Parathyroid hormone detection kit and use and preparation method thereof

Abstract

The invention discloses a parathyroid hormone detection kit and a use and preparation method, and belongs to the field of immunodetection analysis, the parathyroid hormone detection kit comprises a reagent card and a diluent, the reagent card comprises a reagent strip, the reagent strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, the lower end of the sample pad is overlapped with the upper end of the combination pad, and the upper end of the nitrocellulose membrane is overlapped with the lower end of the PVC bottom plate. The lower end of the combination pad is overlapped with the upper end of the nitrocellulose membrane; the upper end of the absorbent paper is overlapped with the lower end of the nitrocellulose membrane; the parathyroid hormone (PTH) in a sample is combined with the colloidal gold labeled anti-PTH antibody on the combination pad to form a compound, chromatography is carried out towards the absorbent paper end under the capillary effect, the anti-PTH antibody at the detection line on the film is captured, the redundant unreacted colloidal gold labeled antibody is subjected to chromatography to the quality control line, and whether PTH exists in the sample is reflected through the color change of the quality control line and the detection line. The PTH qualitative analysis is simple and convenient to operate, high in sensitivity, low in cost and fast in detection.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay analysis, and in particular relates to a parathyroid hormone detection kit and a use and preparation method thereof. Background Art

[0002] Parathyroid hormone has the function of secreting parathyroid hormone, and plays a vital role in regulating serum calcium concentration and maintaining the balance of calcium and phosphorus metabolism in the human body. Hypocalcemia caused by hypoparathyroidism after thyroidectomy has become the most common complication in thyroid surgery. The main reason is the damage to the parathyroid glands and poor blood flow during thyroidectomy. In the past three decades, with the continuous development of thyroid and parathyroid surgery, the issue of parathyroid function protection has received increasing attention, and autologous parathyroid transplantation has also been widely used in clinical practice. However, the parathyroid gland has a special anatomical position and complex shape. Even if the surgeon carefully operates and separates it during thyroidectomy, insufficient secretion of parathyroid hormone often occurs due to misjudgment and failure to perform autologous transplantation in time.

[0003] Currently, frozen tissue sectioning technology is often used in clinical practice to assist in determining whether parathyroid glands are present in tissues, but this method takes 30 minutes to complete, which delays the best time for autologous transplantation to a certain extent, and also causes certain damage to the parathyroid tissue that needs autologous transplantation. Currently, there are enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECLIA), etc. for detecting PTH. However, these methods are time-consuming, labor-intensive, and expensive, and require professional operators to use specific instruments for detection. Therefore, a non-destructive rapid detection method is needed. Summary of the invention

[0004] The purpose of the present invention is to provide a parathyroid hormone detection kit and a method for using and preparing the kit, so as to solve the problem that hypocalcemia caused by hypoparathyroidism after thyroidectomy has become a common complication, and the existing clinical detection methods such as frozen tissue section technology are time-consuming and damaging to parathyroid tissue, and enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay, etc. are time-consuming, labor-intensive and expensive and require professionals to use specific instruments for detection, and lack a non-destructive rapid detection method.

[0005] To achieve the above object, the present invention provides the following technical solution: a parathyroid hormone detection kit, comprising a reagent card and a diluent, wherein the reagent card comprises a reagent strip, and the reagent strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, wherein the lower end of the sample pad overlaps with the upper end of the conjugation pad, the lower end of the conjugation pad overlaps with the upper end of the nitrocellulose membrane, and the upper end of the absorbent paper overlaps with the lower end of the nitrocellulose membrane;

[0006] The nitrocellulose membrane is coated with anti-parathyroid hormone antibody and goat anti-mouse IgG, and the conjugate pad is coated with anti-parathyroid hormone antibody labeled with colloidal gold.

[0007] Furthermore, the reagent card also includes an upper card shell and a lower card shell, the upper card shell has a visual window and a sample addition hole, and a parathyroid hormone reagent strip is fixed in the card shell slot of the lower card shell, and the upper card shell and the lower card shell are fixed by snap connection.

[0008] Furthermore, the upper and lower ends of the conjugate pad overlap with the lower end of the sample pad and the upper end of the nitrocellulose membrane by 1-2 mm respectively, and the upper end of the absorbent paper overlaps with the lower end of the nitrocellulose membrane by 1-2 mm.

[0009] Furthermore, the diluent is 0.9% sodium chloride solution.

[0010] A method for using a parathyroid hormone detection kit, using a 2mL sterile disposable syringe with a 22G needle (0.7# black) to puncture the tissue to be tested, and perform 2 to 3 punctures at different locations, with a cumulative puncture depth of about 8 to 10 mm, eluate the needle of the syringe in 1mL of diluent, mix the eluent, draw 60μL of the eluent and add it to the sample well of the reagent card of the detection kit for detection, and obtain the test result within 3-10 minutes.

[0011] A method for preparing a parathyroid hormone detection kit comprises the following steps:

[0012] Step S1: Preparation of conjugate pad;

[0013] Step S2: nitrocellulose membrane preparation;

[0014] Step S3: preparation of sample pad;

[0015] Step S4: Assembling.

[0016] Furthermore, the preparation of the conjugate pad comprises the following steps:

[0017] Step S11, preparation of colloidal gold solution: add 1 mL of 1% HAuCl4 (W / V) aqueous solution to 100 mL of purified water, heat to boiling on an electromagnetic heating stirrer while stirring, then quickly add 2 mL of 1% Na3C6H5O7 (W / V) aqueous solution, continue stirring and heating for 20-30 minutes, until the colloidal gold solution turns wine red, stop heating, and cool to room temperature;

[0018] Step S12, colloidal gold solution labeling: adjust the solution pH to 8.0-8.5 with 0.2M K2CO3 solution, add 0.5-1mg anti-parathyroid hormone antibody 2, stir and mix for 15min, then add 11mL 10% BSA solution, stir and mix for 30min, centrifuge the labeled solution at 14000g for 30min at 2-8℃, remove the supernatant, and resuspend with 10mL labeling preservation solution;

[0019] Step S13, spreading and drying: the colloidal gold conjugate is spread on the treated conjugate pad at 2-4 μL / cm, and dried at 45° C. for 12 hours.

[0020] Furthermore, the nitrocellulose membrane preparation comprises the following steps:

[0021] Step S21: placing the nitrocellulose membrane in an environment with a temperature of 23-27° C. and a humidity of 40-60% for equilibration for 30-60 minutes;

[0022] Step S22: diluting anti-parathyroid hormone antibody 1 and goat anti-mouse IgG to 1.5 mg / mL respectively with coating buffer;

[0023] Step S23: Coat the detection line (T) and quality control line (C) of the nitrocellulose membrane at 0.7 μL / cm, and dry at 45° C. for 16 hours.

[0024] Furthermore, the step S3 is to spread 20 mmol / L phosphate buffer containing 2% BSA and 2% sucrose on the glass fiber membrane, dry it at 45° C. for 24 hours, and seal it and store it at room temperature.

[0025] Furthermore, step S4 is to sequentially stick the sample pad, the colloidal gold binding pad, the nitrocellulose membrane coated with the detection line (T) and the quality control line (C), and the absorbent paper on the PVC bottom plate, cut them into 2.85 mm wide reagent strips with a CNC chopper, and assemble them with an upper card shell and a lower card shell.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The parathyroid hormone detection kit uses a sterile disposable syringe to puncture the tissue to be tested, elutes the needle in a diluent, and then adds the eluent to the sample well of the reagent card. The parathyroid hormone (PTH) in the sample combines with the anti-PTH antibody labeled with colloidal gold on the binding pad to form a complex, which is chromatographed toward the absorbent paper end under the capillary effect and captured by the anti-PTH antibody at the detection line on the membrane. The excess unreacted colloidal gold labeled antibody is chromatographed to the quality control line. The color changes of the quality control line and the detection line reflect whether PTH exists in the sample, and the qualitative analysis of PTH is simple to operate, highly sensitive, low cost, and fast detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the minimum detection limit test results;

[0029] Figure 2 Schematic diagram of specificity test results. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments.

[0031] The following examples are used to illustrate the present invention, but they cannot be used to limit the scope of protection of the present invention. The conditions in the examples can be further adjusted according to specific conditions. Simple improvements to the method of the present invention under the premise of the concept of the present invention belong to the scope of protection claimed in the present invention.

[0032] See also Figure 1 The present invention provides a parathyroid hormone detection kit, comprising a reagent card and a diluent.

[0033] Reagent card: The reagent card includes a reagent strip and a card shell; the reagent strip includes a sample pad, a binding pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, the lower end of the sample pad overlaps with the upper end of the binding pad by 1-2 mm, the lower end of the binding pad overlaps with the upper end of the nitrocellulose membrane by 1-2 mm, the upper end of the absorbent paper overlaps with the lower end of the nitrocellulose membrane by 1-2 mm, the nitrocellulose membrane takes the end close to the sample pad as the starting end and the end close to the absorbent paper as the ending end, and is coated with anti-parathyroid hormone antibodies and goat anti-mouse IgG in sequence, the binding pad is coated with anti-parathyroid hormone antibodies labeled with colloidal gold, the card shell includes an upper card shell and a lower card shell, the upper card shell has a visual window and a sample addition hole, the card slot of the lower card shell is fixed with a parathyroid hormone reagent strip, and the upper and lower card shells are fixed by snapping.

[0034] Diluent: The diluent is 0.9% sodium chloride solution.

[0035] Preparation of parathyroid hormone detection kit

[0036] The preparation of the parathyroid hormone detection kit mainly includes the preparation of the parathyroid hormone reagent strip, which in turn includes the preparation of the binding pad, the preparation of the nitrocellulose membrane and the preparation of the sample pad.

[0037] The preparation of the conjugate pad includes the following steps:

[0038] Step 1 S11, preparation of colloidal gold solution: add 1 mL of 1% HAuCl4 (W / V) aqueous solution to 100 mL of purified water. Heat to boiling on an electromagnetic heating stirrer while stirring, then quickly add 2 mL of 1% Na3C6H5O7 (W / V) aqueous solution, continue stirring and heating for 20-30 minutes, until the colloidal gold solution turns wine red, stop heating, and cool to room temperature;

[0039] Step 2 S12, colloidal gold solution labeling: adjust the solution pH to 8.0-8.5 with 0.2M K2CO3 solution, add 0.5-1mg anti-parathyroid hormone antibody 2, stir and mix for 15 minutes, then add 11mL 10% BSA solution, stir and mix for 30 minutes. The labeled solution is centrifuged at 14000g for 30 minutes at 2-8°C, and the supernatant is removed. Resuspend with 10mL labeling preservation solution.

[0040] Step 3 S13, spreading and drying: Spread the colloidal gold conjugate on the treated conjugate pad at 2-4 μL / cm and dry at 45° C. for 12 hours.

[0041] Preparation of nitrocellulose membrane includes the following steps

[0042] Step 1 S21: Place the nitrocellulose membrane in an environment with a temperature of 23-27° C. and a humidity of 40-60% for 30-60 minutes;

[0043] Step 2 S22: dilute the anti-parathyroid hormone antibody 1 and goat anti-mouse IgG to 1.5 mg / mL respectively using coating buffer.

[0044] Step 3 S23: Coat the detection line (T) and quality control line (C) of the nitrocellulose membrane at 0.7 μL / cm, and dry at 45° C. for 16 hours.

[0045] The preparation of the sample pad includes the following steps

[0046] Step 1 S31: Spread 20 mmol / L phosphate buffer (pH 7.4) containing 2% BSA and 2% sucrose onto the glass fiber membrane, dry at 45° C. for 24 hours, and seal and store at room temperature.

[0047] Assembly: Paste the sample pad, colloidal gold binding pad, nitrocellulose membrane coated with test line (T) and quality control line (C), and absorbent paper on the PVC base plate in sequence. Cut into 2.85 mm wide reagent strips using a CNC chopper and assemble with upper and lower card shells.

[0048] Method for using the above parathyroid hormone detection kit

[0049] Instructions: During thyroidectomy, use a 2mL sterile disposable syringe with a 22G needle (0.7# black) to puncture the tissue to be tested, and perform 2 to 3 punctures at different locations. The cumulative depth of the puncture is about 8 to 10mm. Elute the needle of the syringe in 1mL of diluent, mix the eluent, draw 60μL of the eluent and add it to the sample well of the reagent card of the test kit, and obtain the test results within 3-10 minutes.

[0050] Result judgment: If both the T line and the C line of the reagent card show red lines, the sample is positive for parathyroid hormone (PTH); if the C line shows a red line and the T line does not show a red line, the sample is negative for parathyroid hormone (PTH); if the T line shows a red line and the C line does not show a red line, the result is invalid.

[0051] Parathyroid hormone test kit performance test

[0052] Minimum detection limit test: dilute the PTH standard to 0 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.5 ng / mL and 5.0 ng / mL with 20 mmol / L PBS buffer, and then use this reagent card for detection;

[0053] The results are as follows Figure 1 As shown in the figure, when the concentration of PTH standard is as low as 0.5 ng / mL, the reagent card can still stably detect positive reactions, and the minimum detection limit of this kit is not higher than 0.5 ng / mL.

[0054] Specificity test: Take 60 μL of the elution from the puncture needle of the parathyroid, thyroid, lymph, muscle and adipose tissue and add it to the sample well of the reagent card, wait for 3-10 minutes, and observe the T line and C line;

[0055] The results are as follows Figure 2 As shown in the figure, only the parathyroid puncture needle washout showed red lines on the T line and C line, while the puncture needle washout of non-parathyroid tissue failed to make the T line show red lines, indicating that the kit has good specificity.

[0056] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the embodiments of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description, and these obvious changes or modifications derived from the spirit of the present invention are still within the scope of protection of the present invention.

Claims

1. A parathyroid hormone detection kit, comprising a reagent card and a diluent, characterized in that: The reagent card comprises a reagent strip, which comprises a sample pad, a conjugation pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, wherein the lower end of the sample pad overlaps with the upper end of the conjugation pad, the lower end of the conjugation pad overlaps with the upper end of the nitrocellulose membrane, and the upper end of the absorbent paper overlaps with the lower end of the nitrocellulose membrane; The nitrocellulose membrane is coated with anti-parathyroid hormone antibody and goat anti-mouse IgG, and the conjugate pad is coated with anti-parathyroid hormone antibody labeled with colloidal gold.

2. A parathyroid hormone detection kit according to claim 1, characterized in that: The reagent card also includes an upper card shell and a lower card shell. The upper card shell has a visual window and a sample addition hole. A parathyroid hormone reagent strip is fixed in the card shell slot of the lower card shell. The upper card shell and the lower card shell are fixedly connected.

3. A parathyroid hormone detection kit according to claim 1, characterized in that: The upper and lower ends of the conjugate pad overlap with the lower end of the sample pad and the upper end of the nitrocellulose membrane by 1-2 mm respectively, and the upper end of the absorbent paper overlaps with the lower end of the nitrocellulose membrane by 1-2 mm.

4. A parathyroid hormone detection kit according to claim 1, characterized in that: The diluent used was 0.9% sodium chloride solution.

5. A method for using a parathyroid hormone detection kit, based on a parathyroid hormone detection kit according to any one of claims 1 to 4, characterized in that: Use a 2mL sterile disposable syringe 22G to puncture the tissue to be tested, and perform 2 to 3 punctures at different locations with a cumulative puncture depth of 8-10mm. Elute the needle of the syringe in 1mL of diluent, mix the eluent, and then draw 60μL of the eluent and add it to the sample well of the reagent card of the test kit for testing. The test results will be obtained within 3-10 minutes.

6. A method for preparing a parathyroid hormone detection kit, characterized in that: The following steps are involved: Step S1: Preparation of conjugate pad; Step S2: nitrocellulose membrane preparation; Step S3: preparation of sample pad; Step S4: Assembling.

7. The method for preparing a parathyroid hormone detection kit according to claim 6, characterized in that: The preparation of the conjugate pad comprises the following steps: Step S11, preparation of colloidal gold solution: add 1 mL of 1% HAuCl4 (W / V) aqueous solution to 100 mL of purified water, heat to boiling on an electromagnetic heating stirrer while stirring, then quickly add 2 mL of 1% Na3C6H5O7 (W / V) aqueous solution, continue stirring and heating for 20-30 minutes, until the colloidal gold solution turns wine red, stop heating, and cool to room temperature; Step S12, colloidal gold solution labeling: adjust the solution pH to 8.0-8.5 with 0.2M K2CO3 solution, add 0.5-1mg anti-parathyroid hormone antibody 2, stir and mix for 15min, then add 11mL 10% BSA solution, stir and mix for 30min, centrifuge the labeled solution at 14000g for 30min at 2-8℃, remove the supernatant, and resuspend with 10mL labeling preservation solution; Step S13, spreading and drying: the colloidal gold conjugate is spread on the treated conjugate pad at 2-4 μL / cm, and dried at 45° C. for 12 hours.

8. The method for preparing a parathyroid hormone detection kit according to claim 6, characterized in that: The nitrocellulose membrane preparation comprises the following steps: Step S21: placing the nitrocellulose membrane in an environment with a temperature of 23-27° C. and a humidity of 40-60% for equilibration for 30-60 minutes; Step S22: diluting anti-parathyroid hormone antibody 1 and goat anti-mouse IgG to 1.5 mg / mL respectively with coating buffer; Step S23: Coat the detection line (T) and quality control line (C) of the nitrocellulose membrane at 0.7 μL / cm, and dry at 45° C. for 16 hours.

9. The method for preparing a parathyroid hormone detection kit according to claim 6, characterized in that: The step S3 is to spread 20 mmol / L phosphate buffer containing 2% BSA and 2% sucrose on the glass fiber membrane, dry it at 45° C. for 24 hours, and seal it for storage at room temperature.

10. The method for preparing a parathyroid hormone detection kit according to claim 6, characterized in that: The step S4 is to sequentially stick the sample pad, the colloidal gold binding pad, the nitrocellulose membrane coated with the detection line (T) and the quality control line (C), and the absorbent paper on the PVC bottom plate, cut them into 2.85 mm wide reagent strips with a CNC chopper, and assemble them with an upper card shell and a lower card shell.

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