A colloidal gold labeling method for improving sensitivity, application and product thereof

Abstract

This invention provides a colloidal gold labeling method with improved sensitivity, its applications, and products, relating to the field of immunochromatography technology. The colloidal gold labeling method provided by this invention involves mixing a protein to be labeled, treated with a protein activator, with a colloidal gold solution for labeling reaction to obtain a gold-labeled protein. The protein activator includes a nonionic surfactant, a glycoprotectant, and a reducing agent. Through the synergistic effect of these components, the interactions between the gold-labeled conjugates are effectively reduced, preventing aggregation and improving the stability of the gold-labeled conjugates while protecting protein activity. The pH of the colloidal gold solution is 0.3-0.8 lower than the isoelectric point of the protein to be labeled. Under these conditions, the gold-labeled conjugates formed have good stability and high labeling efficiency. This method is simple to operate, the prepared gold-labeled conjugates have high labeling efficiency and good stability, and the prepared reagent kit has high sensitivity and is less prone to missed detections.

Description

Technical Field

[0001] This invention relates to the field of immunochromatography technology, and in particular to a colloidal gold labeling method with improved sensitivity, its applications, and products. Background Technology

[0002] Colloidal gold binds to proteins primarily through three forces: electrostatic interactions, hydrophobic interactions, and gold-sulfur bonds. The dominant binding forces between colloidal gold and proteins differ at different pH levels, thus affecting labeling efficiency and the stability of the conjugate.

[0003] When the pH value is lower than the isoelectric point of the protein, electrostatic interaction becomes the main force. The protein carries a positive charge, while the colloidal gold carries a negative charge. The protein readily binds to the colloidal gold, and the resulting conjugates tend to aggregate, leading to a phenomenon known as "dead gold." This method results in high labeling efficiency but poor stability of the conjugates.

[0004] When the pH is higher than the isoelectric point of the protein, the protein carries a negative charge and repels the negative charge of the gold particles. At this point, they mainly bind to each other through hydrophobic interactions. As the pH increases, electrostatic repulsion increases, and the hydrophobic interaction decreases due to the increased spatial distance between the protein and the gold particles. Consequently, less protein binds to the colloidal gold, resulting in low labeling efficiency.

[0005] When the pH value is equal to or slightly higher than the isoelectric point of the protein, the total number of positive charges and negative charges carried by the protein are the same, resulting in overall electroneutrality. At this pH, the electrostatic interaction between protein molecules and colloidal gold particles is weak, but the hydrophobic interaction is strongest. Under the influence of hydrophobic forces, the protein adsorbs onto the surface of the gold particles. Because the protein molecules are firmly bound to the surface of the gold particles, forming a protein layer, the colloidal gold particles are prevented from contacting each other, thus stabilizing the colloidal gold. At this point, a good balance is achieved between labeling efficiency and the stability of the conjugate. This method is widely used by most technicians.

[0006] Therefore, colloidal gold labeling usually involves adjusting the pH of the colloidal gold solution to be about 0.5 higher than the isoelectric point of the protein to ensure the stability of the labeled state. However, the labeling efficiency is not high, the activity of the labeled conjugate is not fully displayed, and the sensitivity of the prepared kit is relatively low.

[0007] CN 102183633 discloses a colloidal gold labeling method. This method labels proteins in an acidic environment and then slowly rehydrates them to improve the stability of the labeled material. While this method improves labeling efficiency to some extent, the operation is relatively complex, requiring cooling of the labeled material before slow rehydration. Furthermore, the colloidal gold-protein conjugates before rehydration exhibit strong electrostatic attraction in the acidic environment, easily leading to cross-linking of dead gold and resulting in gold aggregation. Even after rehydration, the aggregated conjugates cannot be redispersed and rehydrated, resulting in low overall efficiency.

[0008] In view of this, the present invention is hereby proposed. Summary of the Invention

[0009] The first objective of this invention is to provide a colloidal gold labeling method to solve at least one of the above-mentioned problems.

[0010] The second objective of this invention is to provide the application of the above-described colloidal gold labeling method in colloidal gold immunochromatographic test strips.

[0011] A third objective of this invention is to provide a gold-labeled composite.

[0012] The fourth objective of this invention is to provide a test strip.

[0013] The fifth objective of this invention is to provide a reagent kit.

[0014] In a first aspect, the present invention provides a colloidal gold labeling method, wherein a pretreated protein to be labeled is mixed with a colloidal gold solution to carry out a labeling reaction, thereby obtaining a gold-labeled protein;

[0015] The pretreatment includes treating the protein to be labeled with a protein activator;

[0016] The protein activator includes nonionic surfactants, glycoprotectants, and reducing agents;

[0017] The nonionic surfactant includes at least one of Triton X-100, ON-870, Tween-20, Tween-80, Brij-35, and Chemal LA9;

[0018] The sugar protectant includes at least one of dextran, sucrose, trehalose, mannose, and lactose;

[0019] The reducing agent includes at least one of ascorbic acid, citric acid and tartaric acid;

[0020] The pH of the colloidal gold solution is 0.3-0.8 lower than the isoelectric point of the protein to be labeled.

[0021] As a further technical solution, the final mass concentration of the nonionic surfactant is 0.005%-0.1%;

[0022] Preferably, the final mass concentration of the sugar preservative is 0.01%-1%;

[0023] Preferably, the final mass concentration of the reducing agent is 0.01%-1%.

[0024] As a further technical solution, the protein activator comprises the following components: ON-870 with a final mass concentration of 0.005%-0.1%, trehalose with a final mass concentration of 0.01%-1%, and tartaric acid with a final mass concentration of 0.01%-1%.

[0025] Preferably, the final mass concentration of the ON-870 is 0.05%;

[0026] Preferably, the final mass concentration of the trehalose is 0.2%;

[0027] Preferably, the final mass concentration of the tartaric acid is 0.1%.

[0028] As a further technical solution, the proteins to be labeled include novel coronavirus N antibody, adenovirus antigen, respiratory syncytial virus antigen, influenza A antibody, influenza B antibody, and anti-human IgM antibody.

[0029] As a further technical solution, the labeling reaction time is 25-35 minutes, preferably 30 minutes.

[0030] As a further technical solution, the labeling reaction also includes a step of adding a blocking agent;

[0031] Preferably, the sealing time is 10-20 minutes;

[0032] Preferably, the blocking agent comprises bovine serum albumin.

[0033] Secondly, the present invention provides an application of a colloidal gold labeling method in the preparation of colloidal gold immunochromatographic products;

[0034] The products include testing reagents, test strips, and kits.

[0035] Thirdly, the present invention provides a gold labeling conjugate prepared by the above-described colloidal gold labeling method.

[0036] Fourthly, the present invention provides a test strip comprising the aforementioned gold-labeled conjugate.

[0037] Fifthly, the present invention provides a reagent kit comprising the above-described test strip.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The colloidal gold labeling method provided by this invention involves treating the protein to be labeled with a protein activator, mixing it with a colloidal gold solution, and performing a labeling reaction to obtain gold-labeled protein. The protein activator includes a nonionic surfactant, a glycoprotective agent, and a reducing agent. The nonionic surfactant lacks ionic groups and does not dissociate in solution. It primarily interacts with the protein through hydrophobic forces, while its hydrophilic end contacts other hydrophobic groups, competitively inhibiting the protein from contacting other hydrophobic groups. Simultaneously, the disulfide bonds of the protein are opened under the action of the reducing agent. Through the synergistic effect of the nonionic surfactant and the reducing agent, the protein structure changes, the total positive charge decreases, and the electrostatic attraction between the protein and colloidal gold weakens, thereby reducing the interaction and aggregation between the gold-labeled conjugates and improving their stability. The glycoprotective agent contains a large number of hydroxyl groups, which can form hydrogen bonds with the protein and simultaneously increase the viscosity of the solution, achieving the effect of protecting protein activity. The pH of the colloidal gold solution is 0.3-0.8 lower than the isoelectric point of the protein to be labeled. Labeling the protein under conditions below its isoelectric point results in gold-labeled conjugates with good stability and high labeling efficiency. This method is simple to operate, and the prepared gold-labeled conjugate has high labeling efficiency and good stability. The prepared kit has high sensitivity and is not prone to missed detection. Compared with the labeling method provided by CN 102183633, it is simple to operate, does not require re-alkali, has high labeling efficiency, good repeatability, and is easy to scale up. Detailed Implementation

[0040] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0041] In a first aspect, the present invention provides a colloidal gold labeling method, wherein a pretreated protein to be labeled is mixed with a colloidal gold solution to carry out a labeling reaction, thereby obtaining a gold-labeled protein;

[0042] The pretreatment includes treating the protein to be labeled with a protein activator;

[0043] The protein activator includes nonionic surfactants, glycoprotectants, and reducing agents.

[0044] Nonionic surfactants do not have ionic groups, do not dissociate in solution, and mainly interact with proteins through hydrophobic forces. Their hydrophilic ends contact other hydrophobic groups, competitively inhibiting the contact between proteins and other hydrophobic groups. In this invention, nonionic surfactants include at least one of Triton X-100, ON-870, Tween-20, Tween-80, Brij-35, and Chemal LA9.

[0045] The glycoprotectant contains a large number of hydroxyl groups, which can form hydrogen bonds with proteins and increase the viscosity of the solution, thereby protecting the protein activity. In this invention, the glycoprotectant includes at least one of dextran, sucrose, trehalose, mannose and lactose.

[0046] The reducing agent can break the disulfide bonds of the protein. Under the synergistic effect of the nonionic surfactant and the reducing agent, the protein structure is changed, the total number of positive charges is reduced, and the electrostatic attraction between the protein and colloidal gold is weakened, thereby reducing the interaction and aggregation between the gold-labeled conjugates and improving the stability of the gold-labeled conjugates. In this invention, the reducing agent includes at least one of ascorbic acid, citric acid and tartaric acid.

[0047] The pH of the colloidal gold solution is lower than the isoelectric point of the protein to be labeled, for example, but not limited to, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8. Labeling the protein under conditions below its isoelectric point results in a gold-labeled conjugate with good stability and high labeling efficiency.

[0048] The colloidal gold labeling method provided by this invention is simple to operate, and the prepared gold-labeled conjugates have high labeling efficiency and good stability.

[0049] In some preferred embodiments, the final mass concentration of the nonionic surfactant may be, for example, but not limited to, 0.005%, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, or 0.1%.

[0050] Preferably, the final mass concentration of the sugar preservative is 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, or 1%.

[0051] Preferably, the final mass concentration of the reducing agent is 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, or 1%.

[0052] By further optimizing and adjusting the concentration of each component in the protein activator, the prepared gold-labeled conjugate has higher labeling efficiency and better stability.

[0053] In some preferred embodiments, the protein activator comprises the following components: ON-870 at a final mass concentration of 0.005%-0.1%, trehalose at a final mass concentration of 0.01%-1%, and tartaric acid at a final mass concentration of 0.01%-1%; wherein, the final mass concentration of ON-870 is preferably 0.05%; the final mass concentration of trehalose is preferably 0.2%; and the final mass concentration of tartaric acid is preferably 0.1%. Under these conditions, the prepared gold-labeled conjugate exhibits the highest labeling efficiency and the best stability.

[0054] In some preferred embodiments, the protein to be labeled includes, but is not limited to, novel coronavirus N antibody, adenovirus antigen, respiratory syncytial virus antigen, influenza A antibody, influenza B antibody, and anti-human IgM antibody.

[0055] It should be noted that the proteins to be labeled are, but are not limited to, antigens or antibodies of novel coronavirus, Legionella pneumophila, Cryptococcus capsular polysaccharide, influenza A virus, influenza B virus, Mycoplasma pneumoniae, respiratory syncytial virus, adenovirus, parainfluenza virus, Coxsackie virus, enterovirus, mumps virus, human herpesvirus, human papillomavirus, human immunodeficiency virus, measles virus, rubella virus, Chlamydia pneumoniae, Streptococcus pneumoniae, Helicobacter pylori, etc.

[0056] In some preferred embodiments, the labeling reaction time can be, for example, but not limited to, 25 min, 27 min, 29 min, 31 min, 33 min or 35 min, preferably 30 min.

[0057] In some preferred embodiments, the step of adding a blocking agent is further included after the labeling reaction;

[0058] Preferably, the sealing time can be, for example, but not limited to, 10 min, 12 min, 14 min, 16 min, 18 min or 20 min;

[0059] Preferably, the blocking agent includes, but is not limited to, bovine serum albumin, or other blocking agents well known to those skilled in the art.

[0060] Secondly, the present invention provides an application of a colloidal gold labeling method in the preparation of colloidal gold immunochromatographic products;

[0061] The products include testing reagents, test strips, and kits.

[0062] The gold-labeled conjugate prepared by the colloidal gold labeling method provided by this invention has high labeling efficiency and good stability, and can be used to prepare colloidal gold immunochromatographic products.

[0063] Thirdly, the present invention provides a gold labeling conjugate prepared by the above-mentioned colloidal gold labeling method, which has high labeling efficiency and good stability.

[0064] Fourthly, the present invention provides a test strip comprising the aforementioned gold-labeled conjugate, which has high detection sensitivity.

[0065] Fifthly, the present invention provides a reagent kit comprising the above-described test strip.

[0066] This kit has higher sensitivity and is less prone to missed detection than kits prepared by conventional labeling methods. It is also simpler to operate than the labeling method provided by CN102183633, requires no re-alkali treatment, has high labeling efficiency, good repeatability, and is easy to scale up.

[0067] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0068] Example 1: Determination of Optimal Labeling pH

[0069] Taking the novel coronavirus N antibody-1 as an example, the isoelectric point of the novel coronavirus N antibody-1 is 7.9. The optimal labeling pH of the novel coronavirus N antibody-1 treated with different protein activators was compared.

[0070] Protein activator 1: Purified water.

[0071] Protein activator 2: ON-870 with a final mass concentration of 0.005%, trehalose with a final mass concentration of 0.2%, tartaric acid with a final mass concentration of 0.1%, and purified water.

[0072] Protein activator 3: ON-870 with a final mass concentration of 0.05%, trehalose with a final mass concentration of 0.2%, tartaric acid with a final mass concentration of 0.1%, and purified water.

[0073] Protein activator 4: ON-870 with a final mass concentration of 0.01%, trehalose with a final mass concentration of 0.2%, tartaric acid with a final mass concentration of 0.1%, and purified water.

[0074] Protein activator 5: ON-870 with a final mass concentration of 0.02%, Brij-35 with a final mass concentration of 0.03%, trehalose with a final mass concentration of 0.2%, tartaric acid with a final mass concentration of 0.1%, and purified water.

[0075] Protein activator 6: Triton X-100 at a final mass concentration of 0.1%, dextran at a final mass concentration of 1%, ascorbic acid at a final mass concentration of 0.01%, and purified water.

[0076] Protein activator 7: Tween-20 with a final mass concentration of 0.05%, sucrose with a final mass concentration of 0.01%, citric acid with a final mass concentration of 1%, and purified water.

[0077] Protein activator 8: Chemal LA9 (final concentration 0.05%), mannose (final concentration 0.1%), lactose (final concentration 0.1%), tartaric acid (final concentration 0.05%), citric acid (final concentration 0.05%), and purified water.

[0078] Take 1 ml of colloidal gold into a centrifuge tube, and add 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution to 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, and 8.4.

[0079] Take 5 centrifuge tubes, add 8 μg of novel coronavirus N antibody-1 to each tube, dilute the novel coronavirus N antibody-1 to 1.0 mg / mL with the above 5 protein activators, mix well and let stand for 10 minutes.

[0080] Diluted novel coronavirus N antibody-1 was added to colloidal gold and mixed thoroughly. After standing for 5 minutes, 200 μl of 10% NaCl was added and mixed thoroughly. After standing for 5 minutes, the color change was observed. The results are shown in Table 1.

[0081] Table 1: Results of novel coronavirus N antibody-1 labeling after treatment with different protein activators

[0082]

[0083]

[0084] Conclusion: The optimal labeling pH for SARS-CoV-2 N antibody-1 diluted directly with purified water is 8.2, which is slightly higher than the isoelectric point of SARS-CoV-2 N antibody-1. The optimal labeling pH for SARS-CoV-2 N antibody-1 treated with the protein activator described in this invention is 7.2-7.6, which is lower than the isoelectric point of SARS-CoV-2 N antibody-1. This indicates that the protein activator described in this invention can label the protein to be labeled under conditions lower than the isoelectric point of the protein to be labeled, and the gold-labeled conjugate is relatively stable.

[0085] Example 2: Preparation of Novel Coronavirus Antigen Detection Kit

[0086] 1. Preparation of gold-labeled conjugates

[0087] Take 10 mL of colloidal gold into a beaker, add 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution to be 0.3-0.8 below the isoelectric point of novel coronavirus N antibody-1, and stir at 250 r / min for 5 minutes;

[0088] Take 80 μg of novel coronavirus N antibody-1, add protein activator, dilute novel coronavirus N antibody-1 to 1.0 mg / mL, mix well and let stand for 10 minutes;

[0089] Add the prepared novel coronavirus N antibody-1 to the colloidal gold and stir for 30 minutes;

[0090] Add 200 μL of 10% bovine serum albumin and continue stirring for 15 minutes;

[0091] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0092] The protein activator comprises the following components: ON-870 at a final mass concentration of 0.05%, trehalose at a final mass concentration of 0.2%, and tartaric acid at a final mass concentration of 0.1%.

[0093] 2. Preparation of the binding pad

[0094] The pretreatment solution was evenly spread on the glass fiber and dried overnight. The pretreatment solution consisted of sucrose, BSA and ON-870, with a final mass concentration of 0.5%-5%, a final mass concentration of 0.1%-2% for BSA and a final mass concentration of 0.1%-2% for ON-870.

[0095] The gold-labeled conjugate was diluted to one-tenth the volume of colloidal gold using a gold diluent, sprayed onto the treated glass fiber, and then freeze-dried under vacuum. The gold diluent consisted of Tris, sucrose, trehalose, BSA, casein, polyethylene glycol, and ON-870, with the following concentrations: Tris molar concentration of 10 mM-50 mM, sucrose final mass concentration of 1%-5%, trehalose final mass concentration of 1%-5%, BSA final mass concentration of 0.1%-5%, casein final mass concentration of 0.1%-5%, polyethylene glycol final mass concentration of 0.1%-5%, and ON-870 final mass concentration of 0.1%-2%.

[0096] 3. Coating of nitrocellulose membranes

[0097] The novel coronavirus N antibody-2 and goat anti-mouse IgG polyclonal antibody were diluted with coating buffer and sprayed onto nitrocellulose membranes. The coating concentration of novel coronavirus N antibody-2 was 1.0 mg / ml, and the coating concentration of goat anti-mouse IgG polyclonal antibody was 2.0 mg / ml. The coating buffer consisted of PBS and BSA, with the PBS molar concentration being 10 mM and the final mass concentration of BSA being 0.5%.

[0098] 4. Preparation of the sample pad

[0099] Glass fibers were soaked in a sample pad treatment solution and dried overnight. The sample pad treatment solution consisted of Tris, NaCl, BSA, casein, polyethylene glycol, and Tween 20, wherein the molar concentration of Tris was 10-50 mM, the final mass concentration of NaCl was 0.1-1.0%, the final mass concentration of BSA was 0.1-1.0%, the final mass concentration of casein was 0.1-1.0%, the final mass concentration of polyethylene glycol was 0.1-0.5%, and the final mass concentration of Tween 20 was 0.1-0.5%.

[0100] 5. Preparation of sample release solution

[0101] The sample release solution consisted of PBS and Tetronic 1307, with a PBS molar concentration of 10-50 mM and a Tetronic 1307 final mass concentration of 0.1-1.0%.

[0102] 6. Assembly, cutting, and packaging of test strips

[0103] Assemble the test strip by attaching the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad to the support base in that order; cut the assembled test strip into strips 4mm wide, pack them into a plastic CT box, press them tightly, add desiccant, seal them in an aluminum foil bag, and dry them for later use.

[0104] Example 3: Preparation of Adenovirus IgM Antibody Detection Kit

[0105] 1. Preparation of gold-labeled conjugates

[0106] Take 10 mL of colloidal gold into a beaker, add 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution to 0.3-0.8 below the isoelectric point of the adenovirus antigen, and stir at 250 r / min for 5 minutes;

[0107] Take 80 μg of adenovirus antigen, add protein activator, dilute the adenovirus antigen to 1 mg / mL, mix well and let stand for 10 minutes;

[0108] Add the prepared adenovirus antigen to the colloidal gold and stir for 30 minutes.

[0109] Add 200 μL of 10% bovine serum albumin and continue stirring for 15 minutes;

[0110] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0111] The protein activator comprises the following components: ON-870 at a final mass concentration of 0.05%, trehalose at a final mass concentration of 0.2%, and tartaric acid at a final mass concentration of 0.1%.

[0112] 2. Preparation of the binding pad

[0113] See Example 2 for the preparation of the binding pad.

[0114] 3. Coating of nitrocellulose membranes

[0115] Anti-human IgM antibody and goat anti-mouse IgG polyclonal antibody were diluted with coating buffer and sprayed onto nitrocellulose membranes. The coating concentration of anti-human IgM antibody was 1.5 mg / ml, and the coating concentration of goat anti-mouse IgG polyclonal antibody was 2.0 mg / ml. The coating buffer consisted of PBS and BSA, with PBS having a molar concentration of 10 mM and BSA having a final mass concentration of 0.5%.

[0116] 4. Preparation of the sample pad

[0117] See the preparation of the sample pad in Example 2.

[0118] 5. Preparation of sample release solution

[0119] See Example 2 for the preparation of the sample release solution.

[0120] 6. Assembly, cutting, and packaging of test strips

[0121] See Example 2 for the assembly, cutting, and packaging of the test strips.

[0122] Example 4: Preparation of Respiratory Syncytial Virus IgM Antibody Detection Kit

[0123] 1. Preparation of gold-labeled conjugates

[0124] Take 10 mL of colloidal gold into a beaker, add 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution to be 0.3-0.8 below the isoelectric point of respiratory syncytial virus antigen, and stir at 250 r / min for 5 minutes;

[0125] Take 80 μg of respiratory syncytial virus antigen, add protein activator, dilute the respiratory syncytial virus antigen to 1.0 mg / mL, mix well and let stand for 10 minutes.

[0126] Add the prepared respiratory syncytial virus antigen to the colloidal gold and stir for 30 minutes.

[0127] Add 200 μL of 10% bovine serum albumin and continue stirring for 15 minutes;

[0128] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0129] The protein activator comprises the following components: ON-870 at a final mass concentration of 0.05%, trehalose at a final mass concentration of 0.2%, and tartaric acid at a final mass concentration of 0.1%.

[0130] 2. Preparation of the binding pad

[0131] See Example 2 for the preparation of the binding pad.

[0132] 3. Coating of nitrocellulose membranes

[0133] Anti-human IgM antibody and goat anti-mouse IgG polyclonal antibody were diluted with coating buffer and sprayed onto nitrocellulose membranes. The coating concentration of anti-human IgM antibody was 1.0 mg / ml, and the coating concentration of goat anti-mouse IgG polyclonal antibody was 2.0 mg / ml. The coating buffer consisted of PBS and BSA, with PBS having a molar concentration of 10 mM and BSA having a final mass concentration of 0.5%.

[0134] 4. Preparation of the sample pad

[0135] See the preparation of the sample pad in Example 2.

[0136] 5. Preparation of sample release solution

[0137] See Example 2 for the preparation of the sample release solution.

[0138] 6. Assembly, cutting, and packaging of test strips

[0139] See Example 2 for the assembly, cutting, and packaging of the test strips.

[0140] Example 5: Preparation of Influenza A / B Antigen Detection Kit

[0141] 1. Preparation of gold-labeled conjugates

[0142] Take 10 mL of colloidal gold into a beaker, add 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution to be 0.3-0.8 below the isoelectric point of influenza A antibody-1 and 0.3-0.8 below the isoelectric point of influenza B antibody-1, and stir at 250 r / min for 5 minutes;

[0143] Take 50 μg of influenza A antibody-1 and 50 μg of influenza B antibody-1 respectively and add them to the protein activator. Dilute influenza A antibody-1 and influenza B antibody-1 to 1.0 mg / mL, mix well and let stand for 10 minutes.

[0144] Add the treated influenza A antibody-1 and influenza B antibody-1 to the colloidal gold separately, and stir for 30 minutes;

[0145] Add 200 μL of 10% bovine serum albumin and continue stirring for 15 minutes;

[0146] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0147] The protein activator comprises the following components: Triton X-100 at a final mass concentration of 0.1%, dextran at a final mass concentration of 1%, and ascorbic acid at a final mass concentration of 0.01%.

[0148] 2. Preparation of the binding pad

[0149] See Example 2 for the preparation of the binding pad.

[0150] 3. Coating of nitrocellulose membranes

[0151] Influenza A antibody-2, influenza B antibody-2, and goat anti-mouse IgG polyclonal antibody were diluted with coating buffer and sprayed onto nitrocellulose membranes. The coating concentration of influenza A antibody-2 was 0.8 mg / ml, the coating concentration of influenza B antibody-2 was 1.0 mg / ml, and the coating concentration of goat anti-mouse IgG polyclonal antibody was 2.0 mg / ml. The coating buffer consisted of PBS and BSA, with PBS having a molar concentration of 10 mM and BSA having a final mass concentration of 0.5%.

[0152] 4. Preparation of the sample pad

[0153] See the preparation of the sample pad in Example 2.

[0154] 5. Preparation of sample release solution

[0155] See Example 2 for the preparation of the sample release solution.

[0156] 6. Assembly, cutting, and packaging of test strips

[0157] See Example 2 for the assembly, cutting, and packaging of the test strips.

[0158] Example 6: Preparation of Mycoplasma pneumoniae IgM antibody detection kit

[0159] 1. Preparation of gold-labeled conjugates

[0160] Take 10 mL of colloidal gold into a beaker, add 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution to the isoelectric point of the anti-human IgM antibody (0.3-0.8), and stir at 250 r / min for 5 minutes;

[0161] Take 50 μg of anti-human IgM antibody, add protein activator, dilute the anti-human IgM antibody to 1.0 mg / mL, mix well and let stand for 10 minutes;

[0162] Add the prepared anti-human IgM antibody to the colloidal gold and stir for 30 minutes;

[0163] Add 200 μL of 10% bovine serum albumin and continue stirring for 15 minutes;

[0164] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0165] The protein activator comprises the following components: ON-870 at a final mass concentration of 0.005%, trehalose at a final mass concentration of 0.2%, and tartaric acid at a final mass concentration of 0.1%.

[0166] 2. Preparation of the binding pad

[0167] See Example 2 for the preparation of the binding pad.

[0168] 3. Coating of nitrocellulose membranes

[0169] Mycoplasma pneumoniae and goat anti-mouse IgG polyclonal antibody were diluted with coating buffer and sprayed onto nitrocellulose membranes. The coating concentration of Mycoplasma pneumoniae was 1.2 mg / ml, and the coating concentration of goat anti-mouse IgG polyclonal antibody was 2.0 mg / ml. The coating buffer consisted of PBS and BSA, with PBS having a molar concentration of 10 mM and BSA having a final mass concentration of 0.5%.

[0170] 4. Preparation of the sample pad

[0171] See the preparation of the sample pad in Example 2.

[0172] 5. Preparation of sample release solution

[0173] See Example 2 for the preparation of the sample release solution.

[0174] 6. Assembly, cutting, and packaging of test strips

[0175] See Example 2 for the assembly, cutting, and packaging of the test strips.

[0176] Comparative Example 1

[0177] The difference from Example 2 is that the protein was directly diluted with purified water during the preparation of the gold-labeled conjugate, and the pH of the colloidal gold solution was adjusted to 8.2. This pH is the lowest stable pH of the gold-labeled conjugate of novel coronavirus N antibody-1 diluted with purified water, which is higher than the isoelectric point of novel coronavirus N antibody-1.

[0178] Comparative Example 2

[0179] The difference from Example 2 is that the labeling method provided in CN 102183633 is used to label the novel coronavirus N antibody-1. The preparation of the gold-labeled conjugate includes the following steps:

[0180] Take 10 mL of colloidal gold, adjust the pH of the colloidal gold to 7.5 with 1% K2CO3, add 80 μg of novel coronavirus N antibody-1 and mix thoroughly, adjust the pH to 6.5, and react for 30 minutes;

[0181] After cooling, the pH of the gold standard solution was adjusted to 8.0 with 1% K2CO3 solution, and then reacted in a 25°C water bath for 30 minutes.

[0182] Add 2 mL of 10% bovine serum albumin and react for 20 minutes;

[0183] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0184] Comparative Example 3

[0185] The difference from Example 3 is that the protein was directly diluted with purified water during the preparation of the gold-labeled conjugate, and the pH of the colloidal gold solution after adjustment was 10.0. This pH is the lowest stable pH of the gold-labeled conjugate of adenovirus antigen diluted with purified water, which is higher than the isoelectric point pH of adenovirus antigen, 9.4.

[0186] Comparative Example 4

[0187] The difference from Example 3 is that the adenovirus antigen is labeled using the labeling method provided in patent CN 102183633. The preparation of the gold-labeled conjugate includes the following steps:

[0188] Take 10 mL of colloidal gold, adjust the pH of the colloidal gold to 7.5 with 1% K2CO3, add 80 μg of adenovirus antigen and mix thoroughly, adjust the pH to 6.5, and react for 30 minutes.

[0189] After cooling, the pH of the gold standard solution was adjusted to 8.0 with 1% K2CO3 solution, and then reacted in a 25°C water bath for 30 minutes.

[0190] Add 2 mL of 10% bovine serum albumin and react for 20 minutes;

[0191] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0192] Comparative Example 5

[0193] The difference from Example 4 is that the protein was directly diluted with purified water during the preparation of the gold-labeled conjugate, and the pH of the colloidal gold solution after adjustment was 9.5. This pH is the lowest stable pH of the gold-labeled conjugate of respiratory syncytial virus antigen diluted with purified water, which is higher than the isoelectric point of respiratory syncytial virus antigen of 9.0.

[0194] Comparative Example 6

[0195] The difference from Example 4 is that the respiratory syncytial virus antigen is labeled using the labeling method provided in patent CN 102183633. The preparation of the gold-labeled conjugate includes the following steps:

[0196] Take 10 mL of colloidal gold, adjust the pH of the colloidal gold to 7.5 with 1% K2CO3, add 80 μg of respiratory syncytial virus antigen, mix thoroughly, adjust the pH to 6.5, and react for 30 minutes.

[0197] After cooling, the pH of the gold standard solution was adjusted to 8.0 with 1% K2CO3 solution, and then reacted in a 25°C water bath for 30 minutes.

[0198] Add 2 mL of 10% bovine serum albumin and react for 20 minutes;

[0199] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0200] Comparative Example 7

[0201] The difference from Example 5 is that the protein was directly diluted with purified water during the preparation of the gold-labeled conjugate. The pH of the colloidal gold solution used to label influenza A antibody-1 was adjusted to 7.2, which is the lowest stable pH of the gold-labeled conjugate of influenza A antibody-1 diluted with purified water, and is higher than the isoelectric point of influenza A antibody-1 (6.8). The pH of the colloidal gold solution used to label influenza B antibody-1 was adjusted to 7.5, which is higher than the isoelectric point of influenza B antibody-1 (7.3).

[0202] Comparative Example 8

[0203] The difference from Example 5 is that the labeling method provided in CN 102183633 is used to label influenza A antibody-1 and influenza B antibody-1. The preparation of the gold-labeled conjugate includes the following steps:

[0204] Take 10 mL of colloidal gold, adjust the pH of the colloidal gold to 7.5 with 1% K2CO3, add 50 μg of influenza A antibody-1 and influenza B antibody-1 respectively, mix thoroughly, adjust the pH to 6.0, and react for 30 minutes;

[0205] After cooling, the pH of the gold standard solution was adjusted to 7.5 with 1% K2CO3 solution, and then reacted in a 25°C water bath for 30 minutes.

[0206] Add 2 mL of 10% bovine serum albumin and react for 20 minutes;

[0207] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0208] Comparative Example 9

[0209] The difference from Example 6 is that the protein was directly diluted with purified water during the preparation of the gold-labeled conjugate, and the pH of the colloidal gold solution was adjusted to 8.0. This pH is the lowest stable pH of the gold-labeled conjugate of anti-human IgM antibody diluted with purified water, which is higher than the isoelectric point of 7.6 of the anti-human IgM antibody.

[0210] Comparative Example 10

[0211] The difference from Example 6 is that the anti-human IgM antibody is labeled using the labeling method provided in patent CN 102183633. The preparation of the gold-labeled conjugate includes the following steps:

[0212] Take 10 mL of colloidal gold, adjust the pH of the colloidal gold to 7.5 with 1% K2CO3, add 50 μg of anti-human IgM antibody and mix thoroughly, adjust the pH to 6.5, and react for 30 minutes.

[0213] After cooling, the pH of the gold standard solution was adjusted to 8.0 with 1% K2CO3 solution, and then reacted in a 25°C water bath for 30 minutes.

[0214] Add 2 mL of 10% bovine serum albumin and react for 20 minutes;

[0215] Centrifuge at 4℃, 7500 r / min for 35 min, discard the supernatant, measure the absorbance using a UV spectrophotometer, and calculate the OD value and labeling efficiency.

[0216] Test Example 1 Sensitivity Detection

[0217] The inactivated cultures of the novel coronavirus were used to test the sensitivity of Examples 2, 1, and 2 above. The results are shown in Table 2.

[0218] Table 2: Sensitivity test results of the novel coronavirus antigen detection kit

[0219] Dilution <![CDATA[1∶1×10 3 ]]> <![CDATA[1∶2×10 3 ]]> <![CDATA[1∶4×10 3 ]]> <![CDATA[1∶8×10 3 ]]> <![CDATA[1∶16×10 3 ]]> Example 2 + + + + - Comparative Example 1 + + - - - Comparative Example 2 + + + - -

[0220] Note: + indicates positive, - indicates negative.

[0221] Adenovirus IgM antibody-positive serum was collected and the sensitivity of Examples 3, 3, and 4 above was tested. The results are shown in Table 3.

[0222] Table 3: Sensitivity test results of the adenovirus IgM antibody detection kit

[0223] Dilution 1:2 1:4 1:8 1:16 1:32 Example 3 + + + + - Comparative Example 3 + + + - - Comparative Example 4 + + + + -

[0224] Respiratory syncytial virus (RSV) IgM antibody-positive serum was used to test the sensitivity of Examples 4, 5, and 6 above. The results are shown in Table 4.

[0225] Table 4: Sensitivity test results of the respiratory syncytial virus IgM antibody detection kit

[0226] Dilution 1:4 1:8 1:16 1:32 1:64 Example 4 + + + - - Comparative Example 5 + + - - - Comparative Example 6 + + + - -

[0227] Inactivated influenza A and B virus cultures were used to test the sensitivity of Examples 5, 7, and 8 above. The results are shown in Table 5.

[0228] Table 5: Sensitivity Test Results of Influenza A / B Antigen Detection Kits

[0229]

[0230]

[0231] Serum samples positive for Mycoplasma pneumoniae IgM were used to test the sensitivity of the samples from Examples 6, 9, and 10 above. The results are shown in Table 6.

[0232] Table 6: Sensitivity Detection Results of the Mycoplasma pneumoniae IgM Antibody Detection Kit

[0233] Dilution 1:2 1:4 1:8 1:16 1:32 Example 6 + + + + - Comparative Example 9 + + + - - Comparative Example 10 + + + + -

[0234] Conclusion: The novel coronavirus antigen detection kit, adenovirus IgM antibody detection kit, respiratory syncytial virus IgM antibody detection kit, influenza A / B antigen detection kit, and mycoplasma pneumoniae IgM antibody detection kit prepared using the method described in this invention have higher sensitivity than kits prepared by conventional methods, and are comparable in sensitivity to the labeling method provided in patent CN 102183633.

[0235] Experimental Example 2: Marking Efficiency Calculation

[0236] The labeling efficiency was calculated based on the volume and OD value of the gold-labeled conjugate obtained after centrifugation. The labeling efficiencies of Example 2 and Comparative Example 2, Example 3 and Comparative Example 4, and Example 4 and Comparative Example 6 were compared, and the results are shown in Table 7.

[0237] Labeling efficiency = (gold-labeled conjugate volume * gold-labeled conjugate OD value) / (colloidal gold volume * colloidal gold OD value).

[0238] Table 7: Calculation Results of Marking Efficiency

[0239]

[0240] Conclusion: The labeling efficiency of the novel coronavirus N antibody-1, adenovirus antigen, respiratory syncytial virus antigen, influenza A antibody-1, influenza B antibody-1, and anti-human IgM antibody using the method described in this invention is higher than that of the labeling method provided in patent CN 102183633.

[0241] The above embodiments describe preferred embodiments of the present invention. It should be clear that the reagent kits prepared using the method described in the present invention to label antigens or antibodies can achieve the same effect. The detection kits include, but are not limited to, antigen or antibody detection kits for novel coronavirus, Legionella pneumophila, Cryptococcus capsular polysaccharide, influenza A virus, influenza B virus, Mycoplasma pneumoniae, respiratory syncytial virus, adenovirus, parainfluenza virus, Coxsackie virus, enterovirus, mumps virus, human herpesvirus, human papillomavirus, human immunodeficiency virus, measles virus, rubella virus, Chlamydia pneumoniae, Streptococcus pneumoniae, and Helicobacter pylori.

[0242] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A colloidal gold labeling method for improving sensitivity, characterized in that, The pretreated protein to be labeled is mixed with a colloidal gold solution to perform a labeling reaction, and gold-labeled protein is obtained; The pretreatment includes treating the protein to be labeled with a protein activator; The protein activator includes a non-ionic surfactant, a sugar protective agent, and a reducing agent; The non-ionic surfactant includes at least one of Triton X-100, ON-870, Tween-20, Tween-80, Brij-35, and Chemal LA9; The sugar protective agent includes at least one of dextran, sucrose, trehalose, mannose, and lactose; The reducing agent includes at least one of ascorbic acid, citric acid, and tartaric acid; The pH of the colloidal gold solution is 0.3-0.8 lower than the isoelectric point of the protein to be labeled; The final mass concentration of the non-ionic surfactant is 0.005%-0.1%; The final mass concentration of the sugar protective agent is 0.01%-1%; The final mass concentration of the reducing agent is 0.01%-1%.

2. The method of claim 1, wherein, The protein activator includes the following components: ON-870 with a final mass concentration of 0.005%-0.1%, trehalose with a final mass concentration of 0.01%-1%, and tartaric acid with a final mass concentration of 0.01%-1%.

3. The method of claim 2, wherein, The final mass concentration of the ON-870 is 0.05%; The final mass concentration of the trehalose is 0.2%; The final mass concentration of the tartaric acid is 0.1%.

4. The method of claim 1, wherein, The protein to be labeled includes a novel coronavirus N antibody, an adenovirus antigen, a respiratory syncytial virus antigen, an influenza A antibody, an influenza B antibody, and an anti-human IgM antibody.

5. The colloidal gold labeling method according to claim 1, characterized in that, The labeling reaction time is 25-35 min.

6. The colloidal gold labeling method according to claim 1, characterized in that, The labeling reaction time is 30 min.

7. The colloidal gold labeling method according to claim 1, characterized in that, After the labeling reaction, a step of adding a blocking agent is further included; The blocking time is 10-20 min; The blocking agent includes bovine serum albumin.

8. Use of the colloidal gold labeling method according to any one of claims 1-7 in the preparation of a colloidal gold immunochromatographic product; The product includes a detection reagent, a test strip, and a kit.

9. A gold conjugate, characterized in that, Prepared by using the colloidal gold labeling method according to any one of claims 1-7.

10. A test strip, characterized in that The gold-labeled conjugate according to claim 9 is included.

11. A kit characterized in that, The test strip according to claim 10 is included.

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