A colloidal gold kit for qualitative detection of adiponectin and preparation method thereof

Abstract

The present invention belongs to the technical field of in vitro diagnosis, and discloses a colloidal gold kit for qualitative detection of adiponectin and a preparation method thereof. The kit comprises a PVC plate and a sample pad, an antibody marker binding pad, a nitrocellulose membrane and a blotting paper which are overlapped with each other in sequence on the PVC plate. The antibody marker binding pad is prepared by adding BSA to block the chitosan-loaded colloidal gold particles and the adiponectin antibody after labeling reaction, and then resuspending and diluting the colloidal gold particles with trehalose using a marker buffer and spraying them on a glass fiber membrane and drying. The kit of the present invention uses colloidal gold loaded by porous chitosan microspheres as an antibody marker carrier, which has a stronger and more stable binding force with the antibody, and overcomes the defects of the traditional colloidal gold particles being too small and easy to self-aggregate and the defects of poor hydrophilicity, and has the advantages of high detection accuracy and detection efficiency, high detection sensitivity and good storage stability for qualitative detection of adiponectin.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis, and specifically relates to a colloidal gold kit for qualitatively detecting adiponectin and a preparation method thereof. Background Art

[0002] In recent decades, the incidence of diabetes has increased rapidly. Prediabetes is a transitional stage before diabetes occurs. About 30% of prediabetes patients eventually develop into diabetes. Once diabetic patients have high blood sugar, it cannot be reversed, and patients need to take medication for life. However, if it is found in the prediabetes stage, the disease can be reversed through lifestyle intervention, that is, drug intervention, thereby achieving the purpose of preventing diabetes. A large number of studies on different populations have found that the adiponectin level of patients with prediabetes is significantly lower than that of healthy controls, and the lower the adiponectin level, the higher the risk of diabetes. Therefore, by detecting adiponectin, patients with prediabetes can be screened and identified. And by monitoring the level of adiponectin, the control of the disease and the development trend of complications in diabetic patients can be observed.

[0003] In addition, monitoring adiponectin levels can also be used for: 1) monitoring the therapeutic effect of insulin sensitizers. The higher the adiponectin level after taking the medicine, the better the patient responds to the treatment; 2) it can be used as an auxiliary diagnosis for the classification of type 2 diabetes. Type 2 diabetes patients with low adiponectin levels have high insulin resistance. Conversely, patients with high adiponectin levels may have insufficient insulin secretion; 3) judging the risk of cardiovascular disease; 4) patients with metabolic functions such as liver and kidney abnormalities will have abnormal adiponectin levels, resulting in higher results.

[0004] At present, the main methods for detecting adiponectin are chemiluminescence, latex-enhanced immunoturbidimetry, and immunocolloidal gold or latex labeling technology. Chemiluminescence method: excellent performance indicators, high precision and sensitivity. It is the most widely used methodology in clinical practice, but the supporting instruments are expensive, and it takes a long time to get the results, which is difficult to carry out. Typical products are luminescent products such as Yahuilong. Adiponectin assay kit (latex-enhanced immunoturbidimetry): good sensitivity, but it needs to be matched with instruments and quality control curves, which is more cumbersome. Immunocolloidal gold labeling technology and immunolatex labeling technology have become the development trend of in vitro diagnostic reagents in recent years because of their simple operation, no need for any special instruments, no need for special training of operators, intuitive test results, and convenience for grassroots units and on-site use. It is especially suitable for home self-testing.

[0005] Colloidal gold is usually spherical gold nanoparticles (AuNPs) with a diameter of 5 to 50 nm and good dispersion. When the gold nanoparticles aggregate, the resonance absorption wavelength is red-shifted, and the solution appears purple or purple-red. By directly observing the color change with the naked eye, it can be determined whether the gold nanoparticles have aggregated. By modifying the surface of the gold nanoparticles with adiponectin antibodies, it can specifically bind to the adiponectin in the sample to be tested, move to the detection area and quality control area under the action of absorbent paper, and bind and aggregate with specific antibodies to form an "antibody-adiponectin-antibody" sandwich complex deposition. The adiponectin level can be qualitatively detected by its color change.

[0006] However, since the gold nanoparticles prepared by the conventional method have a small particle size and a high surface chemical activity, they are prone to spontaneous aggregation, resulting in reagent failure and poor storage stability. In addition, ordinary colloidal gold is generally a negatively charged hydrophobic colloid solution, which has poor hydrophilicity and is easily adsorbed and deposited on the membrane material during detection, resulting in low detection sensitivity and prone to false negative results. The particle size of colored latex microspheres is larger than that of colloidal gold particles (the particle size of colored latex microspheres is 200nm-400nm) and has a higher relative sensitivity. However, the dye coating of colored latex microspheres is relatively complex, and the visual color change resolution is poor, which is prone to false positive results; and in order to enhance the binding with antibodies, surface modification is required, which increases the preparation cost. Summary of the invention

[0007] In view of the shortcomings and deficiencies of the above prior art, the primary purpose of the present invention is to provide a method for preparing a colloidal gold kit for qualitative detection of adiponectin.

[0008] Another object of the present invention is to provide a colloidal gold kit prepared by the above method.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing a colloidal gold kit for qualitatively detecting adiponectin comprises the following preparation steps:

[0011] (1) Preparation of porous chitosan microspheres:

[0012] The chitosan and PVA are dissolved in an acetic acid solution to obtain a mixed solution, and the mixed solution is spray-dried to obtain chitosan microspheres; the obtained chitosan microspheres are added to an alkaline solution for alkaline dissolution treatment to remove the PVA component, and after filtering and washing, the mixed solution is cross-linked with glutaraldehyde vapor in a closed drying oven to obtain porous chitosan microspheres;

[0013] (2) Preparation of chitosan-loaded colloidal gold:

[0014] The porous chitosan microspheres are added to the chloroauric acid solution and stirred to mix evenly, and then heated to 70-100° C., and then trisodium citrate solution is added and stirred to react, and the reaction product is filtered, washed, and dried to obtain chitosan-loaded colloidal gold particles;

[0015] (3) Preparation of antibody marker binding pad:

[0016] Chitosan-loaded colloidal gold particles are added to a K2CO3 solution and dispersed evenly, and then adiponectin antibody is added for labeling reaction. After the reaction is completed, BSA (bovine serum albumin) is added for blocking, and the precipitate is collected by centrifugation to obtain an antibody marker; then the precipitate is resuspended and diluted with trehalose using a marker buffer and sprayed on a glass fiber membrane, and then dried to obtain an antibody marker binding pad;

[0017] (4) Preparation of test lines and quality control lines:

[0018] A nitrocellulose membrane is attached to a PVC plate, and a detection area working solution and a quality control area working solution are drawn on the membrane, and after drying, a detection line and a quality control line are obtained respectively; the detection area working solution is a coating solution diluent of adiponectin monoclonal antibody, and the quality control area working solution is a coating solution diluent of goat anti-mouse IgG;

[0019] (5) Preparation of sample pad:

[0020] The sample pad treatment liquid is coated on the glass fiber membrane, and the sample pad is obtained after drying;

[0021] (6) Preparation of colloidal gold test kit:

[0022] The absorbent paper, nitrocellulose membrane, antibody marker binding pad and sample pad are sequentially attached to the PVC board in overlapping order; they are sequentially connected from one end to the other end of the PVC board, and then cut into reagent strips of appropriate width, and the reagent strips are assembled into a test kit (card).

[0023] Furthermore, the amount of PVA added in step (1) is 1% to 15% of the mass of chitosan. In the present invention, PVA is used as a porogen for chitosan microspheres, and the PVA component is removed by subsequent alkaline dissolution treatment, so that a porous structure can be formed on the surface of the chitosan microspheres, and the positive charge and porous structure of chitosan are used to adsorb negatively charged and small-sized gold nanoparticles, thereby completing the stable loading of gold nanoparticles.

[0024] Furthermore, the alkaline solution in step (1) refers to a KOH aqueous solution with a concentration of 0.1 to 1 mol / L.

[0025] Furthermore, in step (2), the porous chitosan microspheres, chloroauric acid and trisodium citrate are added in a mass ratio of 10:0.5-2:1-5.

[0026] Furthermore, the pH value of the K2CO3 solution in step (3) is 7.5-8.5; and the amount of the adiponectin antibody added is 1%-5% of the mass of the chitosan-loaded colloidal gold particles.

[0027] Furthermore, the marker buffer in step (3) refers to a PBS buffer containing 8-12wt% PEG, 3-8wt% sucrose, 0.2-1wt% casein, 0.1-0.5wt% preservative, and 1-3wt% surfactant.

[0028] Furthermore, the amount of trehalose added in step (3) is 20% to 100% of the amount of the antibody marker.

[0029] Furthermore, the coating solution in step (4) refers to a PBS buffer solution containing 0.5-4 wt % sucrose and 0.1-0.5 wt % preservative.

[0030] Furthermore, the sample pad treatment solution in step (5) refers to a PBS buffer solution containing 0.1-1.0 mg / mL of mouse IgG, 1-6 wt% of K2CO3, 0.05-0.5 wt% of surfactant, 1-5 wt% of BSA, 0.1-0.5 wt% of preservative and 0.1-0.5 mg / mL of red blood cells (RBC).

[0031] A colloidal gold kit for qualitatively detecting adiponectin is prepared by the above method.

[0032] The schematic diagram of the colloidal gold kit for qualitatively detecting adiponectin obtained in the present invention is as follows: Figure 1 It comprises a PVC plate and a sample pad, an antibody marker binding pad, a nitrocellulose membrane and absorbent paper which are overlapped with each other in sequence on the PVC plate; the nitrocellulose membrane is marked with a T line (detection line) and a C line (quality control line).

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The kit of the present invention uses colloidal gold loaded by porous chitosan microspheres as an antibody marker carrier, which has a stronger and more stable binding force with the antibody and a better labeling effect.

[0035] (2) The colloidal gold loaded on the porous chitosan microspheres of the present invention overcomes the defect that the gold nanoparticles are too small and easily aggregate spontaneously, and improves the defect that the gold nanoparticles have poor hydrophilicity and are easily adsorbed and deposited on the membrane material during detection, thereby significantly enhancing its storage stability and detection sensitivity.

[0036] (3) The colloidal gold loaded on the porous chitosan microspheres of the present invention has the characteristic of color change due to aggregation of gold nanoparticles, and its color change is obvious and has high visual accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure is a schematic diagram of the structure of the colloidal gold kit for qualitatively detecting adiponectin obtained in the present invention.

[0038] Figure 2 This is a schematic diagram of the result determination of the adiponectin antigen detection by the kit of the present invention.

[0039] Figure 3 This is a diagram showing the results of the kit of the present invention detecting adiponectin antigen.

[0040] Figure 4 This is a graph showing the results of testing ordinary people using the kit of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] Example 1

[0043] A colloidal gold kit for qualitative detection of adiponectin in this embodiment is prepared by the following method:

[0044] (1) Preparation of porous chitosan microspheres:

[0045] Chitosan and PVA are dissolved in an acetic acid solution with a concentration of 1.5wt% to obtain a mixed solution, the amount of PVA added is 10% of the mass of chitosan, and spray drying is performed to obtain chitosan microspheres; the obtained chitosan microspheres are added to a KOH aqueous solution with a concentration of 0.5mol / L for alkaline dissolution treatment to remove the surface PVA component, and after filtering and washing, they are cross-linked with glutaraldehyde vapor and dried in a closed drying oven to obtain porous chitosan microspheres.

[0046] (2) Preparation of chitosan-loaded gold nanoparticles:

[0047] The porous chitosan microspheres were added to a 1wt% chloroauric acid solution and stirred to mix evenly. After heating and boiling for about 1 minute, trisodium citrate solution was added and stirred to react. The mass ratio of the porous chitosan microspheres, chloroauric acid and trisodium citrate was 10:1:2. The color of the solution changed from bright yellow to purple-black, and then to wine red for a short time. After boiling for 3 minutes, it was cooled. The reaction product was filtered, washed and dried to obtain chitosan-loaded gold nanoparticles.

[0048] (3) Preparation of antibody marker binding pad:

[0049] Chitosan-loaded gold nanoparticles were added to K2CO3 solution and dispersed evenly, and then adiponectin antibody was added for labeling reaction (the appropriate pH was adjusted by K2CO3 solution, and a certain volume of adiponectin antibody was added in proportion. The color of different pH values ​​changed from gray to blue to purple to purple-red. When the color no longer changed when the pH was gradually increased to about 8, the pH was the best labeling pH. After the antibody labeling was completed, the labeling result was checked with 10% NaCl solution. When NaCl solution was added at a volume ratio of 1:10, the minimum amount of antibody when no black precipitate appeared was about 3% of the mass of chitosan-loaded gold nanoparticles, which was the best labeling amount). After 30 minutes of labeling reaction, the mixture was diluted with 1:50 (v / v BSA / v 标记物溶液 ) was added with 10wt% BSA (bovine serum albumin) at a ratio of 10000rpm for blocking, and the blocking time was 20min. Centrifuge at 10000rpm for 20min, discard the supernatant, collect the precipitate to obtain the antibody marker; then the antibody marker and trehalose were resuspended and diluted (20wt% antibody marker conjugate, 10wt% trehalose) with a marker buffer (take 80mL 50mM PBS pH8.0 buffer, add PEG2000010g, sucrose 5g, casein 0.5g, ProClin3000.5mL, Tween 20 2g, and finally use the corresponding buffer to make the volume to 100mL, and filter with a 0.22um microporous filter membrane to prepare the marker buffer; wherein PEG2000010%, sucrose 5%, casein 0.5%, ProClin3000.5%, Tween 20 2%), and then sprayed on the glass fiber membrane at a speed of 3.0μL / cm, and dried overnight to obtain the antibody marker conjugate pad.

[0050] (4) Preparation of test lines and quality control lines:

[0051] Take 80mL 50mM PBS pH7.4 buffer, add 2g sucrose and 0.5mL ProClin300, finally make up to 100mL with corresponding buffer, and filter with 0.22um microporous filter membrane to prepare coating solution; sucrose 2%, ProClin3000 0.5%.

[0052] Stick the nitrocellulose membrane on the PVC plate, and draw lines on the membrane with the working solution of the detection area (dilute adiponectin 2 to 1.5 mg / ml with the coating solution) and the working solution of the quality control area (dilute the goat anti-mouse IgG solution to 1.5 mg / ml with the coating solution). The speed of the membrane drawing is 1.2 μL / cm. Place it at 50°C and dry it for 48 hours to obtain the detection line and quality control line, respectively.

[0053] (5) Preparation of sample pad:

[0054] The sample pad treatment solution (50mM PBS solution pH 8.0, mouse IgG 0.5mg / mL, K2CO3 5%, TW20 0.2%, BSA 2%, ProClin3000.5% and RBC 0.5mg / mL) was coated on the glass fiber membrane at a ratio of 5cm×5cm / mL and dried overnight to obtain the sample pad.

[0055] (6) Preparation of colloidal gold test kit:

[0056] The absorbent paper, nitrocellulose membrane, antibody marker binding pad and sample pad are sequentially attached to the PVC plate in overlapping order; they are sequentially connected from one end to the other end of the PVC plate, and then cut into reagent strips of appropriate width (3.5 mm), and the reagent strips are assembled into a test kit (card).

[0057] 1. Using the kit obtained in this example to detect adiponectin antigen:

[0058] 1. Dilute the antigen into samples with different concentration gradients: 0 mg / L, 1.0 mg / L, 2.0 mg / L, 4.0 mg / L, 8.0 mg / L, 16.0 mg / L;

[0059] 2. Pipette 5 μL of sample into buffer (1 mL) and mix thoroughly for 30 seconds to 1 minute;

[0060] 3. Use a pipette to draw 75 μL from the above solution and add it to the sample well of the test card;

[0061] 4. Observe the displayed results within 5 to 15 minutes. The results will be invalid after 15 minutes.

[0062] 5. The results are as follows Figure 2 shown.

[0063] 6. Test results such as Figure 3 shown.

[0064] Depend on Figure 3 The test results show that samples with concentrations of 0mg / L, 1.0mg / L, and 2.0mg / L are all negative, while samples with concentrations of 4.0mg / L, 8.0mg / L, and 16.0mg / L are all positive.

[0065] 2. Use the kit obtained in this example to test ordinary people and compare with biochemical reagents:

[0066] 1. Select 21 ordinary people for testing;

[0067] 2. Open the aluminum foil bag along the tear edge, take out the test card and lay it flat;

[0068] 3. Use a whole blood sampler to pierce the sealing film;

[0069] 4. Wash the hands for blood collection and disinfect the blood collection site;

[0070] 5. Aim the blood collection needle at the finger and gently press it to pierce the finger, and use the sampler to take a certain amount of blood;

[0071] 6. Insert the collected blood sample into the buffer bottle and mix thoroughly for 30 seconds to 1 minute;

[0072] 7. Turn the sampler upside down and drip the first 2 drops of solution into the sampler cover, making sure there are no bubbles at the sample tip, then drip 2 drops vertically into the sample well of the test card.

[0073] 8. Observe the displayed results within 5 to 15 minutes. Results displayed after 15 minutes have no clinical significance.

[0074] The test results of 21 people were compared with the concentration of biochemical reagents. Figure 4 And as shown in Table 1.

[0075] Table 1

[0076]

[0077]

[0078] The yin-yang coincidence rate results are shown in Table 2 below.

[0079] Table 2

[0080]

[0081] From the above results, it can be seen that the positive coincidence rate, negative coincidence rate and total coincidence rate of the test kit of the present invention for ordinary people are all 100%, and the detection accuracy is high.

[0082] Comparative Example 1

[0083] In this comparative example, conventional colloidal gold was used to replace the chitosan-loaded colloidal gold particles in Example 1, and the rest was the same.

[0084] 1. The kits obtained in Example 1 and Comparative Example 1 were tested for the minimum detection limit:

[0085] The enterprise reference products with concentrations of 2.80 mg / L, 3.20 mg / L and 3.50 mg / L were tested respectively and repeated 5 times. The test results were considered valid if at least 4 of them were consistent. The test results are shown in Table 3 below.

[0086] Table 3

[0087] Test sample 2.80mg / L 3.20mg / L 3.50mg / L Example 1 Negative Positive Positive Comparative Example 1 Negative Negative Positive

[0088] It can be seen from the results in Table 3 that the detection sensitivity of the kit of the present invention is higher than that of the conventional colloidal gold kit.

[0089] 2. Batch Difference Test:

[0090] 20 kits were used to test weak positive (3.50 mg / L) enterprise reference products, and the results showed that the positive compliance rate of the test results of the kit in Example 1 was 100%, and the positive compliance rate of the test results of the kit in Comparative Example 1 was 90%, indicating that the detection accuracy and reliability of the kit of the present invention are high.

[0091] 3. Detection efficiency test:

[0092] 10 test kits were used to test the positive (10.0 mg / L) and weak positive (3.50 mg / L) enterprise reference products, and the time when the color change of the test line was visually observed was recorded, and the average value was taken. The results showed that the effective reaction time of the test kit of Example 1 to the positive enterprise reference product was 5.2 minutes, and the effective reaction time to the weak positive enterprise reference product was 6.5 minutes; the effective reaction time of the test kit of Comparative Example 1 to the positive enterprise reference product was 7.0 minutes, and the effective reaction time to the weak positive enterprise reference product was 12.4 minutes. This shows that the detection efficiency of the test kit of the present invention is higher.

[0093] 4. Stability test:

[0094] After the test kit was stored in a 37°C incubator for 6 months, the positive (10.0 mg / L) and weakly positive (3.50 mg / L) enterprise reference products were tested respectively. Each sample was measured 20 times, and the test results were statistically analyzed for effectiveness and positive rate. The results showed that the test kit of Example 1 had a detection effectiveness of 100% and a positive rate of 100% for positive enterprise reference products; a detection effectiveness of 95% and a positive rate (based on effective results) of 100% for weakly positive enterprise reference products. The test kit of Comparative Example 1 had a detection effectiveness of 80% and a positive rate of 100% for positive enterprise reference products; a detection effectiveness of 75% and a positive rate of 66.7% for weakly positive enterprise reference products. This shows that the storage and detection stability of the test kit of the present invention is better.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a colloidal gold kit for qualitative detection of adiponectin, characterized in that: The method comprises the following preparation steps: (1) Preparation of porous chitosan microspheres: The chitosan and PVA are dissolved in an acetic acid solution to obtain a mixed solution, and the mixed solution is spray-dried to obtain chitosan microspheres; the obtained chitosan microspheres are added to an alkaline solution for alkaline dissolution treatment to remove the PVA component, and after filtering and washing, the mixed solution is cross-linked with glutaraldehyde vapor in a closed drying oven to obtain porous chitosan microspheres; (2) Preparation of chitosan-loaded colloidal gold: The porous chitosan microspheres are added to the chloroauric acid solution and stirred to mix evenly, and then heated to 70-100° C., and then a trisodium citrate solution is added and stirred to react, and the reaction product is filtered, washed, and dried to obtain chitosan-loaded colloidal gold particles; (3) Preparation of antibody marker binding pad: Chitosan-loaded colloidal gold particles are added to a K2CO3 solution and dispersed evenly, and then adiponectin antibody is added for labeling reaction. After the reaction is completed, BSA is added for blocking, and the precipitate is collected by centrifugation to obtain an antibody marker; then the antibody marker and trehalose are resuspended and diluted with a marker buffer and sprayed on a glass fiber membrane, and then dried to obtain an antibody marker binding pad; (4) Preparation of test lines and quality control lines: A nitrocellulose membrane is attached to a PVC plate, and a detection area working solution and a quality control area working solution are drawn on the membrane, and after drying, a detection line and a quality control line are obtained respectively; the detection area working solution is a diluent obtained by diluting adiponectin monoclonal antibody with a coating solution, and the quality control area working solution is a diluent obtained by diluting goat anti-mouse IgG with a coating solution; (5) Preparation of sample pad: The sample pad treatment liquid is coated on the glass fiber membrane, and the sample pad is obtained after drying; (6) Preparation of colloidal gold test kit: Adhere absorbent paper, nitrocellulose membrane, antibody marker binding pad, and sample pad to the PVC board in overlapping order; connect them from one end to the other end of the PVC board in order, then cut them into reagent strips of appropriate width, and assemble the reagent strips into a test kit; The amount of PVA added in step (1) is 1% to 15% of the mass of chitosan; The marker buffer in step (3) refers to a PBS buffer having a pH value of 8.0 and containing 8-12 wt% PEG, 3-8 wt% sucrose, 0.2-1 wt% casein, 0.1-0.5 wt% preservative, and 1-3 wt% surfactant.

2. The method for preparing a colloidal gold kit for qualitative detection of adiponectin according to claim 1, characterized in that: The alkaline solution in step (1) refers to a KOH aqueous solution with a concentration of 0.1-1 mol / L.

3. The method for preparing a colloidal gold kit for qualitative detection of adiponectin according to claim 1, characterized in that: The mass ratio of the porous chitosan microspheres, chloroauric acid and trisodium citrate added in step (2) is 10:0.5~2:1~5.

4. The method for preparing a colloidal gold kit for qualitative detection of adiponectin according to claim 1, characterized in that: The pH value of the K2CO3 solution in step (3) is 7.5-8.5; the amount of the adiponectin antibody added is 1%-5% of the mass of the chitosan-loaded colloidal gold particles.

5. The method for preparing a colloidal gold kit for qualitative detection of adiponectin according to claim 1, characterized in that: The amount of trehalose added in step (3) is 20% to 100% of the amount of the antibody marker.

6. The method for preparing a colloidal gold kit for qualitative detection of adiponectin according to claim 1, characterized in that: The coating solution in step (4) refers to a PBS buffer solution containing 0.5-4 wt% sucrose and 0.1-0.5 wt% preservative.

7. The method for preparing a colloidal gold kit for qualitative detection of adiponectin according to claim 1, characterized in that: The sample pad treatment solution in step (5) refers to a PBS buffer solution containing 0.1-1.0 mg / mL mouse IgG, 1-6 wt% K2CO3, 0.05-0.5 wt% surfactant, 1-5 wt% BSA, 0.1-0.5 wt% preservative, and 0.1-0.5 mg / mL RBC.

8. A colloidal gold kit for qualitative detection of adiponectin, characterized in that: It is prepared by the method according to any one of claims 1 to 7.

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