Claims
- 1. A diagnostic kit for simultaneously detecting multiple infectious diseases consisting of an immunogold filtration assay device, buffer and the mixed solution of the colloidal gold conjugates,
wherein said immunogold filtration assay device comprises a nitrocellulose membrane with HBsAg monoclonal antibody, HCV antigen, syphilitic antigen, HIV antigen and goat anti-mouse IgG antibody for quality control dotted thereon separately; wherein the mixed solution of the colloidal gold conjugates comprises four kinds of colloidal gold conjugates, including the colloidal gold conjugates of HBsAg monoclonal antibody, HCV antigen, syphilitic antigen and HIV antigen; the protein concentrations of the HBsAg monoclonal antibody, HCV antigen, syphilitic antigen and HIV antigen, when used to form the colloidal gold conjugates, are 20-50 μg/ml, 90-120 μg/ml, 90-120 μg/ml and 80-120 μg/ml, respectively; the diameter of the colloidal gold particles for preparing each colloidal gold conjugates is 20-30 nm; these four kinds of conjugates are mixed in a volume ratio of 1:1:1.2-2.5:1.2-2.5 to form said mixed solution of the colloidal gold conjugates.
- 2. The diagnostic kit for simultaneously detecting multiple infectious diseases according to claim 1, wherein said syphilitic antigen is a single syphilitic antigen or the mixture of multiple syphilitic antigens; said HIV antigen is HIV-1 antigen, HIV-2 antigen or the mixture of HIV-1 antigen and HIV-2 antigen.
- 3. The diagnostic kit for simultaneously detecting multiple infectious diseases according to claim 1, wherein said buffer is the PBS buffer comprising 0.03-0.05% Tween 20 at pH7.2-8.8.
- 4. A method for preparing the diagnostic kit for simultaneously detecting multiple infectious diseases according to claim 1, said method comprises the following steps:
A. preparing the analytical device for immunogold filtration assay, which comprises the following steps:
1). preparing the samples dotted onto the membrane, comprising dissolving the HBsAg monoclonal antibody, the HCV antigen, the syphilis antigen and the HIV antigen with PBS (0.02M, pH8.0), respectively; dialyzing overnight at 4° C.; diluting each dialyzed proteins to 0.2-1.0 mg/ml with PBS (0.02M, pH8.0); 2). applying the samples onto the membrane, comprising pipetting the above described HBsAg monoclonal antibody, HCV antigen, syphilitic antigen and HIV antigen and carefully dotting them onto the specific positions on the nitrocellulose membrane in a volume of 0.3-3 μl for each proteins; pipetting goat anti-mouse IgG antibody for labeling a mark, which may be a dot, a line or in other forms, at the position far from the above dotting positions on the same nitrocellulose membrane; 3). post-treating the membrane, comprising
drying the above nitrocellulose membrane in an oven at 37° C. for 30 minutes; leaving it at room temperature for over 20 minutes; immersing it in a blocking solution at 37° C. for 20 minutes; washing and vibrating in a washing solution for 5-10 minutes at room temperature; repeating the above washing step for several times; and air drying said membrane; 4). assembling the device, comprising
putting two layers of water-absorbing filter paper under the nitrocellulose membrane; placing and immobilizing both the membrane and the filter paper in the plastic reaction device; B. preparing the mixed solution of the colloidal gold conjugates, which comprises the following steps:
1). preparing the colloidal gold particles, comprising heating 0.01% chloroauric acid solution to the boiling point; adding 1% sodium citrate solution quickly; keeping the solution boiling for 5 minutes to make the size of the colloidal gold particles to be 20-30 nm; 2). preparing the colloidal gold conjugates, comprising
2.1). preparing the colloidal gold-labeled HBsAg monoclonal antibody, comprising the following procedures:
adding the HBsAg monoclonal antibody slowly into the colloidal gold solution, of which the particle diameter is, 20-30 nm under magnetic stirring to a final concentration of 20-50 μg/ml; stirring it for 30 minutes at room temperature; adding 10% BSA solution to make the antibody a final concentration of 0.2-1.0%; stirring the mixture for 5 minutes at room temperature; adding 10% PEG20000 solution to make the antibody a final concentration of 0.1-0.5%; stirring the mixture for 5 minutes at room temperature; centrifuging the mixture at 12000-15000 r/min for 60 minutes; discarding the supernatant; dissolving the precipitate in a storing solution; filtering the solution through a 0.45 mm filter membrane; and storing the mixture at 4° C. for further use; 2.2). preparing the colloidal gold-labeled HCV antigen, comprising the following procedures:
adding the HCV antigen slowly into the colloidal gold solution, of which the particle diameter is 20-30 nm, under magnetic stirring to a final concentration of 90-120 μg/ml; stirring the mixture for 30 minutes at room temperature; adding 10% BSA solution to make the antigen a final concentration of 0.2-1.0%; stirring the mixture for 5 minutes at room temperature; adding 10% PEG20000 solution to make the antigen a final concentration of 0.1-0.5%; stirring the mixture for 5 minutes at room temperature; centrifuging the mixture at 12000-15000 r/min for 60 minutes; discarding the supernatant; dissolving the precipitate in a storing solution; filtering the solution through a 0.45 mm filter membrane; and storing the mixture at 4° C. for further use; 2.3). preparing the colloidal gold-labeled syphilitic antigen, comprising the following procedures:
adding the syphilitic antigen slowly into the colloidal gold solution of which the particle diameter is 20-30 nm, under magnetic stirring, to a final concentration of 90-120 μg/ml; stirring the mixture for 30 minutes at room temperature; adding 10% BSA solution to make the antigen a final concentration of 0.2-1.0%; stirring the mixture for 5 minutes at room temperature; adding 10% PEG20000 solution to make the antigen a final concentration of 0.1-0.5%; stirring the mixture for 5 minutes at room temperature; centrifuging the mixture at 12000-15000 r/min for 60 minutes; discarding the supernatant; dissolving the precipitate in a storing solution; filtering the solution through a 0.45 mm filter membrane; and storing the mixture at 4° C. for further use; 2.4). preparing the colloidal gold-labeled HIV antigen comprising the following procedures:
adding the HIV antigen slowly into a colloidal gold solution of which the particle diameter is 20-30 nm, under magnetic stirring, to a final concentration of 80-120 μg/ml; stirring the mixture for 30 minutes at room temperature; adding 10% BSA solution to make the antigen a final concentration of 0.2-1.0%; stirring the mixture for 5 minutes at room temperature; adding 10% PEG20000 solution to make the antigen a final concentration of 0.1-0.5%; stirring the mixture for 5 minutes at room temperature; centrifuging the mixture at 12000-15000 r/min for 60 minutes; discarding the supernatant; dissolving the precipitate in a storing solution; filtering the solution through a 0.45 mm filter membrane; and storing the mixture at 4° C. for further use; 3). preparing the mixed solution of the colloidal gold conjugates by mixing the colloidal gold conjugates separately prepared as above in the volume ratio of the HBsAg monoclonal antibody:HCV antigen:syphilitic antigen:HIV antigen being 1:1:1.2-2.5:1.2-2.5; C. preparing the buffer by adding Tween 20 into PBS (pH7.2-8.8) to a final concentration of 0.03-0.05%.
- 5. The use of the diagnostic kit according to claim 1 for simultaneously detecting Hepatitis B, Hepatitis C, syphilis and AIDS.
Priority Claims (1)
| Number |
Date |
Country |
Kind |
| CN 01126500.0 |
Aug 2001 |
CN |
|
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent Application PCT/CN01/01519, filed on Oct. 30, 2001, which claims priority to Chinese Patent Application CN 01126500.0, filed on Aug. 17, 2001, the contents of which are hereby incorporated in their entirety by reference.
Continuations (1)
|
Number |
Date |
Country |
| Parent |
PCT/CN01/01519 |
Oct 2001 |
US |
| Child |
10393555 |
Mar 2003 |
US |
Patent Application
20030165970
