Colloidal carbon test strip for detecting swine transmissible gastroenteritis virus based on nano antibody and application of colloidal carbon test strip

Abstract

The invention belongs to the technical field of virus immunodetection, and relates to a colloidal carbon test strip for detecting swine transmissible gastroenteritis virus based on a nano antibody and application of the colloidal carbon test strip. According to the TGEV colloidal carbon detection method established by the invention, Mab 12H6 is screened as a carbon labeled antibody, the concentration of N protein coated by a T line is 0.7 mg / mL, the use amount of a probe is 6 [mu] L, and the detection time is 10 min, a virus solution (5 * 10 < 4 > TCID50) with the antigen content of 1 / 256 can be sensitively detected, the specificity is good, and the detection coincidence rate of clinical test products is 95.2%. The test strip has the advantages of high specificity, high sensitivity, simplicity and convenience in operation, low production cost and the like, and can be applied to rapid detection of TGEV in a porcine diarrhea sample clinically.

Description

Technical Field

[0001] This invention belongs to the field of viral immunoassay technology and relates to a test strip for detecting porcine transmissible gastroenteritis virus. Background Technology

[0002] Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by the transmissible gastroenteritis virus (TGEV). It is characterized by severe vomiting, severe diarrhea, and high mortality in piglets, posing a significant threat to the pig industry. TGEV belongs to the family Coronaviridae, genus Coronavirus. While the virus is not highly resistant to environmental factors, it is resilient to low temperatures, making outbreaks more likely in winter and early spring. Pigs of all ages are susceptible, but piglets under 10 days old have the highest morbidity and mortality rates, often reaching 100%. Finishing pigs and adult pigs often recover from infection, but it can cause severe growth retardation and feed waste. The virus leads to a large number of newborn piglet deaths, resulting in direct economic losses. It can also prolong the growth period of surviving pigs, reduce feed conversion ratios, and extend the time to market. Furthermore, its clinical symptoms are very similar to those of porcine epidemic diarrhea (PED) and rotavirus infection, making misdiagnosis very easy and delaying optimal prevention and control.

[0003] While traditional TGEV laboratory testing methods each have their advantages, they also have significant shortcomings in practical production applications. For example, application CN120366519A discloses a reagent and its application for porcine epidemic diarrhea virus nucleic acid extraction and amplification. This detection method requires nucleic acid amplification, necessitates specialized instruments, has a long testing time, and cannot achieve real-time, large-scale testing. It fails to meet the modern livestock industry's diagnostic needs for "early, fast, accurate, and simple" diagnosis.

[0004] To address the shortcomings of traditional testing methods, the development of a rapid, accurate, simple, and low-cost on-site testing tool is urgently needed. Immunochromatographic assay strips (ICA), also known as rapid test strips, are an ideal solution to meet this need. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanoantibodies and its application.

[0006] The technical solution of this invention is implemented as follows: A colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobodies includes a base plate and a sample pad, a conjugate pad, a detection pad, and an absorbent pad stacked sequentially on the base plate. The detection pad has a control line (C line) and a detection line (T line). The conjugate pad contains colloidal carbon-labeled Mab 12H6 antibody.

[0007] Furthermore, the aforementioned Mab 12H6 antibody includes a Mab 12H6 light chain and a Mab 12H6 heavy chain. The Mab 12H6 light chain includes CDR1 as in SEQ ID No. 1, CDR2 as in SEQ ID No. 2, and CDR3 as in SEQ ID No. 3. The Mab 12H6 heavy chain includes CR1 as in SEQ ID No. 4, CR2 as in SEQ ID No. 5, and CR3 as in SEQ ID No. 6.

[0008] Specifically, the Mab 12H6-light chain further includes VL-FR1 as in SEQ ID No. 7, VL-FR2 as in SEQ ID No. 8, and VL-FR3 as in SEQ ID No. 9. The Mab 12H6-heavy chain further includes VH-FR1 as in SEQ ID No. 10, VH-FR2 as in SEQ ID No. 11, and VH-FR3 as in SEQ ID No. 12.

[0009] Preferably, the amino acid sequence of the Mab 12H6-light chain is shown in SEQ ID No. 13, and the amino acid sequence of the Mab 12H6-heavy chain is shown in SEQ ID No. 14.

[0010] The amount of colloidal carbon-labeled Mab 12H6 antibody added to the above-mentioned binding pad was 6 μL, and the antibody concentration was 1 mg / mL.

[0011] The detection line T contains TGEN N protein, and the control line C contains goat anti-mouse IgG antibody.

[0012] The concentration for the detection line T is 0.7 mg / mL; the concentration for the control line C is 1 mg / mL.

[0013] The above-mentioned colloidal carbon test strip is used in the preparation of products for detecting porcine transmissible gastroenteritis virus. The detection time using the test strip of this application is 10 minutes. The present invention has the following beneficial effects: The TGEV colloidal carbon detection method established in this invention, using Mab 12H6 as the carbon-labeled antibody, with a T-line coated N protein concentration of 0.7 mg / mL, a probe volume of 6 μL, and a detection time of 10 min, achieves a sensitivity capable of detecting viral fluid with an antigen content of 1 / 256 (5 × 10⁻⁶). 4 TCID 50 The test strip exhibits high specificity and good specificity, with a clinical sample detection accuracy rate of 95.2%. It boasts advantages such as high specificity, high sensitivity, ease of operation, and low production cost, making it suitable for rapid detection of TGEV in porcine diarrhea samples in clinical settings. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 Determination of antibody screening for TGEV N colloidal carbon probes.

[0016] Figure 2 To determine the concentration of TGEV N protein coating.

[0017] Figure 3 Determination of the amount of Mab 12H6 antibody added.

[0018] Figure 4 Determining the amount of Mab 12H6 probe used.

[0019] Figure 5 Determining the optimal color development time for TGEV.

[0020] Figure 6 Sensitivity testing of TGEV colloidal carbon test strips.

[0021] Figure 7 This is for the specific detection of TGEV colloidal carbon test strips. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0027] Example 1: Screening of antibodies and determination of coating concentration on colloidal carbon test strips 1. Preliminary screening of antibodies for TGEV colloidal carbon test strips Preliminary antibody screening was conducted using six existing antibodies: Mab 12H6, TGEV polyclonal antibody, Mab 5B4, Mab 6H9, Mab 6H10, and Mab 13G7. These antibodies were used as probes and bound to colloidal carbon. The pH was adjusted before adding an appropriate amount of antibody. The mixture was incubated with shaking for 30 min, followed by the addition of 10% BSA blocking agent and another 30 min of shaking incubation. Six probe antibodies were prepared. TGEV N protein (0.7 mg / mL) was drawn along the T line on the NC membrane, and goat anti-mouse or goat anti-rabbit antibody was drawn along the C line. The sample consisted of TGEV virus solution (10...). 7 TCID 50 The virus solution was mixed with different probe antibodies, and the preliminarily prepared test strips were inserted into the different mixtures. The color development results were recorded after ten minutes. A deeper T-line indicated better pairing, and the antibody with the deepest T-line was used for subsequent condition optimization. Results are as follows: Figure 1 As shown in the results, Mab 12H6 performed best in antigen detection, with its colorimetric signal intensity significantly higher than that of other antibodies.

[0028] The sequence of the Mab 12H6 antibody was sequenced, and the results are as follows: Mab 12H6 antibody light chain: FR1 amino acid: DIVLTQSPASLAVSLGQRATISCRAS; FR1 nucleotide: ttgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatatcctgcagagccagtgaa; CDR1 amino acid: ESVDRYGNSF; CDR1 nucleotide: agttgatcgttatggcaatagttttatg; FR 2 amino acids: MHWFQQKPGQPPKLLIY; FR 2 nucleotide: cactggttccagcagaaaccaggacagccacccaaactcctcatctatcg; CDR2 amino acid: RAS; CDR2 nucleotide: tgcatccaa; FR3 amino acids: NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC; FR3 nucleotides: cctagaatctgggatccctgccaggttcagtggcagtgggtctaggacagacttcaccctcaccattaatcctgtggaggctgatgatgttgcaacctattactgt; CDR3 amino acid: QQSNEDPYT; CDR3 nucleotide: cagcaaagtaatgaggatccgtacacg; The amino acid sequence of the Mab 12H6- light chain is: DIVLTQSPASLAVSLGQRATISCRASESVDRYGNSF MHWFQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYT.

[0029] Mab 12H6 antibody heavy chain: FR1 amino acids: QVQLQQSGAELMRPGASVKLSCKAS; FR1 nucleotide: tccagctgcagcagtctggagctgagctgatgaggcctggagcttcagtgaagctgtcctgcaaggcttctggc; CR1 amino acid: GFTFTSSW; CR1 nucleotide: ttcaccttcaccagctcctggatg; FR2 amino acid: MHWAKQRPGQGLEWIGE; FR2 nucleotide: cactgggcgaagcagaggcctggacaaggccttgagtggattggagaaat; CR2 amino acid: IHPNSINT; CR2 nucleotide: tcatcctaatagtattaatactaa; FR3 amino acids: NYNEKFKGKATLTVDTSSSTAYVDLSRLTSEDSAVYYC; FR3 nucleotides: Ctacaatgaaaaattcaagggcaaggccacactgactgtagacacatcctccagcacagcctacgtggatctcagtcgtctgacatctgaggactctgcggtctattactgt; CR3 amino acid: AREEVRGLDY; CR3 nucleotide: gcaagagaggaggtacgaggtttggactac; The amino acid sequence of the Mab 12H6- heavy chain is: QVQLQQSGAELMRPGASVKLSCKASGFTFTSSW MHWAKQRPGQGLEWIGEIHPNSINTNYNEKFKGKATLTVDTSSSTAYVDLSRLTSEDSAVYYCAREEVRGLDY.

[0030] 2. Selection of TGEV N protein coating concentration Mab 12H6 antibody was used as a monoclonal antibody that binds to colloidal carbon. Different concentrations of TGEV N protein were drawn along the T line, and goat anti-mouse antibody was drawn along the C line. The antibody and probe concentrations were fixed, as was the antibody concentration along the C line. The T line concentrations were set to 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, and 1 mg / mL, respectively. TGEV virus solution (10... 7 TCID 50The sample and probe antibody were mixed. Different pre-prepared test strips were inserted into the mixture and incubated for 10 min. The color development results were recorded; the darkest T-line indicated the optimal streak concentration. The streak concentration with the darkest T-line was used for subsequent condition optimization. Results are as follows... Figure 2 As shown, the colorimetric effect is best when the concentration of TGEV N protein coated on the detection line is 0.7 mg / mL and the concentration of goat anti-mouse IgG coated on the control line is 1 mg / mL.

[0031] Example 2: Selection of Mab 12H6 antibody and probe dosage 1. Selection of Mab 12H6 addition amount Mab 12H6 was used as a monoclonal antibody that binds to colloidal carbon, and different amounts of antibody were added during probe preparation. 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of antibody were added to 1 mL of colloidal carbon solution, respectively. The amount of probe added, the concentration of antibody at the C-line, and the concentration at the T-line were kept constant. TGEV virus solution (10... 7 TCID 50 The sample and probe antibody were mixed, and the preliminarily prepared test strips were inserted into the mixture and incubated for 10 min. The color development results were recorded; the darkest T line indicated the optimal antibody dosage. The antibody dosage with the darkest T line was used for subsequent condition optimization. Results are as follows: Figure 3 As shown in the figure, it can be observed by the naked eye that the T and C lines of the test strip show the best color development when 6 μL of antibody is added to each milliliter of colloidal carbon solution. Therefore, the optimal amount of antibody added to the test strip is 6 μL.

[0032] 2. Selection of Mab 12H6 probe dosage Mab 12H6 was used as the antibody binding to colloidal carbon. 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of probe were added to 200 μL of sample solution, respectively. The amount of probe added, the concentration of antibody at the C-line, and the concentration at the T-line were kept constant. TGEV virus solution (10... 7 TCID 50 The sample and different probe antibodies were mixed, and the preliminarily prepared test strips were inserted into the mixture and incubated for 10 min. The color development results were recorded. The darkest T-line indicated the optimal amount of probe antibody added. The amount of probe with the darkest T-line was used for subsequent condition optimization. Results are as follows: Figure 4As shown, a gradient of probe usage was established, with 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of probe added to 200 μL of sample solution, respectively. The figures show test strips prepared with different probe-antibody amounts. The results indicate that a probe usage of 10 μL yields the best results. The darkest T-line indicates the optimal probe-antibody dosage; the probe-antibody dosage with the darkest T-line was used for subsequent condition optimization.

[0033] Example 3: Determination of the optimal color development time for colloidal carbon and sensitivity testing of TGEV colloidal carbon test strips 1. Determination of the optimal color development time for colloidal carbon Mab 12H6 was used as the antibody to bind to colloidal carbon, and a fixed amount of probe antibody was added when preparing the probe. 10 μL of probe antibody was added to 200 μL of sample solution. The preliminarily prepared test strips were inserted into the mixture, and different incubation times were set: 8 min, 10 min, 12 min, and 15 min. The color development results were recorded; the time with the deepest T-line color development indicated the best color development time, and this time was used for subsequent condition optimization. Results are as follows... Figure 5 As shown, when the reaction proceeds for 10 minutes, the detection results reach a stable state, at which point the color intensity of both the T line and the C line is significant and stable.

[0034] 2. Sensitivity testing of TGEV colloidal carbon test strips Using Mab 12H6 antibody as a monoclonal antibody that binds to colloidal carbon, a fixed amount of probe antibody was added when preparing the probe. 10 μL of probe was added to 200 μL of sample solution. TGEV virus solution (10...) 7 TCID 50 Using this as a sample, different dilution factors were set to create a gradient. The probe and antibody mixture was fixed, and the pre-prepared test strips were inserted into the mixture. The same incubation time of 10 minutes was set. The color development results were recorded, and the closest fold between T color development, NC, and positive was used as the cutoff point, which is also the limit of detection for the lowest antigen amount. Results are as follows: Figure 6 As shown, a series of dilutions of TGEV virus stock solution were performed: the virus stock solution was serially diluted 10 times at an initial concentration of 1:2 (dilution range 1:2-1:1024), with DMEM culture medium used as a negative control. It was found that when the virus was diluted to a maximum of 1:256, the T-line color intensity of the test strip was lower than that of the negative control. Therefore, the limit of detection for TGEV virus on this test strip was determined to be a dilution of 1:256. This result indicates that the test strip has high detection sensitivity and can effectively identify low concentrations of virus samples. It is known that a virus solution with an antigen content of 1 / 256 (5 × 10⁻⁶) is... 4 TCID 50 ).

[0035] Example 4: Specificity detection and clinical sample testing of TGEV colloidal carbon test strips 1. Specificity detection of TGEV colloidal carbon test strips Mab 12H6 was used as the antibody binding to colloidal carbon, and a fixed amount of probe antibody was added when preparing the probe. 6 μL of probe antibody was added to 200 μL of sample solution. The amount of probe added, the concentration of antibody at the C-line, and the concentration at the T-line were kept constant. TGEV virus solution (10...) 7 TCID 50 Using different viral solutions as samples, PDCoV, PEDV, TGEV, PSV, RV, MRV, and GETV viral solutions were taken respectively. Probe antibodies were fixed and mixed, and the preliminarily prepared test strips were inserted into the mixture. The same incubation time of 10 min was set. The colorimetric results were recorded. The results are as follows: Figure 7 As shown, the test strip of this application can specifically detect TGEV.

[0036] 2. Clinical sample testing Clinical stool samples were collected using sterile cotton swabs, vortexed in 1 mL of sample diluent for 10 min, centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The prepared test strips were then used to test the clinical samples. The results are shown in Table 1. Table 1 shows that the TGEV colloidal carbon detection method established in this invention, when Mab 12H6 was selected as the carbon-labeled antibody, the T-line coated N protein concentration was 0.7 mg / mL, the probe volume was 6 μL, and the detection time was 10 min, showed that the sensitivity could detect viral fluid with an antigen content of 1 / 256 (4 × 10⁻⁶). 4 TCID 50 It has good specificity, and the clinical sample detection accuracy rate of the RT-PCR method is 95.2%. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody, comprising a base plate and a sample pad, a conjugate pad, a detection pad, and an absorbent pad sequentially stacked on the base plate, wherein the detection pad has a control line (C-line) and a detection line (T-line), characterized in that: The binding pad contains colloidal carbon-labeled Mab 12H6 antibody.

2. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 1, characterized in that: The Mab 12H6 antibody comprises a Mab 12H6 light chain and a Mab 12H6 heavy chain. The Mab 12H6 light chain includes CDR1 as in SEQ ID No. 1, CDR2 as in SEQ ID No. 2, and CDR3 as in SEQ ID No.

3. The Mab 12H6 heavy chain includes CR1 as in SEQ ID No. 4, CR2 as in SEQ ID No. 5, and CR3 as in SEQ ID No.

6.

3. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 2, characterized in that: The Mab 12H6-light chain also includes VL-FR1 as in SEQ ID No. 7, VL-FR2 as in SEQ ID No. 8, and VL-FR3 as in SEQ ID No.

9.

4. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 3, characterized in that: The Mab 12H6-heavy chain also includes VH-FR1, such as SEQ ID No. 10, VH-FR2, such as SEQ ID No. 11, and VH-FR3, such as SEQ ID No.

12.

5. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 4, characterized in that: The amino acid sequence of the Mab 12H6-light chain is shown in SEQ ID No. 13, and the amino acid sequence of the Mab 12H6-heavy chain is shown in SEQ ID No.

14.

6. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to any one of claims 1-5, characterized in that: The amount of colloidal carbon-labeled Mab 12H6 antibody added to the conjugate pad was 6 μL, and the antibody concentration was 1 mg / ml.

7. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 6, characterized in that: The detection line T contains TGEN N protein, and the control line C contains goat anti-mouse IgG antibody.

8. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 7, characterized in that: The concentration of the detection line T is 0.7 mg / mL.

9. The colloidal carbon test strip for detecting porcine transmissible gastroenteritis virus based on nanobody according to claim 7, characterized in that: The concentration for the C line of the quality control line is 1 mg / mL.

10. The use of the colloidal carbon test strip according to claim 1 in the preparation of products for detecting porcine transmissible gastroenteritis virus.

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