transition pad becomes a barrier for resisting the flow of the solution and a switch for releasing the labeled antibody, and the conjugate pad starts to infiltrate and release the labeled antibody at a specific time; delaying the release of the labeled antibody can effectively separate the competitive reaction between the analyte to be detected and the immobilized antibody and the labeled antibody, thereby eliminating the hook effect at high analyte concentration.

The invention also provides a preparation method of the immunochromatographic test strip without the hook effect, which comprises the following steps:

the method comprises the following steps: fixing a test line and a control line on the nitrocellulose membrane, and drying for 2 hours at 36 ℃ or until the nitrocellulose membrane is completely dried;

step two: blocking the nitrocellulose membrane with a 0.01MPBS buffer solution containing 2% w/v BSA and 0.02% w./v.tween-20 at room temperature, and drying at 36 ℃ for 2h or until the nitrocellulose membrane is completely dried;

step three: adding sucrose in a supersaturated amount into deionized water, and stirring at room temperature for 3 to 4 days to obtain a saturated aqueous solution of sucrose; removing sucrose precipitate in the solution, and diluting the saturated sucrose solution to a desired concentration by using deionized water;

step four: cutting the nitrocellulose membrane, and soaking the nitrocellulose membrane in a sucrose solution from the side of the nitrocellulose membrane until the nitrocellulose membrane is completely soaked in the sucrose solution; any air bubbles can not be remained on the nitrocellulose membrane; completely drying the nitrocellulose membrane;

step five: immobilizing the pre-coupled labeled antibody on a conjugate pad, and completely drying at 36 ℃;

step six: assembling an LFIA test strip: firstly, a nitrocellulose membrane is stuck on a bottom plate, then a transition pad, an absorption pad and a sample pad are stuck in sequence, and a conjugate pad is stuck on the transition pad.

Wherein the pH of the 0.01M PBS buffer solution in the second step is 7.2.

Wherein the labeled antibody is a fluorescence labeled antibody or a nanogold labeled antibody.

The invention has the following advantages:

1. under the condition of no need of additional manual operation, the hook effect in the LFIA test strip of the traditional three-Ming-and-technology method is solved at low cost, the false negative in the test result is effectively eliminated, and the application limit of the traditional LFIA test strip is broken; the novel immune lateral flow chromatography test strip shows extremely high detection specificity aiming at the analyte;

2. in conventional LFIA test strips, the analyte binds to both the labeled antibody and the detection antibody. Among these, the analyte generally has the highest affinity for the labeled antibody in solution. When the analyte is in excess (at too high a concentration), most of the labeled antibody will preferentially bind to the analyte, reaching equilibrium, and forming a labeled antibody-analyte complex. At this time, the complex and the unlabeled detection object form a competitive reaction with the detection antibody of the T line, and the residual labeled antibody in the solution sample is insufficient to label the detection object on the T line, resulting in a false negative result. By introducing the transition pad with adjustable infiltration rate, the release time of the labeled antibody is controlled, and the competitive reaction among the analyte, the labeled antibody and the detection antibody in the traditional test strip is effectively avoided. By optimizing the release time, the hook effect is resolved by maximizing the binding of unlabeled analyte to the T-line and then releasing the labeled antibody to label the analyte on the T-line. In addition, the method for treating the cellulose membrane by using the sucrose solution is simple and easy to implement. The transition pad treated by the chemical method can effectively block the flow of the solution, and the switch effect is realized. By contrast, under the same optimized condition, the novel immune lateral flow chromatography test strip with the transition pad has a detection range 100 times wider than that of the traditional test strip.

Drawings

Fig. 1 is a schematic structural diagram of an immunochromatographic test strip without a hook effect provided in an embodiment of the present invention.

FIG. 2 shows the test curves (a and b) and the test results (C and d) of the novel LFIA test strip with a transition pad, which are detected by using C-reactive protein as an example;

FIG. 3 is a test curve of a conventional LFIA test strip for comparison, which is provided by an embodiment of the present invention and uses C-reactive protein as an example for detection;

fig. 4 is a test of the wetting rate of the transition pad treated with sucrose solution provided by the present invention.

The reference numerals are explained below:

1. a base plate; 2. a nitrocellulose membrane; 3. a sample pad; 4. an absorbent pad; 5. a transition pad; 6. conjugate pad.

Detailed Description

The present invention will be further described below, but the present invention is not limited to these.

Examples

The utility model provides an immunochromatographic test strip of no hook form effect, includes bottom plate 1, and the top of bottom plate 1 is provided with nitrocellulose membrane 2, sample pad 3 and absorption pad 4, and nitrocellulose membrane 2 is located between sample pad 3 and the absorption pad 4, and 2 tops of nitrocellulose membrane are provided with transition pad 5, test wire, control line, and the test wire setting is between transition pad 5 and control line, and the top of transition pad 5 is provided with conjugate pad 6, and the top of conjugate pad 6 is provided with the mark antibody.

Wherein, the transition pad 5 is a film processed by a chemical method or a film processed by a physical method and with gradient distribution of porosity.

The invention also provides a preparation method of the immunochromatographic test strip without the hook effect, which comprises the following steps:

the method comprises the following steps: fixing a test line and a control line on the nitrocellulose membrane 2, and drying for 2 hours at 36 ℃ or until the nitrocellulose membrane 2 is completely dried;

step two: blocking the nitrocellulose membrane 2 with a 0.01MPBS buffer solution ph7.2 containing 2% w/v BSA and 0.02% w./v. tween-20 at room temperature, drying for 2h at 36 ℃ or until the nitrocellulose membrane 2 is completely dried;

step three: adding sucrose in a supersaturated amount into deionized water, and stirring at room temperature for 3 to 4 days to obtain a saturated aqueous solution of sucrose; removing sucrose precipitate in the solution, and diluting the saturated sucrose solution to a desired concentration by using deionized water;

step four: cutting the nitrocellulose membrane 2, and soaking the nitrocellulose membrane 2 in a sucrose solution from the side of the nitrocellulose membrane 2 until the nitrocellulose membrane 2 is completely soaked in the sucrose solution; any air bubbles cannot remain on the nitrocellulose membrane 2; completely drying the nitrocellulose membrane 2;

step five: fixing the pre-coupled fluorescence labeled antibody or nanogold labeled antibody on the conjugate pad 6, and completely drying at 36 ℃;

step six: assembling an LFIA test strip: firstly, a nitrocellulose membrane 2 is pasted on a bottom plate 1, then a transition pad 5, an absorption pad 4 and a sample pad 3 are pasted in sequence, and a conjugate pad 6 is pasted on the transition pad 5.

It is noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.