CN113281526A - Sample pad treatment reagent of free thyroxine detection reagent strip - Google Patents
Sample pad treatment reagent of free thyroxine detection reagent strip Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention provides a novel sample pad treatment reagent of a free thyroxine detection reagent strip, which is characterized in that a sample pad treatment solution prepared by adopting high polymer urea-formaldehyde resin with specific molecular weight and content can completely separate the free thyroxine in a sample to be detected, the sample pad treated by the sample pad and the further prepared free thyroxine detection reagent strip are used for detecting the free thyroxine by a competition method, so that the free thyroxine can be fully captured and fully reacted, the detection sensitivity of the free thyroxine is greatly improved, the detection sensitivity reaches pg 10/ml, and the clinical detection requirements are met. The application of the urea-formaldehyde resin in preparing free thyroxine detection reagent strips or sample pad treatment reagents of the free thyroxine detection reagent strips is also provided.
Description
Technical Field
The invention belongs to the technical field of biological detection, relates to an immunochromatography detection reagent strip, and particularly relates to a sample pad treatment reagent of a free thyroxine detection reagent strip.
Background
The fluorescence immunochromatography technique, which is the most commonly used POCT technique, has the characteristics of simple operation, high reaction speed, small instrument requirements and the like, and generally, the detection reagent strip consists of a sample pad, a conjugate pad (containing labeled antibodies), an NC membrane (containing T-line and C-line coated with antibodies/antigens), a water absorption pad and the like. During testing, a sample is added into a sample pad, flows into a binding pad, hydrates a labeled antibody of the binding pad, then flows to a detection line T line and a detection line C line on an NC membrane, the concentration of a detected object in the sample is calculated according to a signal value of T/C, generally, a signal of the C line tends to be stable, the difference between reagent strips is reduced as a control measure, and the signal value of the T line is generally used as a judgment basis when the detection reagent strips are evaluated.
Free thyroxine (abbreviated as free T4 or FT4) is used as a marker for detecting thyroid function, chemiluminescence detection (such as CN102095881A, CN101551389A and the like) is mainly used at present, and no product for detecting free thyroxine by using fluorescence immunochromatography technology is commercially available, mainly because the free T4 cannot be fully captured in the fluorescence immunochromatography process, so that the reaction is insufficient, and the sensitivity is low.
Generally, the sample pad and the conjugate pad of the detection reagent strip need to be pretreated, but the sample pad treated by using the reagent for treating the sample pad can only detect free T4 with the concentration of 100pg/mL, while the free T4 is used as an important index for thyroid function detection, the normal value ranges from 8 to 15pg/mL, so the detection sensitivity of 100pg/mL cannot meet the current requirements, and higher sensitivity must be considered during detection.
Therefore, the development of a fluorescence immunochromatographic assay more suitable for detecting free thyroxine is urgently needed, and the detection sensitivity is improved, so that the field rapid detection of the free thyroxine with more convenience, rapidness and high accuracy is realized.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a novel sample pad treatment reagent of a free thyroxine detection reagent strip, which is characterized in that a sample pad treatment solution is prepared by adopting high polymer urea-formaldehyde resin with specific molecular weight and content, so that the free thyroxine in a sample to be detected can be better separated, the sample pad treated by the sample pad treatment solution is used for further preparing the free thyroxine detection reagent strip for detecting the free thyroxine by a competition method, the free thyroxine can be fully captured and fully reacted, the detection sensitivity of the free thyroxine is greatly improved, and the clinical detection requirements are met.
In one aspect, the present invention provides a sample pad treatment reagent for a free thyroxine detection reagent strip, said reagent comprising urea formaldehyde resin.
In an immunochromatography detection device, a conventional sample pad treatment solution generally comprises a system buffer solution, protein, a high polymer, salt ions and a surfactant, wherein the high polymer mostly adopts a non-ionic high molecular compound such as PVP-k30, but when the high polymer is used for detecting free thyroxine, the free thyroxine cannot be well separated from other impurities in a blood sample to be detected, so that accurate detection is difficult to realize.
Through a large number of experiments, the research group surprisingly discovers that the detection sensitivity of the free thyroxine can be greatly improved by using the urea resin as a sample pad treatment reagent prepared from the high polymer. This is probably because the addition of urea resin makes the liquid viscosity become bigger, and the sample chromatographic speed becomes slower on the sample pad, and free thyroxine is separated better, makes the antibody catch free thyroxine more easily, makes free thyroxine more easily bound, and antigen-antibody reaction is more sufficient.
Further, the molecular weight of the urea resin is 10000-.
Further, the content of the urea-formaldehyde resin is 1-2%.
Research proves that the larger the concentration of the urea-formaldehyde resin is, the larger the molecular weight is, the more viscous the liquid is, but the chromatography speed of the excessively viscous solution is too low during detection, so that the detection time is too long, and the detection sensitivity is adversely affected. Therefore, the molecular weight and concentration must be selected appropriately. Urea-formaldehyde resins with molecular weights of 10000 and 20000 are selected by screening, and the concentrations are 1% and 2% respectively.
Further, the sample pad processing reagent further comprises a system buffer, a protein, a salt ion and a surfactant.
Further, the system buffer is 50mM boric acid buffer, the protein is 1% BSA, the salt ion is 0.1M NaCl, and the surfactant is 1% surfactant S9; the pH of the sample pad treatment reagent was 8.2.
In another aspect, the present invention provides a free thyroxine detecting reagent strip, wherein the sample pad of the detecting reagent strip is treated with the sample pad treatment reagent as described above.
Further, the reagent strip comprises a sample pad, a binding pad, an NC membrane, absorbent paper and a bottom plate, wherein the binding pad is fixed with a T4 antibody-microsphere marker, and the NC membrane is fixed with a BSA-T4 antigen complex.
The T4 antibody can be combined with free thyroxine in human body, and can be used for detecting the free thyroxine in human body.
The free thyroxine is tested by a competition method, and the concentration of the tested substance in the sample is inversely proportional to the signal value.
In another aspect, the present invention provides a method for preparing the free thyroxine detection reagent strip, comprising the following steps:
step one, sample pad preparation
1) Preparing a boric acid buffer solution, and adjusting the pH value to 8.2;
2) preparing a sample pad treatment solution containing 50mM boric acid buffer solution, 1% BSA, 0.1M NaCl, 1% surfactant S9 and 1% -2% urea-formaldehyde resin with the molecular weight of 10000-;
3) uniformly dispersing the uniformly mixed sample pad treatment solution on glass fibers, and uniformly absorbing 50mL of treatment solution by each glass fiber;
4) drying in an oven at 37 deg.C for 12 hr;
step two: bond pad preparation
1) Mixing a fluorescent microsphere solution with EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, which is used for activating carboxyl of the microsphere and enabling the microsphere to be coupled with an antibody), and a T4 antibody uniformly to react to obtain a T4 antibody-microsphere marker, storing the T4 antibody-microsphere marker in a Tris buffer solution with the pH of 8.050 mM as a marking stock solution, wherein the microsphere content of the marking stock solution is 0.5%;
2) diluting the marking standby solution by 10 times and spraying a film;
3) the combination pad is made of glass fiber;
4) after film spraying, drying at 45 ℃ overnight;
step three: NC film preparation
1) BSA-T4 antigen complex was diluted to 1 mg/mL;
2) scribing an NC film at a speed of 10 muL/cm by using a scriber;
3) after the film is scratched, drying at 45 ℃ overnight;
step four: reagent strip assembly
1) Cutting the sample pad processed in the step one into strips with the width of 17mm, cutting the bonding pad obtained in the step two into strips with the width of 10mm, cutting the NC film processed in the step three into strips with the width of 30mm, cutting the absorbent paper into strips with the width of 17mm, sequentially adhering the strips on a bottom plate, and firmly lapping 1-2mm between every two layers to obtain a large card;
3) cutting the large card into test cards with the width of 4mm by using a cutting machine, and filling the test cards into a card shell to form a detection reagent strip;
3) packaging into aluminum foil bags, sealing and storing to be tested.
In another aspect, the present invention provides a method for detecting free thyroxine, comprising the following steps:
1) removing the strip of any one of claims 6 to 7 and rewarming at 15-30 ℃;
2) and adding 60 microliters of a sample to be tested into the reagent strip, placing the reagent strip on a horizontal desktop at room temperature for reaction, reading a test result by a fluorescence immunoassay analyzer after 15 minutes, and repeating the step for 5 times.
In yet another aspect, the invention provides the use of urea formaldehyde resin for preparing a free thyroxine detection reagent strip or a sample pad treatment reagent for a free thyroxine detection reagent strip, wherein the urea formaldehyde resin has a molecular weight of 10000-
Further, the content of the urea-formaldehyde resin is 1-2%.
The invention has the beneficial effects that:
1. the sample pad treating solution is prepared by adopting the high polymer urea-formaldehyde resin with specific molecular weight and content, so that the free thyroxine in a sample to be detected can be completely separated and obtained, and the detection sensitivity is improved from 100pg/ml to 10 pg/ml;
2. the novel free thyroxine detection reagent strip is used for detecting free thyroxine by a competition method, a sample pad of the reagent strip is treated by a sample pad treatment reagent containing urea resin with specific molecular weight and content, so that the free thyroxine can be fully captured and fully reacted, the detection sensitivity of the free thyroxine is greatly improved, the detection sensitivity reaches 10pg/ml, and the clinical detection requirement is met.
3. The use of urea formaldehyde resin for preparing free thyroxine detecting reagent strip or sample pad treating reagent of free thyroxine detecting reagent strip is provided.
Drawings
FIG. 1 is a schematic view of the structure of a free thyroxine-detecting reagent strip in example 1
FIG. 2 is a graph showing the trend of the gradient of the mean value of the T-line detection signal of the detection reagent strip prepared from the sample pad treated with the sample pad treatment solution according to examples 1 to 5
Detailed Description
The present invention is described in further detail below with reference to examples, which are intended to facilitate the understanding of the present invention without limiting it in any way. The reagents not specifically mentioned in this example were all known products and were obtained by purchasing commercially available products.
Example 1 sample pad treatment reagent formulation a: the high polymer is 1 percent PVP-k30
Preparing a boric acid buffer solution, and adjusting the pH to 8.2; a sample pad treatment solution containing 50mM boric acid buffer, 1% BSA, 0.1M NaCl, 1% surfactant S9, and 1% PVP-k30 was prepared by mixing 1000ml of boric acid buffer, 10g BSA, 5.8g NaCl, 10ml surfactant S9, and 10g PVP-k 30.
Example 2 sample pad treatment reagent formulation B1: the high polymer is urea-formaldehyde resin with 1% molecular weight of 10000
Preparing a boric acid buffer solution, and adjusting the pH to 8.2; 1000ml of boric acid buffer solution, 10g of BSA, 5.8g of NaCl, 10ml of surfactant S9 and 10g of urea-formaldehyde resin with the molecular weight of 10000 are uniformly mixed to prepare the sample pad treating fluid containing 50mM of boric acid buffer solution, 1% of BSA, 0.1M of NaCl, 1% of surfactant S9 and 1% of urea-formaldehyde resin with the molecular weight of 10000.
Example 3 sample pad treatment reagent formulation B2: the high polymer is urea-formaldehyde resin with 2% molecular weight of 10000
Preparing a boric acid buffer solution, and adjusting the pH to 8.2; 1000ml of boric acid buffer solution, 10g of BSA, 5.8g of NaCl, 10ml of surfactant S9 and 20g of urea-formaldehyde resin with the molecular weight of 10000 are uniformly mixed to prepare the sample pad treating fluid containing 50mM of boric acid buffer solution, 1% of BSA, 0.1M of NaCl, 1% of surfactant S9 and 2% of urea-formaldehyde resin with the molecular weight of 10000.
Example 4 sample pad treatment reagent formulation B3: the high polymer is urea-formaldehyde resin with 1% molecular weight of 20000
Preparing a boric acid buffer solution, and adjusting the pH to 8.2; a sample pad treatment solution containing 50mM boric acid buffer, 1% BSA, 0.1M NaCl, 1% surfactant S9, and 1% urea resin having a molecular weight of 20000 was prepared by mixing 1000ml of boric acid buffer, 10g BSA, 5.8g NaCl, 10ml surfactant S9, and 10g urea resin having a molecular weight of 20000 uniformly.
Example 5 sample pad treatment reagent formulation B4: the high polymer is urea-formaldehyde resin with 2% of molecular weight of 20000
Preparing a boric acid buffer solution, and adjusting the pH to 8.2; a sample pad treatment solution containing 50mM boric acid buffer solution, 1% BSA, 0.1M NaCl, 1% surfactant S9, and 2% urea resin having a molecular weight of 20000 was prepared by mixing 1000ml of 10g BSA, 5.8g NaCl, 10ml surfactant S9, and 20g urea resin having a molecular weight of 20000 uniformly.
Example 6 sample pad treatment reagent formulation C: the high polymer is 1 percent of polyacrylamide
Preparing a boric acid buffer solution, and adjusting the pH to 8.2; a sample pad treatment solution containing 50mM boric acid buffer solution, 1% BSA, 0.1M NaCl, 1% surfactant S9, and 1% polyacrylamide with molecular weight of 10000 was prepared by uniformly mixing 1000ml of boric acid buffer solution, 10g BSA, 5.8g NaCl, 10ml surfactant S9, and 10g polyacrylamide with molecular weight of 10000.
EXAMPLE 7 preparation of free thyroxine detection reagent strips
The preparation steps of the test reagent strip of this example are as follows:
the method comprises the following steps: sample pad preparation
1) The sample pad treated solution provided in example 6 was uniformly dispersed on glass fibers (30 cm. times.30 cm, length. times.width) and 50mL of the treated solution was uniformly absorbed by each glass fiber to obtain a sample pad treated by formulation A and a sample pad treated by formulation B1-B4.
4) And (3) drying the sample pad obtained by the treatment in an oven at 37 ℃ for 12 hours, taking out, and sealing in an aluminum foil bag for later use.
Step two: bond pad preparation
1) And (3) adjusting the fluorescent microsphere solution to a fixed pH value, uniformly mixing the fluorescent microsphere solution with EDC and an antibody for reaction to obtain a T4 antibody-microsphere marker, and storing the marker in a pH 8.050 mM Tris buffer solution for later use, wherein the microsphere content of a marking solution for later use is 0.5%.
2) Diluting the marking standby solution by 10 times and spraying a film;
3) the combination pad is made of glass fiber;
4) after spraying the film, it was dried overnight at 45 ℃.
Step three: NC film preparation
1) BSA-T4 antigen complex was diluted to 1 mg/mL;
2) scribing an NC film at a speed of 10 muL/cm by using a scriber;
3) after scratching, oven dry at 45 deg.C overnight.
Step four: reagent strip assembly
1) Cutting the sample pad processed in the step one into strips with the width of 17mm, cutting the bonding pad obtained in the step two into strips with the width of 10mm, cutting the NC film processed in the step three into strips with the width of 30mm, cutting the absorbent paper into strips with the width of 17mm, sequentially adhering the strips on a bottom plate, and firmly connecting the strips by 1-2 mm.
2) And (3) cutting the stuck large card into test cards with the width of 4mm by using a cutting machine, and filling the test cards into a card shell to form the detection reagent strip.
3) Packaging into aluminum foil bags, sealing and storing to be tested.
EXAMPLE 8 Effect of different sample pad treatment reagents on the results of free thyroxine detection
In this example, the test reagent strip provided in example 7 was used to detect free thyroxine, and the sample pad treatment reagents were used to perform sample pad treatments using the sample pad treatment reagents provided in examples 1-6, respectively.
The sample to be detected is respectively diluted into concentrations of 0, 10, 20, 40, 70, 100 and 200pg/mL by adopting a T4 pure product, theoretically, a T line signal value detected by a matched instrument after the sample to be detected with different concentrations is added into the detection reagent strip can present a gradient effect according to different concentrations, but the gradient effect cannot be presented due to sensitivity reasons when the concentration is low, and the corresponding concentration cannot be calculated according to the T line signal values with different gradients. For example, assuming that X1, X2, X3 and X4 are samples corresponding to a concentration sequence, there is no difference in T-line signal values detected by X1 and X2, and a significant gradient in T-line signal values detected by X2, X3 and X4, the concentration corresponding to X3 is considered to be the lowest concentration that can be detected, i.e., the corresponding sensitivity, and in this experiment, a difference of 10% is considered to be a significant difference.
The specific experimental process is as follows:
1) the detection reagent strip is rewarming at 15-30 ℃.
2) Phosphate buffer was prepared and the pH was adjusted to 7.4.
3) Diluting the T4 pure product (2ng/mL) with the phosphate buffer solution in 2) to 10 times, 20 times, 28.6 times, 50 times, 100 times and 200 times respectively, and taking one copy of the phosphate buffer solution without adding the T4 pure product to obtain samples with the concentrations of 0pg/mL, 10pg/mL, 20pg/mL, 40pg/mL, 70pg/mL, 100pg/mL and 200 pg/mL.
4) Each sample in 3) was divided into five equal parts, one of which was used for the test strip prepared from the sample pad treated with the sample pad treatment solution of formulation a, and the other four of which were used for the test strip prepared from the sample pads treated with the sample pad treatment solutions of formulations B1 to B4, respectively.
5) 60 microliters of each sample in 4) was added to the corresponding test strip, and the strip was placed on a horizontal table at room temperature for reaction, and after 15 minutes, the test results were read by a fluorescence immunoassay analyzer and repeated 5 times.
The test results are shown in tables 1-7:
table 1, results of tests performed on test strips prepared from sample pads treated with sample treatment reagent (formulation A) provided in example 1
Results of tests performed on test strips prepared from sample pads treated with the sample treatment reagent (formulation B1) provided in Table 2 and example 2
Results of tests performed on test strips prepared from sample pads treated with the sample treatment reagent (formulation B2) provided in Table 3 and example 3
Table 4, results of tests performed on test strips prepared from sample pads treated with the sample treatment reagent (formulation B3) provided in example 4
Results of tests performed on test strips prepared from sample pads treated with the sample treatment reagent (formulation B4) provided in Table 5 and example 5
Results of tests performed on test strips prepared from sample pads treated with the sample treatment reagents (formulation C) provided in Table 6 and example 6
TABLE 7 Effect of different sample pad treatment reagents on the results of free thyroxine detection
| Sample concentration (pg/mL) | Formulation A | Formulation B1 | Formulation B2 | Formulation B3 | Formulation | Formulation C | |
| 0 | 11407.2 | 20184.9 | 22216.4 | 23830.1 | 25167.3 | 12200.6 | |
| 10 | 11528.0 | 18876.7 | 19844.7 | 20598.8 | 22233.0 | 12408.1 | |
| 20 | 11619.9 | 16441.2 | 18178.7 | 18641.8 | 20140.3 | 12164.2 | |
| 40 | 11480.8 | 14081.2 | 16016.3 | 16047.9 | 17418.5 | 12151.7 | |
| 70 | 11465.3 | 10847.4 | 12695.4 | 12796.8 | 13634.9 | 11957.5 | |
| 100 | 10085.4 | 8119.0 | 8276.8 | 8375.0 | 9970.7 | 10939.3 | |
| 200 | 4657.7 | 4110.4 | 4777.0 | 4728.4 | 4887.3 | 10771.4 |
As can be seen from tables 1 to 7, the test reagent strips prepared from the sample pad treated with the sample pad treatment solution (containing 1% PVP-k30) provided in example 1 have no significant gradient between 0pg/mL and 70pg/mL, and can be calculated only when there is a gradient division between the two concentrations before and after a certain concentration to be calculated, that is, there is no gradient division between the test concentrations corresponding to 10pg/mL, 20pg/mL, 40pg/mL and 70pg/mL, and thus the corresponding test results cannot be calculated, and the test results have significant gradients only between 70pg/mL, 100pg/mL and 200pg/mL, that is, the test result concentration corresponding to 100pg/mL can be calculated at least.
The test reagent strip prepared from the sample pad treated by the sample pad treating solution (containing 1% polyacrylamide) provided in example 6 has no obvious gradient between 0pg/mL and 200pg/mL, so that the corresponding test result cannot be calculated. It can be seen that the polyacrylamide with molecular formula of 10000 can also increase the liquid viscosity and slow down the chromatography speed of the sample on the sample pad, but has no obvious effect of improving the detection sensitivity of the free thyroxine.
On the other hand, the test strips prepared from the sample pads treated with the sample pad treatment solutions (containing urea-formaldehyde resins) provided in examples 2 to 5 showed a great change in signal value from 0pg/mL to 70pg/mL, and showed a sharp gradient, and the concentration was calculated by dividing the gradient, so that the lowest detectable concentration was increased to 10 pg/mL. Particularly, in the formula example 5, when the molecular weight of the urea resin is 20000 and the content is 2%, the gradient trend of the mean value of the T line test signal of the samples with different concentrations corresponding to each formula is strongest.
FIG. 2 is a graph showing the gradient trend of the mean value of the T-line detection signal of the detection reagent strip prepared from the sample pad treated with the sample pad treatment solution provided in examples 1 to 5. It can be concluded from the following results by combining tables 2-5 and fig. 2 that the detection signal of free thyroxine can be amplified significantly by using the sample pad treatment solution prepared from urea-formaldehyde resin, and that the greater the molecular weight, the greater the signal enhancement, and the greater the signal enhancement for the same type of urea-formaldehyde resin, the higher the concentration. However, the molecular weight and concentration of the urea-formaldehyde resin are increased continuously, so that the chromatography time is prolonged, and the molecular weight of the urea-formaldehyde resin is 20000 and the content of the urea-formaldehyde resin is 2% which is an optimal choice in comprehensive consideration.
All patents and publications mentioned in the specification of the invention are indicative of the techniques disclosed in the art to which this invention pertains and are intended to be applicable. All patents and publications cited herein are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The invention described herein may be practiced in the absence of any element or elements, limitation or limitations, which limitation or limitations is not specifically disclosed herein. For example, the terms "comprising", "consisting essentially of … …" and "consisting of … …" in each instance herein may be substituted for the remaining 2 terms of either. The word "a" or "an" herein means only "one", and does not exclude only one, but may mean 2 or more. The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described, but it is recognized that various modifications and changes may be made within the scope of the invention and the claims which follow. It is to be understood that the embodiments described herein are preferred embodiments and features and that modifications and variations may be made by one skilled in the art in light of the teachings of this disclosure, and are to be considered within the purview and scope of this invention and the scope of the appended claims and their equivalents.
Claims (10)
1. A sample pad treatment reagent of a free thyroxine detection reagent strip is characterized by containing urea formaldehyde resin.
2. The sample pad treatment reagent as claimed in claim 1, wherein the urea formaldehyde resin has a molecular weight of 10000-.
3. The sample pad treatment reagent of claim 1 or 2, wherein the urea formaldehyde resin is present in an amount of 1% to 2%.
4. The sample pad processing reagent of claim 3, further comprising a system buffer, a protein, a salt ion, and a surfactant.
5. The sample pad processing reagent of claim 4, wherein the system buffer is 50mM boric acid buffer, the protein is 1% BSA, the salt ion is 0.1M NaCl, and the surfactant is 1% surfactant S9; the pH of the sample pad treatment reagent was 8.2.
6. A free thyroxine detection reagent strip wherein the sample pad of said strip is treated with a sample pad treatment reagent according to any of claims 1 to 5.
7. The reagent strip of claim 6, wherein the reagent strip comprises a sample pad, a conjugate pad, an NC membrane, absorbent paper, and a bottom plate, wherein the conjugate pad has T4 antibody-microsphere labels immobilized thereon, and the NC membrane has BSA-T4 antigen complex immobilized thereon.
8. A method of making a reagent strip according to any one of claims 6 to 7, comprising the steps of:
step one, sample pad preparation
1) Preparing a boric acid buffer solution, and adjusting the pH value to 8.2;
2) preparing a sample pad treatment solution containing 50mM boric acid buffer solution, 1% BSA, 0.1M NaCl, 1% surfactant S9 and 1% -2% urea-formaldehyde resin with the molecular weight of 10000-;
3) uniformly dispersing the uniformly mixed sample pad treatment solution on glass fibers, and uniformly absorbing 50mL of treatment solution by each glass fiber;
4) drying in an oven at 37 deg.C for 12 hr;
step two: bond pad preparation
1) Uniformly mixing the fluorescent microsphere solution with EDC and T4 antibodies for reaction to obtain a T4 antibody-microsphere marker, storing the marker in a pH 8.050 mM Tris buffer solution as a marking standby solution, wherein the microsphere content of the marking standby solution is 0.5%;
2) diluting the marking standby solution by 10 times and spraying a film;
3) the combination pad is made of glass fiber;
4) after film spraying, drying at 45 ℃ overnight;
step three: NC film preparation
1) BSA-T4 antigen complex was diluted to 1 mg/mL;
2) scribing an NC film at a speed of 10 muL/cm by using a scriber;
3) after the film is scratched, drying at 45 ℃ overnight;
step four: reagent strip assembly
1) Cutting the sample pad processed in the step one into strips with the width of 17mm, cutting the bonding pad obtained in the step two into strips with the width of 10mm, cutting the NC film processed in the step three into strips with the width of 30mm, cutting the absorbent paper into strips with the width of 17mm, sequentially adhering the strips on a bottom plate, and firmly lapping 1-2mm between every two layers to obtain a large card;
2) cutting the large card into test cards with the width of 4mm by using a cutting machine, and filling the test cards into a card shell to form a detection reagent strip;
3) packaging into aluminum foil bags, sealing and storing to be tested.
9. A method for detecting free thyroxine, comprising the steps of:
1) removing the strip of any one of claims 6 to 7 and rewarming at 15-30 ℃;
2) and adding 60 microliters of a sample to be tested into the reagent strip, placing the reagent strip on a horizontal desktop at room temperature for reaction, reading a test result by a fluorescence immunoassay analyzer after 15 minutes, and repeating the step for 5 times.
10. The application of the urea-formaldehyde resin in preparing the free thyroxine detection reagent strip or the sample pad treatment reagent of the free thyroxine detection reagent strip is characterized in that the molecular weight of the urea-formaldehyde resin is 10000-20000.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110293228.4A CN113281526B (en) | 2021-03-18 | 2021-03-18 | Sample pad treatment reagent of free thyroxine detection reagent strip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202110293228.4A CN113281526B (en) | 2021-03-18 | 2021-03-18 | Sample pad treatment reagent of free thyroxine detection reagent strip |
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001021563A (en) * | 1999-07-06 | 2001-01-26 | Nitto Denko Corp | Specific bonding body |
| WO2006107339A2 (en) * | 2005-03-31 | 2006-10-12 | Georgetown University | Free thyroxine and free triiodothyronine analysis by mass spectrometry |
| CN104101708A (en) * | 2013-04-11 | 2014-10-15 | 中国科学院化学研究所 | Up-conversion fluorescent/magnetic nanoparticles-based immune-chromatographic test paper and making method thereof |
| CN105891468A (en) * | 2016-04-08 | 2016-08-24 | 四川新健康成生物股份有限公司 | Sample release pad treatment solution |
| CN108548931A (en) * | 2018-05-09 | 2018-09-18 | 南京岚煜生物科技有限公司 | Free thyroxine fT4 kits and preparation and detection method are detected based on micro-fluidic chip |
| CN109085334A (en) * | 2018-06-22 | 2018-12-25 | 东南大学 | A kind of colloidal gold fast detecting test paper strip and its preparation method and application of SFTSV nucleoprotein antigen |
| US20190064160A1 (en) * | 2016-08-09 | 2019-02-28 | Sekisui Medical Co., Ltd. | Immunochromatographic detection kit |
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
| CN211554026U (en) * | 2019-12-31 | 2020-09-22 | 深圳市活水床旁诊断仪器有限公司 | Dry-type immunochromatography reagent card |
-
2021
- 2021-03-18 CN CN202110293228.4A patent/CN113281526B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001021563A (en) * | 1999-07-06 | 2001-01-26 | Nitto Denko Corp | Specific bonding body |
| WO2006107339A2 (en) * | 2005-03-31 | 2006-10-12 | Georgetown University | Free thyroxine and free triiodothyronine analysis by mass spectrometry |
| CN104101708A (en) * | 2013-04-11 | 2014-10-15 | 中国科学院化学研究所 | Up-conversion fluorescent/magnetic nanoparticles-based immune-chromatographic test paper and making method thereof |
| CN105891468A (en) * | 2016-04-08 | 2016-08-24 | 四川新健康成生物股份有限公司 | Sample release pad treatment solution |
| US20190064160A1 (en) * | 2016-08-09 | 2019-02-28 | Sekisui Medical Co., Ltd. | Immunochromatographic detection kit |
| CN108548931A (en) * | 2018-05-09 | 2018-09-18 | 南京岚煜生物科技有限公司 | Free thyroxine fT4 kits and preparation and detection method are detected based on micro-fluidic chip |
| CN109085334A (en) * | 2018-06-22 | 2018-12-25 | 东南大学 | A kind of colloidal gold fast detecting test paper strip and its preparation method and application of SFTSV nucleoprotein antigen |
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
| CN211554026U (en) * | 2019-12-31 | 2020-09-22 | 深圳市活水床旁诊断仪器有限公司 | Dry-type immunochromatography reagent card |
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