CN111521827A - Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing - Google Patents
Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides an immunofluorescence test strip for rapidly determining the titer of a monoclonal antibody based on antigen tracing, which comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially arranged, wherein fluorescence-labeled bacteria are sprayed on the combination pad, goat anti-mouse antibodies are sprayed on the nitrocellulose membrane to form a detection line, and positive serum or antibodies are sprayed on a C line to form a quality control line. In addition, the invention also provides application of the immunofluorescence test strip for rapidly determining the titer of the monoclonal antibody based on antigen tracing in rapidly detecting the titer of the monoclonal antibody. The test strip has high sensitivity and short detection time.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an immunofluorescence test strip for rapidly determining the titer of a monoclonal antibody based on antigen tracing.
Background
The monoclonal antibody is mainly secreted by an effector B cell, and the B cell is fused with a myeloma cell, so that the monoclonal antibody not only maintains the capacity of secreting a specific antibody of the effector B cell, but also has the capacity of unlimited proliferation of the myeloma cell. The existing monoclonal antibody has hybridoma monoclonal antibody, recombinant monoclonal antibody, phage display monoclonal antibody and the like according to a preparation mode, and the methods for preparing the monoclonal antibody by establishing the strains have advantages and disadvantages, but have the common points of complicated preparation process, long preparation period, and need of multiple positive test screening and positive determination after establishing the strains. Meanwhile, for the obtained monoclonal antibody, the titer needs to be measured so as to have a better dilution guide range, thereby ensuring the use of the antibody.
At present, the screening and titer determination of monoclonal antibodies rely on an indirect ELISA method, which is mainly characterized in that an antigen is fixed on a solid phase carrier, sealed and eluted, and is incubated with a target antibody, eluted, and is incubated with an enzyme-labeled secondary antibody and eluted to form a ternary complex of the antigen-antibody-enzyme-labeled secondary antibody, a catalytic substrate forms a colored substance, the amount of a product is directly related to the amount of the antibody, and each determination time needs 5-24 hours. This time and labor consuming assay has become one of the important bottlenecks that restrict the preparation and use of monoclonal antibodies.
Therefore, in order to detect the positive and titer of an antibody, it is necessary to design a test strip with high sensitivity using antigen labeling.
Disclosure of Invention
The invention aims to solve the problems and provide an immunofluorescence test strip for rapidly determining the titer of a monoclonal antibody based on antigen tracing.
The invention provides an immunofluorescence test strip for rapidly determining the titer of a monoclonal antibody based on antigen tracing, which is provided with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are arranged in sequence and has the characteristics that: wherein, the fluorescence-labeled bacteria are sprayed on the bonding pad, the goat anti-mouse antibody is sprayed on the nitrocellulose membrane to form a detection line, and the positive serum or the antibody is sprayed on the C line to form a quality control line.
The invention also provides application of the immunofluorescence test strip for rapidly determining the titer of the monoclonal antibody based on antigen tracing in rapidly detecting the titer of the monoclonal antibody.
Action and Effect of the invention
According to the immunofluorescence test strip for rapidly determining the titer of the monoclonal antibody based on antigen tracing, the positive and the titer of the antibody can be conveniently and rapidly detected, the screening and preparation efficiency of the monoclonal antibody can be greatly improved, and the high-quality antibody can be rapidly applied.
Drawings
FIG. 1 is a structural diagram of rapid determination of titer of monoclonal antibodies based on antigen tracing in the example of the present invention;
FIG. 2 is a graph showing the results of the different labeling methods for antigens according to the example of the present invention;
FIG. 3 is a graph showing the results of flow cytometry testing of FITC-labeled gram-negative bacteria Escherichia coli O157H 7 at different concentrations in the examples of the present invention;
FIG. 4 is a graph of flow cytometry results for gram-positive bacteria Staphylococcus aureus labeled with FITC at various concentrations in accordance with embodiments of the present invention;
FIG. 5 is a graph showing the results of observation under a fluorescence microscope of the gram-negative bacterium Escherichia coli O157: H7 after labeling in the examples of the present invention;
FIG. 6 is a graph of test results of different concentrations of fluorescently labeled bacteria sprayed on the conjugate pad in an embodiment of the present invention;
FIG. 7 is a graph showing the results of testing the antibody titer of the supernatant of the bloody Escherichia coli O157H 7 cells based on the antigen-labeled fluorescent test strip in the example of the present invention;
FIG. 8 is a graph showing the results of testing the antibody titer in the ascites fluid of Escherichia coli hemorrhagic disease O157: H7 based on the antigen-labeled fluorescent test strip in the example of the present invention;
FIG. 9 is a graph showing the results of antibody titer after purification of Escherichia coli O157H 7 tested on the basis of the antigen-labeled fluorescent test strip in the example of the present invention.
Detailed Description
In order to make the technical means and functions of the present invention easy to understand, the present invention is specifically described below with reference to the embodiments and the accompanying drawings.
Example (b):
the main reagents and main instruments used in this example are as follows:
primary reagent
PVP, TWEEN-20 national drug group chemical reagents GmbH; the sample pad and the water absorption pad are made of Hai jin-labeled Biotech Co., Ltd; BSA shanghaije a biotechnology limited; FITC Shanghai Solebao Biotechnology Ltd; nitrocellulose membrane (NC membrane) 95 sartorius; sheep resistance mouse Luoyang Baiotton experimental materials center.
Main instrument
Autoclave is available from TOMY, japan; microplate reader SpectraMax M2 was purchased from Molecular Devices; nanodrop 2000C was purchased from Thermo Scientific; spotting apparatus AD6010 was purchased from BIO-DOT; the constant temperature culture shaking table SPH-100B is purchased from Shanghai Shiping; BioLogic protein purification instrumentTMLP 358BR5057 is available from BIO-RAD; single person decontamination workbench model SW-SJ-2D was purchased from Suzhou decontamination. Flow cytometry was purchased from bidi medical instruments (shanghai) ltd.
First, the fluorescent test strip for measuring the potency is constructed
1. Test strip assembly
FIG. 1 is a structural diagram of rapid determination of monoclonal antibody titer based on antigen tracing in the example of the present invention.
As shown in fig. 1, the titer-measuring fluorescent test strip comprises: the device comprises a sample pad 1, a combination pad 2, a nitrocellulose membrane 3, a detection line 4, a quality control line 5, a water absorption pad 6 and a PVC bottom plate 7. PVC7 is in the lower floor, and secondly is cellulose nitrate membrane 2(NC membrane), and the cellulose nitrate membrane is in the centre, and both ends are being connected respectively and are being bound pad 2 and absorption of water pad 5, and sample pad 1 is being linked to the one end that NC membrane 3 was kept away from to binding pad 2, has detection line 3 and matter control line 4 on the NC membrane 3, and detection line 3 is close to binding pad 2 one side.
In addition, the fluorescence-labeled bacteria are sprayed on the binding pad 2, the goat anti-mouse antibody is sprayed on the nitrocellulose membrane 3 to form a detection line 4, and the positive serum or antibody is sprayed on the C line to form a quality control line 4.
2. Screening by labeling methods
Coli in vivo labeling: activating Escherichia coli O157H 7, picking single colony, inoculating in liquid culture medium, adding FITC dissolved in DMSO to make its final concentration 40mg/mL, culturing at constant temperature shaking table 180rpm/min for 16H, inactivating, taking bacterial suspension, centrifuging at 5000rpm for 5min, re-suspending with sterile PBS until the centrifuged supernatant is clear, and concentrating.
The Escherichia coli dead body labeling method comprises the following steps: first, the liquid is expanded to culture larger than 1 x 108CFU/mL bacteria, centrifugation, use cross-linking buffer washing three times, heavy suspension. The preparation method of the crosslinking solution comprises weighing 7.56g NaHCO31.06g of NaCO3 and 7.36g of NaCl, and adding water to the mixture to make the volume of the mixture reach 1L. FITC was then dissolved in DMSO at a concentration of 1mg/mL and was freshly prepared for each cross-linking. Adding the prepared FITC solution into the bacterial solution cleaned by the cross-linking solution, and placing the solution in a dark place at 4 ℃ for solution treatment. Then, 5moL/L of NH4Cl was added to a final concentration of 50mmoL/L, and the reaction was terminated at 4 ℃ for 2 hours. Finally, the cross-linked material was centrifuged at 5000rpm for 5min and resuspended in PBS until the supernatant was clear after centrifugation.
FIG. 2 is a graph showing the results of the different labeling methods for antigens according to the present invention.
In order to research a suitable labeling method of an antigen in a test strip, a vital staining method and a dead staining method are adopted, and the concentration of a goat anti-mouse antibody on a T line is optimized, wherein the concentration of the T line antibody of the No. 1/2 test strip is 1mg/mL, the concentration of the T line antibody of the No. 3/4 test strip is 2mg/mL, the concentration of the T line antibody of the No. 5/6 test strip is 3mg/mL, the concentration of the T line antibody of the No. 7/8 test strip is 4mg/mL, and the concentration of the T line antibody of the No. 9/10 test strip is 5 mg/mL. As shown in FIG. 2(a), in the method of living body labeling, test strips No. 1, 3, 5, 7 and 9 all show bands, which indicates that the method of living body labeling can cause the test strips to show false positive results. By adopting a dead body staining method, as shown in fig. 2(b), no strip appears on test strips No. 1, 3, 5, 7 and 9, no false positive test strips appear on the test strips, no strip appears on test strips No. 2, 4, 6, 8 and 10, and the fluorescence intensity of the T line is not obviously increased when the concentration of the T line is 1-5mg/mL antibody. Therefore, the bacteria were labeled by a dead body staining method, and the T-line antibody concentration was 1 mg/mL.
3. Fluorescent dye concentration optimization
FIG. 3 is a flow cytometer test result chart of FITC labeled gram negative bacteria Escherichia coli O157: H7 at different concentrations in the example of the present invention, FIG. 4 is a flow cytometer test result chart of FITC labeled gram positive bacteria Staphylococcus aureus at different concentrations in the example of the present invention, and FIG. 5 is a result chart of observation of labeled gram negative bacteria Escherichia coli O157: H7 under a fluorescence microscope in the example of the present invention.
The washed gram-positive and gram-negative bacteria were labeled with FITC at final concentrations of 200 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ g/mL, 600 μ g/mL, respectively, and blocked with ammonium chloride, washed, diluted, and measured by flow cytometry, the gram-negative bacteria escherichia coli O157: the fluorescence labeling result of H7 is shown in FIG. 3, and the fluorescence labeling result of gram-positive hemorrhagic Staphylococcus aureus is shown in FIG. 4. As shown in FIG. 3, Escherichia coli O157: the control group of H7 fluorescence labeling efficiency was 0, and the fluorescence labeling efficiencies were 97.8%, 77.87%, 75.68%, 77.01% and 77.44% at other concentrations, respectively. As shown in FIG. 4, the control group of the fluorescence labeling efficiency of Staphylococcus aureus was 0, and the concentrations of the other fluorescence labeling efficiencies were 69.49%, 70.31%, 66.09%, 67.44% and 54.28%, respectively. Therefore, the concentration of the hemorrhagic Escherichia coli O157: H7 FITC marker is 200 mu g/mL-300 mu g/mL, wherein the concentration of 200 mu g/mL FITC is optimal, and the concentration of the Staphylococcus aureus marker is 200 mu g/mL-300 mu g/mL, wherein the concentration of 300 mu g/mLFITC is optimal. And a certain diluted concentration of fluorescence-labeled Escherichia coli O157: H7 is observed under a fluorescence microscope, and the result is shown in FIG. 5, and the bacteria have obvious whole body fluorescence phenomenon.
4. Optimization of conjugate pad fluorescence labeled bacteria concentration
FIG. 6 is a graph showing the results of testing the conjugate pads sprayed with fluorescent-labeled bacteria of different concentrations in the examples of the present invention.
According to the optimized marking method, marking the hemorrhagic escherichia coli O157: H7, washing with PBS, respectively preparing FITC marked bacteria with different concentrations, determining the OD600 absorbance values as shown in Table 1, soaking the combined pad in containers containing fluorescence marked bacteria with different concentrations, drying for 2 hours at 50 ℃, assembling test strips, and testing, wherein the results are shown in figure 6, and 1, 3, 5, 7 and 9 are negative test results and have no false positive. 2. Test strips No. 4, 6, 8 and 10 are positive test results, and test strip No. 2 has a weaker band, and test strip No. 3 and the following test strips are brighter, wherein the test strip No. 8 has the brightest concentration, and the OD600 has an absorbance value of 1.2293.
TABLE 1 results of fluorescence-labeled bacteria-soaked conjugate pad testing at different concentrations
| Numbering | 1/2 | 3/4 | 5/6 | 7/8 | 9/10 |
| A600 | 0.4233 | 0.7069 | 0.9782 | 1.2293 | 1.481 |
Thirdly, rapid determination of the potency of monoclonal antibodies based on antigen tracing
1. Measuring the titer of cell supernatant
A matched diluent based on the antigen tracing fluorescent test strip is prepared, and the components of the diluent comprise 0.5% of PVP, 0.05% of Tween 20, 1% of sucrose and 1% of BSA, and the diluent is dissolved by TB buffer solution. E.coli hybridoma O157: H7D 3 hybridoma cell supernatants were assayed at 1:10, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 fold gradient was diluted, and 100. mu.L of each dilution gradient was used for indirect ELISA for titer determination, while 200. mu.L of antigen-labelled fluorescent test strips were used for assay.
The steps of indirect ELISA determination of the titer of the monoclonal antibody after cell supernatant, ascites and purification are as follows:
step 14, adding 100uL of TMB color development liquid (the prepared color development liquid is colorless, and cannot be used after color change);
and step 15, incubating for 10-15 minutes at room temperature and 37 ℃ in a dark place, adding 50uL of stop solution to stop the reaction, and reading the OD value at 450nm (wiping the bottom water and the finger mold of the micropore by lint-free cloth before reading).
FIG. 7 is a graph showing the results of testing the antibody titer of the supernatant of the bloody Escherichia coli O157H 7 cells based on the antigen-labeled fluorescent test strip in the example of the present invention.
The indirect ELISA test results are shown in Table 2, and the positive determination standard is that the positive OD value/negative OD value is more than 2.1 times, and the indirect ELISA test result is that the titer of E7 cell supernatant is 400 times. The test titer result based on the antigen tracing fluorescent test strip is shown in fig. 7, when the dilution ratio is 1600 times, the test strip No. 7 has weak strips, the test strip No. 12 (negative) has no strips, and the titer of the antigen tracing test strip for measuring cell supernatant is 1600 times.
TABLE 2 results of indirect ELISA assay of antibody titer in cell supernatants
2. Ascites titer measurement
FIG. 8 is a graph showing the results of the antibody titer test in the ascites fluid of Escherichia coli hemorrhagic disease O157H 7 based on the antigen-labeled fluorescent test strip in the example of the present invention.
Ascites prepared from E.coli hybridoma O157: H7D 3 was treated according to the ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1: 64000. 1:128000, 1:256000, 1: 512000, 1: 1024000 gradients were diluted, and 100. mu.L of each dilution was used for indirect ELISA for titer determination, as described above, while 200. mu.L was used for antigen-labelled fluorescent strip testing. The indirect ELISA test results are shown in table 3, and the positive determination standard is that the positive OD value/negative OD value is more than 2.1 times, and the indirect ELISA test result is that the E7 cell supernatant titer is 128000 times. The test titer results based on the antigen-labeled fluorescent test strip are shown in fig. 8, and when the dilution ratio is 256000 times, the test strip No. 9 has a weak strip, the test strip No. 12 (negative) has no strip, and the titer of the antibody in the ascites measured by the antigen-labeled test strip is 256000 times.
TABLE 3 results of indirect ELISA assay for antibody titer in ascites
3. Measuring the antibody titer after purification
FIG. 9 is a graph showing the results of antibody titer after purification of Escherichia coli O157H 7 tested on the basis of the antigen-labeled fluorescent test strip in the example of the present invention.
Ascites prepared from E.coli hybridoma O157: H7D 3 was treated according to the ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1: 64000. 1:128000, 1:256000, 1: 512000, 1: 1024000 gradients were diluted, and 100. mu.L of each dilution was used for indirect ELISA for titer determination, as described above, while 200. mu.L was used for antigen-labelled fluorescent strip testing. The indirect ELISA test results are shown in table 4, and the positive determination standard is that the positive OD value/negative OD value is more than 2.1 times, and the indirect ELISA test result is that the E7 cell supernatant titer is 256000 times. The test titer results based on the antigen tracer test strip are shown in fig. 9, in the case of the dilution ratio of 256000 times, the test strip No. 11 has a weak band, the test strip No. 12 (negative) has no band, and the titer of the antibody in the ascites measured by the antigen tracer test strip is 1024000 times.
TABLE 4 results of the indirect ELISA assay of antibody titers after purification
Effects and effects of the embodiments
In the above embodiment, the new test strip is adopted, so that the experimental result shows that, compared with the indirect ELISA method, the sensitivity of rapidly determining the titer of the monoclonal antibody titer by the immunofluorescence test strip based on antigen tracing is high, and the time is only 10-15 min.
In addition, when the antibody is used as the recognition molecule to detect macromolecules, such as pathogenic bacteria, usually, the signal molecule carried by the antibody is used for tracing, so that the purpose of detection is achieved, and when the signal molecule carried by the macromolecules is used for tracing, under the condition that the recognition of the antibody antigen is not influenced, the fluorescent quantity carried by the antigen is far greater than the molecular weight of the antibody due to the large molecular weight of the macromolecular antigen, so that under the condition of equal combination, the detection method using the sensitivity of antigen tracing is far higher than the detection method using the antibody tracing.
Therefore, the immunofluorescence test strip for rapidly determining the titer of the monoclonal antibody based on antigen tracing can conveniently and rapidly detect the positivity and the titer of the antibody, can greatly improve the screening and preparation efficiency of the monoclonal antibody, and can rapidly apply the high-quality antibody.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (2)
1. The utility model provides an immunofluorescence test paper strip of monoclonal antibody titer based on antigen trace spot test, has sample pad, combination pad, nitrocellulose membrane and the pad that absorbs water that arranges in proper order, its characterized in that:
wherein, the fluorescence-labeled bacteria are sprayed on the bonding pad, the goat anti-mouse antibody is sprayed on the nitrocellulose membrane to form a detection line, and the positive serum or the antibody is sprayed on the C line to form a quality control line.
2. The use of the immunofluorescence test strip for rapid determination of monoclonal antibody titer based on antigen tracking according to claim 1 for rapid detection of monoclonal antibody titer.
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