CN111077311B - ELISA spot detection kit and detection method thereof - Google Patents
ELISA spot detection kit and detection method thereof Download PDFInfo
- Publication number
- CN111077311B CN111077311B CN201911403515.5A CN201911403515A CN111077311B CN 111077311 B CN111077311 B CN 111077311B CN 201911403515 A CN201911403515 A CN 201911403515A CN 111077311 B CN111077311 B CN 111077311B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- concentration
- well
- tested
- hole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2307—Interleukin-7 (IL-7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2315—Interleukin-15 (IL-15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2321—Interleukin-21 (IL-21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an ELISA spot detection kit, which comprises an experimental hole: a culture medium comprising anti-CD28, IL-2, a polypeptide, a cell suspension; negative control: culture medium containing anti-CD28, IL-2, DMSO, cell suspension; positive control: culture medium containing anti-CD28 and IL-2, PMA+Ionomycin, and cell suspension; standard curve: culture medium containing anti-CD28 and IL-2, and cell suspension with each concentration of anti-CD 3. The method has the advantages of improving the sensitivity of the ELISA spot detection method and ensuring that a stable and reliable detection result can be obtained.
Description
Technical Field
The invention belongs to the technical field of enzyme-linked immunosorbent assay (ELISA) spot detection, and particularly relates to an ELISA spot detection kit and a detection method thereof.
Background
ELISA spot detection ELISpot (Enzyme-Linked ImmunoSpot assay) is one of the most widely used single cell function detection methods at present. In the research of tumor vaccines, functional immunology detection is an essential component of vaccine development and clinical tests, and ELISPot detection is used as a powerful experimental technique, and is mainly applied to the identification of new immunogenicity targets, the verification of the existence of antigen-specific T cells and the evaluation of vaccine-induced immune responses, thereby being helpful for understanding the immunogenicity, efficacy and effectiveness of the vaccine.
ELISpot requires a relatively low number of T cells to be able to detect peptide-specific T cell responses simply and quickly. ELISpot analysis is generally considered a highly sensitive method, allowing detection of one of 10000 cells. Despite the high sensitivity of ELISpot detection, it is still very difficult to detect immune responses to neoantigen-specific T cells in peripheral blood mononuclear cells of patients or healthy donors because the frequency of these T cells can be very low.
ELISpot analysis will generate potentially important data, and it is extremely important to obtain comparable results in all assays for longitudinal follow-up immune response monitoring, which is not addressed by the current commercially available ELISpot kits.
Disclosure of Invention
The invention provides an ELISA spot detection kit and a detection method thereof, aiming at improving the sensitivity of the ELISA spot detection method by adopting antibodies, cytokines and the like, shortening the in-vitro stimulation time and ensuring that the detection result is stable and reliable.
In order to achieve the above purpose, the technical scheme provided by the invention is that the ELISA spot detection kit provided by the invention comprises an experiment hole: adding 96 μl of culture medium of PBMC cells to be tested, 4 μl of polypeptide, each peptide having a concentration of 1-10 μg/ml, and 100 μl of 1-5×10 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml;
negative control: mu.L of culture medium of PBMC cells to be tested, 1 mu LDMSO, and 100 mu.L of 1-5×10 concentration were added to the negative control wells 6 PBMC cell suspension to be tested/ml, finalThe volume is 200 mu L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml;
positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 1-5×10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml;
Standard curve containing anti-CD3 gradient concentration: adding 95 μl of culture medium of PBMC cells to be tested into each standard curve well, 5 μl of anti-CD3 with each concentration, and 100 μl of 1-5×10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml; the gradient of the anti-CD3 concentration is set to start with a concentration of 0.001-0.005 mug/ml, and the anti-CD3 concentration is diluted in a continuous multiple ratio.
Further, the culture medium of PBMC cells to be tested used in the experimental hole, the negative control, the positive control and the standard curve also comprises IL-21, and when the culture medium contains anti-CD28, IL-2 and IL-21, the concentration of each component in the culture medium is 0.1-5 mug/ml, IL-2 is 1-30U/ml and IL-21 is 10-50ng/ml.
Further, the culture medium of PBMC cells to be tested used in the experimental hole, the negative control, the positive control and the standard curve also comprises IL-7, and when the culture medium contains anti-CD28, IL-2 and IL-7, the concentration of each component in the culture medium is 0.1-5 mug/ml, IL-2 is 1-30U/ml and IL-7 is 1-30ng/ml.
Further, the culture medium of PBMC cells to be tested used in the experimental hole, the negative control, the positive control and the standard curve also comprises IL-7 and IL-15, and when the culture medium contains anti-CD28, IL-2, IL-7 and IL-15, the concentration of each component in the culture medium is 0.1-5 mug/ml, IL-2 is 1-30U/ml, IL-7 is 1-30ng/ml and IL-15 is 1-50ng/ml.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) Taking the 96-well ELISPot plate coated with the detection antibody in advance out of the refrigerator, opening the plate in a safety cabinet, and adding PBS into each well for washing;
(2) Adding a culture medium of PBMC cells to be tested into each hole, and incubating at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into each standard curve hole, and obtaining a final volume of 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing in an incubator at 37 ℃ for 12-48 hours;
(5) Taking the ELISPot plate out of the incubator after 12-48 hours, pouring the culture medium in the holes, adding 200 mu L of PBS into each hole for washing, and finally buckling on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing, and finally buckling on the absorbent paper for the last time;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Further, the step (1) further comprises the step (0) of in vitro stimulation:
(01) The cell concentration of PBMC to be tested is adjusted to 2-8×10 by using a culture medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100 mu L of PBMC cell culture medium to be tested, 0.4 mu L of polypeptide, wherein the concentration of each peptide in the polypeptide is 1-10 mu g/ml, and the concentration of 100 mu L is 2-8 multiplied by 10 6 Ml PBMC cell suspension to be tested, negative well: 100 mu L of PBMC cell culture medium to be tested and 100 mu L of PBMC cell culture medium with concentration of 2-8 multiplied by 10 6 /ml of PBMC cell suspension to be tested;
(03) Placing the 96-well U-bottom cell culture plate prepared in the step (02) in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
By adopting the technical scheme, the invention has the beneficial effects that: 1. subjective judgment of ELISpot results is one of the biggest obstacles to the global standardization of ELISpot. The anti-CD3 internal reference standard curve is established, so that the consistency among single experimental results is enhanced, the reliability and comparability of ELISPot results are improved, and the analysis change among sample follow-up time points is reduced.
2. In ELISPOT plating, T cells are co-stimulated with anti-CD28 and IL-2 (or anti-CD28, IL-2 and IL-7; or anti-CD28, IL-2, IL-7 and IL-15; or anti-CD28, IL-2 and IL-21), which can increase sensitivity over conventional ELISPots.
3. The PBMC is pre-stimulated by the antigen in vitro for 5 days, and compared with the traditional method for pre-stimulating in vitro for more than 10 days, the method can achieve the same experimental effect, effectively save time and reduce the interference caused by long-time culture and complex operation.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The starting materials or reagents used are all commercially available. The concentration of PBMC to be tested in the invention is adjusted by using a culture medium for culturing the PBMC to be tested. The detection antibody is anti-IFN-gamma-biotin or anti-Granzyme B-biotin or anti-IL-2-biotin carried by the kit.
Embodiment one: the invention provides an ELISA spot detection kit, which comprises an experiment hole: 96. Mu.L of the medium of PBMC cells to be tested, 4. Mu.L of the polypeptide, and 100. Mu.L of the medium at a concentration of 1X 10 were added to the experimental wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1 mug/ml, and the concentration of the IL-2 in the culture medium is 1U/ml;
negative control: mu.L of medium of PBMC cells to be tested, 1. Mu.L of LDMSO, and 100. Mu.L of 1X 10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1 mug/ml, and the concentration of the IL-2 in the culture medium is 1U/ml;
positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 1X 10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1 mug/ml, and the concentration of the IL-2 in the culture medium is 1U/ml;
standard curve containing anti-CD3 gradient concentration: adding 95 mu L of culture medium of PBMC cells to be tested into each standard curve hole, 5 mu L of anti-CD3 with each concentration,100 mu L concentration of 1X 10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1 mug/ml, and the concentration of the IL-2 in the culture medium is 1U/ml; the gradient of anti-CD3 concentration was set to start at a concentration of 0.001 μg/ml, serially doubling the dilution.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) Taking the 96-well ELISPot plate coated with the detection antibody in advance out of the refrigerator, opening the plate in a safety cabinet, adding 200 mu L of PBS into each well, and washing for 5 times;
(2) 200. Mu.L of culture medium was added to each well and incubated for 30min at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into a standard curve hole, wherein the final volume is 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing for 12 hours at 37 ℃ in an incubator;
(5) After 12 hours, the ELISPot plate is taken out from the incubator, the culture medium in the holes is poured, 200 mu L of PBS is added into each hole for washing, and finally the culture medium is buckled on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole, washing for 5 times, and finally buckling on the absorbent paper for drying;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing for 5 times, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Using the above assay, the assay antibody of this example was anti-IFN-gamma-biotin, and the peptides used were peptides comprising CMV, influenza A and EBV, each at a concentration of 1 μg/ml, and the following partial comparison experiments were performed to demonstrate that the selected combinations of cytokines and amounts of cytokines used in the present invention can help the assay of the present invention to increase sensitivity, as shown in the following Table:
from this, we can see that when we stimulated PBMC with peptides containing CMV, influenza and EBV, the number of IFN-gamma spots was increased by a factor of 2-5 compared to the prior art without the addition of cytokines or with some cytokines alone or in combination.
Embodiment two: the invention provides an ELISA spot detection kit, which comprises an experiment hole: 96. Mu.L of culture medium of PBMC cells to be tested, 4. Mu.L of polypeptide, and 100. Mu.L of 5X concentration were added to the experimental wells10 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 5 mug/ml, the concentration of the IL-2 in the culture medium is 30U/ml, and the concentration of the IL-21 in the culture medium is 10ng/ml;
negative control: mu.L of medium of PBMC cells to be tested, 1. Mu.L of LDMSO, and 100. Mu.L of 5X 10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 5 mug/ml, the concentration of the IL-2 in the culture medium is 30U/ml, and the concentration of the IL-21 in the culture medium is 10ng/ml;
positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 5X 10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 5 mug/ml, the concentration of the IL-2 in the culture medium is 30U/ml, and the concentration of the IL-21 in the culture medium is 10ng/ml;
standard curve containing anti-CD3 gradient concentration: mu.L of culture medium of PBMC cells to be tested is added to each standard curve well, 5 mu.L of anti-CD3 with each concentration, and 100 mu.L of culture medium with concentration of 5 multiplied by 10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium comprises anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 5 mug/ml, the concentration of the IL-2 in the culture medium is 30U/ml, and the concentration of the IL-21 in the culture medium is 10ng/ml; the gradient of anti-CD3 concentration was set to start at a concentration of 0.005 μg/ml, serially doubling the dilution.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) Taking the 96-well ELISPot plate coated with the detection antibody in advance out of the refrigerator, opening the plate in a safety cabinet, adding 200 mu L of PBS into each well, and washing for 5 times;
(2) 200. Mu.L of culture medium was added to each well and incubated for 30min at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into a standard curve hole, wherein the final volume is 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing in an incubator at 37 ℃ for 48 hours;
(5) After 48 hours, the ELISPot plate is taken out of the incubator, the culture medium in the holes is poured, 200 mu L of PBS is added into each hole for washing, and finally the culture medium is buckled on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole, washing for 5 times, and finally buckling on the absorbent paper for drying;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing for 5 times, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Further, the step (1) further comprises the step (0) of in vitro stimulation:
(01) The concentration of PBMC cells was adjusted to 8X 10 with medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100. Mu.L of PBMC cell culture medium to be tested, 0.4. Mu.L of polypeptide, 100. Mu.L of concentration of 8X 10 6 Ml PBMC cell suspension to be tested, negative well: 100. Mu.L of PBMC cell culture medium to be tested and 100. Mu.L of PBMC cell culture medium with concentration of 8X 10 6 /ml of PBMC cell suspension to be tested;
(03) Place 96-well U-bottom cell culture plate in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
With the above detection method, the detection antibody of this embodiment is anti-Granzyme B-biotin, the peptides used are peptides comprising CMV, influenza a and EBV, the concentration of each peptide is 5 μg/ml, the peptides used for 5 days in vitro stimulation are consistent with those in the detection method, and the following list is a partial comparison experiment to demonstrate that the cytokine combination selected in the present invention and the amount of the cytokine used can help the detection method of the present invention to improve sensitivity, and the experimental results are shown in the following table:
it can be seen that when we stimulated PBMC with peptides containing CMV, influnza A and EBV, the number of Granzyme B spots was increased 3-6 fold over the prior art without the addition of cytokines or with one cytokine alone or with both cytokines.
Embodiment III: the invention provides an enzyme-linked immune systemThe epidemic spot detection kit comprises an experimental hole: 96. Mu.L of the medium of PBMC cells to be tested, 4. Mu.L of the polypeptide, and 100. Mu.L of the medium at a concentration of 3X 10 were added to the experimental wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 0.5 mug/ml, the concentration of the IL-2 in the culture medium is 5U/ml, and the concentration of the IL-21 in the culture medium is 50ng/ml;
negative control: mu.L of medium of PBMC cells to be tested, 1. Mu.L of LDMSO, and 100. Mu.L of 3X 10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 0.5 mug/ml, the concentration of the IL-2 in the culture medium is 5U/ml, and the concentration of the IL-21 in the culture medium is 50ng/ml;
positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 3X 10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 0.5 mug/ml, the concentration of the IL-2 in the culture medium is 5U/ml, and the concentration of the IL-21 in the culture medium is 50ng/ml;
Standard curve containing anti-CD3 gradient concentration: mu.L of culture medium of PBMC cells to be tested is added to each standard curve well, 5 mu.L of anti-CD3 with each concentration, and 100 mu.L of 3X 10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium contains anti-CD28, IL-2 and IL-21, wherein the concentration of the anti-CD28 in the culture medium is 0.5 mug/ml, the concentration of the IL-2 in the culture medium is 5U/ml, and the concentration of the IL-21 in the culture medium is 50ng/ml; the gradient of anti-CD3 concentration was set to start at a concentration of 0.003. Mu.g/ml, serial multiple dilution.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) Taking the 96-well ELISPot plate coated with the detection antibody in advance out of the refrigerator, opening the plate in a safety cabinet, adding 200 mu L of PBS into each well, and washing for 5 times;
(2) 200. Mu.L of medium was added to each well and incubated for 30 min at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into a standard curve hole, wherein the final volume is 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing for 24 hours at 37 ℃ in an incubator;
(5) After 24 hours, the ELISPot plate is taken out of the incubator, the culture medium in the holes is poured, 200 mu L of PBS is added into each hole for washing, and finally the culture medium is buckled on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole, washing for 5 times, and finally buckling on the absorbent paper for drying;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing for 5 times, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Further, the step (1) further comprises the step (0) of in vitro stimulation:
(01) The concentration of PBMC cells was adjusted to 8X 10 with medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100. Mu.L of PBMC cell culture medium to be tested, 0.4. Mu.L of polypeptide, 100. Mu.L of concentration of 8X 10 6 Ml PBMC cell suspension to be tested, negative well: 100. Mu.L of PBMC cell culture medium to be tested and 100. Mu.L of PBMC cell culture medium with concentration of 8X 10 6 /ml of PBMC cell suspension to be tested;
(03) Place 96-well U-bottom cell culture plate in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
With the above detection method, the antibody for detection in this example is anti-IL-2-biotin, the peptides used are peptides comprising CMV, influenza A and EBV, the concentration of each peptide is 10 μg/ml, the peptides used for in vitro 5 days stimulation are identical to those in the detection method, and the following list is a partial comparison test to illustrate that the combination of cytokines selected in the present invention and the amount of cytokines used can help the detection method of the present invention to improve the sensitivity, and the experimental results are shown in the following table:
It follows that when we stimulated PBMC with peptides comprising CMV, influenza and EBV, the number of IL-2 spots was increased by a factor of 1.5-2.4 compared to the prior art without the addition of cytokines or with one cytokine alone or with both cytokines.
Embodiment four: the detection antibodies of the following examples are all exemplified by the common anti-Granzyme B-biotin antibody, which is disclosed in the inventionThe ELISA spot detection kit provided by the invention comprises an experimental hole: 96. Mu.L of the medium of PBMC cells to be tested, 4. Mu.L of the polypeptide, and 100. Mu.L of the medium at a concentration of 1X 10 were added to the experimental wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2 and IL-7, wherein the concentration of the anti-CD28 in the culture medium is 1 mug/ml, the concentration of the IL-2 in the culture medium is 10U/ml, and the concentration of the IL-7 in the culture medium is 1ng/ml;
negative control: mu.L of medium of PBMC cells to be tested, 1. Mu.L of LDMSO, and 100. Mu.L of 1X 10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2 and IL-7, wherein the concentration of the anti-CD28 in the culture medium is 1 mug/ml, the concentration of the IL-2 in the culture medium is 10U/ml, and the concentration of the IL-7 in the culture medium is 1ng/ml;
Positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 1X 10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2 and IL-7, wherein the concentration of the anti-CD28 in the culture medium is 1 mug/ml, the concentration of the IL-2 in the culture medium is 10U/ml, and the concentration of the IL-7 in the culture medium is 1ng/ml;
standard curve containing anti-CD3 gradient concentration: mu.L of culture medium of PBMC cells to be tested is added to each standard curve well, 5 mu.L of anti-CD3 with each concentration, and 100 mu.L of 1X 10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium comprises anti-CD28, IL-2 and IL-7, wherein the concentration of the anti-CD28 in the culture medium is 1 mug/ml, the concentration of the IL-2 in the culture medium is 10U/ml, and the concentration of the IL-7 in the culture medium is 1ng/ml; the gradient of anti-CD3 concentration was set to start at a concentration of 0.002. Mu.g/ml, serial multiple dilution.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) Taking the 96-well ELISPot plate coated with the detection antibody in advance out of the refrigerator, opening the plate in a safety cabinet, adding 200 mu L of PBS into each well, and washing for 5 times;
(2) 200. Mu.L of culture medium was added to each well and incubated for 30min at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into a standard curve hole, wherein the final volume is 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in a CO2 incubator to culture for 12 hours at 37 ℃;
(5) After 12 hours, the ELISPot plate is taken out from the incubator, the culture medium in the holes is poured, 200 mu L of PBS is added into each hole for washing, and finally the culture medium is buckled on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole, washing for 5 times, and finally buckling on the absorbent paper for drying;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing for 5 times, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Further, the step (1) further comprises the step (0) of in vitro stimulation:
(01) The concentration of PBMC cells was adjusted to 5X 10 with the medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100. Mu.L of PBMC cell culture medium to be tested, 0.4. Mu.L of polypeptide, 100. Mu.L of concentration 5X 10 6 Ml PBMC cell suspension to be tested, negative well: 100. Mu.L of PBMC cell culture medium to be tested and 100. Mu.L of PBMC cell culture medium at a concentration of 5X 10 6 /ml of PBMC cell suspension to be tested;
(03) Place 96-well U-bottom cell culture plate in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
Fifth embodiment: the invention provides an ELISA spot detection kit, which comprises an experiment hole: 96. Mu.L of the medium of PBMC cells to be tested, 4. Mu.L of the polypeptide, and 100. Mu.L of the medium at a concentration of 1X 10 were added to the experimental wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 2 mug/ml, the concentration of the IL-2 in the culture medium is 15U/ml, the concentration of the IL-7 in the culture medium is 30ng/ml, and the concentration of the IL-15 in the culture medium is 1ng/ml;
negative control: mu.L of medium of PBMC cells to be tested, 1. Mu.L of LDMSO, and 100. Mu.L of 1X 10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 2 mug/ml, the concentration of the IL-2 in the culture medium is 15U/ml, the concentration of the IL-7 in the culture medium is 30ng/ml, and the concentration of the IL-15 in the culture medium is 1ng/ml;
Positive control: in the positive direction96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 1X 10 concentration were added to the control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 2 mug/ml, the concentration of the IL-2 in the culture medium is 15U/ml, the concentration of the IL-7 in the culture medium is 30ng/ml, and the concentration of the IL-15 in the culture medium is 1ng/ml;
standard curve containing anti-CD3 gradient concentration: mu.L of culture medium of PBMC cells to be tested is added to each standard curve well, 5 mu.L of anti-CD3 with each concentration, and 100 mu.L of 1X 10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 2 mug/ml, the concentration of the IL-2 in the culture medium is 15U/ml, the concentration of the IL-7 in the culture medium is 30ng/ml, and the concentration of the IL-15 in the culture medium is 1ng/ml; the gradient of anti-CD3 concentration was set to start at a concentration of 0.001 μg/ml, serially doubling the dilution.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) The 96-well ELISPot plate coated with the detection antibody in advance is taken out of the refrigerator, opened in a safety cabinet, and washed 5 times by adding 200 mu L of PBS into each well;
(2) 200. Mu.L of culture medium was added to each well and incubated for 30min at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into a standard curve hole, wherein the final volume is 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing for 12 hours at 37 ℃ in an incubator;
(5) After 12 hours, the ELISPot plate is taken out from the incubator, the culture medium in the holes is poured, 200 mu L of PBS is added into each hole for washing, and finally the culture medium is buckled on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole, washing for 5 times, and finally buckling on the absorbent paper for drying;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing for 5 times, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Further, the step (1) further comprises the step (0) of in vitro stimulation:
(01) The concentration of PBMC cells was adjusted to 8X 10 with medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100. Mu.L of PBMC cell culture medium to be tested, 0.4. Mu.L of polypeptide and 100. Mu.L of polypeptideThe concentration is 8 multiplied by 10 6 Ml PBMC cell suspension to be tested, negative well: 100. Mu.L of PBMC cell culture medium to be tested and 100. Mu.L of PBMC cell culture medium with concentration of 8X 10 6 /ml of PBMC cell suspension to be tested;
(03) Place 96-well U-bottom cell culture plate in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
Example six: the invention provides an ELISA spot detection kit, which comprises an experiment hole: 96. Mu.L of the medium of PBMC cells to be tested, 4. Mu.L of the polypeptide, and 100. Mu.L of the medium at a concentration of 1X 10 were added to the experimental wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 4 mug/ml, the concentration of the IL-2 in the culture medium is 20U/ml, the concentration of the IL-7 in the culture medium is 15ng/ml, and the concentration of the IL-15 in the culture medium is 50ng/ml;
negative control: mu.L of medium of PBMC cells to be tested, 1. Mu.L of LDMSO, and 100. Mu.L of 1X 10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 4 mug/ml, the concentration of the IL-2 in the culture medium is 20U/ml, the concentration of the IL-7 in the culture medium is 15ng/ml, and the concentration of the IL-15 in the culture medium is 50ng/ml;
positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 1X 10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 4 mug/ml, the concentration of the IL-2 in the culture medium is 20U/ml, the concentration of the IL-7 in the culture medium is 15ng/ml, and the concentration of the IL-15 in the culture medium is 50ng/ml;
standard curve containing anti-CD3 gradient concentration: mu.L of culture medium of PBMC cells to be tested is added to each standard curve well, 5 mu.L of anti-CD3 with each concentration, and 100 mu.L of 1X 10 6 Per ml of PBMC cell suspension to be tested, the final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of anti-CD3 gradient concentrations are obtainedA standard curve hole; the culture medium comprises anti-CD28, IL-2, IL-7 and IL-15, wherein the concentration of the anti-CD28 in the culture medium is 4 mug/ml, the concentration of the IL-2 in the culture medium is 20U/ml, the concentration of the IL-7 in the culture medium is 15ng/ml, and the concentration of the IL-15 in the culture medium is 50ng/ml; the gradient of anti-CD3 concentration was set to start at a concentration of 0.001 μg/ml, serially doubling the dilution.
The invention also provides a method for detecting ELISA spots by using the kit, which comprises the following steps:
(1) The 96-well ELISPot plate coated with the detection antibody in advance is taken out of the refrigerator, opened in a safety cabinet, and washed 5 times by adding 200 mu L of PBS into each well;
(2) 200. Mu.L of culture medium was added to each well and incubated for 30min at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve control well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into a standard curve hole, wherein the final volume is 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing in an incubator at 37 ℃ for 48 hours;
(5) After 48 hours, the ELISPot plate is taken out of the incubator, the culture medium in the holes is poured, 200 mu L of PBS is added into each hole for washing, and finally the culture medium is buckled on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole, washing for 5 times, and finally buckling on the absorbent paper for drying;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing for 5 times, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
Further, the step (1) further comprises the step (0) of in vitro stimulation:
(01) The concentration of PBMC cells was adjusted to 8X 10 with medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100. Mu.L of PBMC cell culture medium to be tested, 0.4. Mu.L of polypeptide, 100. Mu.L of concentration of 8X 10 6 Ml PBMC cell suspension to be tested, negative well: 100. Mu.L of PBMC cell culture medium to be tested and 100. Mu.L of PBMC cell culture medium with concentration of 8X 10 6 /ml of PBMC cell suspension to be tested;
(03) Place 96-well U-bottom cell culture plate in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
Using the above assay, the conventional antibody anti-Granzyme B-biotin was used in examples four to six, the peptides used were peptides comprising CMV, influenza A and EBV, each peptide concentration was 10. Mu.g/ml, the peptides used for 5 days of in vitro stimulation were identical to the peptides used for the assay, and the following examples were used in part of the comparative experiments to demonstrate that the combinations of cytokines selected according to the invention, and the amounts of cytokines used were able to help the assay of the invention to increase sensitivity, the experimental results were shown in the following Table:
it follows that when we stimulated PBMC with peptides comprising CMV, influenza and EBV, the number of spots was increased by a factor of 1.6-3.6 compared to the prior art without the addition of cytokines or with one cytokine alone or with both cytokines.
In order to save space and reduce unnecessary repetition, specific test data are not listed one by one, the method can be used for various detection antibodies of the ELISPOT kit in the prior art, and compared with the detection method in the prior art, the sensitivity is improved by 1.5-6 times, so that the detection method has higher sensitivity compared with the traditional ELISPOT method.
The invention establishes anti-CD3 internal reference, can revise the result of the current experiment, and ensures the reliability of the result. The prior art has no internal reference, and the number of the added cells is directly used as a denominator, but the reactivity of the cells is also influenced by factors such as different samples, different batch operations and the like. The standard curve is prepared by using the same sample, if the whole reaction is too high and the background is too high in the current experiment, the result is not a real cell reaction result, and the result can be corrected by comparing the experimental hole with the reaction holes with different concentrations of the standard curve.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. An enzyme-linked immunosorbent assay kit, which is characterized by comprising an experimental hole: adding 96 μl of culture medium of PBMC cells to be tested, 4 μl of polypeptide, each peptide having a concentration of 1-10 μg/ml, and 100 μl of 1-5×10 6 /mL PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, the concentration of the IL-2 in the culture medium is 1-30U/ml, and the used polypeptide is peptide containing CMV, influnza A and EBV;
negative control: mu.L of culture medium of PBMC cells to be tested, 1 mu LDMSO, and 100 mu.L of 1-5×10 concentration were added to the negative control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml;
positive control: 96. Mu.L of medium of PBMC cells to be tested, 4. Mu.LPMA+Ionomycin, and 100. Mu.L of 1-5×10 concentration were added to the positive control wells 6 Per ml of PBMC cell suspension to be tested, the final volume is 200. Mu.L; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml;
Standard curve containing anti-CD3 gradient concentration: adding 95 μl of culture medium of PBMC cells to be tested into each standard curve well, 5 μl of anti-CD3 with each concentration, and 100 μl of 1-5×10 6 The final volume of the liquid in each standard curve hole is 200 mu L, and a plurality of standard curve holes containing anti-CD3 gradient concentration are obtained; the culture medium contains anti-CD28 and IL-2, the concentration of the anti-CD28 in the culture medium is 0.1-5 mug/ml, and the concentration of the IL-2 in the culture medium is 1-30U/ml; the gradient of the anti-CD3 concentration is set to start with a concentration of 0.001-0.005 mug/ml, and the anti-CD3 concentration is diluted in a continuous multiple ratio.
2. The kit for ELISA spot detection according to claim 1, wherein the culture medium of PBMC cells to be tested used in the experimental hole, the negative control, the positive control and the standard curve further comprises IL-21, and when the culture medium contains anti-CD28, IL-2 and IL-21, the concentration of each component in the culture medium is 0.1-5 mug/ml, IL-2 is 1-30U/ml and IL-21 is 10-50ng/ml.
3. The kit for ELISA spot detection according to claim 1, wherein the culture medium of PBMC cells to be tested used in the experimental hole, the negative control, the positive control and the standard curve further comprises IL-7, and when the culture medium contains anti-CD28, IL-2 and IL-7, the concentration of each component in the culture medium is 0.1-5 mug/ml, IL-2 is 1-30U/ml and IL-7 is 1-30ng/ml.
4. The kit according to claim 1, wherein the culture medium of the PBMC cells to be tested used in the experimental hole, the negative control, the positive control and the standard curve further comprises IL-7 and IL-15, and the concentration of each component in the culture medium is 0.1-5 μg/ml of anti-CD28, 1-30U/ml of IL-2, 1-30ng/ml of IL-7 and 1-50ng/ml of IL-15 when the culture medium contains anti-CD28, IL-2, IL-7 and IL-15.
5. A detection method using the enzyme-linked immunosorbent assay kit as claimed in any one of claims 1 to 4, comprising the steps of:
(1) Taking the 96-well ELISPot plate coated with the detection antibody in advance out of the refrigerator, opening the plate in a safety cabinet, and adding PBS into each well for washing;
(2) Adding a culture medium of PBMC cells to be tested into each hole, and incubating at room temperature;
(3) Discarding the culture medium in the 96-well ELISPot plate, and adding the kit components into the experimental well, the negative control well, the positive control well and the standard curve well respectively, namely sequentially adding the culture medium, the polypeptide and the cell suspension into the experimental well, wherein the final volume is 200 mu L; sequentially adding the culture medium, the DMSO and the cell suspension into a negative control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, the PMA+Ionomycin and the cell suspension into a positive control hole, wherein the final volume is 200 mu L; sequentially adding the culture medium, anti-CD3 with each concentration and the cell suspension into each standard curve hole, and obtaining a final volume of 200 mu L;
(4) Placing the 96-well ELISPot plate prepared in the step (3) in CO 2 Culturing in an incubator at 37 ℃ for 12-48 hours;
(5) Taking the ELISPot plate out of the incubator after 12-48 hours, pouring the culture medium in the holes, adding 200 mu L of PBS into each hole for washing, and finally buckling on absorbent paper for the last time;
(6) Diluting the detection antibody to 1 mug/mL by using a PBS and FBS mixed solution, adding 100 mug of the detection antibody to each well, and incubating for 2 hours at 37 ℃, wherein the volume percentage of the PBS in the PBS and FBS mixed solution is 99.5%, and the rest is FBS;
(7) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing, and finally buckling on the absorbent paper for the last time;
(8) Mixing enzyme-labeled avidin with PBS and FBS in a volume ratio of 1: after 1000 dilution, 100 μl of each well was added and incubated at 37deg.C for 1 hr, wherein the volume percentage of PBS in the PBS-FBS mixture was 99.5%, the remainder being FBS;
(9) Pouring the liquid in the holes, adding 200 mu L of PBS into each hole for washing, and finally buckling on the absorbent paper for the last time;
(10) Filtering the color development liquid with a filter membrane with the thickness of 0.45 mu m to remove impurities, adding 100 mu L of the color development liquid into each hole, incubating at room temperature in a dark place, and observing every 3-5 min;
(11) Pouring the liquid in the holes, uncovering the base of the plate, washing the front and the back and the base with tap water, stopping developing, placing the plate at a cool place at room temperature, and closing the base after the plate is naturally dried;
(12) Reading spots on the pore plate membrane on an enzyme-linked spot image analyzer;
(13) And (3) making a standard curve according to the anti-CD3 readings of each concentration, fitting, substituting the sample readings into the standard curve, and obtaining a corrected result.
6. The method according to claim 5, wherein the step (1) is preceded by the step of (0) in vitro stimulation:
(01) The cell concentration of PBMC to be tested is adjusted to 2-8×10 by using a culture medium 6 /ml；
(02) The following were added to a 96-well U-bottom cell culture plate in order: experimental hole: 100 mu L of PBMC cell culture medium to be tested, 0.4 mu L of polypeptide, wherein the concentration of each peptide in the polypeptide is 1-10 mu g/ml, and the concentration of 100 mu L is 2-8 multiplied by 10 6 PBMC (peripheral component parts) fraction/ml to be measuredCell suspension, negative well: 100 mu L of PBMC cell culture medium to be tested and 100 mu L of PBMC cell culture medium with concentration of 2-8 multiplied by 10 6 /ml of PBMC cell suspension to be tested;
(03) Placing the 96-well U-bottom cell culture plate prepared in the step (02) in CO 2 Culturing in an incubator at 37 ℃ for 5 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911403515.5A CN111077311B (en) | 2019-12-30 | 2019-12-30 | ELISA spot detection kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911403515.5A CN111077311B (en) | 2019-12-30 | 2019-12-30 | ELISA spot detection kit and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111077311A CN111077311A (en) | 2020-04-28 |
| CN111077311B true CN111077311B (en) | 2023-05-09 |
Family
ID=70320193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911403515.5A Active CN111077311B (en) | 2019-12-30 | 2019-12-30 | ELISA spot detection kit and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111077311B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5658745A (en) * | 1995-02-17 | 1997-08-19 | E. I. Du Pont De Nemours And Company | Cell enumeration immunoassay |
| CN101273122A (en) * | 2005-08-08 | 2008-09-24 | 塔博尔山圣拉弗尔基金中心 | Use of common gamma chain cytokines for the genetic modification of memory T lymphocytes |
| CN102165057A (en) * | 2008-05-16 | 2011-08-24 | 法国血液机构 | Plasmacytoid dendritic cell line used in active or adoptive cell therapy |
| CN105543170A (en) * | 2015-12-31 | 2016-05-04 | 中山大学 | Composition capable of stimulating expansion of T cells |
| CN106353501A (en) * | 2016-08-19 | 2017-01-25 | 南通表源生物技术有限公司 | Tuberculosis T cell detection kit and detection method thereof |
| CN107247146A (en) * | 2017-05-23 | 2017-10-13 | 中山大学附属第三医院 | Kit, the method and its application of high quick detection T cell and function |
| CN107646038A (en) * | 2015-05-28 | 2018-01-30 | 基因泰克公司 | For detecting the measure based on cell of AntiCD3 McAb homodimer |
-
2019
- 2019-12-30 CN CN201911403515.5A patent/CN111077311B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5658745A (en) * | 1995-02-17 | 1997-08-19 | E. I. Du Pont De Nemours And Company | Cell enumeration immunoassay |
| CN101273122A (en) * | 2005-08-08 | 2008-09-24 | 塔博尔山圣拉弗尔基金中心 | Use of common gamma chain cytokines for the genetic modification of memory T lymphocytes |
| CN102165057A (en) * | 2008-05-16 | 2011-08-24 | 法国血液机构 | Plasmacytoid dendritic cell line used in active or adoptive cell therapy |
| CN107646038A (en) * | 2015-05-28 | 2018-01-30 | 基因泰克公司 | For detecting the measure based on cell of AntiCD3 McAb homodimer |
| CN105543170A (en) * | 2015-12-31 | 2016-05-04 | 中山大学 | Composition capable of stimulating expansion of T cells |
| CN106353501A (en) * | 2016-08-19 | 2017-01-25 | 南通表源生物技术有限公司 | Tuberculosis T cell detection kit and detection method thereof |
| CN107247146A (en) * | 2017-05-23 | 2017-10-13 | 中山大学附属第三医院 | Kit, the method and its application of high quick detection T cell and function |
Non-Patent Citations (4)
| Title |
|---|
| Kenichi Kato 等.Novel interferon-γ enzyme-linked immunoSpot assay using activated cells for identifying hypersensitivity-inducing drug culprits.Journal of Dermatological Science.2017,第86卷(第86期),222-229. * |
| Stefan Quast 等.IL-2 absorption affects IFN-γ and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays.Cellular Immunology .2005,第237卷28-36. * |
| Wim Jennes 等.Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15—the Amplispot assay.Journal of Immunological Methods.2002,第270卷99-108. * |
| 袁娇 ; 崔秋燕 ; 唐维 ; 超晨 ; 周智广 ; 杨琳 ; .IL-2和IL-7对优化检测1型糖尿病患者谷氨酸脱羧酶65特异性T细胞反应的比较.中南大学学报(医学版).2017,第42卷(第11期),1257-1262. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111077311A (en) | 2020-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Slota et al. | ELISpot for measuring human immune responses to vaccines | |
| Lee et al. | Enzyme-linked immunospot assay for interferon-gamma in the diagnosis of tuberculous pleurisy | |
| Ranieri et al. | CTL elispot assay | |
| WO2016095273A1 (en) | Antigen stimulant for detecting mycobacterium tuberculosis infection, kit, and applications of antigen stimulant | |
| CN107904278B (en) | Method for detecting influence of drug on cell proliferation | |
| CN111273017A (en) | Fluorescence immunochromatography kit for rapidly detecting novel coronavirus | |
| AU2007228598A1 (en) | Clinical correlates | |
| Ranieri et al. | CTL ELISPOT assay and T cell detection | |
| CN113801208B (en) | Novel T cell immunodetection method of coronavirus | |
| CN111077311B (en) | ELISA spot detection kit and detection method thereof | |
| CN110716050A (en) | Application of antigen combination in preparation of kit for detecting lung cancer related autoantibody, corresponding kit and detection method | |
| JP4990129B2 (en) | Non-ELISpot assay | |
| US8771971B2 (en) | Methods and kits for measurement of lymphocyte function | |
| Sais et al. | Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia | |
| Fischer et al. | Donor‐specific alloreactive T cells can be quantified from whole blood, and may predict cellular rejection after renal transplantation | |
| McLaughlin et al. | Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms | |
| US20100267009A1 (en) | Method for the in vitro diagnosis and/or in vitro therapy monitoring of infections | |
| CN110514845B (en) | Detection method and detection platform for immunogenicity of tumor neoantigen | |
| US20070202546A1 (en) | Immunoassay Carrier | |
| US20080051297A1 (en) | Method for determining antigen-specific T cell response in high throughput format | |
| Knutson et al. | Laboratory analysis of T-cell immunity | |
| CN112229696A (en) | Fixed film breaking agent and preparation method thereof | |
| Tary‐Lehmann et al. | Validating reference samples for comparison in a regulated ELISPOT assay | |
| Kalyuzhny | Membrane microplates for one-and two-color ELISPOT and FLUOROSPOT assays | |
| Park et al. | Direct ex vivo functional analysis of HCV-specific T cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |