CN107765002B - Colloidal gold immunochromatography test strip and preparation method and application thereof - Google Patents
Colloidal gold immunochromatography test strip and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention relates to a colloidal gold immunochromatographic test strip and a preparation method and application thereof, the colloidal gold immunochromatographic test strip comprises a rubber strip, wherein the rubber strip comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent filter paper which are sequentially overlapped, wherein a gold-marked cryptococcus antibody is embedded on the colloidal gold pad, the cryptococcus antibody is coated on the nitrocellulose membrane to be used as a detection line, an antibody coated with an anti-colloidal gold virus antibody is used as a quality control line, and the colloidal gold immunochromatographic test strip has better sensitivity, specificity, repeatability and stability, has higher recovery rate on a target compound and can provide more accurate and reliable detection results.
Description
Technical Field
The invention belongs to the technical field of immunodetection analysis, relates to a colloidal gold immunochromatography test strip, a preparation method and application thereof, and in particular relates to a colloidal gold immunochromatography test strip for detecting cryptococcus.
Background
Cryptococcus is classified into the phylum of the fungus taxonomy, the order of the genus Cryptococcus, the order of the order Cryptococcus, and Cryptococcus causing human infection is mainly Cryptococcus neoformans and Cryptococcus glaucocalycis. The asexual propagules of the two cryptococcus are single spore saccharomycetes of aseptic silk, are non-capsular or only small capsular in vitro, and form thick capsular quickly after entering human body, so that the diameter of cryptococcus with capsular is obviously increased, and the pathogenicity is obviously enhanced.
Cryptococcus (Cryptococcus) is an important conditional pathogen, often infecting patients with hypoimmunity or immunocompetence, resulting in deep fungal infections, mainly central nervous system infections, with high mortality. A large series of epidemiological studies conducted by the center for disease prevention and control (CDC) in 1992-1993 have shown a annual incidence of deep fungal infections of 178.3/million. Among them, cryptococcosis is 65.5/million, accounting for about 36.7%.
Cryptococcus can infect any tissue and organ of the human body, the most common site being the central nervous system, followed by the lungs and skin. Currently, the incidence of cryptococcus infection in immunosuppressed patients is about 5% to 10%, and in AIDS patients, the rate of cryptococcus infection can be as high as 30%; in the population with normal immune function, the infection rate of cryptococcus is about one ten thousandth.
In recent years, cryptococcosis has increased significantly due to the long-term wide application of broad-spectrum antibacterial drugs, adrenocortical hormone, tumor chemotherapy, radiotherapy and immunosuppressants after organ transplantation, and the prevalence of aids. Cryptococcosis meningitis (hereinafter, referred to as "cryptobrain") is the most common central nervous system disease caused by cryptococcus, and accounts for about 80% of cryptococcosis. The clinical manifestations of the cryptobrain are complex and symptoms are atypical, so that diagnosis is difficult, and about 80% of cryptobrain patients can be misdiagnosed as tubercular meningitis.
At present, the cryptobrain can be diagnosed clinically by adopting methods such as cerebrospinal fluid smear ink staining, fungus culture, cryptococcus neoformans membrane-clamping polysaccharide antigen detection and the like. The cerebrospinal fluid smear ink is simple to dye, has short time, but has sensitivity of only 50-80%, and is easy to be subjectively influenced by operators; fungus cultivation and identification generally takes about 3 days and its sensitivity is about 55% -80%. And cryptococcus capsular polysaccharide (GXM) is the main virulence factor responsible for pathological changes. During the growth and propagation of the cells, GXM is continuously secreted extracellularly, affecting the host's immune system by immunosuppression and immunomodulation. The cryptococcus capsular polysaccharide antigen detection has the characteristics of short time consumption, high sensitivity, high specificity and the like. According to literature reports, cryptococcus capsular polysaccharide antigen detection has sensitivity and specificity of more than 90%, and is listed as the basis of diagnosis of cryptococcus by a plurality of professional guidelines, so that development of immune colloidal gold products with high sensitivity, high specificity, simplicity and rapidness has urgency and necessity according to the conditions and the requirements of the current clinical detection market.
The detection methods of cryptococcus developed internationally at present mainly comprise the following 3 major categories: 1) Indian ink staining method: the method has poor sensitivity, low positive detection rate and large limitation; 2) The method for culturing such as blood culture or cerebrospinal fluid culture comprises the following steps: the method has poor sensitivity and low positive detection rate; 3) Immunological detection method: the detection of cryptococcus neoformans is realized by using the antibody specific to GXM, and the sensitivity of blood and cerebrospinal fluid detection can reach more than 85%.
The sensitivity and specificity of immunological detection methods are both significantly better than the former two methods, and are becoming a common method for cryptococcus detection. The immunodetection technique is a method for realizing qualitative or quantitative detection of antigen or antibody by detecting a marker labeled on a reactant by utilizing specific binding reaction between antigen and antibody. According to the labeling substance, it is classified into Enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection, chemiluminescent immunoassay, immunomicrosphere, and immunocolloidal gold. The immune colloidal gold technology has the characteristics of simple operation, high sensitivity and short detection time, and is widely used in clinical detection and scientific research.
In the clinical detection of novel cryptococcus, there are few immunodetection products on the international market at present, such as: emulsion agglutination method product and colloidal gold method product of IMMY company, emulsion agglutination method product of Bio-Rad company, emulsion agglutination method and ELISA method detection kit product of Meridian company. The ELISA kit of Meridian company adopts a double-antibody sandwich method, and the method comprises the steps of firstly coating a specific polyclonal antibody of cryptococcus on an ELISA plate, then adding a sample to be detected to react with the polyclonal antibody, reacting for 10min at 37 ℃, then adding a monoclonal antibody marked by horseradish peroxidase (HRP), reacting for 10min at 37 ℃, combining an antigen in the sample to be detected with the specific monoclonal antibody to form a sandwich structure, adding a chromogenic substrate to develop color, and making the color depth and the concentration of the antigen to be detected positively correlate, thereby realizing GXM detection. At present, no immunological detection product truly used for clinical detection of novel cryptococcus exists in China.
In the prior art, CN 105651996A discloses a novel cryptococcus capsular polysaccharide antigen immunodetection kit, a preparation method and application thereof, the kit comprises a GXM coated enzyme-labeled carrier, an anti-GXM polyclonal enzyme-labeled antibody and a GXM standard substance, the GXM is extracted and purified from a novel cryptococcus culture solution, the kit is prepared by coating the GXM on an enzyme-labeled plate, competing a sample to be detected or a standard antigen with the coated antigen for binding with a limited antibody binding site, performing a chromogenic reaction on the enzyme and the substrate, and calculating the concentration value of the antigen to be detected according to a determination result. CN 103204927A discloses a preparation method of a novel cryptococcus monoclonal antibody, the invention relates to an antibody, a preparation method and application thereof. In particular, the present invention relates to a method for preparing a mouse IgG-type monoclonal antibody BE-A6 against a novel cryptococcus capsular polysaccharide (GXM) antigen and a monoclonal antibody against the novel cryptococcus capsular polysaccharide GXM antigen, and its use in detection of the novel cryptococcus antigen, which patent only shows a method for preparing a cryptococcus monoclonal antibody and does not show a method for detecting cryptococcus.
Therefore, in the field of clinical detection of novel cryptococcus, there is an urgent need for a domestic immune colloidal gold diagnostic kit product for detecting GXM.
Disclosure of Invention
The invention aims to provide a colloidal gold immunochromatographic test strip, a preparation method and application thereof, wherein the colloidal gold immunochromatographic test strip has better sensitivity, specificity, repeatability and stability, has higher recovery rate on a target compound and can provide more accurate and reliable test results.
In order to achieve the purpose of the invention, the invention provides the following technical scheme:
on one hand, the invention provides a colloidal gold immunochromatographic test strip, which is characterized by comprising a rubber strip, wherein the rubber strip comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent filter paper which are sequentially overlapped, wherein gold-labeled cryptococcus antibodies are embedded on the colloidal gold pad, the cryptococcus antibodies are coated on the nitrocellulose membrane to serve as detection lines, and antibodies coated with anti-colloidal gold virus antibodies serve as quality control lines.
In the invention, the colloidal gold immunochromatography test strip adopts a sandwich method, if a detection sample is positive, the cryptococcus capsular polysaccharide and the cryptococcus antibody marked by colloidal gold are combined to form a complex, the complex moves forward along the strip under the action of chromatography and reacts with the pre-coated cryptococcus antibody when passing through the detection line to form an immune complex to present a red strip, and the free gold marked antibody is combined with the goat anti-mouse antibody to present a red strip on a quality control line; if the detection sample is negative, the cryptococcus capsular polysaccharide is not contained, immune complex is not formed, a strip is not generated at the detection line, and only the quality control line is colored; the quality control line should have stripes when detecting positive samples and negative samples, and the red stripes are the standard for judging whether the chromatographic process is normal or not, and are also used as internal control standards of the reagent.
According to the invention, the gold-labeled cryptococcus antibody has a concentration of 2-8. Mu.g/mL, which may be, for example, 2. Mu.g/mL, 3. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL, 6. Mu.g/mL, 7. Mu.g/mL or 8. Mu.g/mL, preferably 5. Mu.g/mL.
As a preferable technical scheme of the invention, the colloidal gold upper virus antibody is a murine antibody, and the antibody used as a quality control line is goat anti-mouse IgG. Murine antibodies are the most common antibody form in current commercial antibodies, and are widely available, low in cost, and good in specificity and stability. The virus antibody may be a polyclonal antibody or a monoclonal antibody, and methods for preparing the polyclonal antibody or the monoclonal antibody are well known to those skilled in the art, for example, animals such as mice may be immunized with the corresponding virus, and the polyclonal antibody may be purified from animal serum, but the polyclonal antibody may have a problem of less specificity than the monoclonal antibody, and may have a false positive result. In particular, although polyclonal antibodies can be adopted for the virus antibodies, monoclonal antibodies have more obvious advantages, so that the method for preparing the monoclonal antibodies is preferable for the invention, for example, the method can be used for immunizing animals, such as mice, with corresponding viruses, taking spleen B cells of the animals, fusing the spleen B cells with myeloma cells of the mice to form hybridoma cells, and obtaining the monoclonal antibodies with stronger specificity from the hybridoma cells.
According to the invention, the nitrocellulose membrane is also coated with a blocking agent as a blocking line, the blocking line is used for removing interference, and substances with interference in a sample can be combined, and the blocking agent is HAMA blocking agent and/or HBR blocking agent.
Preferably, the HAMA blocker is present at a concentration of 0.5-2mg/ml, which may be, for example, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml, 0.9mg/ml, 1mg/ml, 1.2mg/ml, 1.3mg/ml, 1.5mg/ml, 1.8mg/ml or 2mg/ml.
Preferably, the concentration of the HBR blocking agent is 0.3-1mg/ml, which may be, for example, 0.3.mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml, 0.9mg/ml or 1mg/ml.
According to the invention, the colloidal gold pad further comprises a separating agent, wherein the separating agent is any one or a combination of at least two of casein, BSA or PEG6000 (polyethylene glycol 6000), and preferably casein.
In the invention, the inventor finds that the separation agent is added on the colloidal gold pad, so that the combination of the cryptococcus antibody and the colloidal gold particles is more stable, the detection limit of the gold immunochromatographic test strip is further reduced, and the inventor unexpectedly found that when the casein is used as the separation agent, the resolubility and the detection performance of the gold label are further improved, and the minimum detection limit reaches 0.5ng/mL.
According to the invention, the final concentration of casein is 0.1-1% by mass, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably 0.25-0.8%, and more preferably 0.5%.
According to the present invention, the final concentration of BSA by mass is 0.1 to 1%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably 0.25 to 0.8%, and more preferably 0.1%.
According to the present invention, the final concentration of PEG6000 is 0.1-1%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably 0.25-0.8%, and more preferably 1%.
According to the invention, the cryptococcus antibody is a monoclonal antibody against cryptococcus capsular polysaccharide antigen and/or a polyclonal antibody against cryptococcus capsular polysaccharide antigen, preferably a monoclonal antibody against cryptococcus capsular polysaccharide antigen.
Preferably, the preparation method of the cryptococcus antibody comprises the following steps: the cryptococcus capsular polysaccharide antigen is used as a specific cell strain obtained by immunizing animals with immunogens, and ascites obtained by immunizing the animals with the specific cell strain is separated and purified to obtain the cryptococcus capsular polysaccharide antigen.
Preferably, the purification method is saturated ammonium sulphate precipitation and/or affinity chromatography.
According to the invention, the sample pad is a glass fiber film or a polyester fiber film, preferably a glass fiber film.
Preferably, the colloidal gold pad is a glass fiber membrane.
Preferably, the colloidal gold immunochromatographic test strip further comprises a bottom plate, and the adhesive tape is arranged on the bottom plate.
Preferably, the bottom plate is a polyvinyl chloride rubber plate.
In a second aspect, the present invention provides a method for preparing the colloidal gold immunochromatographic test strip according to the first aspect, comprising: antibody coating, preparation of a colloidal gold pad and assembly of a colloidal gold test strip.
In the present invention, the antibody coating is a conventional technical means in the art, and is not particularly limited herein, and one skilled in the art can select and prepare according to actual needs.
Preferably, the antibody-coated antibody coating buffer is any one or a combination of at least two of PBS buffer, PB buffer or CBS buffer (carbonate buffer).
Preferably, the molar concentration of the PBS buffer is 0.01-0.3mol/L, for example, 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.05mol/L, 0.08mol/L, 0.1mol/L, 0.12mol/L, 0.15mol/L, 0.18mol/L, 0.2mol/L, 0.22mol/L, 0.25mol/L, 0.28mol/L or 0.3mol/L, preferably 0.01-0.2mol/L.
Preferably, the pH of the PBS buffer is 6-8.5, which may be, for example, 6, 6.1, 6.2, 6.3, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2 or 8.5, preferably 7-8.
Preferably, the PB buffer has a molar concentration of 0.01-0.1mol/L, for example 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/L, 0.07mol/L, 0.08mol/L, 0.09mol/L or 0.1mol/L, preferably 0.01-0.05mol/L.
Preferably, the PB buffer has a pH of 7-8, for example 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, preferably 7.2-7.4.
Preferably, the molar concentration of the CBS buffer is 0.01-0.1mol/L, for example, 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/L, 0.07mol/L, 0.08mol/L, 0.09mol/L or 0.1mol/L, preferably 0.03-0.05mol/L.
Preferably, the CBS buffer has a pH of 8.5 to 9.8, for example, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7 or 9.8, preferably 9 to 9.5.
According to the invention, the preparation of the colloidal gold pad specifically comprises the following steps:
(1) Adjusting the pH value by adopting colloidal gold particles, adding a separating agent, adding a cryptococcus antibody, and standing;
(2) Adding a sealing liquid into the colloidal gold solution obtained in the step (1) for sealing;
(3) Centrifuging the colloidal gold solution sealed in the step (2) to remove supernatant, and re-dissolving and precipitating by adopting a re-dissolving solution to obtain a cryptococcus antibody marking solution;
(4) Spraying the cryptococcus antibody marking liquid in the step (3) onto a glass fiber membrane, drying and sealing to obtain the colloidal gold pad.
According to the present invention, the colloidal gold particles of step (1) have a particle diameter of 30 to 40nm, and may be, for example, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm or 40nm.
Preferably, the pH in step (1) is 6-7, and may be, for example, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7, preferably 6.3-6.5.
Preferably, the blocking solution in step (2) is a casein and/or BSA solution.
Preferably, the casein is present in a concentration of 0.1-4% by mass, for example 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.6%, 1.8%, 2%, 2.2%, 2.3%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8% or 4%, preferably 0.5-2%.
Preferably, the BSA solution has a mass concentration of 0.5-5%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.6%, 1.8%, 2%, 2.2%, 2.3%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.3%, 4.5%, 4.8% or 5%, preferably 1-4%.
Preferably, the complex solution in step (3) is any one or a mixture of at least two of Tris, sucrose, trehalose and BSA.
Preferably, the molar concentration of Tris is 10-30mM, for example, 10mM, 12mM, 15mM, 16mM, 18mM, 20mM, 22mM, 23mM, 25mM, 26mM, 28mM or 30mM, preferably 20mM.
Preferably, the sucrose is present in a mass concentration of 5-15%, for example 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, preferably 10%.
Preferably, the trehalose is present in a mass concentration of 3-10%, for example 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably 5%.
Preferably, the BSA has a mass concentration of 0.1 to 5%, for example, 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.6%, 1.8%, 2%, 2.2%, 2.3%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8% or 5%, preferably 1%.
Preferably, the pH of the complex solution is 8-9, for example, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9, preferably 8.5.
In a third aspect, the invention provides a detection kit comprising a colloidal gold immunochromatographic strip according to the first aspect.
In a fourth aspect, the present invention provides a colloidal gold immunochromatographic test strip according to the first aspect and/or a detection kit according to the third aspect for detecting cryptococcus.
Preferably, the cryptococcus comprises any one or a combination of at least two of cryptococcus serotype a, cryptococcus serotype B, cryptococcus serotype C, or cryptococcus serotype D.
In the invention, the using method of the test strip is as follows:
(1) Sucking 80 μl of sample to be detected, and slowly dripping the sample into a sample-adding hole of the test strip;
(2) Timing, reading the result in 15-20 minutes, and abnormality may occur in the result after 20 minutes.
Analysis of results:
positive (+): two red bands appear, one located in the detection line (T) and the other located in the quality control line (C), indicating the presence of cryptococcus capsular polysaccharide in the sample.
Negative (-). Only one red band appears on the control line (C) and no red band appears on the detection line (T), indicating no capsular polysaccharide or capsular polysaccharide below the detection level in the sample.
Invalidation: the control line (C) does not appear as a red band, possibly due to incorrect operation or reagent failure. In any case, the test should be retested. If the problem still exists, the use of the lot number product should be stopped immediately and contacted with the local supplier.
Note that: the red bands of the test line (T) will appear in different shades of color due to differences in capsular polysaccharide titer in the sample. However, the test result of the reagent cannot be used as a basis for judging the titer of the capsular polysaccharide in the sample.
In a fifth aspect, the invention provides the use of a colloidal gold immunochromatographic strip according to the first aspect for preparing a diagnostic kit for detecting meningoencephalitis.
Preferably, the meningoencephalitis is a cryptococcus-induced meningoencephalitis.
In the invention, the cryptococcus meningoectomy is the most common meningoectomy caused by fungus intracranial infection, and the colloidal gold immunochromatography test strip realizes the detection of the meningoectomy by detecting the cryptococcus.
Compared with the prior art, the method has the following beneficial effects:
(1) The colloidal gold immunochromatographic test strip adopts a sandwich method to detect cryptococcus, has good sensitivity, specificity, repeatability and stability, has high recovery rate on target compounds, and has accurate and reliable test result;
(2) According to the colloidal gold immunochromatography test strip, the separation agent is added into the colloidal gold pad, so that the combination of the cryptococcus antibody and the colloidal gold particles is more stable, the detection limit of the colloidal gold immunochromatography test strip is further reduced, and the minimum detection limit can reach 0.5ng/mL;
(3) The colloidal gold immunochromatographic test strip has higher sensitivity and specificity for four serotype strains of cryptococcus A, B, C, D.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold immunochromatographic test strip according to the present invention, in which a 1-sample pad, a 2-colloidal gold pad, a 3-blocking line, a 4-detection line (T line), a 5-quality control line (C line), a 6-absorbent filter paper, a 7-PVC base plate, and an 8-nitrocellulose membrane (NC membrane).
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only preferred embodiments of the present invention for better understanding of the present invention, and thus should not be construed as limiting the scope of the present invention.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless specified, are all purchased from conventional biochemical reagent manufacturers.
Sources of reagents and instrumentation used in the examples: goat anti-mouse IgG antibodies were purchased from beijing boaosen biotechnology limited; nitrocellulose membranes were purchased from merck milbo; the three-dimensional planar film drawing instrument is purchased from Shanghai gold mark biotechnology limited company; slitter was purchased from Shanghai gold mark biotechnology limited; PVC plywood, absorbent paper, glass fiber film and cardboard were purchased from Shanghai gold standard biotechnology Co.
Example 1 preparation of colloidal gold immunochromatographic test strip
The colloidal gold immunochromatographic test strip can be prepared according to the following method, and specifically has the structure shown in figure 1:
and (3) attaching a sample pad, a colloidal gold pad, a nitrocellulose membrane and water-absorbing filter paper to one end of a polyvinyl chloride (PVC) rubber plate in a sequential and overlapping manner, cutting the PVC rubber plate and attached materials into test strips with the width of 4+/-0.2 mm, and placing the test strips into a clamping shell, wherein a sample adding area and a color development area (observation area) are formed on the clamping shell.
Wherein the sample pad is a glass fiber membrane; wherein gold-labeled cryptococcus antibody with the concentration of 2-8 mug/mL is embedded on the colloidal gold pad; the nitrocellulose membrane comprises: the blocking line is a mixture of HAMA blocking agent and HBR blocking agent, the concentration of the HAMA blocking agent is 1mg/ml, the concentration of the HBR blocking agent is 0.8mg/ml, the T line (detection limit) is a coating antibody of cryptococcus antibody, and the C line (quality control line) is goat anti-mouse IgG antibody.
(1) Preparation of cryptococcus antibody:
the specific cell strain obtained by immunizing animals with cryptococcus capsular polysaccharide antigen as immunogen, and separating and purifying ascites obtained by immunizing animals with the specific cell strain;
(2) Antibody coating:
(1) diluting cryptococcus antibody and sheep anti-mouse antibody to 1mg/ml by PB with pH of 7.2-7.4 in coating buffer solution of 0.01-0.05mol/L to obtain C-line sheep anti-mouse coating solution and T-line cryptococcus antibody diluent;
(2) scribing the diluted solution obtained in the step (1) to an NC (nitrocellulose) film by using a metal spraying scribing instrument, and scribing according to the condition of 1 mu L/cm;
(3) placing the NC film in the step (2) in a baking oven at 37 ℃ for drying for 4 hours, and adding a drying agent for sealing and preserving after drying;
(3) Preparing a colloidal gold pad:
(1) adjusting pH to 6.5 with colloidal gold particles, adding casein separating agent with final concentration of 0.5%, adding cryptococcus antibody with final concentration of 5 μg/ml, and standing for 60min;
(2) adding 10% BSA blocking solution into the colloidal gold solution obtained in the step (1) to a final concentration of 1% for blocking for 60min;
(3) centrifuging the colloidal gold solution obtained in the step (2) for 40min at the temperature of 8000r, removing the supernatant after centrifugation, and re-dissolving and precipitating by using a complex solution to obtain a cryptococcus antibody marking solution;
(4) spraying the cryptococcus antibody marking liquid obtained in the step (3) on a glass fiber membrane according to 15 mu l/cm by using a metal spraying and film scratching instrument;
(5) drying the glass fiber membrane in the step (4) for 180min at 37 ℃, adding a drying agent after drying, and sealing and preserving to obtain a gold mark pad;
(4) Assembling a colloidal gold test strip:
(1) assembling the coated NC film, the coated gold mark pad, the coated water absorbing paper and the coated sample pad on a PVC plate;
(2) cutting the assembly plate obtained in the step (1) into a colloidal gold test strip of 4+0.2mm by using a strip cutting device;
(3) and (3) placing the test strip obtained in the step (2) in a sealed bag, and adding a drying agent for preservation.
As shown in FIG. 1, the detection principle of the colloidal gold immunochromatographic test strip of the present invention is specifically as follows:
after the sample is applied to the sample application area (sample pad), the cryptococcus is detected in the area by capillary action: the antigen-antibody-colloidal gold conjugate in the sample moves toward one end of the absorbent paper. If the detected sample contains the antigen of the cryptococcus capsular polysaccharide, the sample moves to a T line, namely a coating line of the coating antibody of the cryptococcus antibody marked by the colloidal gold, the antigen-antibody-colloidal gold conjugate is captured, and the antibody-antigen-antibody-colloidal gold conjugate is generated at the T line, so that a red strip is displayed; when the sample continues to flow to the C line, namely the goat anti-mouse IgG antibody, a red strip appears, and the effectiveness of the test strip is proved. If the antigen of cryptococcus capsular polysaccharide is not present in the actual sample tested, immune complex is not formed, red band is not displayed at T line, and color is only developed at C line. As long as the line C does not develop, the test strip is proved to be ineffective and the actual sample needs to be re-detected.
The preparation and detection of the colloidal gold immunochromatographic test strip of the present invention are described below by way of examples. It should be understood that these embodiments are merely exemplary and should not limit the scope of the present invention.
Example 2: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.1% casein.
Example 3: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.25% casein.
Example 4: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 1% casein.
Example 5: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.1% BSA.
Example 6: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.25% BSA.
Example 7: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.5% BSA.
Example 8: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 1% BSA.
Example 9: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.1% PEG6000.
Example 10: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is PEG6000 at 0.25%.
Example 11: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 0.5% PEG6000.
Example 12: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the colloidal gold pad was prepared: the separating agent shown is 1% PEG6000.
Example 13: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, the antibody coating was performed with 0.01-0.20mol/L PBS buffer solution at pH 7.0-8.0; in the preparation of the colloidal gold pad: the pH was adjusted to 6 with colloidal gold particles and blocking solution was added to a final concentration of 4% BSA for blocking.
Example 14: preparation of colloidal gold immunochromatography test strip
In contrast to example 1, antibody coating was performed with 0.05mol/L CBS buffer at pH 9.0.0-9.5; in the preparation of the colloidal gold pad: the pH value is regulated to 7 by using colloidal gold particles, and a blocking solution is added into casein with a final concentration of 0.5-2% for blocking.
Comparative example 1: preparation of colloidal gold immunochromatography test strip
In comparison with example 1, no separating agent was added in the preparation of the colloidal gold pad.
Example 15 colloidal gold immunochromatographic test strip detection
The colloidal gold immunochromatographic test strips in examples 1 to 13 and comparative example 1 were used for detection, and the specific procedures were as follows:
(1) Sucking 80 μl of bacterial liquid 3 groups, and slowly dripping into the sample adding holes of the test strip;
(2) Timing, reading results from 15 to 20 minutes, observing results after 20 minutes, and the results are shown in table 1:
TABLE 1 Effect of different stabilizers on colloidal gold-labeled antibodies
Calculation of detection limit: diluting the specific concentration of the antigen, diluting the detection antigen by 0.1, 0.25, 0.5, 1, 2, 4 and 8ng/ml, detecting by using test paper prepared by different separating agents, repeating for 3 times, and obtaining the lowest concentration of the detection line as the detection limit.
As can be seen from Table 1, the detection limit is 3-5ng/mL without using a separating agent, and BSA is used as the separating agent, and the reagent sensitivity can be remarkably reduced although the gold label has better resolubility; PEG6000 is used as a separating agent, the gold-labeled substance has good re-solubility, but reagent false positive is easy to cause, casein with the final concentration of 0.5% is used as the separating agent, the re-solubility of the gold-labeled substance and the reagent detection performance are good, the detection limit is as low as 0.5ng/mL, and therefore 0.5% BSA is selected as the separating agent of the colloidal gold labeled antibody.
Example 16: detection of different serotypes of bacteria by cryptococcus capsular polysaccharide immunochromatography test strip
(1) Diluting bacterial solutions of 4 different serotypes with physiological saline in a gradient manner;
(2) The bacterial liquids were tested by using the colloidal gold test strip prepared in example 1, and the results are shown in Table 2.
TABLE 2 test strip test results of bacterial liquids
As can be seen from the results in Table 2, the colloidal gold test paper has higher sensitivity to all 4 serotypes, and particularly has high sensitivity to the strains of serotype A.
In conclusion, the colloidal gold immunochromatography test strip adopts a sandwich method to detect cryptococcus, and the combination of the cryptococcus antibody and colloidal gold particles is more stable by adding the separating agent into the colloidal gold pad, so that the detection limit of the gold immunochromatography test strip is further reduced, and the minimum detection limit can reach 0.5ng/mL; the colloidal gold immunochromatographic test strip has good sensitivity, specificity, repeatability and stability, high recovery rate of target compounds, accurate and reliable test results, and high sensitivity and specificity for four serotype strains of cryptococcus A, B, C, D.
The applicant states that the detailed features and detailed methods of the present invention are described by way of the above examples, but the present invention is not limited to the detailed features and detailed methods described above, i.e., it is not meant that the present invention must rely on the detailed features and detailed methods to practice the present invention. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected components, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the invention and the scope of the disclosure.
Claims (52)
1. The preparation method of the cryptococcus detection colloidal gold immunochromatographic test strip is characterized in that the colloidal gold immunochromatographic test strip comprises an adhesive tape, and the adhesive tape comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane and water-absorbing filter paper which are sequentially overlapped;
wherein, gold-labeled cryptococcus antibody is embedded on the colloidal gold pad, the cryptococcus antibody is coated on the nitrocellulose membrane as a detection line, and the antibody coated with anti-colloidal gold virus antibody is used as a quality control line; the colloidal gold pad also comprises a separating agent, wherein the separating agent is casein with the mass final concentration of 0.5%;
the preparation method comprises the following steps: antibody coating, preparation of a colloidal gold pad and assembly of a colloidal gold test strip;
the antibody coating comprises the following steps: diluting the cryptococcus antibody and the antibody of the anti-colloidal gold upper virus antibody by using an antibody coating buffer solution to obtain an antibody diluent of the quality control line anti-colloidal gold upper virus antibody and a cryptococcus antibody diluent of a detection line, respectively scratching the antibody diluent of the anti-colloidal gold upper virus antibody and the cryptococcus antibody diluent onto a nitrocellulose membrane by using a metal spraying and scratching instrument, drying and sealing for preservation;
the preparation of the colloidal gold pad comprises the following steps:
(1) Adjusting the pH value by adopting colloidal gold particles, adding a separating agent, adding a cryptococcus antibody, and standing;
(2) Adding a sealing liquid into the colloidal gold solution obtained in the step (1) for sealing;
(3) Centrifuging the colloidal gold solution sealed in the step (2) to remove supernatant, and re-dissolving and precipitating by adopting a re-dissolving solution to obtain a cryptococcus antibody marking solution;
(4) Spraying the cryptococcus antibody marking liquid in the step (3) onto a glass fiber membrane, and drying and sealing to obtain the colloidal gold pad;
the assembly of the colloidal gold test strip comprises the following steps: and assembling the coated nitrocellulose membrane, the sample pad, the colloidal gold pad and the water-absorbing filter paper, and cutting, drying and preserving after assembling.
2. The method for preparing the colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 1, wherein the concentration of the gold-labeled cryptococcus antibody is 2-8 μg/mL.
3. The method for preparing the colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 2, wherein the concentration of the gold-labeled cryptococcus antibody is 5 μg/mL.
4. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 1, wherein the cryptococcus antibody is a murine antibody, and the antibody against the colloidal gold upper virus antibody is goat anti-mouse IgG.
5. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that a blocking agent is further coated on the nitrocellulose membrane as a blocking line.
6. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 5, wherein the blocking agent is a HAMA blocking agent and/or a HBR blocking agent.
7. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 6, wherein the concentration of the HAMA blocking agent is 0.5-2mg/ml.
8. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 6, wherein the concentration of the HBR blocking agent is 0.3-1mg/ml.
9. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 1, wherein the cryptococcus antibody is a monoclonal antibody against cryptococcus capsular polysaccharide antigen and/or a polyclonal antibody against cryptococcus capsular polysaccharide antigen.
10. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 9, wherein the cryptococcus antibody is a monoclonal antibody against a cryptococcus capsular polysaccharide antigen.
11. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 1, which is characterized by comprising the following steps: the cryptococcus capsular polysaccharide antigen is used as a specific cell strain obtained by immunizing animals with immunogens, and ascites obtained by immunizing the animals with the specific cell strain is separated and purified to obtain the cryptococcus capsular polysaccharide antigen.
12. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic strip according to claim 11, wherein the purification method is a saturated ammonium sulfate precipitation method and/or an affinity chromatography method.
13. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic test strip according to claim 1, wherein the sample pad is a glass fiber membrane or a polyester fiber membrane.
14. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 13, wherein the sample pad is a glass fiber membrane.
15. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic test strip according to claim 1, wherein the colloidal gold pad is a glass fiber membrane.
16. The method for preparing the colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 1, further comprising a bottom plate, wherein the adhesive tape is arranged on the bottom plate.
17. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic test strip according to claim 16, wherein the bottom plate is a polyvinyl chloride gel plate.
18. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 1, wherein the antibody coating buffer is any one or a combination of at least two of PBS buffer, PB buffer and CBS buffer.
19. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 18, wherein the molar concentration of the PBS buffer solution is 0.01-0.3mol/L.
20. The method for preparing the cryptococcus detection colloidal gold immunochromatographic strip according to claim 19, wherein the molar concentration of the PBS buffer is 0.01-0.2mol/L.
21. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 18, wherein the pH of the PBS buffer is 6-8.5.
22. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 21, wherein the pH of the PBS buffer is 7-8.
23. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 18, wherein the molar concentration of the PB buffer is 0.01-0.1mol/L.
24. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 23, wherein the molar concentration of the PB buffer is 0.01-0.05mol/L.
25. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 18, wherein the pH of the PB buffer is 7-8.
26. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 25, wherein the pH of the PB buffer is 7.2-7.4.
27. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic strip according to claim 18, wherein the molar concentration of the CBS buffer is 0.01-0.1mol/L.
28. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 27, wherein the molar concentration of the CBS buffer is 0.03-0.05mol/L.
29. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 18, wherein the pH of the CBS buffer is 8.5-9.8.
30. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 29, wherein the pH of the CBS buffer is 9-9.5.
31. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 1, wherein the particle size of the colloidal gold particles in the step (1) is 30-40nm.
32. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 1, wherein the pH value in the step (1) is 6 to 7.
33. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 32, wherein the pH value in the step (1) is 6.3 to 6.5.
34. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic strip according to claim 1, wherein the blocking solution in the step (2) is casein and/or BSA solution.
35. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 34, wherein the mass concentration of casein is 0.1 to 4%.
36. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 35, wherein the mass concentration of casein is 0.5 to 2%.
37. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 34, wherein the mass concentration of the BSA solution is 0.5-5%.
38. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 37, wherein the mass concentration of the BSA solution is 1-4%.
39. The method for preparing a colloidal gold immunochromatographic test strip for detecting cryptococcus according to claim 1, wherein the complex solution in the step (3) is any one or a mixture of at least two of Tris, sucrose, trehalose and BSA.
40. The method of claim 39, wherein the molar concentration of Tris is 10-30mM.
41. The method of claim 40, wherein the molar concentration of Tris is 20mM.
42. The method for preparing a colloidal gold immunochromatographic test strip for detecting Cryptococcus as defined in claim 39, wherein the mass concentration of sucrose is 5-15%.
43. The method for preparing a colloidal gold immunochromatographic test strip for detecting Cryptococcus as defined in claim 42, wherein the sucrose has a mass concentration of 10%.
44. The method for preparing a colloidal gold immunochromatographic test strip for detecting Cryptococcus as defined in claim 39, wherein the mass concentration of trehalose is 3-10%.
45. The method for preparing a colloidal gold immunochromatographic test strip for detecting Cryptococcus as defined in claim 44, wherein the mass concentration of trehalose is 5%.
46. The method for preparing a colloidal gold immunochromatographic test strip for detecting Cryptococcus as defined in claim 39, wherein the mass concentration of BSA is 0.1-5%.
47. The method for preparing a colloidal gold immunochromatographic test strip for detecting Cryptococcus as defined in claim 46, wherein the mass concentration of BSA is 1%.
48. The method for preparing the cryptococcus detecting colloidal gold immunochromatographic strip according to claim 1, wherein the pH value of the complex solution is 8-9.
49. The method of claim 48, wherein the pH of the reconstituted solution is 8.5.
50. A kit for detecting cryptococcus, comprising a colloidal gold immunochromatographic test strip prepared by the preparation method of any one of claims 1 to 49.
51. The kit for detecting a cryptococcus according to claim 50, wherein the cryptococcus comprises any one or a combination of at least two of cryptococcus serotype A, cryptococcus serotype B, cryptococcus serotype C, or cryptococcus serotype D.
52. The application of the colloidal gold immunochromatographic test strip in preparing a kit for detecting meningoectoencephalitis, which is characterized in that the colloidal gold immunochromatographic test strip is prepared by adopting the preparation method of any one of claims 1-49, and the meningoectoencephalitis is a meningoectoencephalitis caused by cryptococcus.
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