CN115629059A - Application of cell, kit and method for improving chemiluminescence detection sensitivity - Google Patents
Application of cell, kit and method for improving chemiluminescence detection sensitivity Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
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- 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
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- 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
Abstract
The invention relates to the technical field of biochemical detection, and particularly provides application of cells, a kit and a method for improving chemiluminescence detection sensitivity.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to application of cells, a kit and a method for improving chemiluminescence detection sensitivity.
Background
The chemiluminescence detection is mainly based on the principle that the concentration of an object to be detected in a chemical detection system and the chemiluminescence intensity of the system are in a linear quantitative relation under a certain condition, and the chemiluminescence intensity of the system is detected by using an instrument, so that trace analysis of the content of the object to be detected is determined. In the membrane protein array technology, a membrane protein plasmid needs to be transfected into a tool cell in a transient transfection mode, so that the expression of membrane protein is completed, a cell array of the membrane protein is formed on the basis, then a protein to be detected is added into the cell array for immunoreaction, and finally whether a specific binding relationship exists between the membrane protein and the protein to be detected is confirmed by an immunofluorescence, flow cytometry or chemiluminescence method.
However, in the Membrane Protein Array (MPA) experiment, because the transient expression of the Membrane protein is completed in a 384-well plate or a 1536-well plate, the problems of small cell number and low transient expression protein amount exist, so that the detection sensitivity and stability of the immunofluorescence, flow cytometry or chemiluminescence immunoassay technology cannot meet the experimental requirements.
Disclosure of Invention
In view of the above, the present invention provides a cell application, a kit, a method for improving chemiluminescence detection sensitivity, and a membrane protein array using the method or the kit.
In order to achieve the purpose, the invention adopts the following technical scheme:
use of a cell for increasing the sensitivity of chemiluminescence detection.
In the present invention, the cell is a mammalian cell, the mammalian cells include CAKI-1 cells, CFPEO cells, DI-TNC1 cells, FADU cells, HCAEC cells, HCN-1A cells, HCS-2 cells, HUH-7 cells, LNCAP cells, MEF cells, MRC-5 cells, PANC1 cells, VERO cells, SK-N-MC cells, SKNAS cells, WERI-RB-1 cells, PC-12 cells, 3LL cells, 293T cells, hep G2 cells, HELA cells, CHO-K1 cells, COS-7 cells, NIH3T3 cells, A204 cells, A549 cells, D-407 cells, CHO cells at least one of HCS-2 cells, HT-29 cells, U87 cells, sf9 cells, HPAC cells, jurkat cells, KC cells, P19 cells, WEHI-3 cells, BV-2 cells, CV-1 cells, JEG-3 cells, M17 cells, UT-7 cells, SF-17 cells, yac-1 cells, HCT-8 cells, HFF cells, HFTF cells, HFSF cells, A375, HKC cells, HSF cells, WISF cells, HK-2 cells, DU145 cells, 3T3 cells, T84 cells, TF1 cells, THP1 cells, 3T3L1 cells, and chromaffin cells.
The stromal cells are common adherent cells or suspension cells, and mainly have the effect of increasing cell density, so that the loss of positive cells after transfection can be reduced in the cell rinsing and supernatant discarding processes, and more positive cells are reserved for immunoassay.
In some embodiments of the invention, 293T cells, CHO cells, HPAC cells, jurkat cells have been shown to improve chemiluminescence detection sensitivity with and without antibody incubation.
In the invention, after the cells are added into the cells to be detected, 200 mu L PBS is added for washing twice and then labeling the primary antibody, so as to reduce background interference. Furthermore, the addition amount of the stromal cells is 1E 5-1E 6 per hole.
The cells provided by the invention can be used for increasing the sensitivity of various chemical detection methods.
The invention also provides a kit for chemiluminescence detection, which comprises the cell.
In the present invention, the cell is a mammalian cell, the mammalian cells include CAKI-1 cells, CFPEO, DI-TNC1 cells, FADU cells, HCAEC cells, HCN-1A cells, HCS-2 cells, HUH-7 cells, LNCAP cells, MEF cells, MRC-5 cells, PANC1 cells, VERO cells, SK-N-MC cells, SKNAS cells, WERI-RB-1 cells, PC-12 cells, 3LL cells, 293T cells, hep G2 cells, HELA cells, CHO-K1 cells, COS-7 cells, NIH3T3 cells, A204 cells, A549 cells, D-407 cells, CHO cells at least one of HCS-2 cells, HT-29 cells, U87 cells, sf9 cells, HPAC cells, jurkat cells, KC cells, P19 cells, WEHI-3 cells, BV-2 cells, CV-1 cells, JEG-3 cells, M17 cells, UT-7 cells, SF-17 cells, yac-1 cells, HCT-8 cells, HFF cells, HFTF cells, HFSF cells, A375, HKC cells, HSF cells, WISF cells, HK-2 cells, DU145 cells, 3T3 cells, T84 cells, TF1 cells, THP1 cells, 3T3L1 cells, and chromaffin cells.
In the kit, the number of the cells is 1E5 to 1E6, and more preferably 1E6.
In the present invention, the cells are washed, preferably centrifuged, before use, and the supernatant is discarded.
In the present invention, the kit further comprises a chemiluminescent substrate; the chemiluminescent substrate is preferably an enzymatic luminescent substrate or a direct luminescent substrate; the enzymatic luminogenic substrate comprises peroxide and luminol; the peroxide is preferably hydrogen peroxide.
Luminol (Luminol), also known as Luminol. The chemical name is 3-amino-benzenedicarboxhydrazide. Is a pale yellow powder at normal temperature, and is a relatively stable artificially synthesized organic compound. Has a chemical formula of C 8 H 7 N 3 O 2 . Luminol is useful in immunoassays as both a label and a substrate for peroxidase. Luminol can be oxidized by some oxidants under alkaline condition to generate chemiluminescence reaction and radiationGiving chemiluminescence with a maximum emission wavelength of 428 nm.
The kit of the invention also comprises horseradish peroxidase.
Horse Radish Peroxidase (HRP) is a glycoprotein, each molecule contains a hemin (protohemin) region as a prosthetic group, has the advantages of low molecular weight, simple labeling method, stable property and the like, and is a commonly used enzyme in clinical test reagents. Horse radish peroxidase can catalyze luminol to be subjected to oxidative degradation, and a light signal is emitted.
The kit of the invention also comprises an auxiliary agent, preferably sodium hydroxide or potassium hydroxide.
The invention has no special requirement on the chemiluminescence substrate, and can adopt the chemiluminescence substrate which is common in the field. In some embodiments of the invention, the chemiluminescent substrate in the kit is present in solution; the solvent is hydrochloride buffer solution or phosphate buffer solution, and the solvent is DMSO and/or DMF.
The invention also provides a method for improving the chemiluminescence detection sensitivity, which comprises the following steps: the cells are mixed with a chemiluminescent substrate and the chemiluminescent value is detected.
Furthermore, the luminescence kit of the present invention can be used for general chemiluminescence detection, and can also be used for Membrane Protein Array (MPA) luminescence detection, which is not limited in the present invention.
In the present invention, the cell is a mammalian cell including at least one of CAKI-1 cell, CFPEO, DI-TNC1 cell, FADU cell, HCAEC cell, HCN-1A cell, HCS-2 cell, HUH-7 cell, LNCAP cell, MEF cell, MRC-5 cell, PANC1 cell, VERO cell, SK-N-MC cell, SKNAS cell, WERI-RB-1 cell, PC-12 cell, 3LL cell, 293T cell, hep G2 cell, HELA cell, CHO-K1 cell, COS-7 cell, NIH3T3 cell, A204 cell, A549 cell, D-407 cell, CHO cell, HCS-2 cell, HT-29 cell, U87 cell, sf9 cell, HPAC cell, jurkat cell, chromaffin cell.
In the present invention, the number of the cells is 1E5 to 1E6.
In the present invention, the cells are washed, preferably centrifuged, before use, and the supernatant is discarded.
The sample to be tested is preferably labeled with horseradish peroxidase.
The method for improving the chemiluminescence detection sensitivity of the invention preferably uses the kit or the commercial ECL detection kit for detection, and can carry out detection under the conditions of incubation of antibodies, no incubation of antibodies or membrane protein array test.
In one embodiment of the invention, the chemiluminescent detection comprises: centrifuging the cells, discarding the supernatant, then resuspending the cells with PBS, incubating the antibody, incubating a sample to be detected, then washing with PBS, centrifuging, discarding the supernatant, and detecting with an ECL detection kit, wherein the chemiluminescence value is detected by an enzyme-linked immunosorbent assay (ELISA); the number of said cells is in the range of 1E5 to 1E 6; the antibody is a BCMA antibody or a CD70 antibody.
In one embodiment of the invention, the chemiluminescent detection comprises: centrifuging the cells, discarding the supernatant, and detecting by using an ECL detection kit, wherein the chemiluminescence value is detected by using an enzyme-labeling instrument; the number of cells is in the range of 1E 5-1E 6.
In one embodiment of the invention, the chemiluminescent detection comprises:
step 1, transfecting plasmids (antigens) into cells to be detected to obtain detection target cells, mixing the detection target cells with the cells, and washing the cells twice with PBS to obtain mixed cells. Plasmids include, but are not limited to, CD70, BCMA, CD19, CD22, CD7, PSCA, ABCA1, HCST, NDUFB1, ARC, BST1, GNB2, STX5.
And 2, adding primary antibody into the mixed cells, and incubating at 4 ℃ for 30min to wash out the primary antibody.
Step 3, labeling a chemiluminescent substrate, incubating at 4 ℃ for 30min, washing with PBS, centrifuging, discarding the supernatant, and detecting with an ECL detection kit, wherein the chemiluminescent value is detected by an enzyme-linked immunosorbent assay (ELISA) instrument; the number of said cells is in the range of 1E5 to 1E 6; the antigen is BCMA or CD70; the chemiluminescent substrate is preferably horseradish peroxidase-labeled streptavidin (SA-HRP).
The invention also provides the application of the kit and/or the method in the membrane protein array technology. The membrane protein array can be used for screening target substances; the target substance includes, but is not limited to, drugs, antibodies, proteins, polypeptides, or other small molecule substances, and the invention is not limited thereto.
In the present invention, the target substance may be free or produced by a host cell; the host cell may be a host cell transfected or transformed with a gene fragment or plasmid vector, or may be a naturally occurring host cell; the method for transformation comprises the following steps: chemical conversion and electrical conversion; the transfection method comprises calcium phosphate coprecipitation, an artificial liposome method and virus transfection. The virus transfection comprises adenovirus transfection, adeno-associated virus transfection, lentivirus transfection and the like.
The chemiluminescence detection sensitivity in the invention refers to a chemiluminescence value/negative control chemiluminescence value of a sample to be detected, namely a signal background ratio (S/B). The cells are added in the chemiluminescence detection, the chemiluminescence value measured in the embodiment is obviously reduced, but in the actual detection process, the chemiluminescence value/negative control chemiluminescence value of a sample to be detected, namely a signal background ratio (S/B), is usually calculated, and the negative control chemiluminescence value is lower along with the increase of the number of the cells, namely the S/B is higher, which means that the sensitivity of the chemiluminescence detection method is improved; the improved chemiluminescence detection method is applied to the membrane protein array, so that the detection accuracy of the membrane protein array can be improved.
Drawings
FIG. 1 is a chemiluminescence detection value of example 1 of the present invention;
FIG. 2 is a chemiluminescence detection value of example 2 of the present invention;
FIG. 3 is a chemiluminescence detection value of example 3 of the present invention;
FIG. 4 shows the chemiluminescence detection values of example 4 of the present invention;
FIG. 5 shows the chemiluminescence detection values of example 5 of the present invention;
FIG. 6 is a chemiluminescence detection value of example 6 of the present invention;
FIG. 7 shows the chemiluminescence detection values of example 7 of the present invention;
FIG. 8 is a value of chemiluminescence detection in example 8 of the present invention;
FIG. 9 shows the values of chemiluminescence detection in example 9 of the present invention;
FIG. 10 shows the values of chemiluminescence detection in example 10 of the present invention;
FIG. 11 shows the values of chemiluminescence detection in example 11 of the present invention;
FIG. 12 shows the values of chemiluminescence detection in example 12 of the present invention;
FIG. 13 shows the values of chemiluminescence detection in example 13 of the present invention;
FIG. 14 shows the values of chemiluminescence detection in example 14 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
To further illustrate the present invention, the following examples are provided for illustration. The starting materials used in the following examples of the present invention are all commercially available products.
1. Effect of different matrix cells on chemiluminescence detection with incubation of antibodies
Example 1
293T stromal cells are used, the number is within the range of 1E 5-1E 6, the cells are added into a centrifuge tube for 3 minutes at 1000rpm, the cells are resuspended by 100ul PBS after discarding the supernatant, the BCMA antibody is added, incubation is carried out for 30 minutes at 4 ℃, washing is carried out by 1ml PBS, centrifugation is carried out for 3 minutes at 1000rpm, the supernatant is discarded after repeating twice, ECL detection is carried out (ECL detection kit, manufacturer: changzhou Tiandi Ming He, product number: S32500), solution I25 ul and solution II 25ul are successively added, and chemiluminescence values are detected by an enzyme reader, as shown in figure 1 and table 1.
TABLE 1
Example 2
The 293T stromal cells of example 1 were replaced with CHO cells, and other conditions were performed by referring to the procedure of example 1, and the results of the measurement are shown in FIG. 2 and Table 2.
TABLE 2
Example 3
The 293T stromal cells in example 1 were replaced with Jurkat cells, and other conditions were performed by referring to the procedure in example 1, and the results of the measurement are shown in FIG. 3 and Table 3.
TABLE 3
Example 4
The 293T stromal cells in example 1 were replaced with HPAC cells, and other conditions were performed by referring to the procedure in example 1, and the results of the assay are shown in FIG. 4 and Table 4.
TABLE 4
Example 5
The BCMA antibody in example 1 was replaced with the CD70 antibody, and other conditions were performed with reference to the procedure in example 1, and the results of the detection are shown in fig. 5 and table 5.
TABLE 5
Example 6
The BCMA antibody in example 1 was replaced with the CD70 antibody, and other conditions were performed with reference to the procedure in example 1, and the results of the detection are shown in fig. 6 and table 6.
TABLE 6
Example 7
The BCMA antibody in example 2 was replaced with the CD70 antibody, and other conditions were performed with reference to the procedure in example 1, and the results of the detection are shown in fig. 7 and table 7.
TABLE 7
Example 8
The BCMA antibody in example 3 was replaced with the CD70 antibody, and other conditions were performed with reference to the procedure in example 1, and the results of the detection are shown in fig. 8 and table 8.
TABLE 8
In the embodiments 1 to 8 of the present invention, it is selected that, in the case of incubating two different antibodies (CD 70 and BCMA), it is determined that the number of different cells added to different stromal cells has no significant difference to the chemiluminescence detection result, although the chemiluminescence value in part of data is significantly reduced as the number of cells increases, in the actual detection process, the chemiluminescence value/negative control chemiluminescence value of the sample to be detected, i.e. Signal to background ratio (S/B), is usually calculated, and the negative control chemiluminescence value is lower as the number of cells increases, i.e. S/B is higher, thereby improving the sensitivity of the detection method.
2. Investigation of the Effect of different stromal cells on the chemiluminescent detection without incubating the antibody
Example 9
Jurkat cells with the number of 1E 5-1E 6 are added into a centrifuge tube to be centrifuged at 1000rpm for 3 minutes, the supernatant is discarded, ECL detection (ECL detection kit, manufacturer: changzhou Tiandi ren He, cat # S32500) is carried out, solution I25 ul and solution II 25ul are added in sequence, chemiluminescence values are detected by an enzyme-labeling instrument, and the detection results are shown in FIG. 9 and Table 9.
TABLE 9
Example 10
The Jurkat cells of example 9 were replaced with CHO cells, and other conditions were performed by referring to the procedure of example 9, and the results of the measurement are shown in FIG. 10 and Table 10.
Watch 10
Example 11
The Jurkat cells of example 9 were replaced with 293T cells, and other conditions were performed by referring to the procedure of example 9, and the results of the measurements are shown in FIG. 11 and Table 11.
TABLE 11
Example 12
The Jurkat cells in example 9 were replaced with HPAC cells, and other conditions were performed by referring to the procedure in example 9, and the results of the measurements are shown in FIG. 12 and Table 12.
TABLE 12
As can be seen from examples 9 to 12, except that the chemiluminescence group with the 1E6 cell as the stromal cell is significantly decreased from other groups, the rest of the groups have no significant effect on chemiluminescence detection, and the chemiluminescence value of the sample to be detected divided by the chemiluminescence value of the negative control in chemiluminescence detection is the Signal to background ratio (S/B), so that the chemiluminescence value is significantly decreased after the addition of the stromal cell 1E6, and the Signal background ratio is significantly increased, which can improve the detection sensitivity of the chemiluminescence detection method, and thus examples 9 to 12 of the present invention demonstrate: the addition of the 1E6 matrix cells can improve the detection sensitivity of the method more obviously, and other types and orders of magnitude of matrix cells have no obvious difference on chemiluminescence detection.
3. Transient transformation of CD70 or BCMA, addition of matrix cells to detect the effect of chemiluminescence (improved chemiluminescence was applied to membrane protein arrays)
Example 13
a) Fluorescence detection transfection
b) The BCMA antigen is transfected into 293T cells through lipofection, wherein the specific steps of transfection are as follows:
(1) irradiating with ultraviolet on a clean bench for 30min, adding NEAA and 1 Xpancreatin in a proportion of Opti-MEM, DMEM +10% FBS +1%, and preheating in a water bath at 37 deg.C;
(2) after sterilization, putting required reagents, plasmid BCMA, cell culture plates and the like into a superclean workbench;
(3) adding plasmid and transfection reagent into 384-hole cell culture plate, and timing;
(4) centrifuging the shot, and incubating for 30min at room temperature;
(5) add 20. Mu.L cells/well (1.8E + 04) (incubation gap treated cells);
(6) centrifuging the shot;
(7) observing the cells under a microscope, and marking abnormal holes;
(8) culturing in carbon dioxide incubator for 48 hr;
(9) after 48h, the cultured cells were removed and observed under a microscope, wells with contamination and a smaller number of cells were marked,
opening an enzyme label reader at the red mark, setting a detection step, and detecting ZsGreen or mcherry values.
c) Chemiluminescence detection
(1) Adding pancreatin into a 384-well plate, and blowing and beating four corners of each well (generating no bubbles as much as possible);
(1) transferring the cells to a 384-deep-well plate, adding 1E 5-1E 6 293T stromal cells/well, and setting a control without stromal cells;
(2) centrifuging, inverting and centrifuging to discard the supernatant;
(3) adding 200 mu L PBS/hole, and suspending;
(4) repeating the step (2);
(2) Labeling a primary antibody
(1) Diluting the antibody with PBS to the required concentration, adding the antibody/hole BCMA, and resuspending;
note that: the lyophilized antibody is diluted with purified water before use, and activated at room temperature for 30min for use
②4℃，30min；
(3) Add 100uL PBS/well;
(4) centrifuging, inverting, centrifuging and discarding the supernatant;
(5) adding PBS/hole, and suspending;
(6) repeating the step (4);
(3) Marker SA-HRP
(1) Diluting SA-HRP to required concentration with PBS, adding antibody/hole, and resuspending;
②4℃，30min；
(3) PBS/well is added;
(4) centrifuging, inverting, centrifuging and discarding the supernatant;
(5) adding PBS/hole, and suspending;
(6) repeating the step (4), observing cells in each hole, and recording the hole with less cell quantity;
( 4) Chemiluminescence detection was performed (ECL detection kit, manufacturer: changzhou heaven and earth, cargo number: s32500 )
(1) Adding 25 mu.L solution I/hole, resuspending cells, and transferring to a detection white plate (operation in dark place);
(2) opening the microplate reader, and setting a detection step;
(3) adding 25 mu L of solution II/hole, incubating at room temperature, loading on a machine, and storing the result after detection.
Watch 13
Example 14
BCMA in example 13 was replaced with CD70, and the remaining conditions were performed with reference to the procedure in example 13, and the test results are shown in fig. 14 and table 14.
TABLE 14
From examples 13 and 14, it can be seen that, the addition of different types and amounts of stromal cells has no significant difference to the negative control sample, or the chemiluminescence value decreases with increasing cell mass, the present invention intends to prove that the use of the method of adding stromal cells in the immunoassay (specifically, membrane protein array) can improve the detection sensitivity, and the results show that the addition of stromal cells to the positive sample can improve the chemiluminescence value of the detection, improve the detection sensitivity, and that the chemiluminescence value of the positive control without stromal cells in the set group is significantly different from that of the control group with stromal cells, and the S/B is also significantly different. Therefore, the sensitivity and stability of chemiluminescence immunoassay can be improved by adding the matrix cells.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (11)
1. Use of a cell for increasing the sensitivity of chemiluminescence detection.
2. The use of claim 1, wherein the cell is a mammalian cell.
3. The use according to claim 1, wherein the cells are stromal cells.
4. Use according to claim 3, wherein the stromal cells are selected from one or more of 293T cells, CHO cells, HPAC cells, jurkat cells.
5. A kit for use in chemiluminescent detection comprising a cell, a chemiluminescent substrate.
6. The kit according to claim 5, wherein the number of the cells is 1E5 to 1E6.
7. The kit according to claim 5 or 6, wherein the cells are selected from one or more of 293T cells, CHO cells, HPAC cells, jurkat cells.
8. A method of increasing the sensitivity of chemiluminescence detection, comprising: the cells are mixed with a chemiluminescent substrate and the chemiluminescent value is detected.
9. The method according to claim 8, wherein the number of the cells is 1E5 to 1E6.
10. The method according to claim 8, wherein the cells are selected from one or more of 293T cells, CHO cells, HPAC cells, jurkat cells.
11. Use of a kit according to any one of claims 5 to 7, and/or a method according to any one of claims 8 to 10 in membrane protein array technology.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6406840B1 (en) * | 1999-12-17 | 2002-06-18 | Biomosaic Systems, Inc. | Cell arrays and the uses thereof |
| US20030189850A1 (en) * | 2002-04-09 | 2003-10-09 | Toyo Boseki Kabushiki Kaisha | Cell array system |
| EP1651225A1 (en) * | 2003-07-15 | 2006-05-03 | Chanxpress, Inc. | High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins |
| CN1878553A (en) * | 2003-07-15 | 2006-12-13 | 钱克斯普瑞斯公司 | High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins |
| CN102183512A (en) * | 2011-02-15 | 2011-09-14 | 杭州师范大学 | Living cell detection method based on chemiluminescence principle |
| CN103033625A (en) * | 2012-12-19 | 2013-04-10 | 中国科学院生物物理研究所 | Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof |
-
2022
- 2022-10-17 CN CN202211284612.9A patent/CN115629059A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6406840B1 (en) * | 1999-12-17 | 2002-06-18 | Biomosaic Systems, Inc. | Cell arrays and the uses thereof |
| US20030189850A1 (en) * | 2002-04-09 | 2003-10-09 | Toyo Boseki Kabushiki Kaisha | Cell array system |
| EP1651225A1 (en) * | 2003-07-15 | 2006-05-03 | Chanxpress, Inc. | High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins |
| CN1878553A (en) * | 2003-07-15 | 2006-12-13 | 钱克斯普瑞斯公司 | High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins |
| CN102183512A (en) * | 2011-02-15 | 2011-09-14 | 杭州师范大学 | Living cell detection method based on chemiluminescence principle |
| CN103033625A (en) * | 2012-12-19 | 2013-04-10 | 中国科学院生物物理研究所 | Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| LIHUA DING等: "A Novel Cell-Assisted Enhanced Chemiluminescence Strategy for Rapid and Label-Free Detection of Tumor Cells in Whole Blood", ACS SENS, vol. 5 * |
| ROBERTO BELLUCCI等: "Graft-versus-tumor response in patients with multiple myeloma is associated with antibody response to BCMA, a plasma-cell membrane receptor", BLOOD, vol. 105, pages 2 * |
| 张修彦: "化学发光免疫分析技术", 重庆医药, vol. 16, no. 6, pages 40 * |
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