LU101938B1 - A Test Strip for Testing Infectious Laryngotracheitis Virus, the Preparation Method, Test Method and Kit - Google Patents
A Test Strip for Testing Infectious Laryngotracheitis Virus, the Preparation Method, Test Method and Kit Download PDFInfo
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- LU101938B1 LU101938B1 LU101938A LU101938A LU101938B1 LU 101938 B1 LU101938 B1 LU 101938B1 LU 101938 A LU101938 A LU 101938A LU 101938 A LU101938 A LU 101938A LU 101938 B1 LU101938 B1 LU 101938B1
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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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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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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
- 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
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies 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
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Abstract
The invention discloses a test strip for testing infectious laryngotracheitis virus (ILTV), the preparation method, test method and kit, which relate to the technical field of ILTV testing, specifically comprising a base plate and the water absorbing material adhered to the upper edge of the base plate, with a sample pad, a colloidal gold binding pad and a nitrocellulose membrane pre-spotted with T line and C line successively laid on the upper surface of the base plate along the direction of chromatography, and the upper edge of the nitrocellulose membrane is overlapped with the lower edge of the water absorbing material, the lower edge of the nitrocellulose membrane is bonded with the upper edge of the colloidal gold binding pad, the lower edge of the colloidal gold binding pad is bonded with the upper edge of the sample pad, and the T line is coated with ILTV monoclonal antibody.The test strip is characterized by high specificity, good repeatability and high sensitivity for testing of ILTV.
Description
| 1 LU101938 A Test Strip for Testing Infectious Laryngotracheitis Virus, the Preparation Method, Test Method and Kit Technical Field The invention relates to the technical field of ILTV testing, in particular to a test strip for testing ILTV, the preparation method, test method and kit.
Background to the Invention At present, avian infectious laryngotracheitis (AILT) is an acute and contagious upper respiratory tract infectious disease caused by infectious laryngotracheitis virus (ILTV), which spreads rapidly.Since the discovery of the disease in Rhode Island, USA in 1925, it has now spread throughout chicken raising areas around the world.
The main lesions of the disease are in the laryngeal and tracheal tissues and are clinically characterized by dyspnea, cough, and expectoration of blood like exudates.
Once a susceptible chicken flock is infected with the disease, the disease will spread rapidly, with an infection rate of more than 90% and a mean mortality rate of 10% ~ 20%. The egg production of laying hens will decrease after infection, causing great economic losses to the chicken industry.
The clinical symptoms and pathological changes of the disease are easily confused with diseases caused by other viruses such as infectious bronchitis virus (IBV) and newcastle disease virus (NDV). Misdiagnosis is likely, which brings some difficulties to clinical diagnosis.
Therefore, it is usually necessary to confirm the diagnosis with the help of laboratory methods, which take a long time to confirm the diagnosis and are not conducive to rapid testing in actual production.
Immune colloidal gold technique is a novel immunolabelling technique applied to antigen-antibody with colloidal gold as the tracing marker.Colloidal gold is formed by the polymerization of chloric acid (HAuCl4) into gold particles of specific size under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, and tannic acid, and maintains a stable colloidal state due to electrostatic action, which is called colloidal gold.Colloidal gold is negatively charged in a weak base environment and can firmly bind with the positively charged groups of protein molecules.
Because the binding is electrostatic, it does not affect the biological characteristics of proteins.In addition to binding with proteins, colloidal gold can also bind with many other biological macromolecules, such as SPA, PHA, and ConA.According to some physical characteristics of colloidal gold, such as high electron density, particle size, shape and color reaction, coupled with the immune and biological | characteristics of conjugates, colloidal gold is widely used in the fields of immunology, LU101938 histology, pathology and cell biology, with the advantages of convenience, high efficiency, low cost, wide application scope, etc. Summary of the Invention The first objective of the invention is to provide a test strip for testing ILTV. The test strip can be used for rapid testing of ILTV by colloidal gold immunochromatography.
The second objective of the invention is to provide the preparation method of the test strip for testing ILTV.
The third objective of the invention is to provide a method to test ILTV with the test strip, which is characterized by high specificity, good repeatability and high sensitivity.
The fourth objective of the invention is to provide an ILTV test kit, which has the advantages of convenient operation, broad testing scope of samples, fast testing speed and high sensitivity.
The invention is realized as follows: A test strip for testing ILTV comprising a base plate and the water absorbing material adhered to the upper edge of the base plate, with a sample pad, a colloidal gold binding pad and a nitrocellulose membrane pre-spotted with T line and C line successively laid on the upper surface of the base plate along the direction of chromatography, and the upper edge of the nitrocellulose membrane is overlapped with the lower edge of the water absorbing material, the lower edge of the nitrocellulose membrane is bonded with the upper edge of the colloidal gold binding pad, the lower edge of the colloidal gold binding pad is bonded with the upper edge of the sample pad, and the T line is coated with ILTV monoclonal antibody.
In the good embodiment of the invention, colloidal gold-ILTV monoclonal antibody conjugate is added on the colloidal gold binding pad, the colloidal gold-ILTV monoclonal antibody conjugate is the diluent of colloidal gold-ILTV monoclonal antibody conjugate, with the dilution ratio of 1:3.0-4.2 or 1:2.0-2.9, and the minimum antibody amount required for stabilizing 1 mL of colloidal gold by infectious laryngotracheitis virus monoclonal antibody is 10 ug/ml; Preferably, the dilution ratio is 1:4.
In the good embodiment of the invention, the adding amount of ILTV monoclonal antibody on the T line is 36-51 pg or 30-35pg, the C line is coated with X anti-mouse IgG antibody, the X is any of goat, rabbit, pig, horse and monkey, and the adding amount of X anti-mouse IgG antibody is 24-30 ug or 20-23 ug.
Preferably, the adding amount of ILTV monoclonal antibody on the T line is 45 pg. The LU101938 C line is coated with goat anti-mouse IgG antibody, and the adding amount of goat anti-mouse IgG antibody is 30 pg.
In the good embodiment of the invention, the nitrocellulose membrane is any of s Millipore HF135, Whatman Immunopore PR, Whatman PRIMA40 and Sartorius CN140; Preferably, the nitrocellulose membrane is Sartorius CN140.
A preparation method of the test strip for testing ILTV comprising the following steps: (1) Providing water absorbing material, nitrocellulose membrane pre-spotted with T line and C line, colloidal gold binding pad and sample pad; (2) Laying the water absorbing material, nitrocellulose membrane, colloidal gold binding pad and sample pad successively on the base plate to form the big plate of test strip.
In the good embodiment of the invention, the preparation of nitrocellulose membrane pre-spotted with T line and C line specifically includes the following steps: first, the ILTV monoclonal antibody and X anti-mouse IgG antibody are respectively diluted to 1.2-1.7 mg/ml and 0.8-1.1 mg/ml with PBS, then the ILTV monoclonal antibody is sprayed on the nitrocellulose membrane with a spotting volume of 1 ul/cm as the T line, and the X anti-mouse IgG antibody is sprayed with a spotting volume of 1 pl/cm as the C line, while ensuring that the distance between the T line and the C line is 0.5-0.8 cm; Preferably, the distance between the T line and the C line is 0.5 cm.
In the good embodiment of the invention, the preparation method also comprises the preparation of colloidal gold binding pad, in particular, labeling the ILTV monoclonal antibody with colloidal gold at pH 8.0-8.1; Preferably, the ILTV monoclonal antibody is marked by colloidal gold at pH 8.0.
A method to test ILTV with a test strip comprising the following steps: adding 20-100 pl of sample to be tested on the sample pad, and observing the color development result 5-10 min after sample addition.
In the good embodiment of the invention, the sample to be tested is any of serum, plasma, cell culture supernatant, urine, animal tissue lysate and plant tissue lysate.
A kit for testing ILTV comprising a test strip, with a detection limit of 100ng/ml.
The invention has the following beneficial effects: The invention provides a test strip for testing ILTV, the preparation method and test method. The test strip has the advantages of broad testing scope of samples, fast testing speed, high sensitivity and strong specificity, which is conducive to effective prevention and control -—___________________ _— — — —_— —_————
at the early stage of ILTV outbreak In addition, the test strip provided by the invention is easy LU101938 to operate, with low requirement for the professional level of the operator and high specificity, thus avoiding misdiagnosis. Brief Description of the Figures Figure 1 is the structure diagram of the test strip; Figure 2 is the result determination diagram of the test strip (2a: positive control, 2b: negative control, and 2c and 2d: invalid test strip); Figure 3 is the specificity test result diagram of the test strip (3a: 5000 ng/ml, 3b: 500 ng/ml, 3c: 50 ng/ml, 3d: PBS negative control); Figure 4 is the result diagram of repeatability experiment of the test strip (4a-d: samples of different batches, 4e and 4f: negative PBS control group). Detailed Description of the Invention Embodiment 1 The invention provides a test strip for testing ILTV. As shown in Figure 1, the test strip comprises a base plate and the water absorbing material adhered to the upper edge of the base plate.In this embodiment, the base plate is preferably a PVC plywood. The sample pad, colloidal gold binding pad and nitrocellulose membrane pre-spotted with T line and C line are successively laid on the upper surface of the bottom plate along the direction of chromatography, and the upper edge of nitrocellulose membrane is overlapped with the lower edge of the water absorbing material; the lower edge of the nitrocellulose membrane is bonded with the upper edge of the colloidal gold binding pad; the lower edge of colloidal gold binding pad is bonded with the upper edge of the sample pad; T line is coated with ILTV monoclonal antibody. The test line T and quality control line C in Figure 1 represent the T line and C line respectively.
Further, when preparing the test strip for the testing of ILTV, the following parameters need to be determined: the optimal pH for antibody labeling, the optimal labeling amount of antibody, the concentration of working solution on the colloidal gold binding pad, the type of nitrocellulose membrane, and the optimal test conditions of T line and C line.
A preparation method of the test strip for testing ILTV comprising the following steps: (1) Determination of optimal pH for antibody labeling.
The ILTV monoclonal antibody is independently developed.The ILTV monoclonal antibody 2D4 1D7 (McAb 1) is labeled with colloidal gold with a mass volume fraction of |
\ ibod cor the binding between fe antiboy nd the optimal pH 10 “fic operation LU101938 : cle size of 20 NM. a dient method. The spect p
0.02% and a partic ned by colloidal gold gras . i {erm and the colloidal gold 15 dete | of prepared . ing ml oI pre steps are as follows: 11 glass test tubes, and respectively adding 570 Taking à PUY © PE dusting the pH of the colloidal gold solution to 6.2, 7% son: tively adjus . 5 colloidal gold solution; respec > JL potassium carbonate solution; taking 50 ul of 1 ith 0.2 MO . 75, 8.0, 8.5, 9.0 and 9.5 WI! ely adding into the test tubes containing colloidal gold, ectively . mg/ml ILTV McAb | and resP 0 min: then respectively adding d allowing to stand at room temperature for 20 mn; P À Oo mixing well and ® . vine well and allowing to stand at room 100 wl of 10% NaCl solution to each tube, MIXING £ colloidal gol, and recordin the lowest temperature for 1-2 h; observing the color change © © the | di ° . . + . pH to keep the colloidal gold red: then adjusting the pH to thelowest gradient pH + 0.1; repeating the above test;recording the lowest PH to keep red, whid is the optimal pH. The optimal pH of ILTV McAb 1 bound with colloidal goldis determined to be 8.0 by colloidal gold gradient test. 15 . (2) Selection of optimal labeling amount for labeling antibodyın colloidal gold. The optimal concentration of ILTV McAb | for bindin with colloi dal gold is determined by protein gradient method.The specific operation tM as follows: i i 11 glass test tub tively ad ; Selecting a plurality of we g = = | S or y ea 7 1ml of colloidal gold i timal pH is adjusted to 8.0; diluti Cc solution whose * me P 5 A © i" , ne 280 Loft em with purified water, respective awing 0, J, LU, ZU, 3U, 40, an ul 0 € oP ’ ° . "NE of ILTV McAb | and adding into the small test tubes containing colloidal gold solution. | ‘equence, mixing well; allowing to stand for 10 min, adding 0.1 ml of 10% NaCl aqueous sc. 2 . 0 in each small test tube, mixing well, allowing to stand at room temperature for 1-2 h, : \bserving the color change of small test tubes. In the blank control group, coagulation can be observed with the color ch, fr to blue, because the amount of protein added is insufficient to stabilize the om red colloidal gold. When the amount of protein added to the test tube reaches or be of minimum amount required to stabilize the colloidal gold, the color will remain re the out the junction tube in which the colloidal gold solution changes from red to vs amount of protein contained in the tube is the minimum protein amount required to std. ml of colloidal gold.In the actual preparation of colloidal gold probe, the amount of ant added to colloidal gold is often 120% — 130% of the minimum protein amount. The protein gradient test shows that the minimum amount of ILTV McAb 1 required
| stabilize 1 mi of colloidal gold is 10 pg/ml. (8) preparation of colloidal gold probe. Adding 200 V8 of ILTV McAb | into 20 mL of colloidal gold solution with optimal pt 01938 and stirred at room «emperaturé for 30 min to prepare colloidal gold-antibody conjugate solution. Adding another mi of 10% BSA solution into the prepared colloidal gold-antibody conjugate solution (final concentration of BSA: 0.4%), and stirring at room temperature for min; or adding 0.2m} of 10% polyethylene glycol into the colloidal gold-antibody conjugate solution. and stirring at room temperature for 10 min Centrifugins the above stirred solution at 11,000 ¢/min for 40 min, discarding the supernatant; dissolving the precipitate in2 10 ml of colloidal gold-antibod preservation solution, and filtering with 0.43 yum filter membrane» and the obtained solution is colloidal gold-antibody conjugate stock solution.
After 1 mi of 10% NaCl aqueous solution is added to 1 ml of colloidal gold-antibody conjugate stock solution, {he solution is still à yiolaceous liquid without precipitation, indicating that the prepared colloidal gold-antibody conjugate stock solution had good stability 4) Determination of working concentration of colloidal gold-antibody conjugate stock solution and preparation of colloidal gold binding pad. Diluting the colloidal gold-antibody conjugate stock solution by 1:2, 1:4, 1:8 and 1:16 respectively with the working solution;taking 1.4 ml of the diluent of colloidal gold-antibody conjugates evenly adding on the glass fiber membrane, drying at 37°C, and the colloidal gold binding pad is prepared;after the test, determining the optimal working concentration of colloidal gold-labeled antibody. The results show that the prepared colloidal god binding pad has the best test effect whe the working dilution concentration of colloidal gold-antibody conjugate stock solution is 1:4 (5) petermination of the model of nitrocellulose membrane.
Millipore HF135, Whatman Immunoporé PR, Whatman PRIMA40 and Sartorius CN! are selected as pitrocellulose membranes, and the optimal model of nitrocellulose membr is determined to be Sartorius CN140 by plate running function test, test of fluidity, hyste and background residue of colloidal gold solution on different models of nitrocell membranes, reselection test of nitrocellulose membranes of other tests.
6) Optimal test conditions of T line and C line. Setting the following concentration gradients for the coating antibodies of T line line on nitrocellulose membrane: 1 mg/m}, 1.5 mg/ml, 2 mg/ml and 2.5 mg/ml. Select group with optimal color development effect as the application concentration 0
| monoclonal antibody 1D$ 1G3 (Me IN) (X ine) and goat anti-mousé 1gG antibody (C 1ine)- | The results show {hat the group with the optimal color dev etopment effect is as follows the working coneentsatio® of LIV McAb 2 (on T Yime) 18 1.5 mg/d and the W orkMB 5101938 concentration of goat anti-mousé 1gG antibody (on C line) isl mg/m | (M) Potting T line and C ine on the pivrocelhuloSé membrané with à membrané cutting machine- specifically aituting ILTV cho 2 and 207 anti-mouse 186 antibody © 1 mgt and 1 g/mol respectively with 0.01 moVL pH 30 PBS and dotting °° the nitrocel1ul05® membrane with à membrané scribinS machine The distance between the T 110€ and the C line 0 18 0.5 CM.
Spraying goat anti-mousé 1gG antibody at an application yolume of | ul/em as the C line. and spraying [LTV Me Ab 2 ata application volume of 1 pe as the T line, drying the sprayed nitrocellulose membrane at37° C for more than 8 n. (8) preparing the PVC basé plate, {he water absorbing material. the nitrocellulose membrane in step (7), the colloidal gold binding pad and sample pad in step (4). AS shown in Figure b> pasting the pitrocelluloSé membrane in the middle of the PVC base plate, SC that the upper edge of pitrocellulosé membrane 1s maintained at 1.5 CM from the upper edge of the PVC base plate; pasting the colloidal gold pinding pad on the lower edge of the pitrocellulosé membrane so that the colloidal gold binding pad overlaps with the pitrocelluloSé membrane by 0-1 cm, and then pasting a 1.7 cm wide sample pad on the lower edge of colloidal gol binding pad SO that the sample pad overlaps with the colloidal gold pinding pad by Ô em.
Finally: pasting the 17 cm water absorbing material on the upper edge of t pitrocelluloSé membrane (close to fhe C line end of the pitrocelluloSé membrane) go that water absorbing material overlaps with the upper edge of the pitroceliulose membrane by cm.
The plate obtained is the big plate of test strip- Cutting the big plate of test strip INÉC strips Of 2,8 mm wide and 6 CM long.
Embodiment 2 This embodiment provides {he result determination method of ILTV test striP- Randomly taking test strips» and testing OB the standard ILTV negative sample ( diluent) and the standard ILTV positive sample; the test results are shown in Figw results in Figure 7 show that the negative sample (Figure 2b) shows only one rose be at C lime. while the standard Lv positive sample (Figure 22) shows two FOSÉ ber ie.
T line and C line- The result determination principle of the [LIV test strip provided by the inv follows: after the sample © be tested 18 added to the sample pad, the sample
| colloidal gold binding pad under the action of chromatography. If the sample to be tested is LU101938 infected with ILTV, the ILTV in the sample to be tested is specifically recognized by the colloidal gold-labeled antibody in the colloidal gold binding pad, forming the ILTV-antibody-colloidal gold complex. The complex continues to move forward along the direction of chromatography to the nitrocellulose membrane, the antigen binds with the matching antibody on the test T line, forming the T line matching antibody-ILTV-antibody-colloidal gold complex. When the colloidal gold accumulates at the T line matching antibody, red or pink spots are visible to the naked eye on the T line, thus the test and recognition of antigen are realized.Excess sample solution quickly swims the antibody on the colloidal gold binding pad to the C line, so that the antibody on the colloidal gold binding pad specifically binds and aggregates with the goat anti-mouse antibody coated on the C line, so that red or pink spots are visible on the C line. If the sample is not infected with ILTV and does not contain ILTV antigen, when the sample enters the colloidal gold binding pad from the sample pad, since the sample does not carry antigen, it does not bind with the antibody on the colloidal gold binding pad and T line. It moves forward to the C line under the effect of chromatography, and color development can be observed only at the C line. Therefore, it can be seen from Figure 2 that the test strip prepared for this application is effective.lf no color development is observed at the C line, the test strip is determined to be ineffective, regardless of whether color development is observed at the T line. Embodiment 3 This embodiment provides a sensitivity test for the testing of ILTV.Replacing the sample stock solution with 50 ng/ml, 500 ng/ml and 5000 ng/ml of the sample diluent, and using PBS solution as the negative control to determine the specificity of the test strips prepared in Embodiment 1.The determination results are shown in Figure 3. Only 1 rose bengal line appears at the C line for the negative control and sample diluent of 50 ng/ml, as shown in Figures 3c and 3d; 2 rose bengal lines (T line and C line) appear for the 500 ng/ml and 5,000 ng/ml sample diluent, as shown in Figures 3a and 3b. Therefore, based on the total protein concentration of the virus, the test sensitivity of the test strip provided by the invention is determined as 100 ng/ml. Embodiment 4 This embodiment provides a repeatability test for the testing of ILTV. Taking the test strips prepared in Embodiment 1 and testing 5 samples of different batches and 5 samples of the same batch;repeating the test on each sample for 10 times. The test results are completely
A | consistent.
The test results are consistent among different batches and within the same batch. | y101938 Some test results are provided in Figure 4. As shown in Figures 4a, 4b, 4c, 4d, 4e and 4f, the test strip provided by the invention has good repeatability.
Claims (10)
1. A test strip for testing ILTV, characterized in that it comprises a base plate and the water absorbing material adhered to the upper edge of the base plate, with a sample pad, a colloidal gold binding pad and a nitrocellulose membrane pre-spotted with T line and C line successively laid on the upper surface of the base plate along the direction of chromatography, and the upper edge of the nitrocellulose membrane is overlapped with the lower edge of the water absorbing material, the lower edge of the nitrocellulose membrane is bonded with the upper edge of the colloidal gold binding pad, the lower edge of the colloidal gold binding pad is bonded with the upper edge of the sample pad, and T line is coated with ILTV monoclonal antibody.
2. The test strip for testing ILTV as claimed in Claim 1, characterized in that colloidal gold-ILTV monoclonal antibody conjugate is added on the colloidal gold binding pad, the colloidal gold-ILTV monoclonal antibody conjugate is the diluent of the colloidal gold-ILTV monoclonal antibody conjugate, the dilution ratio of the colloidal gold-ILTV monoclonal antibody conjugate is 1:3.0-4.2 or 1:2.0-2.9, and the minimum amount of the ILTV monoclonal antibody labeled with colloidal gold per unit volume is 10 pg.
3. The test strip for testing ILTV as claimed in Claim 1 characterized in that the adding amount of ILTV monoclonal antibody on the T line is 36-51 pg or 30-35ug, the C line is coated with X anti-mouse IgG antibody, the X is any of goat, rabbit, pig, horse and monkey, and the adding amount of X anti-mouse IgG antibody is 24-30 pg or 20-23 pg.
4. The test strip for testing ILTV as claimed in Claim 1, characterized in that the nitrocellulose membrane is any of Millipore HF135, Whatman Immunopore PR, Whatman PRIMA40 and Sartorius CN 140.
5. A method for preparing the test strip for testing ILTV as claimed in Claims 1-4, characterized in that it comprises the following steps: (1) Providing water absorbing material, nitrocellulose membrane pre-spotted with T line and C line, colloidal gold binding pad and sample pad, and the T line is pre-coated with ILTV monoclonal antibody; (2) The water absorbing material, the nitrocellulose membrane, the colloidal gold binding pad and the sample pad are successively laid on the base plate, so that the upper edge of the nitrocellulose membrane is overlapped with the lower edge of the water absorbing material, the lower edge of the nitrocellulose membrane is bonded with the upper edge of the colloidal gold binding pad, and the lower edge of the colloidal gold binding pad is bonded with the | upper edge of the sample pad to form a big plate of test strip. LU101938
6. The preparation method as claimed in Claim 5, characterized in that the preparation of the nitrocellulose membrane pre-spotted with T line and C line specifically includes the following steps: first, the ILTV monoclonal antibody and X anti-mouse IgG antibody are respectively diluted to 1.2-1.7 mg/ml and 0.8-1.1 mg/ml with PBS, then the ILTV monoclonal antibody is sprayed on the nitrocellulose membrane with a spotting volume of 1 pl/cm as the T line, and the X anti-mouse IgG antibody is sprayed with a spotting volume of 1 ul/cm as the C line, while ensuring that the distance between the T line and the C line is 0.5-0.8 cm;
7. The preparation method as claimed in Claim 5, characterized in that it includes the preparation of a colloidal gold binding pad, specifically including the labeling of ILTV monoclonal antibody with colloidal gold at pH 8.0-8.1.
8. A method for testing ILTV with the test strip as claimed in any of Claims 1-4, characterized in that it comprises the following steps: adding 20-100 pl of sample to be tested on the sample pad, and observing the color development result 5-10 min after sample addition.
9. The method for testing ILTV with the test strip as claimed in Claim 8, characterized in that the sample to be tested is any of serum, plasma, cell culture supernatant, urine, animal tissue lysate, and plant tissue lysate.
10. A test kit for ILTV, characterized in that it comprises the test strip as claimed in Claim 1, and the detection limit of the test kit is 100 ng/ml.
LE
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101938A LU101938B1 (en) | 2020-07-21 | 2020-07-21 | A Test Strip for Testing Infectious Laryngotracheitis Virus, the Preparation Method, Test Method and Kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101938A LU101938B1 (en) | 2020-07-21 | 2020-07-21 | A Test Strip for Testing Infectious Laryngotracheitis Virus, the Preparation Method, Test Method and Kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| LU101938B1 true LU101938B1 (en) | 2021-01-27 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LU101938A LU101938B1 (en) | 2020-07-21 | 2020-07-21 | A Test Strip for Testing Infectious Laryngotracheitis Virus, the Preparation Method, Test Method and Kit |
Country Status (1)
| Country | Link |
|---|---|
| LU (1) | LU101938B1 (en) |
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2020
- 2020-07-21 LU LU101938A patent/LU101938B1/en active IP Right Grant
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Effective date: 20210127 |