CN117310163A - Respiratory syncytial virus detection method based on recombinant antibody - Google Patents
Respiratory syncytial virus detection method based on recombinant antibody Download PDFInfo
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- CN117310163A CN117310163A CN202311569493.6A CN202311569493A CN117310163A CN 117310163 A CN117310163 A CN 117310163A CN 202311569493 A CN202311569493 A CN 202311569493A CN 117310163 A CN117310163 A CN 117310163A
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- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The invention discloses a respiratory syncytial virus detection method based on a recombinant antibody, which belongs to the technical field of respiratory syncytial virus detection, and the respiratory syncytial virus detection method is to detect respiratory syncytial virus in a biological sample by using a colloidal gold platform prepared by using an RSV (respiratory syncytial virus) nanometer recombinant antibody. According to the invention, the RSV recombinant nanobody is obtained by constructing a phage display library, fishing the RSV specific nanobody by taking the RSV-F protein with high inducibility as an antigen, and then constructing an RSV specific nanobody eukaryotic expression system by genetic engineering, wherein the RSV recombinant nanobody has specific recognition and binding capacity on the RSV-F protein, does not react with other nonspecific cross-reactive proteins, and has wide application value.
Description
Technical Field
The invention belongs to the technical field of respiratory syncytial virus detection, and particularly relates to a respiratory syncytial virus detection method based on a recombinant antibody.
Background
Respiratory Syncytial Virus (RSV) belongs to the family paramyxoviridae, pneumovirinae, is the most dominant RNA virus for lower respiratory tract infection, RSV infection is extremely broad and parent, thus infection cannot be completely prevented, infection of children under 2 years old is extremely common and the infection rate is up to 97%, and RSV is transmitted mainly in 3 modes: the spray, direct contact and excrete excretions gradually change from cough, nasal discharge and other upper respiratory tract symptoms to lower respiratory tract infection after infection, cause pathological changes such as bronchus and the like, and more serious severe manifestations such as asthma, respiratory failure and the like, and no safe and effective product is applied to clinical treatment of RSV at present, can rapidly identify infants infected with RSV and take effective preventive measures to reduce the morbidity and the disease rate of the infants infected with RSV.
RSV is an enveloped virus containing a single-stranded, non-segmented negative-strand RNA encoded by a 15.2kb RSV genome, 150-300nm in size, divided into two subtypes A, B. The RSV genome contains 10 genes, encoding 11 proteins: three envelope glycoproteins G, F proteins (G protein responsible for adhesion, F protein responsible for fusion and membrane penetration), small Hydrophobins (SH), five structural proteins: l, N (nucleic acid protein: N protein encases RNA during the whole replication process, preventing its degradation), P, M, M2 (where M2 gene encodes M2-1, M2-2), and two nonstructural proteins NS1, NS24, of which only F protein is essential for RSV infection among the three proteins on the virion surface, and it has also been demonstrated in human serum experiments that F protein is the main target for neutralizing antibodies.
The conventional detection technology of RSV at present comprises a Polymerase Chain Reaction (PCR) -fluorescent probe method, an enzyme-linked immunosorbent assay (ELISA) and a colloidal Gold Immunochromatography (GICA), wherein the PCR-fluorescent probe method combines two technologies of PCR and fluorescent probe, has the characteristics of high sensitivity, low omission ratio and high specificity, but the method requires a professional PCR detection laboratory and professional detection personnel, has strict requirements and high cost, has the longest detection time limit in 3 detection methods, and is not suitable for popularization and use in basic medical units; ELISA is easy to operate, but is easy to cause missed detection, and is not suitable for wide clinical popularization; the GICA method is simple to operate, low in cost and low in test requirement, the detection time limit is shortest among three detection methods, the positive detection rate is high, but the requirements on sensitivity and specificity are high, so that the antibody with good specificity, good tolerance and strong stability is provided, and has important significance for realizing early detection and diagnosis of RSV.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a respiratory syncytial virus detection method based on a recombinant antibody, which is characterized in that a phage display library is independently constructed, RSV specific nanobodies are fished by taking high-inducibility RSV-F protein as an antigen, then an RSV specific nanobody eukaryotic expression system is constructed through genetic engineering to obtain the RSV recombinant nanobodies, the RSV recombinant nanobodies can specifically identify respiratory syncytial virus, and the method has wide application value.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the invention provides a respiratory syncytial virus detection method based on a recombinant antibody, which is to detect respiratory syncytial virus in a biological sample by using a colloidal gold platform prepared by using an RSV (respiratory syncytial virus) nanometer recombinant antibody.
Preferably, the biological sample is selected from one of an in vitro cell infected with RSV, nasal secretion, nasal wash, pharyngeal secretion, bronchial secretion.
Further, the RSV nano-recombinant antibody comprises at least three sets of complementarity determining regions, including CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
Further, the RSV nanorecombinant antibodies comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID nos. 4.
Preferably, the RSV nano-recombinant antibody comprises the amino acid sequence shown in SEQ ID NO. 4.
Further, the RSV nano-recombinant antibody comprises a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO. 4, wherein the nucleotide sequence is shown in SEQ ID NO. 5.
In addition, the invention also provides a nucleic acid molecule and an expression vector of an expression regulatory element thereof, wherein the nucleic acid molecule codes for the RSV nanometer recombinant antibody.
As a further aspect of the invention, the RSV nanorecombinant antibodies are also conjugated to a label that allows their detection, said label being selected from the group consisting of fluorophores, biotin, radioisotopes, metals and enzymes.
Further, the colloidal gold platform comprises a PVC bottom plate, a sample pad, a colloidal gold pad, a test T line, a control C line, a nitrocellulose membrane and water absorption filter paper, wherein the sample pad is fixedly connected to one end of the PVC bottom plate, the water absorption filter paper is fixedly connected to one end of the PVC bottom plate, which is far away from the sample pad, the colloidal gold pad is arranged on one side of the sample pad, the nitrocellulose membrane is arranged on one side of the colloidal gold pad, the test T line is arranged on one side, close to the colloidal gold pad, of the nitrocellulose membrane, and the control C line is arranged on one side, close to the water absorption filter paper, of the nitrocellulose membrane.
Preferably, the sample pad and the colloidal gold pad are overlapped by 1-2mm, the colloidal gold pad and the nitrocellulose membrane are overlapped by 1-2mm, and the nitrocellulose membrane and the absorbent filter paper are overlapped by 1-2mm.
Preferably, the colloidal gold pad contains a solution and an antibody of the recombinant nano antibody of the RSV marked by the colloidal gold, the RSV antigen is coated on the test T line, and the secondary antibody is coated on the control C line.
Preferably, the antibody is selected from one of human IgG, chicken IgY or mouse IgG, and the secondary antibodies include, but are not limited to, mouse anti-human IgG, sheep anti-chicken IgY and sheep anti-mouse IgG, so long as the existing antibodies and the corresponding secondary antibodies that can achieve the same function can be used as an alternative range, which is not limited herein.
Preferably, the nitrocellulose membrane has a climbing speed of one of 95s/4cm, 120s/4cm or 140s/4 cm.
Further, the colloidal gold platform is used by dripping a biological sample on a sample pad, when the biological sample contains RSV antigen, the antigen in the biological sample contacts and combines with the RSV recombinant nanobody in the colloidal gold pad due to the adsorption of the water-absorbing filter paper, so that when the liquid flows to a test T line, the RSV recombinant nanobody cannot combine with the RSV antigen coated on the test T line, thereby not displaying color, and when the liquid flows to a control C line, the secondary antibody combines with the human IgG, chicken IgY or mouse IgG marked by the colloidal gold, and color is generated; thus when the biological sample contains RSV antigen, the test T line does not develop color and the C line is controlled to develop color; when the biological sample does not contain RSV antigen, the test T line develops color and the control C line also develops color; if the control line C does not develop, the detection is invalid.
The beneficial effects obtained by the invention by adopting the structure are as follows:
(1) According to the invention, a phage display library is constructed, RSV specific nano antibody is fished by taking high-inducibility RSV-F protein as an antigen, then an RSV specific nano antibody eukaryotic expression system is constructed through genetic engineering to obtain the RSV recombinant nano antibody, and the RSV recombinant nano antibody has unique CDR1, CDR2 and CDR3 region sequences, so that the nano antibody has specific recognition and binding capacity to the RSV-F protein, does not react with other nonspecific cross-reactive proteins, and has wide application value;
(2) The F protein is required to mediate fusion of a virus envelope and a host cell through conformational change, after the host cell membrane is anchored, the F0 precursor is subjected to amino-terminal glycosylation modification, and is cleaved into two subunits of F1 and F2 connected by disulfide bonds by using tyrosinase, then a hairpin structure of a trimer is formed, so that fusion of two layers of membranes is promoted, and the RSV-F protein used by the invention is protein before conformational change, so that the antibody induction effect is better;
(3) The invention optimizes the RSV codon, is more favorable for the expression of the RSV recombinant nanobody, has simple preparation, does not need serum extraction, has stronger stability, improves the production efficiency, and is suitable for mass production;
(4) The colloidal gold platform constructed by the invention can specifically detect respiratory syncytial virus, has no cross reaction with influenza virus, parainfluenza virus, respiratory adenovirus and mycoplasma pneumoniae, and has strong specificity;
(5) The invention uses the recombinant antibody with high stability and high specificity to be applied to a colloidal gold platform, thereby improving the sensitivity and stability of detection, achieving the purposes of simple operation and low price, realizing detection without professional technicians or large-scale instruments and equipment, and being particularly suitable for being used in remote poverty areas;
(6) The method is applicable to early diagnosis, and the detection result can be read through naked eyes, so that more relevant information is obtained, a clinician is assisted to give correct medical diagnosis and the like, and the method has important clinical significance in early discovery, early diagnosis, early treatment and early recovery.
Drawings
FIG. 1 is a graph of the results of PCR amplification of VHH;
FIG. 2 is a graph showing the expression results of RSV nano-recombinant antibodies;
fig. 3 is a schematic perspective view of a colloidal gold platform, wherein 1, a PVC bottom plate, 2, a sample pad, 3, a colloidal gold pad, 4, a test T line, 5, a control C line, 6, a nitrocellulose membrane, 7, and a water absorbing filter paper;
FIG. 4 is a graph showing the result of antigen reactivity of the RSV nano-recombinant antibodies provided by the present invention to RSV-F protein;
FIG. 5 is a graph showing the result of the specificity test of the RSV nano-recombinant antibody provided by the present invention to RSV-F protein;
FIG. 6 is a graph of a colloidal gold plate specificity test constructed in accordance with the present invention;
FIG. 7 is a graph of the colloidal gold flat sensitivity test constructed by the basic invention.
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the scope of the present application.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials and test strains used in the examples described below, unless otherwise specified, were commercially available.
Example 1
Construction of phage display libraries
Referring to the method in the prior art, a phage display library is built for fishing the RSV nano-recombinant antibody, and the specific operation flow is as follows: mixing 200 mu L of inactivated RSV (purchased from vincbai corporation) with an equal volume of adjuvant, emulsifying, performing multipoint subcutaneous immunization on both sides of alpaca scapula, wherein the immunization dose is 400 mu g each time, total immunization is 4 times, 1 st time is complete Freund's adjuvant (purchased from SIGMA corporation), the last 3 times of boosting is incomplete Freund's adjuvant (purchased from SIGMA corporation), the immunization interval time is 14d, venous blood collection is performed, lymphocytes are separated by using a peripheral blood lymphocyte separation kit (purchased from Soy Biotechnology Co., ltd.), total RNA is extracted by using an RNA extraction kit (purchased from TAKARA corporation), the total RNA is reversely transcribed into cDNA, the VHH fragment is amplified by using nest PCR, the first step is performed by using an F1 primer (Table 1) as a template, the PCR product is subjected to gel cutting recovery, and then the PCR product is subjected to 2 nd round PCR by using a VHH primer (Table 1) to obtain VHH gene; the VHH gene was cloned by homologous recombination into a linearized insert into the pCANTAB-5E vector. The inserted pCANTAB-5E-VHH fragment was electrotransformed into TG1 competent cells and plated on 2YTA + The cells were incubated overnight at 37℃on G solid plates. The plaque was collected and 100. Mu.L was subjected to gradient dilution and then to 2YTA coating + G solid plates were incubated overnight in a 30℃incubator, and the remainder was kept at-80 ℃. Plates cultured overnight were counted and phage library capacity calculated, reference material: shi Zhenzhong, xu Xiaochi, wu Wenhui, xu Jianfeng screening and identification of novel coronavirus-specific nanobodies [ J ]]Journal of immunology, 2022, 38 (08): 720-725.
TABLE 1 primer sequences
| Name of the name | Forward primer | Reverse primer |
| F1 | GGTGGTCCTGGCTGCTCTTCT | GGTACGTGCTGTTGAACTGT |
| VHH | TTTCTATTACTAGGCCCAGCCGGCCGAGTCTGGAGGRRGCTTGGTGCA | AAACCGTTGGCCATAATGGCCTGAGGAGACGRTGACSTSGGTC |
As shown by nested PCR amplification results, VHH fragments with the size of about 400bp (shown in figure 1) are obtained through specific amplification, and the library capacity of phage display antibody library constructed after the VHH fragments are cloned is 8.6X10 8 cfu/mL, 35 monoclonal assays were randomly selected with a VHH gene fragment insertion positive rate of 100% (statistical result).
Example 2
Screening of RSV nanorecombinant antibodies
The elisa plate was coated with 100 ng/well RSV-F protein (purchased from hundred grams company), placed at 4 ℃ overnight, and blocked the next day with skim milk. PEG6000/NaCl solution was added to the primary library supernatant cultured overnight in example 1, and after mixing, the mixture was centrifuged, and the pellet was resuspended in PBS. After the immune tube is blocked, adding 2mL of the primary library bacteria obtained in the embodiment 1 treated by the blocking solution for incubation, eluting phage by using a triethylamine solution, and adding a Tris-HCl solution after shaking to terminate the reaction. Mixing the mixed solution with TG1 bacterial liquid in logarithmic phase, placing in 37 deg.C water bath for 30min, taking out 100 μl for dilution, titrating for eluting phage, and coating the rest bacterial liquid on 2YTA + G, culturing the solid culture plate, and collecting the library, namely, screening the library in the first round.
Three rounds of panning were performed according to the above procedure, 186 single clones were randomly picked from the third round of library plates, inoculated into 96 well plates, shake cultured and transferred, KM13 helper phage was added to each well, and cultured overnight. The next day, the supernatant after centrifugation was taken for ELISA detection: in an RSV-F antigen coated ELISA plate, BSA protein is used as a negative control, centrifugal supernatant and blocking solution are taken to jointly incubate and block for 45min, then the obtained product is respectively added to the RSV-F protein and BSA protein coated 96-well ELISA plate, antigen reactivity screening is carried out, an OD450nm absorbance (A) value is measured, high-reactivity positive clone is selected according to the difference value between the experimental hole (A) value and the control hole (A) value for sequencing, the sequencing result is compared and analyzed by using align software, and an RSV-F specific VHH sequence is selected according to a nano-antibody hypervariable region sequence.
Example 3
Identification, eukaryotic expression and purification of RSV nanorecombinant antibodies
The C end of the selected VHH sequence fragment is added with 6 XHis-tag (DNA sequence synthesis) and inserted into pPICZ alpha A vector, and the recombinant plasmid is transformed into DH5 alpha competence for amplification and extraction. After tangential incubation with SacI enzyme, the strain was transformed into Pichia pastoris GS115 strain, which was plated onto YPD bleomycin resistant plates for cultivation. The single clone was picked up and cultured overnight in YPD liquid medium, then the bacterial liquid was transferred to BMGY medium at a ratio of 1% for continuous culture, and 1% methanol was added for protein induction expression.
After induction, the supernatant is collected, protein expression is verified by SDS-PAGE electrophoresis, and the protein is purified by adopting a nickel column affinity chromatography. The protein eluate was placed in a dialysis bag, immersed in phosphate buffer, and dialyzed overnight at 4 ℃. And (3) spreading PEG6000 powder on the surface of the dialysis bag for concentrating protein to obtain the RSV nanometer recombinant antibody.
As shown in fig. 2, when 85% imidazole was used for elution, RSV nanorecombinant antibodies were obtained with protein concentrations of: 1.8mg/mL, high protein concentration.
Example 4
Competition method colloidal gold platform construction
As shown in fig. 3, the colloidal gold platform comprises a PVC bottom plate 1, a sample pad 2, a colloidal gold pad 3, a test T line 4, a control C line 5, a nitrocellulose membrane 6 and a water absorbing filter paper 7, wherein the sample pad 2 is fixedly connected to one end of the PVC bottom plate 1, the water absorbing filter paper 7 is fixedly connected to one end of the PVC bottom plate 1 far away from the sample pad 2, the colloidal gold pad 3 is arranged on one side of the sample pad 2, the nitrocellulose membrane 6 is arranged on one side of the colloidal gold pad 3, the test T line 4 is arranged on one side of the nitrocellulose membrane 6 near the colloidal gold pad 3, and the control C line 5 is arranged on one side of the nitrocellulose membrane 6 near the water absorbing filter paper 7.
Wherein, overlap 1-2mm between sample pad 2 and the colloidal gold pad 3, overlap 1-2mm between the colloidal gold pad 3 and nitrocellulose membrane 6, overlap 1-2mm between nitrocellulose membrane 6 and absorbent filter paper 7.
The colloidal gold pad 3 contains a solution of a recombinant nano antibody of RSV marked by colloidal gold and chicken IgY, the test T line 4 is coated with RSV antigen (purchased from vinca hundred gram company), the control C line 5 is coated with a secondary antibody, and the secondary antibody is sheep anti-chicken IgY. The climbing speed of the nitrocellulose membrane 6 was 120s/4cm.
The colloidal gold platform is used by dripping a biological sample on the sample pad 2, when the biological sample contains RSV antigen, the antigen in the biological sample is combined with the RSV recombinant nano-antibody in the colloidal gold pad 3 due to the adsorption of the water-absorbing filter paper 7, so that when the liquid flows to the test T line, the RSV recombinant nano-antibody cannot be combined with the coated RSV antigen, thereby not displaying color, and when the liquid flows to the control C line 5, the secondary antibody is combined with the gold-labeled chicken IgY to generate color. Thus when the biological sample contains RSV antigen, test T line 4 does not develop color and control C line 5 develop color; when the biological sample does not contain RSV antigen, test T line 4 develops color and control C line 5 also develops color; if the control C line 5 does not develop, the detection is invalid.
Verification of detection Performance results
Experimental example 1
Affinity assay for RSV nanorecombinant antibodies provided herein
The purified RSV nanometer recombinant antibody detects the specific binding force with RSV-F protein antigen by indirect ELISA: the RSV-F protein is coated on a 96-well ELISA plate at the concentration of 1.2 mug/mL, bovine serum albumin BSA and recombinant human serum albumin RHSA are used as a control, the coating is carried out overnight at 4 ℃, 50 mug of RSV nano recombinant antibodies diluted by 2 times are respectively added after the sealing is finished, and the antigen specific binding force of the RSV nano recombinant antibodies is measured.
As shown in fig. 4 and 5, the ELISA titer of the RSV nano-recombinant antibodies provided by the invention was high and there was no antigen cross-reaction with BSA and RHSA.
Experimental example 2
The invention provides an affinity determination of RSV nanometer recombinant antibody to RSV virus
Coating an ELISA plate with inactivated RSV at 4 ℃ overnight, diluting the screened RSV nanometer recombinant antibody by a doubling ratio of 100 ng/hole to obtain a primary antibody, performing an indirect ELISA test with Fc-HRP antibody as a secondary antibody, measuring D450 value, calculating EC50, and calculating the EC of the RSV nanometer recombinant antibody 50 Is 9.820 mug/mL.
Experimental example 3
Specificity test of respiratory syncytial virus detection method based on recombinant antibody
Collecting nasopharyngeal secretion of a patient by a nasal swab scraping method as a sample, and detecting nasopharyngeal secretion samples of influenza virus, parainfluenza virus, respiratory adenovirus, mycoplasma pneumoniae and respiratory syncytial virus by using the colloidal gold test strip, wherein the result is shown in a figure 6, A is respiratory syncytial virus, B is influenza virus, C is parainfluenza virus, D is respiratory adenovirus, E is mycoplasma pneumoniae, the result shows that the detection result of the syncytial virus sample is C line color development, T line does not color development, and the detection result shows that the respiratory syncytial virus is positive; the detection results of influenza virus, parainfluenza virus, respiratory tract adenovirus and mycoplasma pneumoniae samples are all developed on the C and T lines, which indicates that the detection result is negative to respiratory syncytial virus, and indicates that the test strip has no cross reaction with influenza virus, parainfluenza virus, respiratory tract adenovirus and mycoplasma pneumoniae.
Experimental example 4
Sensitivity test of respiratory syncytial virus detection method based on recombinant antibody
The respiratory virus cultures were diluted 1:10,1:80,1:120,1:140,1:150, and then tested on the colloidal gold platform of the invention, respectively, as shown in FIG. 7, wherein A is 1:10, B is 1:80, C is 1:120, D is 1:140, E is 1:150, and the results indicate that the dilution concentrations of 1:10,1:80,1:120,1:140 can detect RSV positives, and the detection sensitivity is high.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The invention and its embodiments have been described above with no limitation, and the actual construction is not limited to the embodiments of the invention as shown in the drawings. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (10)
1. A respiratory syncytial virus detection method based on recombinant antibodies is characterized in that: the respiratory syncytial virus detection method is that a colloidal gold platform prepared by using an RSV nanometer recombinant antibody is used for detecting respiratory syncytial virus in a biological sample;
the RSV nano-recombinant antibody comprises at least three sets of complementarity determining regions, including CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.
2. The method for detecting respiratory syncytial virus based on recombinant antibody according to claim 1, wherein the method comprises the steps of: the biological sample is selected from one of in vitro cells infected with RSV, nasal secretion, nasal wash, pharyngeal secretion, and bronchial secretion.
3. The method for detecting respiratory syncytial virus based on recombinant antibody according to claim 2, wherein the method comprises the steps of: the RSV nanorecombinant antibodies comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID nos. 4.
4. A method for detecting respiratory syncytial virus based on recombinant antibodies according to claim 3, wherein: the RSV nano-recombinant antibody comprises an amino acid sequence shown in SEQ ID NO. 4.
5. A nucleic acid molecule encoding the RSV nano-recombinant antibody of any one of claims 1-4, said nucleic acid molecule being set forth in SEQ ID No. 5.
6. An expression vector comprising the nucleic acid molecule of claim 5 and an expression control element thereof.
7. Be applied to colloidal gold platform of claim 1, colloidal gold platform includes PVC bottom plate (1), sample pad (2), colloidal gold pad (3), test T line (4), control C line (5), nitrocellulose membrane (6) and filter paper (7) that absorbs water, sample pad (2) fixed connection is in the one end of PVC bottom plate (1), filter paper (7) that absorbs water fixed connection keeps away from the one end of sample pad (2) on PVC bottom plate (1), one side of sample pad (2) is located to colloidal gold pad (3), one side of colloidal gold pad (3) is located to nitrocellulose membrane (6), one side that is close to colloidal gold pad (3) on nitrocellulose membrane (6) is located to test T line (4), one side that is close to filter paper (7) that absorbs water on nitrocellulose membrane (6) is located to control C line (5).
8. The colloidal gold bench according to claim 7, wherein: the sample pad (2) and the colloidal gold pad (3) are overlapped by 1-2mm, the colloidal gold pad (3) and the nitrocellulose membrane (6) are overlapped by 1-2mm, and the nitrocellulose membrane (6) and the water absorbing filter paper (7) are overlapped by 1-2mm.
9. The colloidal gold bench according to claim 8, wherein: the colloidal gold pad (3) contains a solution of the RSV recombinant nano-antibody marked by colloidal gold, and the RSV antigen is coated on the test T line (4).
10. A colloidal gold bench according to claim 9 wherein: the RSV nanorecombinant antibodies are also conjugated to a label that allows for their detection, the label being selected from the group consisting of fluorophores, biotin, radioisotopes, metals, and enzymes.
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