CN116068207A - Kit for detecting NK cell activity - Google Patents
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- CN116068207A CN116068207A CN202310294454.3A CN202310294454A CN116068207A CN 116068207 A CN116068207 A CN 116068207A CN 202310294454 A CN202310294454 A CN 202310294454A CN 116068207 A CN116068207 A CN 116068207A
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- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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Abstract
The invention relates to the technical field of biological detection, in particular to a kit for detecting NK cell activity, which comprises a kit body and a kit body, wherein the kit body comprises a kit body and a kit body: comprises a detection card, a sample diluent and an activator; the detection card is internally provided with a test strip, and a binding pad of the test strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with IFN-gamma antibody, the second detection line is coated with TNF-alpha antibody, and the quality control line is coated with rabbit anti-DNP antibody; activators include human interleukin IL-2, IL-12, or a combination of both. The kit adopts fluorescent microspheres combined with a biotin-avidin amplification system, so that the specificity of the kit and the accuracy of a detection result are greatly improved.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for detecting NK cell activity.
Background
Natural Killer (NK) is a large granular lymphocyte that can directly kill target cells, and has antitumor, antiinfectious and immunoregulatory functions. NK cells are group 3 lymphocytes juxtaposed with T, B cells and account for about 5-25% of the total lymphocytes in peripheral blood. NK cell therapy is also an emerging technology for cancer therapy, the key to which is the generation of a sufficient number of immune cells to recognize and kill the tumor, and effector cells to reach the tumor site and be activated around the tumor, and to exert antitumor effects. Besides killing tumor cells, the composition can also replace repair or improve immune function impairment caused by chemotherapy.
The detection of NK cell activity has important guiding significance for early diagnosis of cancer and evaluation of the effect of intervention treatment of cancer. The killing effect is mediated by toxic molecules released after activation, such as gamma-interferon (IFN-gamma) and alpha-tumor necrosis factor (TNF-alpha). NK cell killing target cells are mainly tumor cells, virus-infected cells, larger pathogens (such as fungi and parasites), allograft organs, tissues, etc. It has broad-spectrum anti-tumor effect, especially obvious effect on lymphoma and leukemia cells. Current methods for NK cell activity detection mainly include morphology, flow cytometry techniques and enzyme-linked immunosorbent assay (ELISA). The morphological method is simple and easy to grasp, but the dead cells and the living cells are judged not to be free from subjective factors of the testers, and the slightly damaged cells cannot be counted. The flow cytometry equipment is high in price, the operation is complex, and the accuracy of the result is closely related to the preparation of the sample; the enzyme-linked immunosorbent assay method has low automation degree, long detection time and large influence by human factors. Thus, in view of the above problems, it is necessary to establish a kit for detecting NK cell activity.
Disclosure of Invention
The invention aims at: aiming at the defects existing in the prior art, the kit for detecting the NK cell activity is simple to operate, short in detection time, high in sensitivity, wide in linear range, free of pollution and capable of quantitatively detecting.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
a kit for detecting NK cell activity, the kit comprising a detection card, a sample diluent and an activator; the detection card is internally provided with a test paper strip, and a binding pad of the test paper strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, wherein the first detection line is coated with IFN-gamma antibodies, the second detection line is coated with TNF-alpha antibodies, and the quality control line is coated with rabbit anti-DNP antibodies; the activator comprises human interleukin IL-2, IL-12 or a combination of both.
As an improved technical scheme, the using amount of the activator is 5-10ng/mL.
As an improved technical scheme, the sample diluent is Tris buffer solution containing 0.6-1.8 wt% PEG800, 0.1-1.5 wt% NaCl, 0.75-2 wt% PVA, 0.1-0.5 wt% casein and 0.1-1 wt% S9, wherein the pH value of the Tris buffer solution is 8.0-8.5, and the concentration is 10-50mM.
As an improved technical solution, the method for labeling IFN- γ antibodies and biotin-labeled TNF- α antibodies with biotin comprises the steps of:
(1) Diluting IFN-gamma or TNF-alpha to 1mg/mL with sodium bicarbonate buffer solution with pH of 8.0 and 0.1mol/L or boric acid buffer solution with pH of 8.6 and 0.5mol/L, and continuing dialyzing the diluted IFN-gamma or TNF-alpha with the sodium bicarbonate buffer solution or boric acid buffer solution, wherein the dialyzed IFN-gamma antibody solution or TNF-alpha antibody solution is reserved;
(2) Taking 1ml of the IFN-gamma antibody solution or TNF-alpha antibody solution prepared in the step (1), adding 120 mu L of NHSB solution of 1mg/ml, stirring and preserving heat for 2-4h, adding 9.6 mu L of 1mol/LNH 4 Continuously stirring, dialyzing and passing through Cl solutionAdding sodium azide with final concentration of 0.5g/L, 1.0g/LBSA and glycerol into the liquid, and preserving at-20deg.C.
As an improved technical scheme, the method for labeling avidin by using fluorescent microspheres and labeling DNP-BSA by using fluorescent microspheres comprises the following steps:
(1) Washing the microspheres by adopting MES buffer with pH of 6.0 and 50mM, and carrying out re-dissolution by adopting MES buffer continuously after centrifugation for standby;
(2) Activating microspheres, EDC and Sulfo-NHS according to the mass ratio of 1:0.1-1:0.1-3 for 20-60min, centrifuging, re-dissolving, washing and centrifuging the MES buffer solution in the step (1), re-dissolving the MES buffer solution with pH of 6.5 and 50mM, adding streptavidin or DNP-BSA into the microspheres according to the dosage of 0.1-1mg of streptavidin or DNP-BSA per 1mg of microspheres, performing ultrasound and incubation for 2-3h, quenching the microspheres with glycine with the final concentration of 100mM, continuing to perform incubation for 20-30min, washing with microsphere sealing buffer solution, re-dissolving after centrifugation, sealing for 2-4h, centrifuging, re-dissolving the microspheres with preservation solution, and preserving the microspheres at 4 ℃ for later use;
as an improved technical scheme, the microsphere sealing buffer is PBS buffer containing 1-5wt% BSA and 0.05wt% Tween-20.
As an improved technical scheme, the microsphere preservation solution is potassium carbonate buffer solution containing 0.01wt% of casein and 0.01wt% of sodium azide, wherein the concentration of the potassium carbonate buffer solution is 3mM, and the pH value of the potassium carbonate buffer solution is 8.0-10.0.
As an improved technical scheme, the preparation of the binding pad is to disperse biotin-marked IFN-gamma, TNF-alpha and fluorescent microsphere-marked avidin in a volume ratio of 1-4:1-4:1 into microsphere diluent, re-disperse fluorescent microsphere-marked DNP-BSA in the microsphere diluent in an amount of 2.5% -10% v/v, coat the treated glass fiber with a quantitative metal spraying instrument in a spraying amount of 1-4 mu l/cm, and dry the glass fiber in a blasting way at 37 ℃ for 2 hours and then store the glass fiber for later use.
As an improved technical scheme, the microsphere diluent is Tris-HCl buffer solution containing 0.2% v/v Tween 20, 0.3% wt bovine serum albumin, 0.3% v/v polyethylene glycol, 5% wt sucrose, 5% wt trehalose and 0.5% wt preservative, wherein the pH of the Tris-HCl buffer solution is 8.0-8.2.
As an improved technical scheme, a sealing liquid is adopted for treatment for 30min when the sample adding pad of the test strip is prepared, wherein the sealing liquid is Tris buffer containing 0.5-3wt% of bovine serum albumin, 0.1-0.5wt% of casein, 0.1-5wt% of PVP, 0.02-0.05wt% of monopotassium phosphate and 0.01-0.1wt% of Tween 20, and the concentration of the Tris buffer is 10-50mM.
Compared with the prior art, the invention has the following advantages:
the kit for detecting NK cell activity provided by the invention can realize synchronous and rapid detection of IFN-gamma and TNF-alpha on one test strip, and the antibodies used are monoclonal antibodies, so that the kit has good specificity, no interference among detection of various cytokines, and is simple and rapid. The kit adopts the fluorescent microsphere combined biotin-avidin amplification system, so that the signal sensitivity is improved to the greatest extent, and the specificity of the kit and the accuracy of a detection result are improved greatly.
Drawings
FIG. 1 is a top view of a test card in a kit for detecting NK cell activity of the present invention;
FIG. 2 is a top view of the test strip of FIG. 1;
FIG. 3 is a side view of FIG. 2;
the device comprises a 1-detection card, a 11-sample loading hole, a 12-observation hole, a 13-installation marking hole, a 2-test strip, a 21-loading pad, a 22-binding pad, a 23-nitrocellulose membrane, a 231-first detection line, a 232-second detection line, a 233-quality control line, a 24-water absorption pad, a 241-water absorption paper and a 242-installation mark.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A kit for detecting NK cell activity, comprising a detection card, a sample diluent and an activator; the detection card is internally provided with a test strip, and a binding pad of the test strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with IFN-gamma antibody, the second detection line is coated with TNF-alpha antibody, and the quality control line is coated with rabbit anti-DNP antibody; the activator is human interleukin IL-2, and the dosage of the activator is 5ng/mL.
Wherein the sample dilution is Tris buffer containing 0.6wt% PEG800, 0.1wt% NaCl, 0.75wt% PVA, 0.1wt% casein, 0.1wt% S9, and the pH of the Tris buffer is 8.0 at a concentration of 20mM.
Wherein the binding pad is coated with biotin-labeled IFN-gamma antibody and biotin-labeled TNF-alpha antibody, and the method for labeling IFN-gamma antibody or TNF-alpha antibody with biotin comprises the steps of:
(1) Diluting IFN-gamma or TNF-alpha to 1mg/mL with sodium bicarbonate buffer solution with pH of 8.0 and 0.1mol/L or boric acid buffer solution with pH of 8.6 and 0.5mol/L, and continuing dialyzing the diluted IFN-gamma or TNF-alpha with the sodium bicarbonate buffer solution or boric acid buffer solution, wherein the dialyzed IFN-gamma antibody solution or TNF-alpha antibody solution is reserved;
(2) Taking 1ml of the IFN-gamma antibody solution or TNF-alpha antibody solution prepared in the step (1), adding 120 mu L of NHSB solution of 1mg/ml, stirring and preserving heat for 2h, adding 9.6 mu L of 1mol/LNH 4 And (3) continuously stirring the Cl solution, dialyzing, passing through a molecular sieve column, adding sodium azide with the final concentration of 0.5g/L, 1.0g/LBSA and glycerin into the collected liquid, and preserving at the temperature of minus 20 ℃.
Wherein the binding pad is coated with fluorescent microsphere labeled avidin and fluorescent microsphere labeled DNP-BSA, and the method for labeling the avidin or the DNP-BSA by using the fluorescent microsphere comprises the following steps:
(1) Washing the microspheres by adopting MES buffer with pH of 6.0 and 50mM, and carrying out re-dissolution by adopting MES buffer continuously after centrifugation for standby;
(2) Activating microspheres, EDC and Sulfo-NHS for 20min according to the mass ratio of 1:0.1:0.1, centrifuging, re-dissolving, washing and centrifuging the MES buffer in the step (1), re-dissolving the MES buffer with pH6.5 and 50mM, adding streptavidin or DNP-BSA according to the dosage of coupling 0.1mg streptavidin or DNP-BSA to each 1mg microsphere, carrying out ultrasound and incubation for 2h, quenching with glycine with the final concentration of 100mM, continuing to incubate for 20min, washing with microsphere sealing buffer (PBS buffer containing 1wt% BSA and 0.05wt% Tween-20), re-dissolving after centrifuging, sealing for 2h, centrifuging, re-dissolving with microsphere preservation solution (potassium carbonate buffer containing 0.01wt% casein and 0.01wt% sodium azide, and the concentration of the potassium carbonate buffer is 3mM, pH 8.0), and preserving at 4 ℃;
the preparation of the binding pad is that biotin-marked IFN-gamma, TNF-alpha and fluorescent microsphere marked avidin are dispersed in microsphere diluent (Tris-HCl buffer solution containing 0.2% v/v Tween 20, 0.3% wt. bovine serum albumin, 0.3% v/v polyethylene glycol, 5% wt. sucrose, 5% wt. trehalose and 0.5% wt. preservative according to the volume ratio of 1:1:1), fluorescent microsphere marked DNP-BSA is redispersed in microsphere diluent, and then coated on the treated glass fiber by adopting a quantitative gold spraying instrument according to the spray quantity of 1 mul/cm, and the glass fiber is dried for 2 hours by blowing at 37 ℃ and then stored for standby.
Wherein the sample adding pad of the test strip is treated for 30min by adopting a sealing liquid, the sealing liquid is Tris buffer solution containing 0.5wt% of bovine serum albumin, 0.1wt% of casein, 0.1wt% of PVP, 0.02wt% of monopotassium phosphate and 0.01wt% of Tween 20, and the concentration of the Tris buffer solution is 20mM.
As shown in fig. 1, a detection card 1 in the invention comprises an upper plate block and a lower plate block which are clamped together, wherein one end of the upper plate block is provided with a sample loading hole 11, the other end of the upper plate block is provided with a mounting marking hole 13, and an observation hole 12 is arranged between the sample loading hole 11 and the mounting marking hole 13.
As shown in fig. 1 to 3 together, the test strip 2 in the test card 1 includes a sample adding pad 21, a binding pad 22, a nitrocellulose membrane 23, and a water absorbing pad 24 in order from top to bottom, wherein the sample adding pad 21 corresponds to the sample adding hole 11, the nitrocellulose membrane 23 corresponds to the observation hole 12, and a first detection line 231, a second detection line 232, and a quality control line 233 are sequentially provided on the nitrocellulose membrane 23; a water absorbing paper 241 and a mounting mark 242 are provided above the water absorbing pad 24.
Example 2
A kit for detecting NK cell activity, comprising a detection card, a sample diluent and an activator; the detection card is internally provided with a test strip, and a binding pad of the test strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with IFN-gamma antibody, the second detection line is coated with TNF-alpha antibody, and the quality control line is coated with rabbit anti-DNP antibody; the activator is human interleukin IL-12, and the dosage of the activator is 8ng/mL.
Wherein the sample dilution is Tris buffer containing 1wt% PEG800, 0.5wt% NaCl, 1wt% PVA, 0.2wt% casein, 0.5wt% S9, and the pH of the Tris buffer is 8.2, and the concentration is 40mM.
Wherein the binding pad is coated with biotin-labeled IFN-gamma antibody and biotin-labeled TNF-alpha antibody, and the method for labeling IFN-gamma antibody or TNF-alpha antibody with biotin comprises the steps of:
(1) Diluting IFN-gamma or TNF-alpha to 1mg/mL with sodium bicarbonate buffer solution with pH of 8.0 and 0.1mol/L or boric acid buffer solution with pH of 8.6 and 0.5mol/L, and continuing dialyzing the diluted IFN-gamma or TNF-alpha with the sodium bicarbonate buffer solution or boric acid buffer solution, wherein the dialyzed IFN-gamma antibody solution or TNF-alpha antibody solution is reserved;
(2) Taking 1ml of the IFN-gamma antibody solution or TNF-alpha antibody solution prepared in the step (1), adding 120 mu L of NHSB solution of 1mg/ml, stirring and preserving heat for 3h, adding 9.6 mu L of 1mol/LNH 4 And (3) continuously stirring the Cl solution, dialyzing, passing through a molecular sieve column, adding sodium azide with the final concentration of 0.5g/L, 1.0g/LBSA and glycerin into the collected liquid, and preserving at the temperature of minus 20 ℃.
Wherein the binding pad is coated with fluorescent microsphere labeled avidin and fluorescent microsphere labeled DNP-BSA, and the method for labeling the avidin or the DNP-BSA by using the fluorescent microsphere comprises the following steps:
(1) Washing the microspheres by adopting MES buffer with pH of 6.0 and 50mM, and carrying out re-dissolution by adopting MES buffer continuously after centrifugation for standby;
(2) Activating microspheres, EDC and Sulfo-NHS for 35min according to the mass ratio of 1:0.5:1, centrifuging, re-dissolving, washing and centrifuging the MES buffer solution in the step (1), re-dissolving the MES buffer solution with pH of 6.5 and 50mM, adding streptavidin or DNP-BSA according to the dosage of 0.5mg streptavidin or DNP-BSA coupled to each 1mg microsphere, carrying out ultrasound and incubation for 2.5h, quenching the microspheres with glycine with the final concentration of 100mM, continuing to incubate for 25min, washing the microspheres with a microsphere sealing buffer solution (PBS buffer solution containing 2.5wt% BSA and 0.05wt% Tween-20), re-dissolving and sealing the microspheres for 3h after centrifuging, re-dissolving the microspheres with a microsphere preservation solution (potassium carbonate buffer solution containing 0.01wt% casein and 0.01wt% sodium azide, and the concentration of the potassium carbonate buffer solution being 3mM and pH of 8.8), and preserving the microspheres at 4 ℃ for standby;
the preparation of the binding pad comprises dispersing biotin-labeled IFN-gamma, TNF-alpha and fluorescent microsphere-labeled avidin in a volume ratio of 1:2:1 in microsphere diluent (Tris-HCl buffer containing 0.2% v/v Tween 20, 0.3% w/v polyethylene glycol, 5% sucrose, 5% trehalose and 0.5% by weight preservative, pH of the Tris-HCl buffer being 8.1), re-dispersing fluorescent microsphere-labeled DNP-BSA in the microsphere diluent in an amount of 5% v/v, coating the treated glass fiber with a spray of 2 mu l/cm by adopting a quantitative gold-spraying instrument, and drying in an air blast for 2 hours at 37 ℃ for later use.
Wherein the sample adding pad of the test strip is treated for 30min by adopting a sealing liquid, the sealing liquid is Tris buffer solution containing 1.5wt% of bovine serum albumin, 0.2wt% of casein, 1.5wt% of PVP, 0.03wt% of monopotassium phosphate and 0.05wt% of Tween 20, and the concentration of the Tris buffer solution is 10mM.
Example 3
A kit for detecting NK cell activity, comprising a detection card, a sample diluent and an activator; the detection card is internally provided with a test strip, and a binding pad of the test strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with IFN-gamma antibody, the second detection line is coated with TNF-alpha antibody, and the quality control line is coated with rabbit anti-DNP antibody; the activator is human interleukin IL-12, and the dosage of the activator is 10ng/mL.
Wherein the sample dilution is Tris buffer containing 1.5wt% PEG800, 1wt% NaCl, 1.5wt% PVA, 0.3wt% casein, 0.8wt% S9, and the pH of the Tris buffer is 8.3, at a concentration of 30mM.
Wherein the binding pad is coated with biotin-labeled IFN-gamma antibody and biotin-labeled TNF-alpha antibody, and the method for labeling IFN-gamma antibody or TNF-alpha antibody with biotin comprises the steps of:
(1) Diluting IFN-gamma or TNF-alpha to 1mg/mL with sodium bicarbonate buffer solution with pH of 8.0 and 0.1mol/L or boric acid buffer solution with pH of 8.6 and 0.5mol/L, and continuing dialyzing the diluted IFN-gamma or TNF-alpha with the sodium bicarbonate buffer solution or boric acid buffer solution, wherein the dialyzed IFN-gamma antibody solution or TNF-alpha antibody solution is reserved;
(2) Taking 1ml of the IFN-gamma antibody solution or TNF-alpha antibody solution prepared in the step (1), adding 120 mu L of NHSB solution of 1mg/ml, stirring and preserving heat for 3.5h, adding 9.6 mu L of 1mol/LNH 4 And (3) continuously stirring the Cl solution, dialyzing, passing through a molecular sieve column, adding sodium azide with the final concentration of 0.5g/L, 1.0g/LBSA and glycerin into the collected liquid, and preserving at the temperature of minus 20 ℃.
Wherein the binding pad is coated with fluorescent microsphere labeled avidin and fluorescent microsphere labeled DNP-BSA, and the method for labeling the avidin and the DNP-BSA by using the fluorescent microsphere comprises the following steps:
(1) Washing the microspheres by adopting MES buffer with pH of 6.0 and 50mM, and carrying out re-dissolution by adopting MES buffer continuously after centrifugation for standby;
(2) Activating microspheres, EDC and Sulfo-NHS for 45min according to the mass ratio of 1:0.8:2, centrifuging, re-dissolving, washing and centrifuging the MES buffer in the step (1), re-dissolving the MES buffer with pH6.5 and 50mM, adding streptavidin or DNP-BSA according to the dosage of 0.8mg streptavidin or DNP-BSA coupled to each 1mg microsphere, performing ultrasonic treatment and incubation for 2.8h, quenching the microspheres with glycine with the final concentration of 100mM, continuously incubating for 28min, washing the microspheres with a sealing buffer (PBS buffer containing 3.5wt% BSA and 0.05wt% Tween-20), re-dissolving and sealing the microspheres for 3.5h after centrifuging, re-dissolving the microspheres with a potassium carbonate buffer containing 0.01wt% casein and 0.01wt% sodium azide, and keeping the concentration of the potassium carbonate buffer at 3mM and pH9.5 for standby at 4 ℃;
the preparation of the binding pad comprises dispersing biotin-labeled IFN-gamma, TNF-alpha and fluorescent microsphere-labeled avidin in a volume ratio of 3:2:1 in microsphere diluent (Tris-HCl buffer containing 0.2% v/v Tween 20, 0.3% w/v polyethylene glycol, 5% sucrose, 5% trehalose and 0.5% by weight preservative, pH of the Tris-HCl buffer being 8.2), re-dispersing fluorescent microsphere-labeled DNP-BSA in the microsphere diluent in an amount of 7.5% v/v, coating the treated glass fiber with a spray of 3 μl/cm by adopting a quantitative gold spraying instrument, and air-drying at 37 ℃ for 2 hours for later use.
Wherein the sample adding pad of the test strip is treated for 30min by adopting a sealing liquid, the sealing liquid is Tris buffer containing 2wt% of bovine serum albumin, 0.3wt% of casein, 3wt% of PVP, 0.04wt% of monopotassium phosphate and 0.8wt% of Tween 20, and the concentration of the Tris buffer is 30mM.
Example 4
A kit for detecting NK cell activity, comprising a detection card, a sample diluent and an activator; the detection card is internally provided with a test strip, and a binding pad of the test strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, the first detection line is coated with IFN-gamma antibody, the second detection line is coated with TNF-alpha antibody, and the quality control line is coated with rabbit anti-DNP antibody; the activator comprises human interleukin IL-2 and IL-12 (mixed according to the volume ratio of 1:1). The amount of activator was 8ng/mL.
Wherein the sample dilution is Tris buffer containing 1.8wt% PEG800, 1.5wt% NaCl, 2wt% PVA, 0.5wt% casein, 1wt% S9, and the pH of the Tris buffer is 8.5, and the concentration is 50mM.
Wherein the binding pad is coated with biotin-labeled IFN-gamma antibody and biotin-labeled TNF-alpha antibody, and the method for labeling IFN-gamma antibody or TNF-alpha antibody with biotin comprises the steps of:
(1) Diluting IFN-gamma or TNF-alpha to 1mg/mL with sodium bicarbonate buffer solution with pH of 8.0 and 0.1mol/L or boric acid buffer solution with pH of 8.6 and 0.5mol/L, and continuing dialyzing the diluted IFN-gamma or TNF-alpha with the sodium bicarbonate buffer solution or boric acid buffer solution, wherein the dialyzed IFN-gamma antibody solution or TNF-alpha antibody solution is reserved;
(2) Taking 1ml of the IFN-gamma antibody solution or TNF-alpha antibody solution prepared in the step (1), adding 120 mu L of NHSB solution of 1mg/ml, stirring and preserving heat for 4h, adding 9.6 mu L of 1mol/LNH 4 And (3) continuously stirring the Cl solution, dialyzing, passing through a molecular sieve column, adding sodium azide with the final concentration of 0.5g/L, 1.0g/LBSA and glycerin into the collected liquid, and preserving at the temperature of minus 20 ℃.
Wherein the binding pad is coated with fluorescent microsphere labeled avidin and fluorescent microsphere labeled DNP-BSA, and the method for labeling the avidin or the DNP-BSA by using the fluorescent microsphere comprises the following steps:
(1) Washing the microspheres by adopting MES buffer with pH of 6.0 and 50mM, and carrying out re-dissolution by adopting MES buffer continuously after centrifugation for standby;
(2) Activating microspheres, EDC and Sulfo-NHS for 60min according to the mass ratio of 1:1:3, centrifuging, re-dissolving, washing and centrifuging by adopting the MES buffer solution in the step (1), re-dissolving by adopting the MES buffer solution with pH of 6.5 and 50mM, adding the streptavidin or DNP-BSA according to the dosage of coupling 1mg of the streptavidin or DNP-BSA to each 1mg of microspheres, carrying out ultrasound and incubation for 3h, quenching by adopting glycine with the final concentration of 100mM, continuously incubating for 30min, washing by adopting a microsphere sealing buffer solution (PBS buffer solution containing 5wt% of BSA and 0.05wt% of Tween-20), re-dissolving and sealing for 4h after centrifuging, re-dissolving by adopting a microsphere preservation solution (potassium carbonate buffer solution containing 0.01wt% of casein and 0.01wt% of sodium azide, wherein the concentration of the potassium carbonate buffer solution is 3mM and pH 10), and preserving at 4 ℃ for standby;
the preparation of the binding pad is that biotin-marked IFN-gamma, TNF-alpha and fluorescent microsphere marked avidin are dispersed in microsphere diluent (which is Tris-HCl buffer solution containing 0.2%v/v Tween 20, 0.3%wt bovine serum albumin, 0.3%v/v polyethylene glycol, 5%wt sucrose, 5%wt trehalose and 0.5%wt preservative) according to the volume ratio of 4:4:1, the fluorescent microsphere marked DNP-BSA is redispersed in microsphere diluent according to the volume ratio of 10%v/v, and the fluorescent microsphere marked DNP-BSA is coated on treated glass fiber according to the spray amount of 4 mul/cm by adopting a quantitative gold spraying instrument, and is dried for 2 hours in an air blast at 37 ℃ for standby.
Wherein the sample adding pad of the test strip is treated for 30min by adopting a sealing liquid, the sealing liquid is Tris buffer containing 3wt% of bovine serum albumin, 0.5wt% of casein, 5wt% of PVP, 0.05wt% of monopotassium phosphate and 0.1wt% of Tween 20, and the concentration of the Tris buffer is 50mM.
Example 5
The application method of the kit specifically comprises the following steps:
(1) Taking out the kit from 2-8deg.C, and recovering to room temperature;
(2) Taking 100 mu L of whole blood sample into a centrifuge tube equipped with 300 mu L of activator, fully mixing, standing for 2h, centrifuging at 4000rpm for 5min, and collecting supernatant;
(3) Taking 100 mu L of supernatant, adding the supernatant into a centrifuge tube provided with 200 mu L of sample diluent, and fully and uniformly mixing to obtain sample liquid to be tested;
(4) And adding 100 mu L of the sample liquid to be detected to the sample adding pad of the detection card, and standing for 15min.
(5) And inserting the detection card into the instrument, and reading the detection result.
Production of standard curve
(1) Configuring IFN-gamma quality control according to the concentration of 50pg/mL, 200pg/mL, 500pg/mL, 2000pg/mL and 4000pg/mL, configuring TNF-alpha quality control according to the concentration of 10pg/mL, 50pg/mL, 150pg/mL, 500pg/mL and 1000 pg/mL; three replicates were performed for each spot, 100uL was added dropwise to the wells of the test card of the kit. And detecting by using a fluorescence analyzer after 15min to obtain the corresponding fluorescence signal intensity and T/C ratio of T and C. Using the concentration of a standard substance in a sample as an x axis and the T/C ratio as a y axis, and performing four-parameter curve fitting by using software matched with a fluorescent quantitative analyzer to obtain a standard curve of a corresponding antibody, wherein experimental data are as follows;
the correlation coefficient of IFN-. Gamma.was 0.9999, and the minimum detection limit was 36pg/mL.
The correlation coefficient of TNF-. Alpha.was 0.9999 and the minimum detection limit was 4.2pg/mL.
Example 6
Repeatability test
(1) Taking 10 parts of the same batch of kit (example 3), respectively detecting signals of two concentration points of which the IFN-gamma quality control product concentration is 200pg/mL and 2000pg/mL by using a matched instrument, respectively calculating the total average value and standard deviation (S) of measured values, and calculating the variation Coefficient (CV); the specific results are shown in Table 3.
As can be seen from table 3, the Coefficient of Variation (CV) is less than 10%, satisfying the requirements;
(2) 10 parts of the same batch of kit (example 3) are taken, signals of two concentration points of which the TNF-alpha quality control product concentration is 50pg/mL and 500pg/mL are detected by a matched instrument, and the total average value and standard deviation (S) of the measured values are calculated respectively, wherein the specific results are shown in Table 4.
As can be seen from the data in Table 4, the Coefficient of Variation (CV) is less than 10%, satisfying the requirements.
Example 7
Specificity test
Whole blood samples of 200 cancer patients and 200 healthy subjects were collected, and the samples were tested by the kit of this example 3. The test results are shown in Table 5 below:
as can be seen from the data in table 5, the collected samples have a positive compliance of 99%, and a negative compliance of 99.5%. The kit has better specificity.
Example 8
Stability test
The same batch of the kit (example 2) was divided into 2 groups and each group was placed in an environment of normal temperature and 37 ℃ for accelerated stability investigation. According to the principle of stability experiments, namely an Arrhenius formula: d (Ink)/dT=Ea/RT 2 Ea, stored at normal temperature for 24 months, equivalent to a break of 180 days at 37 ℃. Stability studies were performed on days 0, 5, 10, 15, 30, 60, 100, 150, 180, respectively, and validated with the corresponding quality control, three replicates were performed for each sample, and the average was calculated. Specific experimental results are shown in tables 6 and 7;
as shown in the table, when the product is destroyed for 5 days, the measurement results of the samples slightly change relative to 0 day, but the overall CV value is controllable, the result is acceptable, and the result is basically stable in the later 5-180 days, so that the stability of the product reaches the standard.
In order to better prove that the kit has higher sensitivity and accuracy, 4 comparative examples are given by taking example 3 as a reference, and 200 healthy persons and 200 cancer patients are respectively detected by adopting the kit products in the following 4 comparative examples, and specific detection results are shown in Table 8.
Comparative example 1
Commercial NK cell activity detection kit (enzyme-linked immunosorbent assay);
comparative example 2
Commercial NK cell activity detection kit (fluorescence immunochromatography);
comparative example 3
Unlike the kit of example 3, the sample addition pad was not treated with a blocking solution, and the rest of the operations were the same;
comparative example 4
Unlike the kit of example 3, the microsphere preservation solution was PBS buffer containing 0.01wt% casein and 0.01wt% sodium azide; the rest of the operations are the same;
as can be seen from the data in Table 8, the kit of the present invention has higher sensitivity and specificity than those of comparative examples 1 to 4.
The present patent is not limited to the specific embodiments described above, and various modifications made by those skilled in the art from the above concepts are not subject to inventive effort and are within the scope of the present patent.
Claims (10)
1. A kit for detecting NK cell activity, characterized in that: the kit comprises a detection card, a sample diluent and an activator; the detection card is internally provided with a test paper strip, and a binding pad of the test paper strip is coated with biotin-marked IFN-gamma antibody, biotin-marked TNF-alpha antibody, fluorescent microsphere-marked avidin and fluorescent microsphere-marked DNP-BSA; the nitrocellulose membrane of the test strip is provided with a first detection line, a second detection line and a quality control line, wherein the first detection line is coated with IFN-gamma antibodies, the second detection line is coated with TNF-alpha antibodies, and the quality control line is coated with rabbit anti-DNP antibodies; the activator comprises human interleukin IL-2, IL-12 or a combination of both.
2. A kit for detecting NK cell activity according to claim 1, wherein: the dosage of the activator is 5-10ng/mL.
3. A kit for detecting NK cell activity according to claim 1, wherein: the sample diluent is Tris buffer solution containing 0.6wt% -1.8wt% PEG800, 0.1wt% -1.5wt% NaCl, 0.75wt% -2wt% PVA, 0.1wt% -0.5wt% casein and 0.1wt% -1wt% S9, the pH value of the Tris buffer solution is 8.0-8.5, and the concentration is 10-50mM.
4. A kit for detecting NK cell activity according to claim 1, wherein: the method for labeling IFN-gamma antibody or TNF-alpha antibody by using biotin comprises the following steps:
(1) Diluting IFN-gamma or TNF-alpha to 1mg/mL with sodium bicarbonate buffer solution with pH of 8.0 and 0.1mol/L or boric acid buffer solution with pH of 8.6 and 0.5mol/L, and continuing dialyzing the diluted IFN-gamma or TNF-alpha with the sodium bicarbonate buffer solution or boric acid buffer solution, wherein the dialyzed IFN-gamma antibody solution or TNF-alpha antibody solution is reserved;
(2) Taking 1ml of the IFN-gamma antibody solution or TNF-alpha antibody solution prepared in the step (1), adding 120 mu L of NHSB solution of 1mg/ml, stirring and preserving heat for 2-4h, adding 9.6 mu L of 1mol/LNH 4 And (3) continuously stirring the Cl solution, dialyzing, passing through a molecular sieve column, adding sodium azide with the final concentration of 0.5g/L, 1.0g/LBSA and glycerin into the collected liquid, and preserving at the temperature of minus 20 ℃.
5. A kit for detecting NK cell activity according to claim 1, wherein: the method for labeling avidin or DNP-BSA by using fluorescent microspheres comprises the following steps:
(1) Washing the microspheres by adopting MES buffer with pH of 6.0 and 50mM, and carrying out re-dissolution by adopting MES buffer continuously after centrifugation for standby;
(2) Activating microsphere, EDC and Sulfo-NHS according to the mass ratio of 1:0.1-1:0.1-3 for 20-60min, centrifuging, re-dissolving, washing and centrifuging by adopting the MES buffer solution in the step (1), re-dissolving by adopting the MES buffer solution with pH of 6.5 and 50mM, adding streptavidin or DNP-BSA according to the dosage of 0.1-1mg of streptavidin or DNP-BSA coupled to each 1mg of microsphere, performing ultrasound and incubation for 2-3h, quenching by adopting glycine with the final concentration of 100mM, continuing to incubate for 20-30min, washing by adopting the microsphere sealing buffer solution, re-dissolving after centrifugation, sealing for 2-4h, centrifuging, re-dissolving by adopting microsphere preservation solution, and preserving at 4 ℃ for standby.
6. The kit for detecting NK cell activity of claim 5 wherein: the microsphere blocking buffer is PBS buffer containing 1-5wt% BSA and 0.05wt% Tween-20.
7. The kit for detecting NK cell activity of claim 5 wherein: the microsphere preservation solution is a potassium carbonate buffer solution containing 0.01 weight percent of casein and 0.01 weight percent of sodium azide, and the concentration of the potassium carbonate buffer solution is 3mM, and the pH value of the potassium carbonate buffer solution is 8.0-10.0.
8. A kit for detecting NK cell activity according to claim 1, wherein: the preparation of the binding pad is to disperse biotin-marked IFN-gamma, TNF-alpha and fluorescent microsphere-marked avidin in a volume ratio of 1-4:1-4:1 into microsphere diluent, re-disperse fluorescent microsphere-marked DNP-BSA in the microsphere diluent according to the dosage of 2.5% -10% v/v, coat the treated glass fiber with a quantitative gold spraying instrument in a spraying amount of 1-4 mu l/cm, and dry the glass fiber by blowing at 37 ℃ for 2 hours and then store the glass fiber for later use.
9. The kit for detecting NK cell activity of claim 8, wherein: the microsphere dilution is Tris-HCl buffer solution containing 0.2% v/v Tween 20, 0.3% wt. bovine serum albumin, 0.3% v/v polyethylene glycol, 5% wt. sucrose, 5% wt. trehalose and 0.5% wt. preservative, and the pH of the Tris-HCl buffer solution is 8.0-8.2.
10. A kit for detecting NK cell activity according to claim 1, wherein: the sample adding pad of the test strip is treated for 30min by adopting a sealing liquid, wherein the sealing liquid is Tris buffer solution containing 0.5-3wt% of bovine serum albumin, 0.1-0.5wt% of casein, 0.1-5wt% of PVP, 0.02-0.05wt% of monopotassium phosphate and 0.01-0.1wt% of Tween 20, and the concentration of the Tris buffer solution is 10-50mM.
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| CN116875548A (en) * | 2023-09-08 | 2023-10-13 | 山东康华生物医疗科技股份有限公司 | NK cell activity culture tube and production process thereof |
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