CN115951046A - Human serum amyloid A time-resolved fluorescence immunochromatographic test paper and application - Google Patents

Abstract

The invention relates to the technical field of clinical medical detection, in particular to human serum amyloid A time-resolved fluorescence immunochromatographic test paper and application thereof. Compared with the prior art, the invention has the advantages that the first combination pad and the first envelope film only provided with the detection line but not provided with the quality control line are additionally arranged, when the antigen dose in the sample is higher, a part of SAA can be intercepted, the rest SAA is captured by the envelope film, the situation that the single T-line fluorescence signal intensity is saturated and is difficult to continue to rise is effectively avoided, the linear rear end curve of the test signal T/C tends to be gentle, and even a downward bending state occurs, so that a false low value or even a false negative result occurs.

Description

Human serum amyloid A time-resolved fluorescence immunochromatographic test paper and application

Technical Field

The invention relates to the technical field of clinical medical detection, in particular to human serum amyloid A time-resolved fluorescence immunochromatographic test paper and application thereof.

Background

In the fluorescence immunochromatography method, a double antibody sandwich method is used for detection in a solid-phase immunochromatography mode. The sample serum/plasma/whole blood to be detected diffuses upwards at the sample adding end by capillary force, and the PCT antigen in the sample and the marker are combined into a labeled antibody-antigen compound when the PCT antigen passes through the conjugate pad; the complex diffuses onto the nitrocellulose membrane along with the sample, is intercepted by the regional detection line coated with the PCT antibody to form an immune complex of the labeled antibody-antigen-coated antibody, the label continuously moves forwards along the membrane to a quality control line for reaction, the fluorescence chromogenic intensity at the detection line is in direct proportion to the PCT content, and the PCT concentration is quantitatively determined according to the fluorescence intensity.

Because the complexity of a sample to be detected and the immune reaction process are influenced by sample fluidity, sample matrix influence, protein coating capture difference, cellulose nitrate membrane uniformity, capillary traction left-right difference and the like in the chromatography process, the reaction intensity of a T line can be obviously changed due to the difference in the chromatography process, and a C line has the same influence with the T line, and the difference between test strips can be reduced by using the T/C value due to the test concept of controlling variables. Theoretically, the signal of the combination substance should be in positive correlation with the concentration of the sample, but is influenced by the high dosage of the substance to be detected in the sample and the limitation of the range of the intensity of the fluorescence signal detected by the instrument, the intensity of the T-line fluorescence signal is saturated and is difficult to continue to rise, the T/C linear rear end curve of the test signal tends to be gentle, and even a downward bending state occurs, so that a false low value or even a false negative result occurs, which is a special abnormal phenomenon of a solid-phase determination method, and the phenomenon can cause the linear range of the concentration of the detected sample to be too narrow, and the detection value of the high-concentration sample is inaccurate.

The existing technology for solving the problem basically carries out angle debugging such as increasing the concentration of a fluorescence-labeled monoclonal antibody raw material, reducing the dilution multiple of the fluorescence-labeled monoclonal antibody concentrated solution, increasing the concentration of the monoclonal antibody raw material, reducing the concentration of a sample, improving the purity of the antibody and the like, but the whole debugging process is relatively complicated. And, there is still a problem that the detection value of the high concentration sample is not accurate.

Disclosure of Invention

In order to overcome the defects of the background technology, the invention provides the human serum amyloid A time-resolved fluorescence immunochromatographic test paper with a wider linear range and higher high-end measurement value precision and the application thereof.

The technical scheme adopted by the invention is as follows: human serum amyloid A time resolution fluorescence immunochromatographic test paper and application thereof have the key points that: the test paper comprises a sample pad, a first combination pad, a first coating film, a second combination pad, a second coating film and a water absorption pad which are sequentially overlapped, wherein the first coating film is coated with a T1 detection line, and the second coating film is coated with a T2 detection line and a C line quality control line.

Preferably, the T1 detection line and the T2 detection line are coated with a second antibody, and the concentration ratio of the second antibody coated on the T1 detection line and the T2 detection line is 0.6 to 1:1.

preferably, the first and second conjugate pads are sprayed with the same concentration of fluorescent microsphere-antibody complexes.

Preferably, the fluorescent microsphere-antibody complex is prepared by the following method: diluting the fluorescent microspheres and EDC with a PB solution respectively, quickly vortexing and uniformly mixing, placing in a shaking table at 37 ℃ to shake for 15min, centrifuging at a high speed, removing supernatant, diluting precipitates with the PB solution, and performing ultrasonic dispersion to obtain a fluorescent microsphere solution; and adding the first antibody into the fluorescent microsphere solution, uniformly mixing by vortex, placing in a 37 ℃ shaking table for shaking for 16h, adding 50 mu l of sealant into the fluorescent microsphere solution, uniformly mixing by vortex, placing in a 37 ℃ shaking table for shaking for 2h, centrifuging at high speed, removing supernatant, diluting the precipitate with a treatment solution, and performing ultrasonic dispersion to obtain the fluorescent microsphere solution.

Preferably, the ratio of the concentration of the first antibody on the first and second conjugate pads is 0.6-1:1.

preferably, the treatment liquid consists of the following raw materials: 97.99% PBS, 1% glucose, 0.5% PEG2000, 0.01% TWEEN20, 0.5% BSA solution.

Preferably, the C line quality control line is arranged close to the absorbent pad.

Preferably, the sample pad is prepared by the following method: immersing a glass fiber membrane into a sample pad treatment solution for treatment, and then putting the sample pad treatment solution into a 45 ℃ oven for drying for 8 hours to obtain the sample pad treatment solution, wherein the sample pad treatment solution comprises the following raw materials: 94.3% PBS, 5% glucose, 0.1% PEG2000, 0.1% TWEEN20, 0.5% BSA.

Preferably, the first coating film and the second coating film are made of nitrocellulose membranes, and the first bonding pad and the second bonding pad are made of glass fibers.

Has the advantages that: compared with the prior art, the human serum amyloid A time-resolved fluorescence immunochromatographic test paper and the application thereof provided by the invention have the advantages that by additionally arranging the first binding pad and the first envelope film only provided with the detection line but not the quality control line, when the antigen dose in a sample is higher, a part of SAA can be intercepted, and the rest of SAA is captured by the envelope film, so that the situation that the single T-line fluorescence signal intensity is saturated and is difficult to continue to rise, the linear rear end curve of the test signal T/C tends to be gentle, and even the lower bending state occurs, so that a false low value or even a false negative result occurs, the linear range of the concentration of the detected sample is too narrow, and the detection value of a high-concentration sample is inaccurate is effectively avoided.

Drawings

FIG. 1 is a schematic structural view of the present invention;

FIG. 2 is a schematic diagram of the structure of the kit.

Detailed Description

In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Main outsourcing reagents:

EDC: carbodiimides hydrochloride available from sigma; cellulose nitrate membrane: purchased from sartorius; BSA: bovine serum albumin, purchased from shanghai xibao science and technology ltd; TWEEN20: purchased from sigma; SAA monoclonal antibody 1, purchased from new biotechnology limited, eobai, hangzhou); SAA monoclonal antibody 2 purchased from Hangzhou xian till biotechnology limited; goat anti-mouse IgG antibody purchased from Hangzhou xian till biotechnology limited; fluorescent microspheres, available from Bangs Laboratories, inc.

Self-prepared reagent:

10mM pH7.4 PB: phosphate buffer at 10 mmole/l, PH 7.4;

1 mM pH7.4 PBS: phosphate buffer at a concentration of 10 millimoles per liter, PH7.4, containing sodium chloride at a final concentration of 0.9%;

sample pad treatment solution PBS: phosphate buffer at 10 mmole/l, PH 7.4;

EXAMPLE 1 preparation of test strips

2.1 preparation of sample pad

Immersing the glass fiber membrane into a sample pad treatment solution for treatment, and then putting the sample pad treatment solution into a 45 ℃ oven for drying for 8 hours to obtain the sample pad treatment solution, wherein the sample pad treatment solution comprises the following raw materials: 94.3% PBS, 5% glucose, 0.1% PEG2000, 0.1% TWEEN20, 0.5% BSA.

2.2 preparation of conjugate pads

1) Preparing fluorescent solution microspheres: the fluorescent microspheres were diluted to 0.1% (W/W) with 10mM pH7.4 PB;

2) Activation of microspheres: EDC was dissolved in 10mM pH7.4 PB to 10mg/ml, and then added rapidly to the fluorescent microsphere solution, vortexed and mixed, with the addition of 0.5mg/ml EDC, and shaken in a shaker at 37 ℃ for 15min.

3) High speed centrifugation at 15000rpm for 15min, supernatant was removed, and the precipitate was diluted to 475ul with 10mM pH7.4 PB crosslinking reaction solution and ultrasonically dispersed for 2min.

4) Adding the SAA monoclonal antibody 1 into the fluorescent microsphere solution, uniformly mixing by vortex, and shaking in a shaking table at 37 ℃ for 16h.

5) Adding 50 mul of sealant into the fluorescent microsphere solution, and uniformly mixing by vortex; blocking reagent 10% BSA solution, shake in shaker at 37 ℃ for 2h.

6) Centrifuging at 15000rpm for 15min, removing supernatant, diluting the precipitate with treatment solution to 400ul, and ultrasonically treating the precipitate uniformly, wherein the treatment solution comprises the following raw materials: 97.99% PBS, 1% glucose, 0.5% PEG2000, 0.01% TWEEN20, 0.5% BSA solution.

7) And (3) spraying the fluorescent microsphere solution on a glass fiber membrane, and drying in an oven at 45 ℃ for 8h.

8) Duplicate bond pads, respectively designated as first bond pad 3 and second bond pad 5, were prepared as described above.

2.3 preparation of the first coating film

1, detection line T1: diluting SAA monoclonal antibody 2 with 0.01M PBS (pH7.4), coating, and streaking at a speed of 80mm/s and a concentration of 1 μ L/cm;

2 after scribing, putting the nitrocellulose membrane into a 45 ℃ oven for drying for 8h.

2.4 preparation of the second coating film

1 quality control line C: diluting goat anti-mouse IgG antibody to 3.0mg/mL with 0.01M PBS (pH7.4) for coating, wherein the scribing parameter is 1 μ L/cm, and the speed is 80mm/s;

detection line T2: diluting SAA monoclonal antibody 2 with 0.01M PBS (pH7.4), and coating at scribing parameter of 1 μ L/cm and speed of 80mm/s;

2.4 Assembly

The sample pad 2, the first bonding pad 3, the first coating film 4, the second bonding pad 5, the second coating film 6 and the absorbent pad 7 are assembled in sequence to form a strip cutting and mounting card.

Example 2 human serum amyloid A time-resolved fluorescence immunochromatography kit

As shown in fig. 1-2, the human serum amyloid a time-resolved fluorescence immunochromatographic kit comprises a strip-shaped box body 1 with a hollow structure and a test strip arranged in the strip-shaped box body 1; the test strip was prepared as in example 1. Specifically, the first combination pad and the first envelope membrane are sequentially added between the sample pad and the second combination pad, the added first combination pad can avoid the difficulty in increasing the fluorescence signal intensity caused by the excessively low concentration of a fluorescence labeling antibody, and simultaneously avoid the phenomenon of 'back band effect' caused by the excessively low binding efficiency of the antigen and the antibody due to the excessively high concentration of the antigen and the antibody, the added first envelope membrane with only one detection line T without a quality control line C can divide a high-concentration object to be detected in the sample into two parts to be respectively displayed on the first envelope membrane and the second envelope membrane, so that the problem that the fluorescence signal intensity of the detection line is not increased any more due to the insufficient concentration of envelope raw materials is avoided, and the problem that the linear rising trend of T/C is changed due to the limitation of the fluorescence signal intensity range determined by a fluorescence dry analyzer is solved, two T peaks and one C peak can be scanned, and the T1 peak signal + T2 peak signal/C peak signal can be pulled apart by a larger gradient, 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, the linear range is wider, and the high-end measurement value is higher in precision.

In this example, the T1 detecting line 8 and the T2 detecting line 9 are coated with the second antibodies, and the concentration ratio of the second antibodies coated on the T1 detecting line 8 and the T2 detecting line 9 is 0.6 to 1:1. in a preferred embodiment, the concentration ratio of the second antibody coated on the T1 detection line and the T2 detection line is 1:1, more preferably, the second antibody is SAA monoclonal antibody 2.

In this embodiment, the fluorescent microsphere-antibody complex is sprayed on the first and second binding pads 3 and 5 at the same concentration.

In this embodiment, the concentration ratio of the first antibody on the first conjugate pad 3 and the second conjugate pad 5 is 0.6 to 1:1. in a preferred embodiment, the concentration ratio of the first antibody on the first conjugate pad 3 and the second conjugate pad 5 is 1:1, more preferably, the first antibody is SAA monoclonal antibody 1.

In this embodiment, the top of the strip-shaped box body 1 is covered with a first visual window 11, a second visual window 12 and a sample adding hole 13, the first visual window 11 is located above the first covering film 4, the second visual window 12 is located above the second covering film 6, and the sample adding hole 13 is located above the sample pad 2.

In this embodiment, the second envelope film 6 is further coated with a C-line quality control line 10, and the C-line quality control line 10 is disposed near the absorbent pad 7.

In this embodiment, the first coating film 4 and the second coating film 6 are nitrocellulose films, and the first bonding pad 3 and the second bonding pad 5 are made of glass fibers.

Example 3 evaluation of Properties

The 16 test strips were prepared according to the method of example 1, and the test strips were prepared under the same conditions except that the first antibody concentration ratio on the first and second binding pads on each test strip was different, and the second antibody concentration ratio on the T1 and T2 detection lines on each test strip was different, as shown in table 1.

TABLE 1

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And (3) linear evaluation: SAA positive samples with different concentrations are prepared, each group of test strips are tested, and the fluorescence signal intensities of T1, T2 and C lines are respectively collected through 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. (T1 + T2)/C values or T2/C values were calculated for different SAA positive samples, respectively, as shown in Table 2.

TABLE 2 SAA sample T/C Signal ratio

And (3) precision evaluation: in order to detect the measurement precision of the 16 test strips on the high-concentration SAA positive sample, the 16 test strips are used to measure the SAA sample with the concentration of 120mg/L respectively, the measurement is repeated for 10 times, and the Coefficient of Variation (CV) of each test strip is calculated, and the results are shown in Table 3.

TABLE 3 precision measurement data

Numbering	1	2	3	4	5	6	7	8
									CV	6.57％	8.29％	13.77％	16.68％	9.23％	15.58％	18.87％	22.37％
Numbering
		9	10	11	12	13	14	15	16
CV										14.37％	19.74％	20.22％	22.51％	21.48％	23.27％	25.97％	25.43％

Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.

Claims (10)

1. Human serum amyloid A time-resolved fluorescence immunochromatographic test paper is characterized in that: including overlap joint's sample pad (2), first bonding pad (3), first envelope membrane (4), second bonding pad (5), second envelope membrane (6) and the pad that absorbs water (7) in proper order, first envelope membrane (4) is upwrapped there are T1 detection line (8), second envelope membrane (6) is upwrapped there are T2 detection line (9) and C line matter accuse line (10).

2. The human serum amyloid a time-resolved fluorescence immunochromatographic test strip according to claim 1, characterized in that: the T1 detection line (8) and the T2 detection line (9) are coated with second antibodies, and the concentration ratio of the second antibodies coated on the T1 detection line (8) and the T2 detection line (9) is (0.6-1): 1.

3. the human serum amyloid a time-resolved fluorescence immunochromatographic test strip according to claim 1, characterized in that: the first binding pad (3) and the second binding pad (5) are sprayed with fluorescent microsphere-antibody complexes at the same concentration.

4. The human serum amyloid A time-resolved fluorescence immunochromatographic test strip according to claim 3, characterized in that: the fluorescent microsphere-antibody compound is prepared by the following method: diluting the fluorescent microspheres and EDC with a PB solution respectively, quickly vortexing and uniformly mixing, placing in a shaking table at 37 ℃ to shake for 15min, centrifuging at a high speed, removing supernatant, diluting precipitates with the PB solution, and performing ultrasonic dispersion to obtain a fluorescent microsphere solution; and adding the first antibody into the fluorescent microsphere solution, uniformly mixing by vortex, placing in a 37 ℃ shaking table for shaking for 16h, adding 50 mu l of sealant into the fluorescent microsphere solution, uniformly mixing by vortex, placing in a 37 ℃ shaking table for shaking for 2h, centrifuging at high speed, removing supernatant, diluting the precipitate with a treatment solution, and performing ultrasonic dispersion to obtain the fluorescent microsphere solution.

5. The human serum amyloid A time-resolved fluorescence immunochromatographic test strip according to claim 4, characterized in that: a first antibody concentration ratio on the first conjugate pad (3) and the second conjugate pad (5) is (0.6-1): 1.

6. the human serum amyloid A time-resolved fluorescence immunochromatographic test strip according to claim 4, characterized in that: the treatment fluid consists of the following raw materials: 97.99% PBS, 1% glucose, 0.5% PEG2000, 0.01% TWEEN20, 0.5% BSA solution.

7. The human serum amyloid a time-resolved fluoroimmunochromatographic test strip according to claim 1, wherein said sample pad (2) is prepared by the following method: immersing the glass fiber membrane into a sample pad treatment solution for treatment, and then putting the sample pad treatment solution into a 45 ℃ oven for drying for 8 hours to obtain the sample pad treatment solution, wherein the sample pad treatment solution comprises the following raw materials: 94.3% PBS, 5% glucose, 0.1% PEG2000, 0.1% TWEEN20, 0.5% BSA.

8. The human serum amyloid a time-resolved fluorescence immunochromatographic test strip according to claim 1, characterized in that: the first coating film (4) and the second coating film (6) are nitrocellulose films, and the first combination pad (3) and the second combination pad (5) are made of glass fibers.

9. A test kit comprising a strip according to any one of claims 1 to 8.

10. Use of a strip according to any one of claims 1 to 8 and/or a test kit according to claim 9 in a clinical assay.

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