CN116165378A - High-specificity and high-sensitivity immunochromatography detection test paper, preparation method thereof, detection kit and detection method thereof - Google Patents
High-specificity and high-sensitivity immunochromatography detection test paper, preparation method thereof, detection kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses high-specificity and high-sensitivity immunochromatography detection test paper which comprises a first binding pad, a second binding pad, a nitrocellulose membrane coated with a C line and a T line, a sample absorbing pad and a bottom plate; wherein the binding pad is an antibody A coupled oxidized dextran immobilized with an antigen to be detected; conA-coupling colloidal gold and anti-DNP antibody coupling colloidal gold are fixed on the second bonding pad; t line is coated with antibody B of antigen to be detected; the C-wire is coated with DNP. Also discloses a preparation method of the test paper, a detection kit and a detection method. The invention introduces a dextran and ConA system, wherein ConA is tetrameric globulin, each subunit contains 1 sugar binding site, and each ConA has 4 sugar binding sites, thereby enhancing detection signals, realizing signal amplification and improving detection sensitivity. Is suitable for detecting various product types and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of immunochromatography, in particular to high-specificity and high-sensitivity immunochromatography detection test paper, a preparation method of the test paper, a detection kit and a corresponding detection method.
Background
The detection techniques commonly used for antigens at present are as follows: colloidal gold method, enzyme-linked immunosorbent assay (ELISA), fluorescent immunochromatography, chemiluminescence method and the like. The colloidal gold method is a novel immune labeling technology which uses colloidal gold as a tracer marker to be applied to antigen and antibody, and besides the combination of the colloidal gold with protein, the colloidal gold can also be combined with a plurality of other biological macromolecules, such as SPA, PNA, conA and the like; ELISA has become a leading topic in the field of analytical chemistry at present, is a special reagent analysis method, and is a novel immunoassay technology developed on the basis of an immunoenzymatic technology; the fluorescent immunochromatography technology is a novel membrane detection technology based on antigen-antibody specific immunoreaction, wherein strip-shaped fiber chromatographic materials fixed with detection lines (coated antibodies or coated antigens) and quality control lines (anti-antibodies) are used as stationary phases, test liquid is used as a mobile phase, fluorescent labeled antibodies or antigens are fixed on a connecting pad, and analytes are enabled to move on chromatographic strips through capillary action; the chemiluminescence method is a method for measuring chemiluminescent reactants, catalysts, sensitizers and inhibitors by using chemiluminescence, and coupling the reactants, catalysts and sensitizers in the reaction.
Disclosure of Invention
The invention mainly solves the technical problems existing in the prior art, thereby providing the immunochromatography detection test paper box detection device with high specificity and high sensitivity, and improving the specificity and the sensitivity of the detection of the colloidal gold immunochromatography platform.
The technical problems of the invention are mainly solved by the following technical proposal: a high-specificity and high-sensitivity immunochromatography detection test paper is characterized in that: the sample absorbing pad comprises a first bonding pad, a second bonding pad, a nitrocellulose membrane coated with a C line and a T line, a sample absorbing pad and a bottom plate, wherein the first bonding pad, the second bonding pad, the nitrocellulose membrane coated with the C line and the T line and the sample absorbing pad are sequentially arranged on the bottom plate, the head part of the second bonding pad is overlapped with the tail part of the first bonding pad, the head part of the nitrocellulose membrane is overlapped with the tail part of the second bonding pad, and the head part of the sample absorbing pad is overlapped with the tail part of the nitrocellulose membrane;
wherein the binding pad is an antibody A coupled oxidized dextran immobilized with an antigen to be detected;
ConA-coupling colloidal gold and anti-DNP antibody coupling colloidal gold are fixed on the second bonding pad;
t line is coated with antibody B of antigen to be detected;
the C-wire is coated with DNP.
Preferably, the bottom plate is PVC.
Preferably, the first bonding pad and the second bonding pad are made of glass fiber, non-woven fabric or filter paper.
Preferably, the sample pad is filter paper.
Preferably, the oxidized dextran is oxidized by sodium periodate.
Preferably, the oxidized dextran coupled with the antibody A is prepared by mixing oxidized dextran with the antibody A, adding NaCNBH3 for reaction, adding ethylenediamine for reaction, and adding NaBH4 for reduction.
The invention also discloses an immunochromatography detection kit, which comprises the immunochromatography detection test paper and a cartridge, wherein the test paper is arranged in the cartridge, the cartridge exposes a sample adding hole at a corresponding position of a bonding pad I of the test paper, exposes a diluent adding hole at a corresponding position of a bonding pad II of the test paper, and exposes an observation window at a corresponding position of a nitrocellulose film coated C line and T line of the test paper.
The invention also discloses a preparation method of the immunochromatography detection test paper with high specificity and high sensitivity, which is characterized by comprising the following steps:
1. preparation of bond pad one
1.2 dextran oxidation: oxidizing dextran with sodium periodate, adding excessive glycol after the reaction to terminate the oxidation reaction, and dialyzing to obtain oxidized dextran;
1.2 oxidized dextran-conjugated antibodies: mixing oxidized dextran with antibody A of target antigen, reacting with NaCNBH 3 Reducing into binary conjugate, adding ethylenediamine, ammonifying, and adding NaBH 4 Reducing, and then dialyzing to remove ethylenediamine and NaBH 4 ；
1.3 preparation of bond pad one: fixing the oxidized dextran coupled antibody obtained in the previous step on a glass fiber pad to obtain a first bonding pad;
2. preparation of bonding pad two
2.1 ConA/anti-DNP antibody-conjugated colloidal gold: adding K2CO3 into the colloidal gold solution, then adding ConA or anti-DNP antibody for coupling reaction, then adding a sealing buffer solution for sealing, centrifuging, and placing colloidal gold particles into a preservation solution for standby;
2.2 preparation of a second bonding pad: fixing ConA coupling colloidal gold and anti-DNP antibody coupling colloidal gold on a glass fiber pad to prepare a second bonding pad;
3. preparation of nitrocellulose film coated with C and T lines
3.1, diluting an antibody B of the target antigen by using a coating buffer solution to obtain a T line antibody;
3.2 diluting DNP-BSA with coating buffer to obtain C line antibody;
3.3, respectively coating the C line antibody and the T line antibody on a nitrocellulose membrane, and drying to obtain a nitrocellulose membrane coated with the C line and the T line;
4. assembly
And sequentially placing the first bonding pad, the second bonding pad, the nitrocellulose membrane coated with the C line and the T line and the sample absorbing pad on the bottom plate, wherein the head of the second bonding pad is overlapped with the tail of the first bonding pad, the head of the nitrocellulose membrane is overlapped with the tail of the second bonding pad, and the head of the sample absorbing pad is overlapped with the tail of the nitrocellulose membrane, so that the colloidal gold immunochromatography detection test paper is obtained.
Preferably, the oxidation of dextran in step 1.1 is specifically: dissolving a certain amount of dextran with 20mmol/L phosphate buffer solution with pH of 7.0, preparing 100mg/mL sodium periodate aqueous solution, adding sodium periodate aqueous solution into the dextran solution, and allowing NaIO 4 The molar ratio of the glucose to the monosaccharide units in the glucan is 1:3, the final concentration of the glucan is 40mg/mL, the excess glycol is added to stop the oxidation reaction after weak stirring reaction for 3 hours at the temperature of 30 ℃ in the dark, the mixture is continuously stirred at the temperature of 30 ℃ in the dark for 15 minutes and then taken out, and the mixture is dialyzed by deionized water or 20mmol/L phosphate buffer solution with the pH of 7.0.
Preferably, the oxidized dextran-coupled antibody of step 1.2 is specifically prepared by mixing oxidized dextran with antibody A against the target antigen and reacting for 2 hours, and then using NaCNBH 3 Reducing into binary conjugate, adding ethylenediamine, ammoniating for 2 hr, and adding NaBH 4 Reducing for 2 hours, and then dialyzing to remove ethylenediamine and NaBH 4 。
Preferably, the step 2.1 ConA/anti-DNP antibody-coupled colloidal gold is specifically: taking colloidal gold solution, adding 0.1mol/L K2CO3 to adjust to a proper pH value, adding ConA/anti-DNP antibody, mixing uniformly, standing for reaction, slowly adding a sealing buffer solution after the reaction is finished, mixing uniformly, standing for sealing, centrifuging to remove supernatant, adding a cleaning buffer solution for cleaning, centrifuging to remove supernatant, and adding a preservation solution for mixing uniformly for later use.
Preferably, in step 3.1, the antibody B against the target antigen is diluted to 1.0 to 2.0mg/mL with the coating buffer.
Preferably, DNP-BSA is diluted to 1.0-2.0 mg/mL with coating buffer in step 3.2.
The invention also discloses a high-specificity and high-sensitivity immunochromatography detection method based on the immunochromatography detection kit, which is characterized by comprising the following steps of:
(1) Opening a colloidal gold immunoassay analyzer;
(2) And (3) dripping 50 mu L of a sample to be detected into a sample adding hole of the immunochromatographic test strip, and dripping 100 mu L of diluent into a diluent adding hole after 5-10 min.
(3) And (5) continuing to incubate for 10-15 min, and putting the kit into a colloidal gold immunoassay instrument to read the detection result.
The high-specificity and high-sensitivity fluorescence immunoassay method has the technical route as follows: firstly, coupling an antibody A specific to a specific antigen with oxidized dextran, and fixing the antibody A on a binding pad 1; respectively coupling concanavalin (ConA) with colloidal gold, coupling an anti-DNP antibody with the colloidal gold, mixing two colloidal gold markers, and fixing on a binding pad 2; and then coating another antibody B aiming at the antigen on a specific zone of the nitrocellulose membrane to form a detection line (T line), coating DNP-BSA on the specific zone of the nitrocellulose membrane to form a quality control line (C line), and assembling and preparing the colloidal gold immunochromatographic test paper in parallel with the T line. After the sample is added to the binding pad 1, the oxidized dextran-coupled antibody A reacts with the antigen in the sample to generate an antigen-antibody A-oxidized dextran complex, and the oxidized dextran on the complex is combined with ConA-colloidal gold complex to form the antigen-antibody A-oxidized dextran-ConA-colloidal gold complex when the oxidized dextran continues to move forward through the binding pad 2. At this time, diluent is added at the position of the binding pad 2, the sample continuously moves forwards under the action of the diluent, when the sample is chromatographed to a T line area fixed with another antibody B, the antigen in the sample reacts with the antibody B, and finally an antibody B-antigen-antibody A-oxidized dextran-ConA-colloidal gold complex is generated, and if no antigen to be detected exists in the sample, a fluorescent complex is not formed at the T line; the sample continues to move forward, and whether the sample contains an antigen to be detected or not, the anti-DNP antibody of the labeled colloidal gold can be combined with DNP-BSA coated by the C line. C. The signal intensity of the T line can be read by a matched colloidal gold immunoassay analyzer, and the concentration of the sample is calculated according to a preset standard curve in the SD card, so as to give a quantitative detection result. In the technology, dextran and ConA systems are introduced, conA is tetrameric globulin, each subunit contains 1 sugar binding site, and each ConA has 4 sugar binding sites, so that detection signals are enhanced, signal amplification is realized, and detection sensitivity is improved. The invention is suitable for detecting various product types and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the high-specificity and high-sensitivity immunochromatography detection test paper.
FIG. 2 is a block diagram of an immunochromatographic assay kit of the present invention.
FIG. 3 is a regression equation of the detection result of the S100—β kit.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
The reagents or apparatus used, not specifically identified to the manufacturer, are conventional products commercially available.
Example 1: oxidation of dextran
A solution was dissolved in 20mmol/L phosphate buffer at pH7.0Dextran was quantified and 100mg/mL aqueous sodium periodate (protected from light) was prepared. Adding a certain amount of sodium periodate water solution into the dextran solution to make NaIO 4 The molar ratio of monosaccharide units in the glucan was 1:3, and the final concentration of glucan was 40mg/mL. After the reaction is carried out for 3 hours under weak stirring at the temperature of 30 ℃ in the dark, excessive ethylene glycol is added to stop the oxidation reaction, the reaction is carried out for 15 minutes under stirring at the temperature of 30 ℃ in the dark, and then the reaction is taken out, and the reaction is dialyzed by deionized water or 20mmol/L phosphate buffer solution with the pH of 7.0 (dextran with the molecular weight of 500kd and 70kd is dialyzed by a dialysis bag with the molecular weight of 12000, and dextran with the molecular weight of 10kd is dialyzed by a dialysis bag with the molecular weight of 3500).
Example 2: oxidized dextran conjugated antibody A
Mixing oxidized dextran and antibody in a molar ratio of 1:1, reacting at 4 ℃ for 2 hours, and adding NaCNBH with a protein concentration of 10 times of molar quantity 3 Reacting at 4deg.C for 2 hr, adding ethylenediamine, reacting at 4deg.C for 2 hr, adding 4 times of ethylenediamine molar amount of NaBH 4 Reducing for 2 hours, and then dialyzing to remove ethylenediamine and NaBH 4 。
Example 3: preparation of bond pad 1
The conjugate of example 2 was diluted to 0.02-0.04 mg/mL with conjugate pad fixative and fixed on a glass fiber pad and dried in an oven at 37℃for 24h. The conjugate pad fixative was 50mM PB buffer, pH=7.2-7.4, containing 0.5% Tween-20, 0.5% PVPK-30,5% sucrose, 1% casein-Na,2% protein protectant, 0.02% PC-300.
Example 4: concanavalin (ConA) coupled colloidal gold
The colloidal gold is prepared by adopting a trisodium citrate reduction method, and the steps are as follows: preparing 0.01% HauCl4 water solution, and heating 100ml to boil; adding 6.5ml of 1% trisodium citrate aqueous solution while stirring; heating and boiling for 15min; cooling to room temperature, adding ddH2O to a volume of 500ml to obtain colloidal gold solution, wherein the particle diameter of the colloidal gold is about 30-40nm;
the colloidal gold can also be prepared or purchased into the existing colloidal gold solution finished product by other conventional methods.
Taking 1mL of colloidal gold solution, adding 6-8 mu L of 0.1mol/L K CO3, adding 0.02-0.03 mg of ConA, uniformly mixing, and standing for reaction for one hour. After the reaction, 0.1mL of 10% sodium caseinate was slowly added, mixed well, and left to stand for one hour. Setting a centrifugal force of 6000g at 2-8 ℃ for 40min, removing the supernatant, adding a cleaning buffer solution, uniformly mixing, setting a centrifugal force of 6000g at 2-8 ℃ for 40min, and repeating for 1 time. After centrifugation, the supernatant is removed, and the preservation solution is added and mixed uniformly.
Example 5: anti-DNP antibody-conjugated colloidal gold
Taking 1mL of colloidal gold solution, adding 6-8 mu L of 0.1mol/L K CO3, adding 0.02-0.03 mg of DNP antibody, uniformly mixing, and standing for reaction for one hour. After the reaction, 0.1mL of 10% sodium caseinate was slowly added, mixed well, and left to stand for one hour. Setting a centrifugal force of 6000g at 2-8 ℃ for 40min, removing the supernatant, adding a cleaning buffer solution, uniformly mixing, setting a centrifugal force of 6000g at 2-8 ℃ for 40min, and repeating for 1 time. After centrifugation, the supernatant is removed, and the preservation solution is added and mixed uniformly.
Example 6: preparation of bond pad 2
The conjugates of example 4 and example 5 were mixed and diluted with the conjugate pad fixative solution to a final concentration of 0.02-0.04 mg/mL, fixed on a glass fiber pad, and dried in an oven at 37℃for 24 hours. The conjugate pad fixative was 50mM PB buffer, pH=7.2-7.4, containing 0.5% Tween-20, 0.5% PVPK-30,5% sucrose, 1% casein-Na,2% protein protectant, 0.02% PC-300.
Example 7: preparation of nitrocellulose membrane coated with C and T wires
Diluting the antibody B to 1.0-2.0 mg/mL by using a coating buffer solution; c, preparing a line marking liquid: diluting DNP-BSA to 1.0-2.0 mg/mL by using a coating buffer solution; the coating buffer was 10mM PB containing 1% sucrose. Coating the C line antibody and the T line antibody on a nitrocellulose membrane fixed on a bottom plate by adopting a metal spraying and film drawing instrument; the marked large plate is placed in a blast drying oven to be dried for 24 hours at 37 ℃ and cut into the required size.
Example 8: assembly
As shown in fig. 1, a first bonding pad 1, a second bonding pad 2, a nitrocellulose membrane 5 coated with a C line 3 and a T line 4, and a sample absorbing pad 6 are sequentially placed on a bottom plate 7, the head of the second bonding pad overlaps the tail of the first bonding pad, the head of the nitrocellulose membrane overlaps the tail of the second bonding pad, and the head of the sample absorbing pad overlaps the tail of the nitrocellulose membrane. PVC is selected as the bottom plate, filter paper with good water absorption is selected as the sample absorbing pad, and the preparation of the colloidal gold immunochromatography detection test paper is completed.
As shown in fig. 2, the assembled test paper is loaded into a cartridge 8, a sample loading hole 9 and a diluent adding hole 10 are exposed at a corresponding position of a bonding pad of the test paper, and an observation window 11 is exposed at a corresponding position of a C line and a T line of a nitrocellulose film coating of the test paper, thereby completing the preparation of the colloidal gold immunochromatography detection device.
Example 9: s100-beta kit detection result
The method of the above examples 1-8 is adopted to prepare the S100-beta detection kit, 50 mu L of standard sample is dripped into the sample-adding hole 9 of the immunochromatographic test strip, 100 mu L of dilution liquid is dripped into the dilution liquid-adding hole 10 after 5-10 min, incubation is continued for 10-15 min, and the detection result is read by putting the kit into a colloidal gold immunoassay analyzer. The test results are shown in Table 1. Four-parameter Logistic curve fitting is carried out by taking the concentration as the abscissa and the signal value (T/C) as the ordinate, the R2 value is 0.999, the regression curve is shown in figure 3, and the regression equation is as follows:
four parameter Logistic curve fitting
Equation y= (A-D)/[ 1+ (x/C)/(B) ] +D
A＝78.85141
B＝-0.87812
C＝49.79200
D＝-0.09688
r^2＝0.99964。
TABLE 1 S100-beta kit sample test results
| Concentration ng/mL | Signal value (T/C) |
| 0 | 0.01 |
| 0.051 | 0.11 |
| 0.13 | 0.26 |
| 0.24 | 0.54 |
| 0.67 | 1.49 |
| 1.16 | 2.88 |
| 2.38 | 5.12 |
| 5.66 | 9.97 |
| 7.89 | 12.94 |
| 8.18 | 13.29 |
| 9.44 | 14.98 |
| 10.12 | 15.43 |
。
Wherein the diluent comprises, by weight, 1000 parts of tween-20-5 parts, 1-5 parts of casein sodium salt, 1-5 parts of bovine serum albumin, 1-5 parts of polyethylene glycol 200, 10-50 parts of sucrose, 0.1-0.5 part of PC-300 and the balance of phosphate buffer solution.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any changes or substitutions that do not undergo the inventive effort should be construed as falling within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.
Claims (10)
1. A high-specificity and high-sensitivity immunochromatography detection test paper is characterized in that: comprises a first bonding pad, a second bonding pad, a nitrocellulose membrane coated with a C line and a T line, a sample absorbing pad and a bottom plate;
wherein the binding pad is an antibody A coupled oxidized dextran immobilized with an antigen to be detected;
ConA-coupling colloidal gold and anti-DNP antibody coupling colloidal gold are fixed on the second bonding pad;
t line is coated with antibody B of antigen to be detected;
the C-wire is coated with DNP.
2. The immunochromatographic test strip according to claim 1, wherein: the first bonding pad, the second bonding pad, the nitrocellulose membrane coated with the C line and the T line and the sample absorbing pad are sequentially arranged on the bottom plate, the head of the second bonding pad is overlapped with the tail of the first bonding pad, the head of the nitrocellulose membrane is overlapped with the tail of the second bonding pad, and the head of the sample absorbing pad is overlapped with the tail of the nitrocellulose membrane.
3. The immunochromatographic test strip according to claim 1, wherein: the bottom plate is PVC.
4. The immunochromatographic test strip according to claim 1, wherein: the first bonding pad and the second bonding pad are made of glass fiber, non-woven fabrics or filter paper.
5. The immunochromatographic test strip according to claim 1, wherein: the sample absorbing pad is filter paper.
6. The immunochromatographic test strip according to claim 1, wherein: the oxidized dextran is oxidized by sodium periodate.
7. The immunochromatographic test strip according to claim 1, wherein: the oxidized dextran coupled with the antibody A is prepared by mixing oxidized dextran with the antibody A, and adding NaCNBH 3 Reacting, adding ethylenediamine for reaction, and then adding NaBH 4 And (5) reducing to obtain the product.
8. A high-specificity and high-sensitivity immunochromatography detection kit is characterized in that: an immunochromatographic test strip comprising the immunochromatographic test strip of any one of claims 1 to 7, and a cartridge in which the sample-adding hole and the diluent-adding hole are exposed at a portion corresponding to a bonding pad of the test strip, and the observation window is exposed at a portion corresponding to a C-line and a T-line coated with a nitrocellulose membrane of the test strip.
9. The preparation method of the immunochromatography detection test paper with high specificity and high sensitivity is characterized by comprising the following steps of:
1. preparation of bond pad one
1.1 dextran oxidation: oxidizing dextran with sodium periodate, adding excessive glycol after the reaction to terminate the oxidation reaction, and dialyzing to obtain oxidized dextran;
1.2 oxidized dextran-conjugated antibodies: mixing oxidized dextran with antibody A of target antigen, reacting with NaCNBH 3 Reducing into binary conjugate, adding ethylenediamine, ammonifying, and adding NaBH 4 Reducing, and then dialyzing to remove ethylenediamine and NaBH 4 ；
1.3 preparation of bond pad one: fixing the oxidized dextran coupled antibody obtained in the previous step on a glass fiber pad to obtain a first bonding pad;
2. preparation of bonding pad two
2.1 ConA/anti-DNP antibody-conjugated colloidal gold: adding K2CO3 into the colloidal gold solution, then adding ConA or anti-DNP antibody for coupling reaction, then adding a sealing buffer solution for sealing, centrifuging, and placing colloidal gold particles into a preservation solution for standby;
2.2 preparation of a second bonding pad: fixing ConA coupling colloidal gold and anti-DNP antibody coupling colloidal gold on a glass fiber pad to prepare a second bonding pad;
3. preparation of nitrocellulose film coated with C and T lines
3.1, diluting an antibody B of the target antigen by using a coating buffer solution to obtain a T line antibody;
3.2 diluting DNP-BSA with coating buffer to obtain C line antibody;
3.3, respectively coating the C line antibody and the T line antibody on a nitrocellulose membrane, and drying to obtain a nitrocellulose membrane coated with the C line and the T line;
4. assembly
Sequentially placing a first bonding pad, a second bonding pad, a nitrocellulose membrane coated with a C line and a T line and a sample absorbing pad on a bottom plate, wherein the head of the second bonding pad is overlapped with the tail of the first bonding pad, the head of the nitrocellulose membrane is overlapped with the tail of the second bonding pad, and the head of the sample absorbing pad is overlapped with the tail of the nitrocellulose membrane to obtain colloidal gold immunochromatography detection test paper;
step 1.1 dextran oxidation specifically is: dissolving a certain amount of dextran with 20mmol/L phosphate buffer solution with pH of 7.0, preparing 100mg/mL sodium periodate aqueous solution, adding sodium periodate aqueous solution into the dextran solution, and allowing NaIO 4 The molar ratio of the glucose to the monosaccharide units in the glucan is 1:3, the final concentration of the glucan is 40mg/mL, excessive ethylene glycol is added to terminate the oxidation reaction after weak stirring reaction for 3 hours at the temperature of 30 ℃ in the dark, stirring reaction is continued at the temperature of 30 ℃ in the dark for 15 minutes, and the mixture is taken out and dialyzed by deionized water or 20mmol/L phosphate buffer solution with the pH of 7.0;
step 1.2 mixing the oxidized dextran coupled antibody, specifically oxidized dextran, with an antibody A against the target antigen for 2 hours, and then reacting with NaCNBH 3 Reducing into binary conjugate, adding ethylenediamine, ammoniating for 2 hr, and adding NaBH 4 Reducing for 2 hours, and then dialyzing to remove ethylenediamine and NaBH 4 ；
Step 2.1 ConA/anti-DNP antibody-coupled colloidal gold specifically comprises: taking a colloidal gold solution, adding 0.1mol/L of K2CO3 to adjust the pH to a proper value, adding ConA/anti-DNP antibodies, uniformly mixing, standing for reaction, slowly adding a sealing buffer solution after the reaction is finished, uniformly mixing, standing for sealing, centrifuging to remove the supernatant, adding a cleaning buffer solution for cleaning, centrifuging to remove the supernatant, and adding a preservation solution for uniform mixing;
in the step 3.1, the antibody B of the anti-target antigen is diluted to 1.0-2.0 mg/m by a coating buffer solution;
in step 3.2, DNP-BSA was diluted to 1.0-2.0 mg/mL with coating buffer.
10. A high-specificity and high-sensitivity immunochromatographic detection method based on the high-specificity and high-sensitivity immunochromatographic detection kit according to claim 8, characterized by comprising the following steps:
(1) Opening a colloidal gold immunoassay analyzer;
(2) And (3) dripping 50 mu L of a sample to be detected into a sample adding hole of the immunochromatographic test strip, and dripping 100 mu L of diluent into a diluent adding hole after 5-10 min.
(3) And (5) continuing to incubate for 10-15 min, and putting the kit into a colloidal gold immunoassay instrument to read the detection result.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116735854A (en) * | 2023-06-14 | 2023-09-12 | 之江实验室 | Method for coupling 2, 4-dinitrophenol with antibody |
| CN116754760A (en) * | 2023-06-14 | 2023-09-15 | 之江实验室 | Method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116735854A (en) * | 2023-06-14 | 2023-09-12 | 之江实验室 | Method for coupling 2, 4-dinitrophenol with antibody |
| CN116754760A (en) * | 2023-06-14 | 2023-09-15 | 之江实验室 | Method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of antibody |
| CN116735854B (en) * | 2023-06-14 | 2024-01-26 | 之江实验室 | Method for coupling 2, 4-dinitrophenol with antibody |
| CN116754760B (en) * | 2023-06-14 | 2024-01-26 | 之江实验室 | Method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of antibody |
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