CN113588940B - Method and kit for improving sensitivity of indirect labeling - Google Patents
Method and kit for improving sensitivity of indirect labeling Download PDFInfo
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Abstract
The invention provides a method and a kit for improving the sensitivity of indirect labeling, which adopt a double-antigen sandwich method to detect the content of an antibody to be detected in a sample; the method and the kit can improve the sensitivity of the indirect labeling original mode.
Description
Technical Field
The 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
An "indirect nanoparticle labeling antibody detection double antigen sandwich method and a kit thereof" have been proposed in the chinese patent application No. 200810216400.0, which studied the application of indirect labeling in nanoparticle labels such as colloidal gold. Nanoparticles because of their larger particle size, the final morphology of the label is nanoparticle label-one nanoparticle label surface coupled with multiple antigens, and indirect labeling offers many advantages: firstly, indirect labeling is more beneficial to exposing antigen epitopes because the indirect labeling is equivalent to adding an arm between the nano-particles and the labeled antigen; secondly, the dosage of the labeled antigen can be reduced, and the ideal ratio of the antigen to the nanoparticle label is 1:1.
In practical experiments, the nano-particle markers undergo the oscillation process of the labeled ligand and the labeled antigen, and a 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 further improvement in the labeling method of indirect labeling to improve 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) labeling a nanoparticle label with a ligand, (ii) labeling the nanoparticle label with the ligand obtained in the step (i), and (iii) adding the nanoparticle label with the ligand to a labeling system in a supplementing manner to obtain an antigen labeling complex; wherein the labeling between the nanoparticle-based label and the labeled antigen is an indirect labeling by binding of a label on the antigen to the ligand on the nanoparticle-based label that specifically recognizes the label.
2. The method of item 1, wherein the marker antigen comprises, but is not limited to, an hiv antigen, a hepatitis a virus antigen, a hepatitis b virus antigen, a hepatitis c virus antigen, a hepatitis b virus antigen, a hepatitis e virus antigen, a hepatitis g virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T lymphoblastic leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a west nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an echovirus antigen, a coxsackie virus antigen, a encephalitis b virus antigen, an EB virus antigen, a mumps virus antigen, a treponema pallidum antigen, a borrelia antigen, a chlamydia trachomatis antigen, a chlamydia antigen, a helicobacter pylori antigen, a gonococcus antigen, a plasmodium antigen, a trypanosoma 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-marked microsphere 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 comprises, but is not limited to, a specific antibody to the tag, a substance having affinity for the tag, such as streptavidin or an analog thereof.
6. The method of item 1, wherein the additional addition of the ligand-labeled nanoparticle label is in an amount of 5% to 30% of the volumetric volume of the labeling system; for example, it may be 7.5% -17.5% of the volume of the marking system; preferably 10%.
7. The method of item 7, wherein the marker system uses a marker dilution comprising a composition of 20mM PB,150mM NaCl,1%BSA,0.1%Triton X-100,2% sucrose, 0.01% procrin300.
8. The antigen-labeled complex according to any one of items 1 to 7.
9. A detection kit comprising the antigen-labeled complex according to item 8.
10. The method of any one of items 1 to 7 and/or the use of 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 X fusion protein.
Detailed Description
In some embodiments, the present invention provides an antigen labeling method, wherein the method comprises the steps of (i) labeling a nanoparticle-based label with a ligand, (ii) labeling the antigen-labeled nanoparticle-based label obtained in step (i), and (iii) adding the ligand-labeled nanoparticle-based label to a labeling system in a complementary manner to obtain an antigen-labeled complex; wherein the labeling between the nanoparticle-based label and the labeled antigen is an indirect labeling by binding of a label on the antigen to the ligand on the nanoparticle-based label that specifically recognizes the label.
In some embodiments, the antigen labeling methods of the present invention include, but are not limited to, an hiv antigen, a hepatitis a virus antigen, a hepatitis b virus antigen, a hepatitis c virus antigen, a hepatitis b virus antigen, a hepatitis e virus antigen, a hepatitis g virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T lymphoblastic leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a west nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an ico virus antigen, a coxsackie virus antigen, a encephalitis b virus antigen, an EB virus antigen, a treponema pallidum antigen, a borrelia antigen, a trachoma chlamydia antigen, a tuberculosis antigen, a helicobacter pylori antigen, a helicobacter antigen, a mycoplasma antigen, a cone antigen, a trypanosoma antigen.
In some embodiments, the antibody detection methods of the present invention that indirectly label nanoparticles can be applied to immunodetection with nanoparticles as labels and/or signals; 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 metal, 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 to the N-terminus, the C-terminus, both ends, or within the labeled antigen.
In some embodiments, the ligands of the invention include, but are not limited to, specific antibodies to the tag, substances having affinity for the tag, such as streptavidin or analogs thereof.
In some embodiments, the added amount of the nanoparticle label added with the labeled ligand in a supplementary way can be 5-30% of the volume of the fixed volume of the labeling system; in some embodiments, the amount added may be 7.5% to 17.5% of the volumetric volume of the marking system; in some embodiments, the amount added may be, for example, 5%, such as 7.5%, such as 10%, such as 12.5%, such as 15%, such as 17.5%, such as 20%, such as 22.5%, such as 25%, such as 27.5%, such as 30%, but is not limited thereto.
In some embodiments, the marker dilution used in the marking system of the present invention comprises 20mM PB,150mM NaCl,1%BSA,0.1%Triton X-100,2% sucrose, 0.01% proclin300.
In some embodiments, the 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 formulated as a label-binding pad; in some embodiments, the preparation method includes, but is not limited to, lyophilization, oven drying, and air drying.
In some embodiments, the invention provides a kit (test strip) comprising the antigen-labeled complex of any of the embodiments.
In some embodiments, the invention also provides for the use of the method and/or antigen-labeled complex comprising any of the embodiments in the preparation of an antibody detection kit.
In some embodiments, the sensitivity can be further improved based on the indirect labeling of the original pattern using the methods and/or kits of the present invention.
The present invention will be described in further detail with reference to the following examples. The following examples are provided to illustrate embodiments of the invention and are not intended to limit the invention. The invention may optionally include embodiments not shown by way of example. The cloning vector P2 map used in the present invention is shown in FIG. 1, P2 is not a necessary expression vector in the practice of the present invention, and the enhancer upstream thereof does not play a substantial role in the present invention, and many other expression vectors such as pET-24a (+) (Novagen, U.S.A., 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 antigen of hepatitis C
1.1.1 preparation of hepatitis C coating antigen
The DNA segment corresponding to the full-length 1201-1465aa of the ORF of the hepatitis C virus genome is amplified by PCR, wherein the upstream primer carries a BamHI site, the downstream primer carries an EcoRI site and the coding sequence of 6 His amino acids before the EcoRI site and the termination codon TAA. After the PCR fragment was recovered, it was digested with BamHI and EcoRI, and ligated into the expression vector P2 digested with BamHI and EcoRI to obtain recombinant plasmids P2-NS3, i.e., the antigen-coated recombinant plasmids of the present invention.
ER2566 bacteria were transformed with plasmid P2-NS3, and cultured with 500mL LB medium containing 100. Mu.g/mL kanamycin sulfate at 37℃under shaking to OD 600 About=1.0, induction was performed with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. And (3) centrifuging at 4 ℃ for 20 minutes at 5000g, collecting thalli, re-suspending each liter of thalli by using 20mL of lysis buffer, performing ultrasonic crushing, centrifuging at 12000g at 4 ℃ for 20 minutes, and precipitating part of target proteins after SDS-PAGE electrophoresis identification. The cells per liter of the bacterial solution were resuspended in 10mL of lysis buffer (50 mM Tris-HCl, pH8.0,1mM EDTA,100mM NaCl), sonicated, the inclusion bodies were collected by centrifugation at 12000rpm at 4℃for 20 minutes, resuspended in 2% Triton X-100-containing solution 1 (20 mM Tris-HCl, pH8.5,5mM EDTA,100mM NaCl), collected by centrifugation at 12000rpm at 4℃for 20 minutes, lysed with 4M urea prepared from solution 1, dialyzed against 100 volumes of PB buffer (pH 7.0, 20 mM), thawed 3 times, centrifuged at 12000rpm at 4℃for 20 minutes to remove the precipitate and prepare crude antigen, the crude antigen was equilibrated with the same PB buffer on a Sephacryl S-200 gel column (America Amersham Biosciences Co.), the solution containing the desired protein was collected and pooled, and 5mL of equilibration buffer (10 mM Na. RTM) 2 HPO 4 ，1.8mM KH 2 PO 4 140mM 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, a protein sample was added, unbound protein was washed away with 10 volumes of equilibration buffer, followed by 5 volumes of elution buffer (20 mM Na 2 HPO 4 300mM NaCl,250mM imidazole, pH 8.0), eluting the target protein, determining the protein concentration, and preserving at 4 ℃ or-20 ℃ for later use, wherein the protein is named as P2-NS3.
1.1.2 preparation of hepatitis C marker antigen
1 is used.1.1, and ligating the NS3 fragment to the vector P2-X after double digestion with BamHI and EcoRI to give a recombinant plasmid P2-X-NS3, i.e., a recombinant plasmid of the labeled antigen of the present invention. ER2566 is transformed by the P2-X-NS3 plasmid, a monoclonal colony is selected, inoculated in 500mL LB culture medium containing 100 mug/mL kanamycin sulfate, and shake-cultured at 37 ℃ until OD 600 About=1.0, induction was performed with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. And (3) centrifuging at 4 ℃ for 20 minutes at 5000g, collecting thalli, re-suspending each liter of thalli by using 20mL of lysis buffer, performing ultrasonic crushing, centrifuging at 12000g at 4 ℃ for 20 minutes, and precipitating part of target proteins after SDS-PAGE electrophoresis identification. The cells per liter of the bacterial solution were resuspended in 10mL of lysis buffer (50 mM Tris-HCl, pH8.0,1mM EDTA,100mM NaCl), sonicated, the inclusion bodies were collected by centrifugation at 12000rpm at 4℃for 20 minutes, resuspended in 2% Triton X-100-containing solution 1 (20 mM Tris-HCl, pH8.5,5mM EDTA,100mM NaCl), collected by centrifugation at 12000rpm at 4℃for 20 minutes, lysed with 6M urea prepared from solution 1, dialyzed against 100 volumes of PB buffer (pH 7.0, 20 mM), thawed 3 times, centrifuged at 12000rpm at 4℃for 20 minutes to remove the precipitate and prepare crude antigen, the crude antigen was equilibrated with the same PB buffer on a Sephacryl S-200 gel column (America Amersham Biosciences Co.), the solution containing the desired protein was collected and pooled, and 5mL of equilibration buffer (10 mM Na. RTM.) was used 2 HPO 4 ，1.8mM KH 2 PO 4 140mM 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, a protein sample was added, unbound protein was washed away with 10 volumes of equilibration buffer, followed by 5 volumes of elution buffer (20 mM Na 2 HPO 4 300mM NaCl,250mM imidazole, pH 8.0), eluting the target protein, determining the protein concentration, and preserving at 4 ℃ or-20 ℃ for later use, wherein the protein is named X-NS3.
1.1.3 preparation of immunogens and hybridoma cell screening antigens (protein X)
In P2-X, pinpoint TM Xa-1 (Promega Co., U.S.A., cat# V2031) plasmid transformed E.coli ER2566 was selectively spread on a plasmid containing 100. Mu.g/mL kanamycin sulfate or 100. Mu.g/mL ampicillin depending on the antibiotic gene carried by the plasmidCulturing on LB plate containing penicillin sodium at 37deg.C overnight, picking monoclonal, shake culturing with 500mL LB culture medium containing kanamycin sulfate or ampicillin sodium with the same concentration at 37deg.C to OD 600 About=1.0, induction was performed with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. And (3) centrifuging at 4 ℃ for 20 minutes at 5000g, collecting thalli, re-suspending each liter of thalli by using 20mL of lysis buffer, performing ultrasonic crushing, centrifuging at 12000g at 4 ℃ for 20 minutes, and precipitating part of target proteins after SDS-PAGE electrophoresis identification. The cells per liter of the bacterial solution were resuspended in 10mL of lysis buffer (50 mM Tris-HCl, pH8.0,1mM EDTA,100mM NaCl), sonicated, the inclusion bodies were collected by centrifugation at 12000rpm at 4℃for 20 minutes, resuspended in solution 1 (20 mM Tris-HCl, pH8.5,5mM EDTA,100mM NaCl) containing 2% Triton X-100, the inclusion bodies were collected by centrifugation at 12000rpm at 4℃for 20 minutes, the 100 volumes of PB buffer (pH 7.0, 20 mM) were dialyzed after solubilization with 8M urea prepared from solution 1, the crude antigen was prepared after removal of the precipitate by centrifugation at 12000rpm at 4℃for 3 times, the Sephacryl S-200 gel column (company, U.S. Amersham Biosciences) was equilibrated with the same PB buffer, the solutions containing the target proteins were collected and pooled, and then passed through DEAE-sepharose ion exchange column (company, U.S. Amersham Biosciences) and the non-adsorbed hetero proteins were removed by washing with PBS buffer (pH 7.0, 20mM PB,50mM NaCl), and the proteins were eluted with PBS buffer (pH 7.0, 20mM PB,500mM NaCl) at 4℃or the target concentration was measured at 4℃for 20℃for standby.
1.2 establishment of anti-X hybridoma cell lines and preparation of monoclonal antibodies thereof
1.2.1 recombinant protein X immunized mice
P2-X expressed protein X solution was dialyzed against PBS, diluted to 1.0mg/mL with PBS, mixed with Freund's complete adjuvant in equal volume, and fully emulsified, and back-subcutaneously injected to immunize 6-week-old naive BALB/c mice at a dose of 50. Mu.g/mouse. After 2 weeks, protein X was mixed with equal volumes of Freund's incomplete adjuvant and thoroughly emulsified for intraperitoneal injection at a dose of 50. Mu.g/dose. After 2 weeks, protein X was injected intraperitoneally without adjuvant at a dose of 50 μg/dose. 7 days after the third immunization, mouse tail blood is taken, serum is separated, and the titer is measured by an indirect ELISA method, and the titer is higher than 1:10000, so that the mouse tail blood can be used for fusion. 4 days before fusion, immunization was performed for the fourth time, protein X was injected without adjuvant, tail vein, at a dose of 50. Mu.g/dose.
1.2.2 preparation of hybridoma cell lines
Myeloma cells Sp2/0 (ATCC, cat# CRL-1581) were recovered 12 days before fusion and cultured in 1640 medium containing 10% fetal bovine serum. Cell concentration was adjusted to 3X 10 at 24h prior to fusion 5 /mL. Myeloma cells were collected the next day, centrifuged at 1200g for 5 min, washed 3 times with serum-free medium, and counted. Killing immunized mice, taking out spleen, preparing into cell suspension, counting cells, taking 10 8 Spleen cells, and 2X 10 7 Myeloma cells were mixed, fused with 50% peg1500, and hybridomas were selectively cultured on HAT medium.
1.2.3 selection and cloning of hybridoma cell lines
Antibody activity in supernatants of fused cells of each well was measured by ELISA on day 10 after fusion. 200. Mu.L of each of the supernatant of the fused cell culture was added to a 96-well ELISA plate well, each well being coated with 10. Mu.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 HRP-labeled anti-mouse IgG was added at 1:5000 dilution 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 of a color developer A containing 0.05. Mu.L of urea hydrogen peroxide, 4.76. Mu.L of sodium acetate trihydrate, 0.9. Mu.L of glacial acetic acid, and a color developer B containing 0.32. Mu.L of TMB, 5mM of citric acid, 0.5mM of EDTA-2Na, 5% of methanol, 2. Mu.L of dimethylformamide were added to each well, and developed at 37℃for 30 minutes in the absence of light. The reaction was stopped by adding 50. Mu.L of a stop solution containing 2M sulfuric acid to each well, and the wavelength of 450nm was measured by an ELISA reader. And finally obtaining 12 cell strains which stably secrete anti-X protein through three times of limiting dilution cloning, wherein the cell strain with the highest antibody activity secretes the antibody named 3D4.
Preparation and purification of 1.2.4X protein monoclonal antibody ascites
8-week-old BALB/c mice were given intraperitoneal injections of liquid paraffin, 0.5 mL/mouse. Intraperitoneal injection 1X 10 after 1 week 7 3D4 hybridoma cells. Ascites were collected 7 days after inoculation of the cells, centrifuged at 3000g for 10 minutes, and the supernatant was diluted 3-fold with 0.01M PBS pH 7.4.The sample was eluted with Protein A immunoaffinity column, pH2.8 glycine, on FPLC system. Collecting specific protein peak effluent, immediately correcting with Tris solution with pH of 9.0 to pH7.0, filtering, sterilizing, packaging, and storing at-80deg.C.
1.3 preparation of colloidal gold kit for indirect labeling of hepatitis C
1.3.1 preparation of colloidal gold
100mL of ultrapure water is added into the triangular flask, the mixture is heated to boiling on a magnetic heater, 1mL of 1% chloroauric acid solution is added, 1mL of 1% trisodium citrate aqueous solution is immediately added after boiling, the boiling is continued for 10 minutes, and then natural cooling is carried out.
1.3.2 colloidal gold-labelling
a. 10mL of the gel Jin Fangru beaker was taken, and 150. Mu.L of 0.2: 0.2M K was added with stirring 2 CO 3 Adjusting the pH to 7.0, and continuously stirring for 20 seconds;
b. adding a certain amount of 3D4 monoclonal antibody, and continuously stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 min;
d.5000g was centrifuged for 10 min, the supernatant was aspirated, the pellet was collected into a centrifuge tube and the volume was fixed to 1mL with colloid Jin Xishi solution (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 complex, and the indirect labeled gold-labeled complex is named as X-NS3-3D4-Au.
1.3.3 preparation of gold-labeled pad
And diluting the gold-labeled compound by using colloid Jin Xishi liquid 10 times, soaking a glass fiber membrane, and freeze-drying to obtain the gold-labeled pad.
1.3.4 nitrocellulose Membrane (NC Membrane) coating
The coated antigen P2-NS3 is diluted to 0.8mg/mL by a detection line diluent (10 mM PBS,2% sucrose) to prepare a detection line working solution, the goat anti-mouse monoclonal antibody is diluted to 0.5mg/mL by the same diluent to prepare a control line working solution, the two working solutions are marked on the corresponding positions of a nitrocellulose membrane by a spot film tester, and the nitrocellulose membrane is dried for 1 hour at 37 ℃.
1.3.5 Assembly
And assembling the gold-labeled pad, the coated nitrocellulose membrane, the absorbent paper, the PVC bottom plate, the sample pad and other auxiliary materials into the HCV gold-labeled detection kit.
1.3.6 detection method
The HCV antibodies are detected by adding 100. Mu.L of a sample to be tested (e.g., serum) to the sample pad.
1.4 preparation of gradient-added "nanoparticle-ligand" colloidal gold kit for indirect labeling of hepatitis C
1.4.1 preparation of liver-C labeling Complex
a. 10mL of the gel Jin Fangru of example 1.3.1 was taken in a beaker and 150. Mu.L of 0.2M K was added with stirring 2 CO 3 Adjusting the pH to 7.0, and continuously stirring for 20 seconds;
b. adding a certain amount of 3D4 monoclonal antibody, and continuously stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 min;
d, 5000g is centrifuged for 10 minutes, the supernatant is sucked out, sediment is collected to a centrifuge tube, and colloid Jin Xishi liquid is used for fixing the volume to 1mL;
e. adding a certain amount of labeled antigen X-NS3 into the 1mL colloidal gold labeled 3D4 monoclonal antibody complex;
f. continuing to add colloidal gold labeled with 3D4 mab to the complex, the addition amount is carried out according to 5%, 10%, 15%, 20%, 25% and 30% gradient of the volume (1 mL), the labeling concentration of 3D4 mab is kept consistent with step b, and the gold labeled complex added with 'nano particle-ligand' is named +Au-3D4 (5%) … and so on.
1.4.2 Assembly
See example 1.3.5.
1.5 comparison of the detection effects of the two detection kits for hepatitis C
The results of detection of HCV positive and negative sera with or without the addition of "nanoparticle-ligand" by indirect labeling were compared with the RIBA reagent from Chiron corporation, usa as a confirmation control.
1.5.1 sensitivity
The test of 100 serial dilutions of serum was performed under the same conditions using a colloidal gold kit with indirect labeling of the addition of "nanoparticle-ligand" or not, giving the results of table 1: for the detectable serum with low to high dilution, the sensitivity after Au-3D4 addition is obviously higher than that of the original indirect labeling gold-labeled complex.
Table 1, sensitivity comparison of two detection kits for hepatitis c
Based on the results of table 1, the gradient was further refined, and the same 100 serial dilutions of serum were tested to obtain the results of table 2.
TABLE 2 gradient refinement
According to tables 1 and 2, the group of optimal gradients was selected for the specific amplification experiments.
1.5.2 specificity
Using a colloidal gold kit for indirectly labeling whether the nanoparticle-ligand is added or not, 3000 clinical negative serum is detected under the same conditions: the specificity of the common indirect labeling kit is 99.7%, the specificity of the indirect labeling kit with the gradient addition of 'nano particle-ligand' of 7.5%, 10%, 12.5% and 15% 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 kit of the invention is checked for 7 days at 37 ℃, taken out and stored at 4 ℃ simultaneously, and the kit prepared by the X-NS3-3D4-Au marker without the nanoparticle-ligand and the marker stored at 4 ℃ is tested under the same condition to examine the stability of the kit, and the results are shown in Table 3.
Table 3, stability assessment of markers and kits
Note that: + is weak yang, ++ is medium yang, ++ is the strong yang of the person, -negative.
This experiment shows that the stability of the markers and the kit of the invention is better.
1.5.4 precision
The reagent kit added with 10% gradient nanoparticle-ligand is used for detecting the same known HCV positive specimen, 10 repeated experiments are carried out, the obtained results of each test strip are positive, and the color development degree is not obviously different, so that the reagent kit has better precision.
Example two indirect labeling of nanoparticles of Treponema Pallidum (TP) antibody detection
2.1 preparation of recombinant antigens for syphilis
2.1.1 preparation of syphilis coating antigen
The DNA fragment corresponding to the treponema pallidum 17Kda (TP 17) gene 22-156aa was PCR amplified, with the upstream primer carrying the BamHI site and the downstream primer carrying the EcoRI site followed by the coding sequence of 6 His amino acids and the termination codon TAA. After the PCR fragment was recovered, it was digested with BamHI and EcoRI, and ligated into the expression vector P2 digested with BamHI and EcoRI to obtain recombinant plasmid P2-TP17, i.e., the antigen-coated recombinant plasmid of the present invention. ER2566 bacteria were transformed with plasmid P2-TP17, and cultured with 500mL LB medium containing 100. Mu.g/mL kanamycin sulfate at 37℃under shaking to OD 600 About=1.0, induction was performed with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. The cells were collected by centrifugation at 5000g for 20 min at 4℃and resuspended in 20mL lysis buffer (50 mM Tris-HCl, pH8.0,1mM EDTA,100mM NaCl) per liter of cell, sonicated, and centrifuged at 12000g for 20 min at 4℃and the majority of the target protein was distributed in the lysate supernatant after SDS-PAGE. Collecting supernatant, slowly adding saturated ammonium sulfate solution dropwise until the final concentration of ammonium sulfate is 30%, standing at 4deg.C for 30 minThe supernatant was collected by centrifugation at 12000g at 4℃for 20 minutes, and saturated ammonium sulfate was continuously added dropwise slowly until the final concentration of ammonium sulfate became 60%, and the mixture was allowed to stand at 4℃for 30 minutes, and the precipitate was collected by centrifugation at 12000g at 4℃for 20 minutes, followed by addition of 5mL of equilibration buffer (10 mM Na 2 HPO 4 ，1.8mM KH 2 PO 4 140mM 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, a protein sample was added, unbound protein was washed away with 10 volumes of equilibration buffer, followed by 5 volumes of elution buffer (20 mM Na 2 HPO 4 300mM NaCl,250mM imidazole, pH 8.0), the protein of interest was eluted, the protein concentration was determined, and the protein was stored at-20℃until use, designated as P2-TP17.
2.1.2 preparation of syphilis marker antigen
The recombinant plasmid pGEX-6P-1-TP17, the antigen-labeled recombinant plasmid of the present invention, was obtained by ligating the TP17 fragment of 2.1.1 to the vector pGEX-6P-1 (pharmacia, cat. No. 27-4597-01) after double cleavage with BamHI and EcoRI. The positive clone was inoculated into 500mL LB medium containing 100. Mu.g/mL ampicillin sodium and cultured at 37℃until OD 600 About=1.0, induction was performed with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. The cells were collected by centrifugation at 5000g for 20 min at 4℃and resuspended in 20mL lysis buffer (50 mM Tris-HCl, pH8.0,1mM EDTA,100mM NaCl) per liter of cell, sonicated, and centrifuged at 12000g for 20 min at 4℃and the majority of the target protein was distributed in the lysate supernatant after SDS-PAGE. Collecting the supernatant, slowly adding saturated ammonium sulfate solution dropwise until the final concentration of ammonium sulfate is 25%, standing at 4 ℃ for 30 minutes, centrifuging at 4 ℃ for 20 minutes, collecting the supernatant, continuously slowly adding saturated ammonium sulfate dropwise until the final concentration of ammonium sulfate is 45%, standing at 4 ℃ for 30 minutes, centrifuging at 4 ℃ for 12000g for 20 minutes, collecting the precipitate, and dissolving with 10mL of balance buffer. After the GSTrap affinity column (Amersham, cat. No. 17-5130-02) was equilibrated with 10 volumes of equilibration buffer, a protein sample was added, unbound protein was washed off with 10 volumes of equilibration buffer, and the mesh was eluted with 5 volumes of elution buffer (50 mM Tris-HCl,10mM reduced glutathione, pH 8.0)The protein concentration was measured and stored at-20℃until use, and the protein was designated GST-TP17.
2.1.3 preparation of immunogens and hybridoma cell screening antigens (GST proteins)
Plasmid pGEX-2T (pharmacia, cat. No. 27-4801-01), pGEX-6P-1, pGEX-5X-1 (pharmacia, cat. No. 27-4584-01), pET-41a (Novagen, cat. No. 70556-3) were used to transform E.coli ER2566, and according to the antibiotic gene carried by the plasmid, the plasmid was spread on LB plates containing 100. Mu.g/mL kanamycin sulfate or 100. Mu.g/mL ampicillin sodium, cultured overnight at 37℃to give a single clone, and the single clone was cultured with 500mL LB medium containing the same concentration of kanamycin sulfate or ampicillin sodium at 37℃under shaking until OD 600 About=1.0, induction was performed with IPTG at a final concentration of 0.5mM for 4 hours at 37 ℃. The bacterial cells are collected by centrifugation at 5000g for 20 minutes at 4 ℃, the bacterial cells of each liter of bacterial liquid are resuspended by 20mL of lysis buffer, the bacterial cells are broken by ultrasound, and after centrifugation at 12000g for 20 minutes at 4 ℃, most of target proteins are distributed in the supernatant of the lysis liquid after being identified by SDS-PAGE electrophoresis. Slowly adding saturated ammonium sulfate while rapidly stirring uniformly to make the final concentration of ammonium sulfate 30%, standing at 4deg.C for 30 min, centrifuging at 12000g at 4deg.C for 20 min, collecting supernatant, slowly adding saturated ammonium sulfate while rapidly stirring uniformly to make the cumulative concentration of ammonium sulfate 60%, standing at 4deg.C for 30 min, centrifuging at 12000g at 4deg.C for 20 min, collecting precipitate, and dissolving with 10mL of balance buffer. After balancing GSTrap affinity column with 10 times of column bed volume of balancing buffer, adding protein sample, washing unbound protein with 10 times of medium volume of balancing buffer, eluting target protein with 5 times of volume of eluting buffer, measuring protein concentration, and preserving at-20deg.C for use.
2.2 establishment of anti-GST hybridoma cell Strain and preparation of monoclonal antibody 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 selection and cloning of hybridoma cell lines
Antibody activity in supernatants of fused cells of each well was measured by ELISA on day 10 after fusion. 200. Mu.L of each of the supernatant of the fused cell culture was added to a 96-well ELISA plate well, each well being coated with 10. Mu.g/mL of a different GST protein. For subsequent steps, see 1.2.3, where the antibody activity is highest, the secreted antibody is designated 6F8.
Preparation and purification of 2.2.4GST protein monoclonal antibody
See example 1.2.4 to yield 6F8 mab after purification.
2.3 preparation of colloidal gold kit for indirect labeling of syphilis
2.3.1 preparation of colloidal gold
Colloidal gold was prepared as described in example 1.3.1.
2.3.2 colloidal gold-labelling
a. 10mL of the gel Jin Fangru beaker was taken, and 150. Mu.L of 0.2: 0.2M K was added with stirring 2 CO 3 Adjusting the pH to 7.0, and continuously stirring for 20 seconds;
b. adding a certain amount of 6F8 monoclonal antibody, and continuously stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 min;
d, 5000g is centrifuged for 10 minutes, the supernatant is sucked out, sediment is collected to a centrifuge tube, and colloid Jin Xishi liquid is used for fixing the volume to 1mL;
e. finally, a certain amount of labeled antigen GST-TP17 is added into the 1mL colloidal gold-labeled 6F8 monoclonal antibody complex, and the indirect labeled gold-labeled complex is named GST-TP17-6F8-Au.
2.3.3 preparation of gold-labeled pad
Gold-labeled pads were prepared as described in example 1.3.3.
2.3.4 nitrocellulose film (NC film) coating
Diluting the coated antigen P2-TP17 to 0.8mg/mL with a detection line diluent 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, and marking the two working solutions to the corresponding positions of a nitrocellulose membrane with a spot membrane instrument, and drying at 37 ℃ for 1 hour.
2.3.5 Assembly
See 1.3.5 to assemble the TP gold-labeled detection kit.
2.3.6 detection method
TP antibody was detected as described in example 1.3.6.
2.4 preparation of gradient-added "nanoparticle-ligand" colloidal gold kit for syphilis indirect labeling
2.4.1 preparation of syphilis marker Complex
a. 10mL of the gel Jin Fangru of example 1.3.1 was taken in a beaker and 150. Mu.L of 0.2M K was added with stirring 2 CO 3 Adjusting the pH to 7.0, and continuously stirring for 20 seconds;
b. adding a certain amount of 6F8 monoclonal antibody, and continuously stirring for 10 minutes;
c. 0.1mL of 10% BSA was added and stirring was continued for 5 min;
d, 5000g is centrifuged for 10 minutes, the supernatant is sucked out, sediment is collected to a centrifuge tube, and colloid Jin Xishi liquid is used for fixing the volume to 1mL;
e. adding a certain amount of labeled antigen GST-TP17 into the 1mL colloidal gold labeled 6F8 monoclonal antibody complex;
f. continuing to add colloidal gold labeled with 6F8 mab to the complex, the addition was performed according to a gradient of 5%, 10%, 15%, 20%, 25%, 30% of the volume (1 mL), the labeling concentration of 6F8 mab remained consistent with step b, the gold labeled complex added with "nanoparticle-ligand" was named +Au-6F8 (5%) …, and so on.
2.5 comparison of the detection Effect of the two detection kits for syphilis
The detection results of TP positive serum and negative serum were compared with each other by using the TPPA kit from fuji corporation of japan as a confirmation control, and whether "nanoparticle-ligand" was added or not by indirect labeling.
2.5.1 sensitivity
The test of 100 serial dilutions of serum was performed under the same conditions using a colloidal gold kit with indirect labeling of the addition of "nanoparticle-ligand" or not, giving the results of table 4: for the detectable serum with low to high dilution, the sensitivity after Au-6F8 addition is obviously higher than that of the original indirect labeling gold-labeled complex.
Table 4, sensitivity comparison of two detection kits for syphilis
The group with the optimal gradient (10%) was selected for the specific amplification experiments.
2.5.2 specificity
Using a colloidal gold kit for indirectly labeling whether the nanoparticle-ligand is added or not, 3000 clinical negative serum is detected under the same conditions: the specificity of the common indirect labeling kit is 99.8%, the specificity of the indirect labeling kit with 10% gradient added with 'nano particle-ligand' is 99.8%, and the specificity of the two labeling modes is equivalent.
2.5.3 stability
Finished kits of the invention [ markers: the +Au-6F8 (10%) is checked for 7 days at 37 ℃, the kit stored at 4 ℃ after being taken out and the kit prepared by the label stored at 4 ℃ without adding the GST-TP17-6F8-Au label of the nanoparticle-ligand are tested under the same condition to check the stability of the kit, and the experimental result shows that the stability of the label and the kit of the invention is better.
2.5.4 precision
The same known TP positive specimen is detected by a kit added with 10% gradient nanoparticle-ligand, 10 repeated experiments are carried out, the obtained test strips are positive in result, and the color development degree is not obviously different, so that the kit is good in precision.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (12)
1. An antigen labeling method, characterized in that the method comprises the steps of (i) labeling a nanoparticle label with a ligand, (ii) labeling the nanoparticle label with an antigen, (iii) adding the nanoparticle label labeled with the ligand to a labeling system in a supplementing manner, (iv) adding a sample to be detected, and detecting an antibody to be detected; wherein the labeling between the nanoparticle-based label and the labeled antigen is an indirect labeling by binding a label on the antigen to the ligand on the nanoparticle-based label that specifically recognizes the label;
the nanoparticle labels comprise nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles or rare earth complex nanoparticles;
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;
the ligand comprises a specific antibody against the tag, a substance having affinity for the tag.
2. The method of claim 1, wherein the antibody to be tested comprises, but is not limited to, an hiv antibody, a hepatitis a virus antibody, a hepatitis b virus antibody, a hepatitis c virus antibody, a hepatitis b virus antibody, a hepatitis e virus antibody, a hepatitis g virus antibody, a rubella virus antibody, a human cytomegalovirus antibody, a herpes simplex virus type 1 antibody, a herpes simplex virus type 2 antibody, a rabies virus antibody, a human T lymphoblastic leukemia virus antibody, a dengue virus antibody, a human papilloma virus antibody, a west nile virus antibody, a forest encephalitis virus antibody, a measles virus antibody, an influenza virus antibody, a parainfluenza virus antibody, a varicella virus antibody, an ico virus antibody, a coxsackie virus antibody, an encephalitis b virus antibody, an EB virus antibody, a treponema pallidum antibody, a bordetention antibody, a chlamydia antibody, a mycobacterium tuberculosis antibody, a helicobacter pylori antibody, a coccid antibody, a cone antibody, a trypanosoma antibody.
3. The method of claim 1, wherein the colloid comprises colloidal metal, disperse dyes and dye-labeled microspheres, latex.
4. A method according to claim 3, wherein the colloidal metal comprises colloidal gold, colloidal silver, colloidal selenium.
5. The method of claim 1, wherein the ligand is streptavidin.
6. The method of claim 1, wherein the additional addition of the ligand-labeled nanoparticle labels is in an amount of 5% to 30% of the volumetric volume of the labeling system.
7. The method of claim 1, wherein the additional addition of the ligand-labeled nanoparticle labels is in an amount of 7.5% to 17.5% of the volumetric volume of the labeling system.
8. The method of claim 1, wherein the additional addition of the ligand-labeled nanoparticle labels is at 10% of the volumetric volume of the labeling system.
9. The method of claim 6, wherein the labeling system employs a colloidal gold dilution comprising 20mM PB,150mM NaCl,1%BSA,0.1%Triton X-100,2% sucrose, 0.01% proclin300.
10. The nanoparticle-based label prepared by the antigen labeling method of any one of claims 1 to 9.
11. A test kit comprising the nanoparticle-based label of claim 10.
12. Use of the method of any one of claims 1-9 or the nanoparticle-based label of claim 10 in the preparation of an antibody detection kit.
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