CN113588940A - Method and kit for improving indirect labeling sensitivity - Google Patents
Method and kit for improving indirect labeling sensitivity Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides a method and a kit for improving indirect labeling sensitivity, which adopt a double-antigen sandwich method to detect the content of an antibody to be detected in a sample; by adopting the method and the kit, the sensitivity of indirectly marking the original mode can be improved.
Description
Technical Field
The present invention relates to the field of protein detection. In particular, the present invention relates to methods and kits for increasing the sensitivity of indirect labeling.
Background
In chinese patent No. 200810216400.0, a "double antigen sandwich method for detecting antibody by indirectly labeling nanoparticles and a kit thereof" is proposed, which studies the application of indirect labeling in nanoparticle-based labeling substances such as colloidal gold. Because of the larger particle size of the nanoparticle, the final shape of the label is that the nanoparticle label is used as the core-one nanoparticle label is coupled with a plurality of antigens on the surface, so the indirect labeling brings many advantages: firstly, indirect labeling is more beneficial to exposing epitope because an arm is added between the nanoparticle and the labeled antigen; secondly, the dosage of the labeled antigen can be reduced, and the ratio of the antigen to the nanoparticle-based label is ideally 1: 1.
In practical experiments, the nanoparticle label is subjected to the oscillation process of labeling the ligand and labeling the antigen, and part of the labeled antigen falls off from the ligand and is dissociated in a labeling system, so that the sensitivity is reduced; based on this, the present invention makes a further improvement on the indirectly labeled labeling method to increase the sensitivity.
Disclosure of Invention
In some embodiments, the invention may include one or more of the following:
1. an antigen labeling method, wherein the method comprises the steps of (i) ligand labeling of a nanoparticle-based label, (ii) antigen labeling of the ligand-nanoparticle-based label obtained in step (i), (iii) adding the ligand-labeled nanoparticle-based label to a labeling system to obtain an antigen-labeled complex; wherein the labeling between the nanoparticle-based label and the labeled antigen is indirect labeling which is performed by combining a label on the antigen and the ligand which is labeled on the nanoparticle-based label and can specifically recognize the label.
2. The method of item 1, wherein the marker antigen includes, but is not limited to, HIV antigen, hepatitis A virus antigen, hepatitis B virus antigen, hepatitis C virus antigen, hepatitis D virus antigen, hepatitis E virus antigen, hepatitis G virus antigen, rubella virus antigen, human cytomegalovirus antigen, herpes simplex virus type 1 antigen, herpes simplex virus type 2 antigen, rabies virus antigen, human T lymphocyte leukemia virus antigen, dengue virus antigen, human papilloma virus antigen, West Nile virus antigen, forest encephalitis virus antigen, measles virus antigen, influenza virus antigen, parainfluenza virus antigen, varicella virus antigen, echovirus antigen, coxsackie virus antigen, Japanese encephalitis virus antigen, EB virus antigen, mumps virus antigen, treponema antigen, A Borrelia burgdorferi antigen, a Chlamydia trachomatis antigen, a Chlamydia pneumoniae antigen, a Chlamydia psittaci antigen, a ureaplasma urealyticum antigen, a Mycoplasma pneumoniae antigen, a Mycobacterium tuberculosis antigen, a helicobacter pylori antigen, a gonococcus antigen, a Plasmodium antigen, a Trypanosoma cumini antigen and a Toxoplasma gondii antigen.
3. The method of item 1, wherein the nanoparticle-based label comprises nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, rare earth complex nanoparticles, and their respective derivatives, or a combination of two or more thereof; wherein the colloid comprises colloid metal, disperse dye, dye-labeled microspheres and latex; wherein the colloidal metal comprises colloidal gold, colloidal silver and colloidal selenium.
4. The method of item 1, wherein the tag is a polypeptide or protein selected from the group consisting of: his Tag, T7 Tag, S Tag, Flag Tag, HA Tag, GST fusion protein, Trx fusion protein, SOD fusion protein, CKS fusion protein, DsbA fusion protein, MBP fusion protein, CBD fusion protein, or biotin.
5. The method of item 1, wherein the ligand includes, but is not limited to, a specific antibody against a tag, a substance having an affinity for a tag such as streptavidin or an analog thereof.
6. The method of item 1, wherein the supplementary addition of the nanoparticle-based marker labeled with a ligand can be 5% -30% of the constant volume of the labeling system; for example, the volume of the marking system can be 7.5% -17.5%; preferably 10%.
7. The method of item 7, wherein the labeling system uses a marker dilution comprising 20mM PB, 150mM NaCl, 1% BSA, 0.1% Triton X-100, 2% sucrose, and 0.01% Proclin 300.
8. An antigen-labeled complex according to any one of items 1 to 7.
9. A test kit comprising the antigen-labeled complex of item 8.
10. Use of the method of any one of items 1 to 7 and/or the antigen-labeled complex of item 8 in the preparation of an antibody detection kit.
Drawings
FIG. 1 is a map of vector P2;
FIG. 2 shows the construction of vector P2-X with the X fusion protein.
Detailed Description
In some embodiments, the present invention provides an antigen-labelling method, wherein the method comprises the steps of (i) ligand-labelling a nanoparticle-based label, (ii) labelling the ligand-nanoparticle-based label obtained in step (i) with an antigen, (iii) supplementing the labelling system with the ligand-labelled nanoparticle-based label to obtain an antigen-labelled complex; wherein the labeling between the nanoparticle-based label and the labeled antigen is indirect labeling which is performed by combining a label on the antigen and the ligand which is labeled on the nanoparticle-based label and can specifically recognize the label.
In some embodiments, the antigen labeling methods of the present invention include, but are not limited to, HIV antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis E virus antigens, hepatitis G virus antigens, rubella virus antigens, human cytomegalovirus antigens, herpes simplex virus type 1 antigens, herpes simplex virus type 2 antigens, rabies virus antigens, human T-lymphocyte leukemia virus antigens, dengue virus antigens, human papilloma virus antigens, West Nile virus antigens, forest encephalitis virus antigens, measles virus antigens, influenza virus antigens, parainfluenza virus antigens, varicella virus antigens, echovirus antigens, coxsackie virus antigens, encephalitis B virus antigens, EB virus antigens, mumps virus antigens, treponema antigens, A Borrelia burgdorferi antigen, a Chlamydia trachomatis antigen, a Chlamydia pneumoniae antigen, a Chlamydia psittaci antigen, a ureaplasma urealyticum antigen, a Mycoplasma pneumoniae antigen, a Mycobacterium tuberculosis antigen, a helicobacter pylori antigen, a gonococcus antigen, a Plasmodium antigen, a Trypanosoma cumini antigen and a Toxoplasma gondii antigen.
In some embodiments, the antibody detection method indirectly labeling nanoparticles of the present invention can be applied to an immunoassay using nanoparticles as a label and/or a signal; in some embodiments, the immunoassay is an immunochromatographic assay.
In some embodiments, the nanoparticle-based labels of the present invention include nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, rare earth complex nanoparticles, and their respective derivatives, or a combination of two or more thereof; in some embodiments, the colloid comprises colloidal metals, disperse dyes and dye-labeled microspheres, latex; in some embodiments, the colloidal metal comprises colloidal gold, colloidal silver, colloidal selenium.
In some embodiments, the tag of the invention is a polypeptide or protein selected from the group consisting of: his Tag, T7 Tag, S Tag, Flag Tag, HA Tag, GST fusion protein, Trx fusion protein, SOD fusion protein, CKS fusion protein, DsbA fusion protein, MBP fusion protein, CBD fusion protein, or biotin. In some embodiments, the tag may be attached at the N-terminus, C-terminus, both ends, or within the labeled antigen.
In some embodiments, the ligand of the present invention includes, but is not limited to, a specific antibody against a tag, a substance having an affinity for a tag such as streptavidin or an analog thereof.
In some embodiments, the additive amount of the nanoparticle-based marker supplemented with labeled ligand in the invention can be 5% -30% of the volume of the marker system; in some embodiments, the amount added may be from 7.5% to 17.5% of the volumetric volume of the marking system; in some embodiments, the amount added may be, for example, 5%, e.g., 7.5%, e.g., 10%, e.g., 12.5%, e.g., 15%, e.g., 17.5%, e.g., 20%, e.g., 22.5%, e.g., 25%, e.g., 27.5%, e.g., 30%, but is not limited thereto.
In some embodiments, the marker system of the present invention uses a marker dilution comprising 20mM PB, 150mM NaCl, 1% BSA, 0.1% Triton X-100, 2% sucrose, 0.01% Proclin 300.
In some embodiments, the present invention provides an antigen-labeled complex of any of the embodiments. In some embodiments, the antigen-labeled complex may be a liquid reagent; in some embodiments, the antigen-labeled complex may be made into a label-binding pad; in some embodiments, the preparation means includes, but is not limited to, lyophilization, oven drying, air drying.
In some embodiments, the present invention provides a kit (test strip) comprising the antigen-labeled complex of any of the embodiments.
In some embodiments, the invention also provides a method comprising any one of the embodiments and/or use of the antigen-labeled complex in the preparation of an antibody detection kit.
In some embodiments, using the methods and/or kits of the invention, sensitivity may be further increased based on the indirect labeling of the native pattern.
The present invention will be described in further detail with reference to specific examples. The following examples are provided to illustrate embodiments of the present invention and are not intended to limit the invention. The invention may optionally include embodiments that are not illustrated by the examples. The cloning vector P2 used in the present invention is illustrated in FIG. 1, and P2 is not an essential expression vector in the practice of the present invention, and the upstream enhancer does not play a substantial role in the practice of the present invention, and many other expression vectors such as pET-24a (+) (Novagen, USA, Cat. No. 69749-3) can be used in the practice of the present invention.
Example one, antibody detection of indirectly labeled nanoparticles of Hepatitis C Virus (HCV)
1.1 preparation of recombinant hepatitis C antigen
1.1.1 preparation of hepatitis C coating antigen
PCR amplifies DNA segment corresponding to ORF full length 1201-1465aa of hepatitis C virus genome, its upstream primer has BamHI site, its downstream primer has EcoRI site and coding sequence with 6 His amino acids before EcoRI site and termination code TAA. After the PCR fragment was recovered, it was digested with BamHI and EcoRI, and ligated to the expression vector P2 digested with BamHI and EcoRI to obtain the recombinant plasmid P2-NS3, which is the antigen-coated recombinant plasmid of the present invention.
Plasmid P2-NS3 was transformed into ER2566, which was shake-cultured at 37 ℃ in 500mL LB medium containing 100. mu.g/mL kanamycin sulfate to OD600This was induced with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. Centrifuging at 4 ℃ for 20 minutes at 5000g for collecting thalli, resuspending the thalli of each liter of bacterial liquid by 20mL of lysis buffer, carrying out ultrasonic disruption, centrifuging at 4 ℃ for 20 minutes at 12000g, and after being identified by SDS-PAGE electrophoresis, distributing part of target protein in lysate sediment. The cells of each liter of the bacterial solution were resuspended in 10mL of lysis buffer (50mM Tris-HCl, pH8.0, 1mM EDTA, 100mM NaCl), sonicated, centrifuged at 12000rpm at 4 ℃ for 20 minutes to collect inclusion bodies, resuspended in solution 1(20mM Tris-HCl, pH8.5, 5mM EDTA, 100mM NaCl) containing 2% Triton X-100, centrifuged at 12000rpm at 4 ℃ for 20 minutes to collect inclusion bodies, solubilized with 4M urea prepared in solution 1, dialyzed against 100 volumes of PB buffer (pH7.0, 20mM), changed 3 times, centrifuged at 12000rpm for 20 minutes at 4 ℃ to remove precipitates to prepare crude antigens, equilibrated with the same PB buffer in a Sephacryl S-200 gel column (Amersham Biosciences), passed through the column, collected and pooled solutions containing the target protein, and 5mL of equilibrated buffer (10mM Na) was added2HPO4，1.8mM KH2PO4140mM NaCl, 2.7mM KCl, 25mM imidazole, pH 8.0). After equilibration of the Ni-NTA affinity column (Qiagen, cat # 30210) with 10 bed volumes of equilibration buffer, the protein sample was added, unbound protein was washed off with 10 medium volumes of equilibration buffer, and 5 volumes of elution buffer (20mM Na2HPO4300mM NaCl, 250mM imidazole, pH8.0), eluting the target protein, determining the protein concentration, storing at 4 ℃ or-20 ℃ for later use, and designating the protein as P2-NS 3.
1.1.2 preparation of hepatitis C marker antigen
The NS3 fragment of 1.1.1 is used to be connected into a vector P2-X which is treated by BamHI and EcoRI double digestion to obtain a recombinant plasmid P2-X-NS3 which is the recombinant plasmid of the labeled antigen of the invention. The P2-X-NS3 plasmid was transformed into ER2566, a single colony was picked up, inoculated to 500mL LB medium containing 100. mu.g/mL kanamycin sulfate and shake-cultured at 37 ℃ to OD600This was induced with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. Centrifuging at 4 ℃ for 20 minutes at 5000g for collecting thalli, resuspending the thalli of each liter of bacterial liquid by 20mL of lysis buffer, carrying out ultrasonic disruption, centrifuging at 4 ℃ for 20 minutes at 12000g, and after being identified by SDS-PAGE electrophoresis, distributing part of target protein in lysate sediment. The cells of each liter of the bacterial solution were resuspended in 10mL of lysis buffer (50mM Tris-HCl, pH8.0, 1mM EDTA, 100mM NaCl), sonicated, centrifuged at 12000rpm at 4 ℃ for 20 minutes to collect inclusion bodies, resuspended in solution 1(20mM Tris-HCl, pH8.5, 5mM EDTA, 100mM NaCl) containing 2% Triton X-100, centrifuged at 12000rpm at 4 ℃ for 20 minutes to collect inclusion bodies, solubilized with 6M urea prepared in solution 1, dialyzed against 100 volumes of PB buffer (pH7.0, 20mM), changed 3 times, centrifuged at 12000rpm for 20 minutes at 4 ℃ to remove precipitates to prepare crude antigens, equilibrated with the same PB buffer in a Sephacryl S-200 gel column (Amersham Biosciences), passed through the column, collected and pooled solutions containing the target protein, and 5mL of equilibrated buffer (10mM Na) was added2HPO4，1.8mM KH2PO4140mM NaCl, 2.7mM KCl, 25mM imidazole, pH 8.0). After equilibration of the Ni-NTA affinity column (Qiagen, cat # 30210) with 10 bed volumes of equilibration buffer, the protein sample was added, unbound protein was washed off with 10 medium volumes of equilibration buffer, and 5 volumes of elution buffer (20mM Na2HPO4300mM NaCl, 250mM imidazole, pH8.0), eluting the target protein, determining the protein concentration, storing at 4 ℃ or-20 ℃ for later use, and naming the protein as X-NS 3.
1.1.3 preparation of immunogen and hybridoma cell screening antigen (protein X)
With P2-X, PinpointTMXa-1 (Promega, USA, Cat. V2031) plasmid was transformed into E.coli ER2566, which was transformed into a plasmid containing the antibiotic gene,distribution selection was performed by plating on LB plates containing 100. mu.g/mL kanamycin sulfate or 100. mu.g/mL ampicillin sodium, overnight culturing at 37 ℃, picking out single clones, and shaking culturing to OD at 37 ℃ in 500mL LB medium containing the same concentration of kanamycin sulfate or ampicillin sodium600This was induced with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. Centrifuging at 4 ℃ for 20 minutes at 5000g for collecting thalli, resuspending the thalli of each liter of bacterial liquid by 20mL of lysis buffer, carrying out ultrasonic disruption, centrifuging at 4 ℃ for 20 minutes at 12000g, and after being identified by SDS-PAGE electrophoresis, distributing part of target protein in lysate sediment. Each liter of the bacterial cells were resuspended in 10mL of lysis buffer (50mM Tris-HCl, pH8.0, 1mM EDTA, 100mM NaCl), sonicated, centrifuged at 12000rpm at 4 ℃ for 20 minutes to collect inclusion bodies, resuspended in solution 1(20mM Tris-HCl, pH8.5, 5mM EDTA, 100mM NaCl) containing 2% Triton X-100, centrifuged at 12000rpm at 4 ℃ for 20 minutes to collect inclusion bodies, solubilized with 8M urea prepared in solution 1, dialyzed against 100 volumes of PB buffer (pH7.0, 20mM), changed 3 times, centrifuged at 12000rpm for 20 minutes at 4 ℃ to remove precipitates to prepare crude antigens, equilibrated with the same PB buffer on a Sephacryl S-200 gel column (Amersham Biosciences), passed through a column, collected and pooled solutions containing the target protein, and then passed through a DEAE ion exchange column (Amersham Biosciences PB column), eluted with pHPBS (Amersham Biosciences PB buffer (Amersham Biosciences), 50mM NaCl) to remove the non-adsorbed foreign proteins, eluting the target protein with PBS buffer (pH7.0, 20mM PB, 500mM NaCl), determining the protein concentration, and storing at 4 ℃ or-20 ℃ for later use.
1.2 establishment of anti-X hybridoma cell line and preparation of monoclonal antibody thereof
1.2.1 recombinant X protein immunization of mice
After the P2-X expression X protein solution was dialyzed with PBS, it was diluted to 1.0mg/mL with PBS, mixed with Freund's complete adjuvant in equal volume, and well emulsified, and 6-week-old inbred BALB/c mice were immunized by dorsal subcutaneous injection at a dose of 50. mu.g/mouse. After 2 weeks, protein X was mixed with Freund's incomplete adjuvant in equal volume, emulsified well, and injected intraperitoneally at a dose of 50. mu.g/mouse. After 2 weeks, protein X was injected intraperitoneally without adjuvant at a dose of 50. mu.g/mouse. And 7 days after the third immunization, taking tail blood of the mice, separating serum, and measuring the titer by an indirect ELISA method, wherein the titer is higher than 1:10000, and the mice can be used for fusion. 4 days before fusion, the fourth immunization, protein X without adjuvant, tail vein injection, 50 μ g/mouse.
1.2.2 preparation of hybridoma cell lines
The myeloma cells Sp2/0(ATCC, cat # CRL-1581) were recovered in advance 12 days before the fusion and cultured in 1640 medium containing 10% fetal bovine serum. Cell concentration was adjusted to 3X 10 24h before fusion5and/mL. Myeloma cells were harvested the following day, centrifuged at 1200g for 5 minutes, washed 3 times with serum-free medium and the cells counted. The immunized mice were sacrificed, spleens were removed, a cell suspension was prepared, cells were counted, and 10 cells were collected8Splenocytes, and 2X 107Myeloma cells were mixed, fused with 50% PEG1500, and hybridomas were selectively cultured in HAT medium.
1.2.3 screening and cloning of hybridoma cell lines
After the fusion, the antibody activity in the supernatant of each well of the fused cells was measured by ELISA method on day 10 of culture. 200 μ L of each fusion cell culture supernatant was added to a 96-well ELISA plate well, each well being coated with 10 μ g/mL of a different protein X. The reaction was carried out at 37 ℃ for 1 hour, then washed 5 times with PBS containing 0.05% Tween20 and a 1:5000 dilution of HRP-labeled anti-mouse IgG was added to each well. The reaction was carried out at 37 ℃ for 1 hour, the plate was washed 5 times with the above-mentioned washing solution, and then 50. mu.L each of a developer A containing 0.05% o of urea hydrogen peroxide, 4.76% o of sodium acetate trihydrate, 0.9% o of glacial acetic acid and a developer B containing 0.32% o of TMB, 5mM of citric acid, 0.5mM of EDTA-2Na, 5% of methanol, and 2% o of dimethylformamide was added to each well, and light-shielding development was carried out at 37 ℃ for 30 minutes. Add 50. mu.L to each well, stop the reaction with stop solution containing 2M sulfuric acid, and measure 450nm wavelength of each well with microplate reader. And cloning by limiting dilution for three times to finally obtain 12 cell strains which stably secrete anti-X protein, wherein one cell strain with the highest antibody activity secretes an antibody named 3D 4.
1.2.4 preparation and purification of ascites of protein X monoclonal antibody
The BALB/c mice of 8 weeks old were injected with liquid paraffin 0.5 mL/mouse. Intraperitoneal injection of 1X 10 after 1 week7And 3D4 hybridoma cells. Abdominal Collection 7 days after cell inoculationWater, 3000g centrifugal 10 minutes, the supernatant with 0.01M pH7.4 PBS diluted 3 times. The sample was passed through a Protein A immunoaffinity chromatography column on FPLC system, eluting with glycine of pH 2.8. Collecting specific protein peak effluent, immediately correcting to pH7.0 with Tris solution of pH9.0, filtering, sterilizing, packaging, and storing at-80 deg.C.
1.3 preparation of colloidal gold kit indirectly labeled with hepatitis C
1.3.1 preparation of colloidal gold
Adding 100mL of ultrapure water into a triangular flask, heating the flask to boiling on a magnetic heater, adding 1mL of 1% chloroauric acid solution, immediately adding 1mL of 1% trisodium citrate aqueous solution after boiling, continuing boiling for 10 minutes, and naturally cooling.
1.3.2 colloidal gold labeling
a. 10mL of the above colloidal gold was put into a beaker, and 150. mu.L of 0.2M K was added thereto with stirring2CO3Adjusting the pH value to 7.0, and continuing stirring for 20 seconds;
b. adding a certain amount of 3D4 monoclonal antibody, and continuing stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 minutes;
d.5000g for 10 minutes, sucking out the supernatant, collecting the precipitate to a centrifuge tube, and diluting to 1mL with colloidal gold diluent (20mM PB, 150mM NaCl, 1% BSA, 0.1% Triton X-100, 2% sucrose, 0.01% Proclin 300);
e. finally, a certain amount of labeled antigen X-NS3 is added into the 1mL colloidal gold labeled 3D4 monoclonal antibody compound, and the indirectly labeled gold labeled compound is named as X-NS3-3D 4-Au.
1.3.3 preparation of gold-labeled pad
Diluting the gold-labeled compound by 10 times of colloidal gold diluent, soaking a glass fiber membrane, and freeze-drying to obtain the gold-labeled pad.
1.3.4 nitrocellulose Membrane (NC Membrane) coating
Diluting the envelope antigen P2-NS 3-0.8 mg/mL with a detection line diluent (10mM PBS, 2% sucrose) to prepare a detection line working solution, diluting the goat anti-mouse monoclonal antibody to 0.5mg/mL with the same diluent to prepare a control line working solution, drawing the two working solutions on corresponding positions of a nitrocellulose membrane by using a membrane spotting instrument, and drying at 37 ℃ for 1 hour.
1.3.5 Assembly
And (3) assembling the gold label pad, the coated nitrocellulose membrane, absorbent paper, a PVC base plate, a sample pad and other auxiliary materials into the HCV gold label detection kit.
1.3.6 detection method
HCV antibodies are detected by applying 100. mu.L of a test sample (e.g., serum) to the sample pad.
1.4 preparation of gradient addition 'nanoparticle-ligand' colloidal gold kit indirectly marked by hepatitis C
1.4.1 preparation of hepatitis C marker complexes
a. 10mL of the colloidal gold of example 1.3.1 was placed in a beaker, and 150. mu.L of 0.2M K was added with stirring2CO3Adjusting the pH value to 7.0, and continuing stirring for 20 seconds;
b. adding a certain amount of 3D4 monoclonal antibody, and continuing stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 minutes;
d.5000g, centrifuging for 10 minutes, sucking out supernatant, collecting precipitate to a centrifuge tube, and fixing the volume to 1mL by using colloidal gold diluent;
e. adding a certain amount of labeled antigen X-NS3 into the 1mL of colloidal gold labeled 3D4 monoclonal antibody compound;
f. and (b) continuously adding colloidal gold labeled with the 3D4 monoclonal antibody into the compound, wherein the addition amount is performed according to a gradient of 5%, 10%, 15%, 20%, 25% and 30% of the constant volume (1mL), the labeling concentration of the 3D4 monoclonal antibody is consistent with that of the step b, and the gold labeled compound added with the nanoparticle-ligand is named as + Au-3D4 (5%) …, and so on.
1.4.2 Assembly
See example 1.3.5.
1.5 comparison of detection effects of two detection kits for hepatitis C
The results of HCV positive and negative sera were compared with indirect labeling with "nanoparticle-ligand" using RIBA reagent from Chiron corporation, USA as confirmation control.
1.5.1 sensitivity
Using a colloidal gold kit with indirect labeling of whether "nanoparticle-ligand" was added or not, 100 serial dilutions of serum were tested under the same conditions, yielding the results of Table 1: for the detectable serum with the dilution from low to high, the sensitivity after adding Au-3D4 is obviously higher than that of the original indirectly labeled gold-labeled complex.
TABLE 1 comparison of sensitivity of two detection kits for hepatitis C
From the results in Table 1, the gradient was further refined, and the same serum was tested at 100 serial dilution ratios to obtain the results in Table 2.
TABLE 2 gradient refinement
According to tables 1 and 2, the optimal gradient group is selected for specific amplification experiments.
1.5.2 specificity
3000 clinical negative sera were tested with an indirect labeled colloidal gold kit with "nanoparticle-ligand" addition under the same conditions: the specificity of the common indirect labeling kit is 99.7%, the specificity of the indirect labeling kit with gradient addition of nanoparticle-ligand is 99.7%, 99.7% and 99.6% respectively, and the specificity of the two labeling modes is equivalent.
1.5.3 stability
The finished product kit is examined at 37 ℃ for 7 days, the kit which is taken out and stored at 4 ℃ at the same time, and the kit which is prepared from the marker which is stored at 4 ℃ and added with the X-NS3-3D4-Au marker of 'nano particle-ligand', detect the same negative and positive serums under the same condition to investigate the stability of the kit, and the result is shown in table 3.
TABLE 3 stability assessment of markers and kits
Note: + is weak yang, + is medium yang, + is strong yang, -is negative.
The experiment shows that the marker and the kit have good stability.
1.5.4 precision
The kit added with 10% gradient nanoparticle-ligand is used for detecting the same known HCV positive specimen, 10 times of repeated experiments are carried out, the obtained test strips are positive in result, the color development degree has no obvious difference, and the kit is proved to have good precision.
Example two antibody detection of indirectly labeled nanoparticles of Treponema Pallidum (TP)
2.1 preparation of recombinant antigen of syphilis
2.1.1 preparation of syphilis-coated antigen
PCR amplifies the DNA fragment corresponding to 22-156aa of the 17Kda (TP17) gene of treponema pallidum, the upstream primer of which has BamHI site, the downstream primer has EcoRI site and the coding sequence of 6 His amino acids before the EcoRI site and the termination code TAA. After the PCR fragment was recovered, it was digested with BamHI and EcoRI, and ligated to the expression vector P2 digested with BamHI and EcoRI to obtain the recombinant plasmid P2-TP17, which is the antigen-coated recombinant plasmid of the present invention. Plasmid P2-TP17 was transformed into ER2566 cells, which were shake-cultured at 37 ℃ in 500mL LB medium containing 100. mu.g/mL kanamycin sulfate to OD600This was induced with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. Centrifuging at 4 ℃ for 20 minutes at 5000g, collecting thalli, resuspending the thalli of each liter of bacterial liquid by using 20mL of lysis buffer (50mM Tris-HCl, pH8.0, 1mM EDTA, 100mM NaCl), carrying out ultrasonic disruption, centrifuging at 4 ℃ for 20 minutes at 12000g, and after being identified by SDS-PAGE electrophoresis, distributing most of target protein in supernatant of the lysate. Collecting supernatant, slowly adding dropwiseAdding saturated ammonium sulfate solution to final concentration of ammonium sulfate of 30%, standing at 4 deg.C for 30 min, centrifuging at 4 deg.C 12000g for 20 min, collecting supernatant, adding saturated ammonium sulfate dropwise slowly to final concentration of ammonium sulfate of 60%, standing at 4 deg.C for 30 min, centrifuging at 4 deg.C 12000g for 20 min, collecting precipitate, and adding 5mL balance buffer (10mM Na)2HPO4，1.8mM KH2PO4140mM NaCl, 2.7mM KCl, 25mM imidazole, pH 8.0). After equilibration of the Ni-NTA affinity column (Qiagen, cat # 30210) with 10 bed volumes of equilibration buffer, the protein sample was added, unbound protein was washed off with 10 medium volumes of equilibration buffer, and 5 volumes of elution buffer (20mM Na2HPO4300mM NaCl, 250mM imidazole, pH8.0), eluting the target protein, determining the protein concentration, storing at-20 ℃ for later use, and designating the protein as P2-TP 17.
2.1.2 preparation of syphilis marker antigen
The fragment TP17 of 2.1.1 was ligated to vector pGEX-6P-1 (pharmacia, cat # 27-4597-01) which had been digested with BamHI and EcoRI to obtain recombinant plasmid pGEX-6P-1-TP17, which is a recombinant plasmid for labeling antigen of the present invention. The above positive clones were inoculated into 500mL LB medium containing 100. mu.g/mL ampicillin sodium and shake-cultured at 37 ℃ to OD600This was induced with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. Centrifuging at 4 ℃ for 20 minutes at 5000g, collecting thalli, resuspending the thalli of each liter of bacterial liquid by using 20mL of lysis buffer (50mM Tris-HCl, pH8.0, 1mM EDTA, 100mM NaCl), carrying out ultrasonic disruption, centrifuging at 4 ℃ for 20 minutes at 12000g, and after being identified by SDS-PAGE electrophoresis, distributing most of target protein in supernatant of the lysate. Collecting supernatant, gradually dropwise adding saturated ammonium sulfate solution until the final concentration of ammonium sulfate is 25%, standing at 4 deg.C for 30 min, centrifuging at 4 deg.C for 20 min at 12000g, collecting supernatant, continuously gradually dropwise adding saturated ammonium sulfate until the final concentration of ammonium sulfate is 45%, standing at 4 deg.C for 30 min, centrifuging at 4 deg.C for 20 min at 12000g, collecting precipitate, and dissolving with 10mL of balance buffer solution. After equilibration of the GSTrap affinity column (Amersham, Cat. 17-5130-02) with 10 bed volumes of equilibration buffer, the protein sample was added, unbound protein was washed off with 10 medium volumes of equilibration buffer, and the elution buffer was then diluted 5 volumesWashing solution (50mM Tris-HCl, 10mM reduced glutathione, pH8.0), eluting the target protein, determining the protein concentration, storing at-20 ℃ for later use, and naming the protein as GST-TP 17.
2.1.3 preparation of immunogen and hybridoma cell selection antigen (GST protein)
Escherichia coli ER2566 was transformed with plasmids pGEX-2T (pharmacia, cat # 27-4801-01), pGEX-6P-1, pGEX-5X-1 (pharmacia, cat # 27-4584-01) and pET-41a (Novagen, cat # 70556-3), distributed and selected according to the antibiotic gene carried by the plasmid, spread on LB plate containing 100. mu.g/mL kanamycin sulfate or 100. mu.g/mL ampicillin sodium, cultured overnight at 37 ℃ to pick up a single clone, and cultured with 500mL LB medium containing the same concentration of kanamycin sulfate or ampicillin sodium at 37 ℃ with shaking until OD600This was induced with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. Centrifuging at 4 ℃ for 20 minutes at 5000g for collecting thalli, resuspending the thalli of each liter of bacterial liquid by 20mL of lysis buffer, carrying out ultrasonic disruption, centrifuging at 4 ℃ for 20 minutes at 12000g, and identifying by SDS-PAGE electrophoresis, wherein most of target proteins are distributed in the supernatant of the lysate. Slowly adding saturated ammonium sulfate while rapidly stirring uniformly to make the final concentration of ammonium sulfate be 30%, standing at 4 deg.C for 30 min, centrifuging at 4 deg.C for 20 min, collecting supernatant, slowly adding saturated ammonium sulfate while rapidly stirring uniformly to make the cumulative concentration of ammonium sulfate be 60%, standing at 4 deg.C for 30 min, centrifuging at 4 deg.C for 20 min, collecting precipitate, and dissolving with 10mL of balance buffer solution. After the GSTrap affinity column was equilibrated with 10 bed volumes of equilibration buffer, the protein sample was added, unbound protein was washed off with 10 medium volumes of equilibration buffer, the target protein was eluted with 5 volumes of elution buffer, the protein concentration was determined, and the column was stored at-20 ℃ for future use.
2.2 establishment of anti-GST hybridoma cell lines and preparation of monoclonal antibodies thereof
2.2.1 recombinant GST protein immunized mice
See example 1.2.1, mice were immunized with recombinant GST protein.
2.2.2 preparation of hybridoma cell lines
See example 1.2.2 for cell fusion.
2.2.3 screening and cloning of hybridoma cell lines
After the fusion, the antibody activity in the supernatant of each well of the fused cells was measured by ELISA method on day 10 of culture. 200 μ L of each fusion cell culture supernatant was added to a 96-well ELISA plate well, each well being coated with 10 μ g/mL of a different GST protein. The subsequent steps are shown in 1.2.3, wherein the antibody secreted by the cell with the highest antibody activity is named 6F 8.
2.2.4 preparation and purification of GST protein monoclonal antibody
See example 1.2.4 for 6F8 mab after purification.
2.3 preparation of colloidal gold kit indirectly labeled with syphilis
2.3.1 preparation of colloidal gold
Colloidal gold was prepared as described in example 1.3.1.
2.3.2 colloidal gold labeling
a. 10mL of the above colloidal gold was put into a beaker, and 150. mu.L of 0.2M K was added thereto with stirring2CO3Adjusting the pH value to 7.0, and continuing stirring for 20 seconds;
b. adding a certain amount of 6F8 monoclonal antibody, and continuing stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 minutes;
d.5000g, centrifuging for 10 minutes, sucking out supernatant, collecting precipitate to a centrifuge tube, and fixing the volume to 1mL by using colloidal gold diluent;
e. finally, a certain amount of labeled antigen GST-TP17 is added into the 1mL colloidal gold labeled 6F8 monoclonal antibody compound, and the indirectly labeled gold labeled compound is named as GST-TP17-6F 8-Au.
2.3.3 preparation of gold-labeled pad
See example 1.3.3 for preparation of gold-labeled pads.
2.3.4 nitrocellulose Membrane (NC Membrane) coating
Diluting the envelope antigen P2-TP 17-0.8 mg/mL by using a detection line diluent to prepare a detection line working solution, diluting the goat anti-mouse monoclonal antibody to 0.5mg/mL by using the same diluent to prepare a control line working solution, drawing the two working solutions onto corresponding positions of a nitrocellulose membrane by using a membrane spotting instrument, and drying at 37 ℃ for 1 hour.
2.3.5 Assembly
See 1.3.5 for assembly of a TP gold-labeled assay kit.
2.3.6 detection method
See example 1.3.6 for detection of TP antibody.
2.4 preparation of syphilis indirectly labeled gradient addition 'nanoparticle-ligand' colloidal gold kit
2.4.1 preparation of syphilis-labeled Complex
a. 10mL of the colloidal gold of example 1.3.1 was placed in a beaker, and 150. mu.L of 0.2M K was added with stirring2CO3Adjusting the pH value to 7.0, and continuing stirring for 20 seconds;
b. adding a certain amount of 6F8 monoclonal antibody, and continuing stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 minutes;
d.5000g, centrifuging for 10 minutes, sucking out supernatant, collecting precipitate to a centrifuge tube, and fixing the volume to 1mL by using colloidal gold diluent;
e. adding a certain amount of labeled antigen GST-TP17 into the 1mL of colloidal gold labeled 6F8 monoclonal antibody compound;
f. and (b) continuously adding colloidal gold labeled with the 6F8 monoclonal antibody into the compound, wherein the addition amount is performed according to a gradient of 5%, 10%, 15%, 20%, 25% and 30% of the constant volume (1mL), the labeling concentration of the 6F8 monoclonal antibody is consistent with that of the step b, and the gold labeled compound added with the nanoparticle-ligand is named as + Au-6F8 (5%) …, and so on.
2.5 comparison of detection effects of two syphilis detection kits
TPPA kit of Fuji of Japan was used as a confirmation control, and the results of TP positive serum and TP negative serum were compared with each other with respect to whether "nanoparticle-ligand" was added as an indirect marker.
2.5.1 sensitivity
Using the indirect labeling colloidal gold kit with the addition of "nanoparticle-ligand" or not, 100 serial dilutions of serum were tested under the same conditions, yielding the results in Table 4: for the detectable serum with the dilution from low to high, the sensitivity after adding Au-6F8 is obviously higher than that of the original indirectly labeled gold-labeled complex.
TABLE 4 comparison of sensitivity of two detection kits for syphilis
The group with the optimal gradient (10%) was selected for specific amplification experiments.
2.5.2 specificity
3000 clinical negative sera were tested with an indirect labeled colloidal gold kit with "nanoparticle-ligand" addition under the same conditions: the specificity of a common indirect labeling kit is 99.8 percent, the specificity of a 10 percent indirect labeling kit with gradient addition of nanoparticle-ligand is 99.8 percent, and the specificity of the two labeling modes is equivalent.
2.5.3 stability
The finished kit of the invention [ marker: and the kit is prepared from a kit which is taken out and stored at 4 ℃ at the same time and a marker which is stored at 4 ℃ and is not added with a GST-TP17-6F8-Au marker of 'nanoparticle-ligand', and the same negative and positive serums are detected under the same condition to investigate the stability of the kit, and experimental results show that the marker and the kit have better stability.
2.5.4 precision
The kit added with 10% gradient nanoparticle-ligand is used for detecting the same known TP positive specimen, 10 times of repeated experiments are carried out, the obtained test strips are positive in result, the color development degree has no obvious difference, and the kit is proved to have good precision.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An antigen labeling method, comprising the steps of (i) ligand-labeling a nanoparticle-based label, (ii) labeling the ligand-nanoparticle-based label obtained in step (i) with an antigen, (iii) adding the ligand-labeled nanoparticle-based label to a labeling system to obtain an antigen-labeled complex; wherein the labeling between the nanoparticle-based label and the labeled antigen is indirect labeling which is performed by combining a label on the antigen and the ligand which is labeled on the nanoparticle-based label and can specifically recognize the label.
2. The method of claim 1, wherein the marker antigen comprises, but is not limited to, HIV antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis E virus antigens, hepatitis G virus antigens, rubella virus antigens, human cytomegalovirus antigens, herpes simplex virus type 1 antigens, herpes simplex virus type 2 antigens, rabies virus antigens, human T-lymphocyte leukemia virus antigens, dengue virus antigens, human papilloma virus antigens, West Nile virus antigens, forest encephalitis virus antigens, measles virus antigens, influenza virus antigens, parainfluenza virus antigens, varicella virus antigens, echovirus antigens, coxsackie virus antigens, encephalitis B virus antigens, EB virus antigens, mumps virus antigens, treponema antigens, influenza virus antigens, HIV antigens, a Borrelia burgdorferi antigen, a Chlamydia trachomatis antigen, a Chlamydia pneumoniae antigen, a Chlamydia psittaci antigen, a ureaplasma urealyticum antigen, a Mycoplasma pneumoniae antigen, a Mycobacterium tuberculosis antigen, a helicobacter pylori antigen, a gonococcus antigen, a Plasmodium antigen, a Trypanosoma cumini antigen and a Toxoplasma gondii antigen.
3. The method of claim 1, wherein the nanoparticle-based labels comprise nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, rare earth complex nanoparticles, and their respective derivatives, or a combination of two or more thereof; wherein the colloid comprises colloid metal, disperse dye, dye-labeled microspheres and latex; wherein the colloidal metal comprises colloidal gold, colloidal silver and colloidal selenium.
4. The method of claim 1, wherein the tag is a polypeptide or protein selected from the group consisting of: his Tag, T7 Tag, S Tag, Flag Tag, HA Tag, GST fusion protein, Trx fusion protein, SOD fusion protein, CKS fusion protein, DsbA fusion protein, MBP fusion protein, CBD fusion protein, or biotin.
5. The method according to claim 1, wherein the ligand includes, but is not limited to, a specific antibody against a tag, a substance with affinity to a tag such as streptavidin or an analogue thereof.
6. The method according to claim 1, wherein the additive amount of the nanoparticle-based marker additionally added with the labeled ligand is 5% -30% of the constant volume of the labeling system; for example, the volume of the marking system can be 7.5% -17.5%; preferably 10%.
7. The method of claim 6, wherein the labeling system uses a marker dilution comprising 20mM PB, 150mM NaCl, 1% BSA, 0.1% Triton X-100, 2% sucrose, and 0.01% Proclin 300.
8. The antigen-labeled complex according to any one of claims 1 to 7.
9. A test kit comprising the antigen-labeled complex according to claim 8.
10. Use of the method of any one of claims 1 to 7 and/or the antigen-labelled complex of claim 8 in the preparation of an antibody detection kit.
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