Mab 6B7 antibody, colloidal gold test strip and application

Abstract

The invention belongs to the technical field of virus immunodetection, and relates to a Mab 6B7 antibody, a colloidal gold test strip and application. According to the invention, PDCoV N protein is used as an antigen of a detection line, Mab 6B7 is used as a detection antibody combined with colloidal gold, condition optimization is carried out through different methods, the specificity and sensitivity of the methods are evaluated, and a detection method for detecting the antigen based on an N protein PDCoV colloidal gold competition method is established. The established PDCoV colloidal gold test strip has the advantages of strong specificity, high sensitivity, simplicity and convenience in operation, low production cost and the like, and can be applied to rapid detection of PDCoV in a porcine diarrhea sample clinically.

Description

Technical Field

[0001] This invention belongs to the field of viral immunoassay technology and relates to a colloidal gold test strip. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) was first reported in China in 2012. In 2014, the virus was detected in the feces of piglets and sows with diarrhea at a pig farm in Ohio, USA, leading to an outbreak. Subsequently, many countries reported cases caused by PDCoV, causing significant impact and substantial economic losses on the pig farming industry worldwide. There have been reports of human infection. In recent years, most pig-producing provinces in my country have also reported cases, and the number is steadily increasing, seriously threatening the healthy development of my country's pig farming industry. At the same time, the cross-species transmission of PDCoV and its potential public safety risks have attracted global attention. The main source of PDCoV infection in sick and carrier pigs is through the vomit, feces, or other contaminated materials (such as worker clothing, feed, or transport vehicles), either directly or indirectly. Pigs of all ages can be infected, and it often co-infects with other viruses such as PRRSV, PEDV, and TGEV. The virus spreads very rapidly. Infected suckling piglets will suddenly become ill, causing symptoms such as acute diarrhea and vomiting, leading to rapid dehydration and ultimately death from organ failure. According to incomplete statistics, the morbidity and mortality rates can be as high as 50%-100%. Application CN120446481A discloses a liquid-phase protein chip method for simultaneously detecting three porcine coronaviruses. Although this method can quickly detect the virus, it is costly and does not meet the actual needs of pig farms.

[0003] To meet the needs of rapid clinical diagnosis in pig farms, antigen detection technology, primarily immunochromatographic test strips (colloidal gold or fluorescently labeled), has been developed. This method utilizes monoclonal or polyclonal antibodies against PDCoV proteins (such as the N protein) to directly detect viral antigens in feces or intestinal contents, providing results within 10-15 minutes. It is simple to operate, requires no special equipment, and is highly suitable for initial screening in the field. However, its sensitivity is generally lower than qRT-PCR, and false negative results are common. To further improve the sensitivity of the test strips and promote widespread field testing, our research group has conducted in-depth research. A simple, rapid, and highly specific PDCoV detection method is of great significance for the prevention and control of this disease in clinical practice. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a Mab 6B7 antibody, a colloidal gold test strip, and its applications.

[0005] The technical solution of this invention is implemented as follows: One of the technical solutions of this invention is the screening of gold-labeled antibodies for colloidal gold test strips, selecting Mab 6B7 as the labeling antibody. The Mab 6B7 antibody includes a Mab 6B7 light chain and a Mab 6B7 heavy chain. The Mab 6B7 light chain includes CDR1 as shown in SEQ ID No. 1, CDR2 as shown in SEQ ID No. 2, and CDR3 as shown in SEQ ID No. 3; the Mab 6B7 heavy chain includes CR1 as shown in SEQ ID No. 4, CR2 as shown in SEQ ID No. 5, and CR3 as shown in SEQ ID No. 6.

[0006] The aforementioned Mab 6B7-light chain also includes VL-FR1 as shown in SEQ ID No. 7, VL-FR2 as shown in SEQ ID No. 8, VL-FR3 as shown in SEQ ID No. 9, and VL-FR4 as shown in SEQ ID No. 10. The aforementioned Mab 6B7-heavy chain also includes VH-FR1 as shown in SEQ ID No. 11, VH-FR2 as shown in SEQ ID No. 12, VH-FR3 as shown in SEQ ID No. 13, and VH-FR4 as shown in SEQ ID No. 14.

[0007] Specifically, the amino acid sequence of the Mab 6B7-light chain is shown in SEQ ID No. 15, and the amino acid sequence of the Mab 6B7-heavy chain is shown in SEQ ID No. 16.

[0008] The second technical solution of the present invention provides a product containing the aforementioned Mab 6B7 antibody.

[0009] Taking the colloidal gold test strip as an example, the colloidal gold test strip containing the aforementioned Mab 6B7 antibody of this application includes a base plate and a sample pad, a conjugate pad, a detection pad, and an absorbent pad that are stacked sequentially on the base plate. The conjugate pad contains colloidal gold-labeled Mab 6B7 antibody. The detection pad is provided with a control line C and a detection line T, wherein the control line C contains goat anti-mouse IgG antibody and the detection line T contains PDCoV N protein.

[0010] The T-line concentration is 0.2 mg / mL; the C-line concentration is 1 mg / mL. The optimal antibody addition amount for the test strip is 8 μL. The optimal probe usage amount for the test strip is 8 μL. The optimal color development time for the test strip is 12 min.

[0011] The present invention has the following beneficial effects: The PDCoV colloidal gold detection method established in this invention, when Mab 6B7 was selected as the gold-labeled antibody, the T-line coated N protein concentration was 0.2 mg / mL, the probe volume was 8 μL, and the detection time was 12 min, achieved a sensitivity capable of detecting viral fluid with an antigen content of 1 / 128 (10 8 TCID 50 The test strip exhibits high specificity and good specificity, with a clinical sample detection accuracy rate of 94.2%. It boasts advantages such as high specificity, high sensitivity, ease of operation, and low production cost, making it suitable for rapid detection of PDCoV in porcine diarrhea samples in clinical settings. Attached Figure Description

[0012] 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.

[0013] Figure 1 Determination of PDCoV N colloidal gold probe antibody screening.

[0014] Figure 2 To determine the concentration of PDCoV N protein coating.

[0015] Figure 3 Determination of the amount of Mab 6B7 antibody added.

[0016] Figure 4 Determining the amount of Mab 6B7 probes used.

[0017] Figure 5 Determining the optimal color development time for PDCoV.

[0018] Figure 6 Sensitivity detection of PDCoV colloidal gold test strips.

[0019] Figure 7 This is for the specific detection of PDCoV using colloidal gold test strips. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] Example 1: Screening of antibodies and determination of coating concentration for PDCoV colloidal gold test strips 1. Preliminary screening of antibodies for PDCoV colloidal gold test strips Antibodies were initially screened. The laboratory already possessed four antibodies: Mab 6B7, PDCoV polyclonal antibody, Mab 2E9, and Mab 11E5. These four antibodies were used as probes and bound to colloidal gold. The pH was adjusted first, and then an appropriate amount of antibody was added. The mixture was incubated with shaking for 30 min, followed by the addition of 10% BSA blocking agent and incubation with shaking for another 30 min. Four probe antibodies were prepared: PDCoV N protein (0.2 mg / mL) was drawn onto the T line of the NC membrane, and goat anti-mouse or goat anti-rabbit antibody was drawn onto the C line of the monoclonal antibody. The sample consisted of PDCoV virus solution (10... 8 TCID 50 The viral 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. The antibody with the deepest T-line was used for subsequent condition optimization. The results are as follows: Figure 1 As shown, by Figure 1 It was found that among the four labeled Mab 2E9, Mab 11E5, Mab 6B7, and PDCoV polyclonal antibodies, the Mab 6B7 antibody showed the best performance in the test strip detection, with its color signal intensity being significantly higher than that of the other antibodies.

[0023] The sequence of the Mab 6B7 antibody was sequenced, and the results are as follows: Mab 6B7 antibody light chain: FR1 amino acid: DVVMTQTPLTLSVTIGQPASISCKSS; FR1 nucleotide:gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagtcaagt; 1CDR1 amino acid: QSLLYSNGKAY; CDR1 nucleotide: cagagcctcttatatagtaatggaaaagcctat; FR2 amino acid: LNWLLQRPGQSPKRLIY; FR2 nucleotide: ttgaattggttattacagaggccaggccagtctccaaagcgcctaatctat; 2CDR2 amino acids: LVS; CDR2 nucleotide: ctggtgtct; FR3 amino acids: KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC; FR3 nucleotides: aaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaatcagcagagtggaggctgaggatttgggagtttattactgc; 3CDR3 amino acid: VQGTHFPYT; CDR3 nucleotide: gtgcaaggtacacattttccgtacacg; FR4 amino acid: FGGGTKLEIK; FR4 nucleotide: TTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0024] The amino acid sequence of the 7Mab 6B7-light chain is: DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKAYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPYTFGGGTKLEIK.

[0025] Mab 6B7 antibody heavy chain: FR1 amino acids: EVQLVESGGGLVKPGGSLKLSCAVS; FR1 nucleotide: gaagtgcagctggtggagtctgggggaggcttagtgaagcctggggggtccctgaaactctcctgtgcagtctct; 4CR1 amino acid: GFTFSSYA; CR1 nucleotide: ggattcactttcagtagctatgcc; FR2 amino acid: MSWVRQSPEKRLEWVAE; FR2 nucleotide: atgtcttgggttcgccagtctccagagaagaggctggagtgggtcgcagaa; 5CR2 amino acid: ISSGGSYT; CR2 nucleotide: attachtagtggtggtagttacacc; FR3 amino acids: YYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYC; FR3 nucleotides: tactatccagacactgtgacgggccgattcaccatctccagagacaatgccaagaacaccctgtatttggaaatgagcagtctgaggtctgaggacacggccatgtattactgt; 6CR3 amino acid: VRDGPAWFAY; CR3 nucleotides: gtaagggacggcccggcctggtttgcttac; FR4 amino acids: WGQGTLVTVSA; FR4 nucleotide: tggggccaagggactctggtcactgtctctgca.

[0026] The amino acid sequence of the 8Mab 12H6-heavy chain is: EVQLVESGGGLVKPGGSLKLSCAVSGFTFSSYAMSWVRQSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCVRDGPAWFAYWGQGTLVTVSA.

[0027] 2. Selection of PDCoV N protein coating concentration Mab 6B7 was used as a monoclonal antibody bound to colloidal gold. Different concentrations of PDCoV N protein were streaked along the T line, and goat anti-mouse antibody was streaked along the C line. The antibody and probe concentrations were fixed, and the antibody concentration along the C line was also fixed. The T line concentrations were set to 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively. PDCoV virus solution (10... 8 TCID 50 The 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 best streak concentration, which was used for subsequent condition optimization.

[0028] The results are as follows Figure 2 As shown, by Figure 2It can be seen that among the five T-line concentration gradients set, the colorimetric effect is best when the concentration of PDCoV N protein coated on the detection line is 0.2 mg / mL.

[0029] Example 2: Selection of Mab 6B7 and probe dosage 1. Selection of Mab 6B7 addition amount Mab 6B7 was used as a monoclonal antibody that binds to colloidal gold, 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 gold 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. PDCoV virus solution (10... 8 TCID 50 Mix the sample and probe antibody, then insert the preliminarily prepared test strips into the mixture and incubate for 10 min. Record the color development results; the darkest T line indicates the optimal antibody addition. The antibody addition amount with the darkest T line will be used for subsequent condition optimization.

[0030] The results are as follows Figure 3 As shown, by Figure 3 It can be seen that when 8 μL of antibody is added to each milliliter of colloidal gold solution, the T and C lines of the test strip show the best color development effect.

[0031] 2. Selection of Mab 6B7 probe dosage Mab 6B7 was used as the antibody to bind to colloidal gold. 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of probe were added to 200 μL of sample solution, respectively. PDCoV virus solution (10...) 8 TCID 50 The sample and different amounts of probe 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 indicates the best amount of probe used. The amount of probe used with the darkest T line was used for subsequent condition optimization.

[0032] The results are as follows Figure 4 As shown, by Figure 4 It is known that using 8 μL of probe yields better results.

[0033] Example 3: Determination of the optimal color development time of colloidal gold and detection of the sensitivity of PDCoV colloidal gold test strips 1. Determining the optimal color development time for colloidal gold Mab 6B7 was used as the antibody to bind to colloidal gold, and a fixed amount of probe antibody was added when preparing the probe. 10 μL of probe 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 indicated the best color development time, and this time was used for subsequent condition optimization.

[0034] The results are as follows Figure 5 As shown, by Figure 5 It can be seen that when the reaction proceeds for 12 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. Compared with other detection time points, 12 minutes can ensure both detection sensitivity and meet the requirements of rapid diagnosis. Therefore, 12 minutes was ultimately determined to be the optimal detection reaction time.

[0035] 2. Sensitivity detection of PDCoV colloidal gold test strips Using Mab 6B7 antibody as a monoclonal antibody to bind to colloidal gold, a fixed amount of probe antibody was added when preparing the probe. 10 μL of probe was added to 200 μL of sample solution. PDCoV virus solution (10...) 8 TCID 50 Using this as a sample, set different dilution ratios to create a gradient. Fix the probe-antibody mixture and insert the pre-prepared test strips into the mixture. Set the same incubation time of 10 min. Record the color development results, and use the ratio of T color development to NC and positive as the cutoff point, which is also the limit of detection for the lowest antigen amount.

[0036] The results are as follows Figure 6 As shown, by Figure 6 It was found that when the virus was diluted to a maximum of 1:128, the color intensity of the T line on the test strip was lower than that of the negative control. Therefore, the limit of detection for PDCoV virus on this test strip was determined to be a dilution of 1:128. This result indicates that the test strip has high detection sensitivity and can effectively identify low-concentration virus samples.

[0037] Example 4: Specificity detection and clinical sample testing of PDCoV colloidal gold test strips 1. Specific detection of PDCoV using colloidal gold test strips Using Mab 6B7 as the antibody to bind to colloidal gold, 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 amount of probe added, the concentration of antibody at the C-line, and the concentration at the T-line were kept constant. PDCoV virus solution (10... 8 TCID 50Using different viral solutions as samples, PDCoV, PEDV, TGEV, PSV, RV, MRV, and GETV viral solutions were taken respectively, and probe antibodies were fixed and mixed. The preliminarily prepared test strips were then inserted into the mixture. The same incubation time of 10 min was set. The color development results were recorded.

[0038] The results are as follows Figure 7 As shown, by Figure 7 It was found that the test strip only failed to develop color when reacting with PDCoV samples, while the detection lines for other pathogens all showed color. Tests with other viral samples were all negative, fully demonstrating the high specificity of the test strip.

[0039] 2. Clinical sample testing Take a clinical stool sample using a sterile cotton swab, shake it in 1 mL of sample dilution for 10 min, centrifuge at 12000 rpm / min for 10 min, collect the supernatant, and test the clinical sample using the prepared test strip.

[0040] The results are shown in the table below: As shown in the table above, the PDCoV colloidal gold detection method established in this invention has a clinical sample detection accuracy rate of 94.2% using the RT-PCR method.

[0041] 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 Mab 6B7 antibody, characterized in that: It includes the Mab 6B7-light chain and the Mab 6B7-heavy chain. The Mab 6B7-light chain includes CDR1 as shown in SEQ ID No. 1, CDR2 as shown in SEQ ID No. 2, and CDR3 as shown in SEQ ID No.

3. The Mab 6B7-heavy chain includes CR1 as shown in SEQ ID No. 4, CR2 as shown in SEQ ID No. 5, and CR3 as shown in SEQ ID No.

6.

2. The Mab 6B7 antibody according to claim 1, characterized in that: The Mab 6B7-light chain also includes VL-FR1 as shown in SEQ ID No. 7, VL-FR2 as shown in SEQ ID No. 8, VL-FR3 as shown in SEQ ID No. 9, and VL-FR4 as shown in SEQ ID No.

10.

3. The Mab 6B7 antibody according to claim 2, characterized in that: The Mab 6B7 heavy chain also includes VH-FR1 as shown in SEQ ID No. 11, VH-FR2 as shown in SEQ ID No. 12, VH-FR3 as shown in SEQ ID No. 13, and VH-FR4 as shown in SEQ ID No.

14.

4. The Mab 6B7 antibody according to claim 3, characterized in that: The amino acid sequence of the Mab 6B7-light chain is shown in SEQ ID No. 15, and the amino acid sequence of the Mab 6B7-heavy chain is shown in SEQ ID No.

16.

5. A product containing the Mab 6B7 antibody as described in any one of claims 1-4.

6. A colloidal gold test strip, characterized in that: Contains the Mab 6B7 antibody as described in any one of claims 1-4.

7. The colloidal gold test strip according to claim 6, comprising a base plate and a sample pad, a conjugate pad, a detection pad, and an absorbent pad sequentially stacked on the base plate, characterized in that: The binding pad contains colloidal gold-labeled Mab 6B7 antibody; the test pad has a control line C and a test line T, wherein the control line C contains goat anti-mouse IgG antibody and the test line T contains PDCoV N protein.

8. The colloidal gold test strip according to claim 7, characterized in that: The amount of colloidal gold-labeled Mab6B7 antibody added to the binding pad was 8 μL, and the antibody concentration was 1 mg / mL.

9. The colloidal gold test strip according to claim 7, characterized in that: The concentration for the control line C is 1 mg / mL; the concentration for the detection line T is 0.2 mg / mL.

10. The use of the colloidal gold test strip according to any one of claims 6-9 in the preparation of products for detecting porcine deltacoronavirus.

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