CN111707821A - Colloidal gold test paper for detecting IBV (infectious bronchitis Virus) and preparation method thereof - Google Patents
Colloidal gold test paper for detecting IBV (infectious bronchitis Virus) and preparation method thereof Download PDFInfo
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Classifications
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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
- G01N33/56983—Viruses
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention discloses a colloidal gold test paper for detecting IBV, which comprises a supporting plate, a coating film, a sample pad, a gold-labeled antibody pad, absorbent paper, a detection line and a quality control line, and is characterized in that: the gold-labeled antibody pad is marked with IgG antibodies resisting IBV, the detection line is coated with monoclonal antibodies resisting IBV N protein, and the quality control line is coated with goat anti-mouse IgG antibodies. The invention also discloses an index method of the colloidal gold test paper for detecting IBV. The test paper prepared by the invention is based on the principle of a double-antibody sandwich ELISA antigen detection method, and is used for detecting IBV antigen, so that the detection of virus infection is more visual, rapid, simple and convenient, the specificity is good, and the aim of accurate and rapid detection is fulfilled; can provide technical support for immune monitoring and epidemiological monitoring research of avian infectious bronchitis, and has important significance for prevention and control of avian diseases.
Description
Technical Field
The invention relates to an animal virus detection technology, in particular to colloidal gold test paper for detecting IBV (infectious bursal disease Virus), and a preparation method of the colloidal gold test paper.
Background
Avian Infectious Bronchitis (IB) is an acute and highly contagious infectious disease of chickens caused by Infectious Bronchitis Virus (IBV), and is one of the major infectious diseases seriously harming the poultry industry. IBV is a very variable coronavirus of many serotypes, with a lack of cross-protection between different serotypes. The spread of IBV causes serious economic losses to the poultry industry worldwide. The N gene evolution of IBV is most conserved, and the N protein is an important immunogenic protein for inducing immune responses in the body. The monoclonal antibody against the N protein is prepared, and the rapid diagnosis method of the monoclonal antibody IBV colloidal gold based on the N protein is established, so that the method has important significance for IBV prevention and control.
Enzyme-linked immunosorbent assay (ELISA) is an immunoassay technology widely used for measuring protein, antibody or hormone in a liquid sample, has the characteristics of sensitivity, specificity, simplicity, rapidness, stability, easy automation operation and the like, and is suitable for simultaneous detection of a large number of clinical specimens. At present, a plurality of ELISA methods for IBV infection detection are established at home and abroad; however, the research finds that most of the detection methods are based on antibody detection and have certain limitations; therefore, there is a need to develop a method for detecting IBV antigens to achieve the objective of detecting viral infection more intuitively, rapidly and accurately.
Disclosure of Invention
The invention aims to provide the colloidal gold test paper for detecting IBV and the preparation method thereof, which realize the rapid detection of IBV with simple and rapid operation and clear and easily distinguished result.
In order to achieve the purpose, the invention can adopt the following technical scheme:
the colloidal gold test paper for detecting IBV comprises a supporting plate, a coating film, a sample pad, a gold-labeled antibody pad, absorbent paper, a detection line (T line) and a quality control line (C line), wherein the gold-labeled antibody pad is labeled with an anti-IBV IgG antibody, the detection line is coated with an anti-IBV N protein monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody.
The ELISA titer of the anti-IBV N protein monoclonal antibody is not less than 106。
The subtype of the anti-IBV N protein monoclonal antibody is IgG1 type.
The coating concentration of the anti-IBV N protein monoclonal antibody is 2 mg/mL.
The invention relates to a preparation method of colloidal gold test paper for detecting IBV, which comprises the following specific steps:
firstly, prokaryotic expression is carried out on IBV N protein gene, and exogenous N protein is obtained after protein purification;
secondly, immunizing a BALB/c mouse by taking the N protein purified in the first step as an antigen, screening to obtain hybridoma cell strains of IBV, and selecting the hybridoma cell strains with high titer and strong affinity to prepare the anti-IBV N protein monoclonal antibody;
thirdly, taking IgG resisting IBV as a gold-labeled antibody to be marked on a gold-labeled antibody pad; spraying the anti-IBV N protein monoclonal antibody prepared in the second step onto a detection line on the envelope; spraying/printing goat anti-mouse IgG antibody on the quality control line on the coating film; airing and sealing the bag for later use;
and fourthly, sequentially overlapping and adhering a sample pad, a gold-labeled antibody pad, a coating film and absorbent paper on the supporting plate, and cutting into a designed width to obtain a finished product of the colloidal gold test paper for detecting the IBV.
Wherein the separation distance between the detection line and the quality control line is 4 mm.
And when the gold-labeled antibody pad is manufactured in the third step, firstly, preparing a colloidal gold solution by a trisodium citrate method, treating the colloidal gold solution and the antibody by a stabilizer, adsorbing the colloidal gold solution and the antibody on a glass fiber membrane, and drying in vacuum to obtain a finished gold-labeled antibody pad.
The ratio of the colloidal gold solution to the antibody is 25 mug antibody/mL colloidal gold; the pH value during the adsorption was 7.8.
Compared with the prior art, the beneficial technical effects of the invention are as follows:
1. the test paper prepared by the invention is based on the principle of a double-antibody sandwich ELISA antigen detection method, and is used for detecting IBV antigens, so that the detection of virus infection is more intuitive and rapid.
2. The test paper prepared by the invention can detect IBV, is rapid, simple and convenient, has good specificity, and realizes the aim of accurate and rapid detection.
3. The test paper provided by the invention is simple and rapid to operate, clear in result and easy to distinguish, and is suitable for basic popularization.
4. The test paper prepared by the invention can provide technical support for immune monitoring and epidemiological monitoring research of avian infectious bronchitis, and has important significance for prevention and control of avian diseases.
Drawings
FIG. 1A and FIG. 1B are SDS-PAGE electrophoresis and Western blot detection of the N protein, respectively.
FIG. 2 is a graph showing the results of IPMA experiments with monoclonal antibody 1A 3.
Fig. 3 is a schematic structural diagram of the test strip.
Fig. 4 is a schematic top view of fig. 3.
FIG. 5 is a schematic diagram illustrating test strip result determination.
Detailed Description
The following examples are given to illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.
EXAMPLE 1 immunogen selection and preparation
1. Selection of immunogens
Based on a large number of researches, firstly, in the proteins coded by the genome of the avian infectious bronchitis, the nucleocapsid protein, namely the N protein, is highly conserved; secondly, the N protein is an important immunogenic protein for inducing immune response of the body.
Based on the two points, the N protein is researched and screened to become the first-choice target protein for detecting IBV.
2. N protein preparation of immunogens
(1) Cloning the gene sequence of the avian infectious bronchitis virus N protein, and connecting the sequence to an expression vector pGEX-6p-1 to obtain a recombinant expression vector pGEX-6 p-1-N;
(2) the recombinant expression vector pGEX-6p-1-N is replaced by CaCl2Method for transferring competent cellsE.coliBL21(DE3) to obtain a positive recombinant E.coli BL21(DE 3);
(3) inoculating positive recombinant Escherichia coli BL21(DE3) into LB culture medium, inducing expression at 20 ℃ for 6 hours under the condition that the IPTG concentration is 1mM, collecting samples and purifying the samples through GST columns;
(4) the purified protein was subjected to SDS-PAGE and Western blot detection, and the results are shown in FIGS. 1A and 1B. In FIG. 1A, 1 is Marker, 2 is before induction, 3 is after induction, 4 is supernatant, and 5 is precipitate; in FIG. 1B, 1 is Marker, 2 is recombinant N protein, and 3 is N protein.
The identification result shows that the obtained N protein has higher purity.
EXAMPLE 2 monoclonal antibody preparation and characterization
1. Animal immunization
(1) Adding the immunogen N protein prepared in the example 1 into Freund's complete adjuvant (used for first immunization) or Freund's incomplete adjuvant, and emulsifying to prepare immune antigen;
(2) immunizing female BALB/c mice (3 mice/group) with the age of 4-8 weeks by a back subcutaneous multipoint injection method, wherein the immunizing dose is 50 mu g/mouse;
(3) after the first immunization, BALB/c mice are boosted by the same method and dosage after emulsified with Freund's incomplete adjuvant and immunizing antigen every 2 weeks;
(4) after four times of immunization, 3-4 days before cell fusion, using immunogen without adjuvant to strengthen BALB/c mouse by tail vein injection method, the immunization dose is 100 mug/mouse;
(5) one week after the last boosting immunization, respectively carrying out tail-cutting blood collection on each mouse, and then respectively measuring the titer and the sensitivity of the polyclonal antiserum of 3 mice by an indirect ELISA method and an indirect competitive ELISA method, selecting the mouse with the highest titer No. 1 (the titer can reach 2.56 × 10)4) Cell fusion was performed.
2. Cell fusion and monoclonal antibody preparation
(1) Adopting a polyethylene glycol method, carrying out cell fusion on splenocytes of an immune mouse and myeloma cells SP2/0 of the mouse according to the ratio of the cell number to 8:1, and screening the fused cells by using an HAT selective medium;
(2) subpackaging in 96-well cell culture plate with feeder cells, placing at 37 deg.C and 5% CO2Culturing in an incubator, supplementing 50 mu L of culture medium to each hole on the fifth day after fusion, and after 12 days, taking N protein or polypeptide as a coating antigen, and detecting the OD value of hybridoma cell supernatant through an indirect ELISA method to be used as a primary screen of positive hybridomas;
(3) and performing subcloning screening on the positive hybridoma cells for 3-4 times by a limiting dilution method to finally obtain 13 hybridoma cell strains (1A1, 1A3, 1D2, 1F5, 1G1, 2B3, 2C4, 2E4, 2F8, 3A8, 3A5, 3C6 and 3G1) capable of stably secreting the anti-N protein monoclonal antibody.
3. Immunoperoxidase monolayer cell assay (IPMA)
The IPMA operation process comprises:
inoculating chick embryo kidney Cells (CEK) to a 96-well cell plate, inoculating the cells until the cell confluence is about 80%, and supplementing an M199 culture medium of 2% fetal bovine serum 1h after inoculation; cells were fixed after 48 h: discarding the culture medium in a 96-well plate, washing with PBS for 2-3 times, adding 50 uL/well of a-20 ℃ precooled fixing solution (methanol), and standing for 15min at room temperature; discarding the fixing solution, washing with PBS for 3 times, adding skimmed milk into 200 uL/hole, sealing at 4 deg.C overnight; removing the blocking solution, washing with PBS for 3 times, adding primary antibody with a certain dilution at 50 uL/hole, and keeping the temperature at 37 ℃ for 1 h; discarding the primary antibody, washing with PBS for 5-6 times, patting to dry, adding corresponding secondary antibody diluted by a certain time into 50 uL/hole, and keeping the temperature at 37 ℃ for 1 h; discarding the secondary antibody, washing with PBS for 5-6 times, beating to dry, performing 50 uL/hole, adding AEC color development liquid, and performing color development for 10 min.
By IPMA screening, monoclonal antibodies secreted by hybridoma cell lines 1A3, 1G1, 3A8, 3A5, 2E4, 1A1, 2B3 and 2C4 can be combined with IBV, FIG. 2 shows a graph of IPMA experimental results of monoclonal antibody 1A3, and can be seen from A, B, C three pictures in FIG. 2: with positive chicken serum as a positive control (FIG. 2A) and negative allantoic fluid as a negative control (FIG. 2C), monoclonal antibody 1A3 was able to bind to and develop color from IBV M41 strain (FIG. 2B). And (3) selecting 1A3, 3A8 hybridoma cell strains with strong IBV binding capacity for monoclonal antibody preparation and identification.
4. In vivo induced ascites method for preparing monoclonal antibody
Selecting female Balb/c mice, injecting 500 μ L sterilized paraffin into abdominal cavity, injecting obtained monoclonal hybridoma cells into abdominal cavity again after one week, the injection amount is 2 × 105After one week, ascites is extracted after the abdomen of the mouse is enlarged, the supernatant is centrifuged, and the ascites is purified by ammonium caprylate method.
5. Identification of monoclonal antibody ascites
(1) Measuring the titer of the monoclonal antibody, wherein the ascites induced by SP2/0 cells is used as a negative control, the ascites titer before and after purification is measured by an indirect ELISA method, and the ascites titer of the purified 1A3 monoclonal antibody can reach 1.64 × 106The ascites titer of the purified 3A8 monoclonal antibody can reach 5.12 × 106。
(2) Measuring the affinity of the monoclonal antibody, namely drawing an affinity constant curve through indirect ELISA measurement, and calculating to obtain that the affinity constant Ka of the 1A3 monoclonal antibody is 1.87 × 107L/mol, 3A8 monoclonal antibody has an affinity constant Ka of 6.49 × 108L/mol。
(3) Subtype identification: the subtype of the monoclonal antibody is identified by using a subtype identification kit, and the identification result shows that the monoclonal antibodies 1A3 and 3A8 belong to IgG1 types.
6. Stability characterization
And continuously culturing the established monoclonal hybridoma cell strain for 3 months and repeatedly freezing and storing by liquid nitrogen for resuscitation so as to identify the stability of the hybridoma cell.
EXAMPLE 3 purification of antibodies
The octanoic acid-ammonium sulfate method is adopted for antibody purification, and the operation method comprises the following steps:
(1) and (3) taking out the frozen monoclonal antibody ascites or antiserum, centrifuging at 6000r/min for 30min to obtain supernatant, and diluting by 5 times with sodium acetate buffer (0.06 mol/L pH4.8).
(2) The pH was adjusted to 4.5 with NaOH (5mol/L), stirred slowly at room temperature for 0.5 h, and octanoic acid was added to a final concentration of 25. mu.L/mL.
(3) Centrifuging at 6000r/min at 4 deg.C for 30min, and collecting supernatant.
(4) The resulting supernatant was filtered through a medium speed filter paper, and 10 XPBS buffer (pH7.4) was added thereto in a volume of 1/10, and after mixing well, the pH was adjusted to 7.4.
(5) Solid ammonium sulfate was added to the mixture at 4 ℃ in a ratio of 0.2778 g/mL, and the addition was repeated several times within 30 min.
(6) Centrifuging at 6000r/min for 20min, and discarding supernatant. Adding PBS (pH7.2) with the initial ascites volume of 1/3 for resuspension, dialyzing with PBS buffer solution at 4 ℃ for 24 h, changing the solution 3-4 times, collecting, subpackaging, and storing at-20 ℃.
EXAMPLE 4 preparation of colloidal gold test paper for IBV detection
1. Preparation of colloidal gold-labeled antibody
Firstly, preparing a colloidal gold solution:
preparing colloidal gold solution by a trisodium citrate method, which comprises the following steps:
putting 99mL ddw into a 200mL triangular flask with scales, heating and stirring the triangular flask on a heating magnetic stirrer, then adding 1m L1% chloroauric acid, heating and stirring until the mixture is boiling, then quickly adding 1.6m L1% trisodium citrate, continuously heating and stirring, observing that the color gradually changes from light yellow to dark red until the color does not change, then heating and stirring for 5min, then taking down the triangular flask for cooling, after recovering the room temperature, fixing the volume to 100mL by using sterile ddw, and using ultraviolet scanning to determine that the wavelength of the maximum absorption peak is 530nm and then storing at 4 ℃.
Preparing a gold-labeled antibody pad:
the anti-N protein monoclonal antibody 3A8 purified in example 3 was used as a gold-labeled antibody, the optimal binding pH was 7.8, and the ratio of colloidal gold to antibody was 25 μ g antibody/mL colloidal gold. After being treated by 1% BSA stabilizer, the labeled colloidal gold is uniformly adsorbed on a glass fiber membrane according to the amount of 60 muL per square centimeter, and is dried in vacuum at 37 ℃ for 1 hour, and the cutting width is 7mm, so that the gold-labeled antibody pad is obtained.
3. Preparation of coating film
The concentration of the anti-N protein monoclonal antibody 1A3 purified in example 3 was adjusted to 2mg/mL with a coating buffer, the concentration of goat anti-mouse IgG was adjusted to 1.8mg/mL with a coating buffer, the monoclonal antibody 1A3 was sprayed onto a detection line (T line) on the coating film (nitrocellulose membrane), and the goat anti-mouse IgG was sprayed onto a quality control region C line on the coating film, and the amounts of the monoclonal antibody 1A3 and the goat anti-mouse antibody were 1. mu.L/cm based on the amount of the coating solution. And (3) separating the quality control line and the detection line by 4mm, airing in an oven with the humidity of less than 30% and the temperature of 37 ℃ for 10 hours, and sealing the bag for later use.
4. Sequentially and mutually overlapping and sticking a sample pad (18 mM by 300mM in size, made of glass fiber cotton, and subjected to immersion treatment for 2h by 50mM and pH8.0 Tris-HCl buffer solution), a gold-labeled antibody pad (7 mM by 300mM in size, made of glass fiber cotton), a coating film (25 mM by 300mM in size, made of nitrocellulose) and absorbent paper (16 mM in size) on a bottom lining (a support plate 60 mM by 300mM in size), obtaining a test paper board, and cutting the test paper board into test strips with different widths according to requirements.
The structure of the finished test strip is shown in fig. 3 and 4, wherein: 1 is a support plate, 2 is a sample pad, 3 is a gold-labeled antibody pad, 4 is a coating film, 5 is absorbent paper, 6 is a detection line (T), 7 is a quality control line (C), and 8-1 and 8-2 are plastic coating films.
The sample adding device is assembled in a plastic shell, and a sample adding window (corresponding to the position of the sample pad 2) and a display hole (corresponding to the positions of the detection line T and the quality control line C) are arranged on a plastic upper cover.
Example 5 method of Using colloidal gold test paper for IBV detection and determination of detection result
After a sample to be detected is loaded on the sample pad 2 for filtration, if an object to be detected contains IBV antigen, the sample and the anti-IBV monoclonal antibody 3A8 marked on the gold-labeled antibody pad 3 form a corresponding compound, and the anti-IBV N protein monoclonal antibody 1A3 on the upward coated membrane 4 is intercepted to form a compound, namely a red strip is formed on the detection line 6 (T line); the colloidal gold labeled antibody, with or without IBV antigen, will be intercepted by goat anti-mouse IgG coated on control line 7 (C-line) in the up-flow, forming a red band on control line 7. The strip (line C) is the quality control line, and if the line does not appear, the colloidal gold test strip is invalid.
And (3) judging a detection result: if IBV exists in the sample, the T line (detection line) is colored, the C line (quality control line) is also colored, and if the test paper strip at the lower part in the figure 5 shows that the result is positive; if only C line (quality control line) is developed, and T line (detection line) is not developed, as in the upper test strip in FIG. 5, the result is negative; if the C line (quality control line) and the T line (detection line) do not develop color, the test strip is proved to be invalid.
Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes in the specific parameters of the embodiments may be made without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variations of the present invention, and will not be described in detail herein.
Claims (8)
1. The utility model provides a detect IBV's colloidal gold test paper, includes the backup pad, and the envelope membrane, the sample pad, the gold mark antibody pad, absorbent paper, detection line and quality control line, its characterized in that: the gold-labeled antibody pad is marked with IgG antibodies resisting IBV, the detection line is coated with monoclonal antibodies resisting IBV N protein, and the quality control line is coated with goat anti-mouse IgG antibodies.
2. The colloidal gold test paper for IBV detection according to claim 1, wherein: the ELISA titer of the anti-IBV N protein monoclonal antibody is not less than 106。
3. The colloidal gold test paper for IBV detection according to claim 1 or 2, characterized in that: the subtype of the anti-IBV N protein monoclonal antibody is IgG1 type.
4. The colloidal gold test paper for IBV detection according to claim 1, wherein: the coating concentration of the anti-IBV N protein monoclonal antibody is 2 mg/mL.
5. A preparation method of colloidal gold test paper for detecting IBV is characterized by comprising the following steps: comprises the following steps:
firstly, prokaryotic expression is carried out on IBV N protein gene, and exogenous N protein is obtained after protein purification;
secondly, immunizing a BALB/c mouse by taking the N protein purified in the first step as an antigen, screening to obtain an IBV hybridoma cell strain, and selecting the hybridoma cell strain with strong affinity to prepare an anti-IBV N protein monoclonal antibody;
thirdly, taking IgG resisting IBV as a gold-labeled antibody to be marked on a gold-labeled antibody pad; spraying the anti-IBV N protein monoclonal antibody prepared in the second step onto a detection line on the envelope; spraying/printing goat anti-mouse IgG antibody on the quality control line on the coating film; airing and sealing the bag for later use;
and fourthly, sequentially overlapping and adhering a sample pad, a gold-labeled antibody pad, a coating film and absorbent paper on the supporting plate, and cutting into a designed width to obtain a finished product of the colloidal gold test paper for detecting the IBV.
6. The method for preparing the colloidal gold test paper for detecting IBV according to claim 5, wherein the method comprises the following steps: the separation distance between the detection line and the quality control line is 4 mm.
7. The method for preparing the colloidal gold test paper for detecting IBV according to claim 5, wherein the method comprises the following steps: and when the gold-labeled antibody pad is manufactured in the third step, firstly, preparing a colloidal gold solution by a trisodium citrate method, treating the colloidal gold solution and the antibody by a stabilizer, adsorbing the colloidal gold solution and the antibody on a glass fiber membrane, and drying in vacuum to obtain a finished gold-labeled antibody pad.
8. The method for preparing the colloidal gold test paper for detecting IBV according to claim 7, wherein the method comprises the following steps: the ratio of the colloidal gold solution to the antibody is 25 mug antibody/mL colloidal gold; the pH value during the adsorption was 7.8.
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