CN113481166A - Cryopreserved cell model construction and detection method for immediately detecting MOG-IgG level in serum - Google Patents
Cryopreserved cell model construction and detection method for immediately detecting MOG-IgG level in serum Download PDFInfo
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Abstract
The invention provides a method for constructing a cryopreserved cell model for immediately detecting MOG-IgG level in human serum, which comprises the following steps: step 1, constructing a recombinant GFP-MOG plasmid; step 2, transfecting the GFP-MOG plasmid into an HEK293 cell to establish the GFP-MOG-HEK293 cell; and 3, subpackaging and freezing the GFP-MOG-HEK293 cells to obtain a frozen cell model capable of detecting the serum MOG-IgG level immediately. The invention also provides a detection method for instantly detecting the MOG-IgG level in human serum by using the cryopreserved cell model. According to the technical scheme, the sensitivity and specificity of the MOG-IgG detection on human serum are improved to more than 90%, the frozen cell model can be recovered according to requirements to realize the instant detection on the serum sample, and the detection time is obviously shortened; the longest cryopreservation time of the GFP-MOG-HEK293 cell model prepared by the invention reaches 5 months, and the MOG-IgG level in serum can be still accurately and quantitatively detected; the detection material GFP-MOG-HEK293 cell model prepared by the invention can be used for flow cytometry detection, and qualitatively, accurately and quantitatively analyzing the MOG-IgG level in serum.
Description
Technical Field
The invention relates to the field of serum biomarker detection, and relates to a cryopreserved cell model construction containing GFP-MOG plasmid and a method for detecting MOG-IgG in human serum immediately.
Background
Myelin Oligodendrocyte Glycoprotein (MOG) is a membrane protein of the myelin membrane and the outermost layer of the oligodendrocyte surface. The body's anti-MOG antibodies (MOG-IgG) produced by immune dysregulation, which target MOG, are key factors in inflammatory demyelinating diseases of the central nervous system. At present, the specific pathogenic mechanism of MOG-IgG is not completely elucidated, related researches are carried out on the basis of aspects of serology, immunology, neuropathology and the like, and the MOG-IgG related diseases are preliminarily proved to be possibly used as a new disease entity. Therefore, a' guidance for the proposed diagnosis of MOG-IgG associated diseases (MOG-IgG associated disorders, MOGAD) was published internationally in 2018, and an expert consensus on the diagnosis and treatment of diseases related to anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibodies in China was published in 2020.
Early methods for detecting human serum MOG-IgG are mostly enzyme-linked immunosorbent assay (ELISA) and immunoblotting (Western blot), but sensitivity and specificity are poor; both the MOGAD international guidelines and the Chinese expert consensus recommend a live cell based detection method (CBA) for human serum MOG-IgG detection. However, before various CBA methods applied in the current clinical detection for measuring serum MOG-IgG, a plasmid transfected cell model is mostly required to be reconstructed in advance, the MOG-IgG level in the serum cannot be quantitatively detected in real time and accurately, and the timeliness of the detection result on clinical guidance is reduced.
Therefore, a method for detecting the MOG-IgG level in serum in a real-time, rapid, efficient and accurate quantitative manner is urgently needed, and has very important research significance and clinical application value.
Disclosure of Invention
Aiming at the prior technical problems, the invention provides a cryopreserved cell model construction and detection method for immediately detecting MOG-IgG level in human serum, which aims to solve the problems of long detection time consumption, poor accurate determination quantity and the like in the current detection.
The invention provides a method for constructing a cryopreserved cell model for immediately detecting MOG-IgG level in human serum, which comprises the following steps:
step 1, constructing a recombinant GFP-MOG plasmid;
step 2, transfecting the GFP-MOG plasmid into an HEK293 cell to establish the GFP-MOG-HEK293 cell;
and 3, subpackaging and freezing the GFP-MOG-HEK293 cells to obtain a frozen cell model capable of detecting the serum MOG-IgG level immediately.
Wherein, the step 1 comprises the following steps: adding EcoRI/NheI enzyme cutting sites at two ends of an MOG target gene; inserting EcoRI/NheI enzyme cutting sites into the pIRES2-AcGFP1 vector; thirdly, inserting MOG target genes with EcoRI/NheI enzyme cutting sites added at two ends of the third step into the pIRES2-AcGFP1 vector with the EcoRI/NheI enzyme cutting sites inserted in the third step through a DNA Ligation Kit to obtain GFP-MOG plasmids. And fourthly, identifying the recombinant plasmid by agarose gel electrophoresis by using restriction enzyme Hind III.
Wherein the transfection in the step 2 is selected from the group consisting of transfecting a GFP-MOG plasmid into HEK293 cells by using a Neon electrotransfection system, and establishing the GFP-MOG-HEK293 cells.
The Neon electrotransfection system conditions are pulse Voltage (Voltage)1100V, pulse time (Width)20ms and pulse number (pulse number)2 pulses.
Wherein, the step 3 comprises the following steps: suspending GFP-MOG-HEK293 cells in a cell freezing medium, placing the cell freezing medium in a container, and freezing; and secondly, the container in the step one is transferred to liquid nitrogen for long-term storage, and a frozen cell model capable of detecting the MOG-IgG level of the serum in real time is obtained.
Wherein, in the above-mentioned (I), GFP-MOG-HEK293 cells are expressed by (1-5) × 106cellsThe density of/mL was resuspended in cell culture.
Wherein, the container in the above-mentioned (r) is a freezing storage box capable of controlling cooling rate.
Wherein the cooling rate in the freezing storage box is-1 ℃/minute
Wherein, the freezing condition in the first step is refrigerator for 12 hours at minus 80 ℃.
The invention also provides a detection method for instantly detecting MOG-IgG level in human serum by using the cryopreserved cell model, which comprises the following steps:
the frozen cell model is recovered and mixed with a sample to be tested for the first incubation, and after washing, an APC-labeled anti-human IgG fluorescent antibody (Miltenyi Biotec, Order No. 130-.
Wherein, the recovery is to melt the frozen GFP-MOG-HEK293 cells, quickly add the cells into a test tube, add 10mL of buffer solution, centrifuge and wash.
Wherein, the recovery is to melt the frozen GFP-MOG-HEK293 cells by a water bath constant temperature constant speed oscillator (37 ℃, 5 r/s), quickly add the cells into a test tube with the volume of 15mL, add 10mL of 0.01M phosphate buffer solution (0.01M PBS) buffer solution preheated at 37 ℃, centrifuge (1200rpm/8min), namely complete one-time washing, and complete cell recovery after three times of washing.
Wherein, the first incubation is to mix the serum sample to be detected according to the ratio of 1: 5, diluted with PBS-2 buffer (0.01M PBS, 15mM EDTA, 2% BSA, 0.05% NaN3, 10% Normal coat serum), tubes containing 50. mu.L of the diluted serum sample and GFP-MOG-HEK293 cells were incubated in a rotary incubator at 4 ℃ and 300rpm for 30 min.
Wherein the second incubation is performed with fluorescent antibody at a ratio of 1: 20, diluted with PBS-2 buffer, the tube containing 100. mu.L of diluted fluorescent antibody and GFP-MOG-HEK293 cells was incubated in a rotary incubator at 4 ℃ and 300rpm for 30 min.
The invention has the following beneficial effects:
1. the preparation method can obtain the split-packaged and cryopreserved GFP-MOG-HEK293 cell model for detecting human serum MOG-IgG. Compared with the conventional ELISA method and Western blot method, the detection material disclosed by the invention has the advantages that the sensitivity and specificity for detecting human serum MOG-IgG are improved to more than 90%, the frozen cell model can be recovered according to requirements to realize instant detection of a serum sample, and the detection time is obviously shortened.
2. The detection material GFP-MOG-HEK293 cell model prepared by the invention has the longest freezing time of 5 months, and can still accurately and quantitatively detect the MOG-IgG level in serum.
3. The detection material GFP-MOG-HEK293 cell model prepared by the invention can be used for flow cytometry detection, and qualitatively, accurately and quantitatively analyzing the MOG-IgG level in serum.
Drawings
FIG. 1 shows the result of electrophoretic identification of the recombinant plasmid of the present invention.
FIGS. 2A and 2B shows the results of MOG-IgG assay in human serum using freshly prepared GFP-MOG-HEK293 cells (non-cryopreserved GFP-MOG-HEK293 cells) on the same day, 2A being a negative result and 2B being a positive result;
FIGS. 2C and 2D show the results of MOG-IgG in human serum measured using a GFP-MOG-HEK293 cell model recovered after cryopreservation for 136 days, with 2C being a negative result and 2D being a positive result.
FIG. 3 results of the consistency analysis of the test results of examples 2 and 3.
Detailed Description
The following are specific embodiments and should not be construed as limiting the scope of the invention.
Example 1 frozen cell model construction
The construction of the frozen cell model comprises the following steps:
step 1: recombinant GFP-MOG plasmid
MOG target genes were purchased from Genscript; pIRES2-AcGFP1 vector was purchased from Clontech; inserting the MOG target gene with EcoRI/NheI enzyme cutting sites into pIRES2-AcGFP1 vector to obtain recombinant full-length GFP-MOG plasmid. After identifying the recombinant plasmid by agarose gel electrophoresis, the plasmid can be reused.
Step 2: establishment of GFP-MOG-HEK293 cells
Culturing HEK293 cells: DMEM low-glucose medium was incubated with 56 ℃ inactivated total Bovine Serum (FBS) according to 9: 1, adding 0.2mL kanamycin sulfate (kanamycin) to prepare a DMEM (10% FBS + kanamycin) culture medium; each time the cells were passaged through PET (0.01M PBS, 0.02% EDTA, 0.1% Trypsin), the cells were seeded in 10mL of 100mm cell culture dishes containing DMEM (10% FBS + kanamycin) medium at a cell number of about 1.5X 106cells/mL; the conditions of the incubator are as follows: 37 ℃ and 5% CO2And the humidity is 45-65%.
Calculating the number of HEK293 cells: observing by microscope, when HEK293 cell area in 100mm cell culture dish reaches 70% -80%, removing DMEM (10% FBS + kanamycin) culture medium, adding PET 6mL, slightly washing for 2 times, placing at 37 deg.C and 5% CO2And an incubator with the humidity of 45% -65% for 10 min; adding DMEM (10% FBS + kanamycin) culture medium 6mL, recovering cells, placing in a 15mL test tube, centrifuging for 3min at 25 deg.C and 3000 rpm; removing supernatant, adding 6mL of Opti-MEM (1X) Reduced serum medium; counting with cell counting instrument to keep the cell number at 1.5 × 106cells/mL。
③ 1X 10 cells based on the number of HEK293 cells6cells need a proportioning mode of GFP-MOG plasmid of 5 mu g, the GFP-MOG plasmid is added into HEK293 cells after being processed by a Neon electrotransfection system, and the cells are placed at 37 ℃ and 5% CO2Culturing the cells for 48H in an incubator with the humidity of 45-65 percent to obtain the GFP-MOG-HEK293 cells. Neon electrotransfection system conditions: pulse Voltage (Voltage)1100V, pulse time (Width)20ms, pulse number (pulse number)2 pulses.
And step 3: split charging and freezing GFP-MOG-HEK293 cells:
after 48H, GFP-MOG-HEK293 cells were treated at (1-5). times.106cells/mL were resuspended in cell freezing medium (FBS: Dimethylsulfoxide (DMSO) ═ 9: 1) and further dispensed into cell storage tubes. And (3) moving the cell storage tube to a freezing box filled with isopropanol in advance, placing the freezing box in a refrigerator at the temperature of-80 ℃ for 12 hours (the cooling rate in the freezing box is-1 ℃/minute), taking the cell storage tube out of the freezing box, and placing the cell storage tube in liquid nitrogen for long-term storage.
Example 2 detection of MOG-IgG in human serum 1
The detection of MOG-IgG in human serum comprises the following steps:
the storage tubes (which had been stored in liquid nitrogen for 136 days) of the split-frozen GFP-MOG-HEK293 cell model obtained in example 1 were removed from the liquid nitrogen and placed immediately in a pre-heated shaking water bath at 37 ℃ for 30s at a shaking speed of 5 rpm. After the cell suspension had thawed, it was quickly placed in a clean and dry 15mL tube and washed by adding 10mL of 0.01M PBS buffer pre-warmed at 37 deg.C, centrifuged at 1200rpm for 8min, and the supernatant removed. And (4) repeating the steps to wash the cells for 3 times to complete cell recovery.
1mL of PBS-1 buffer (0.01M PBS, 2mM EDTA, 0.5% BSA, 0.02% NaN3) was added to each GFP-MOG-HEK293 cell tube and incubated under the following conditions: 4 ℃, 300rpm, 10 min.
After centrifugation (4 ℃, 3000rpm, 3min), the supernatant was removed, and after 1mL of 0.01M PBS buffer was added, the mixture was centrifuged again (4 ℃, 3000rpm, 3min), and then the supernatant was removed, i.e., one washing was completed, and the total washing was performed once.
Serum samples were prepared as 1: 5, diluting with PBS-2 buffer (0.01M PBS, 15mM EDTA, 2% BSA, 0.05% NaN3, 10% Normal coat serum), adding 50. mu.L of the diluted serum sample to a GFP-MOG-HEK293 cell tube, incubating in a rotary incubator under the following conditions: 4 ℃, 300rpm, 30 min.
After 0.01M PBS buffer was directly added, centrifugation was performed (4 ℃, 3000rpm, 3min), and then the supernatant was removed, i.e., one washing was completed, and three washes were performed in total.
The fluorescent antibody was applied as 1: 20, diluting with PBS-2 buffer solution, adding 100 μ L of diluted fluorescent antibody into a GFP-MOG-HEK293 cell test tube, and incubating in a rotary incubator at 4 deg.C and 300rpm for 30min
After adding 0.01M PBS buffer directly again, centrifugation (4 ℃, 3000rpm, 3min) was performed, and then the supernatant was removed, i.e., one washing was completed, and three washes were performed in total.
After adding 300. mu.L of 4% paraformaldehyde to a GFP-MOG-HEK293 cell tube, detection was performed by a CantoII flow cytometer, and the results were analyzed.
EXAMPLE 3 detection of MOG-IgG in human serum 2
GFP-MOG-HEK293 cells stored in liquid nitrogen for 136 days in step 1 of example 2 were replaced with non-cryopreserved GFP-MOG-HEK293 cells, and the remaining steps and procedures were identical to those of example 2.
Example 4 the test results were interpreted as follows:
the positive result is interpreted, the cryopreserved cell model for detecting the MOG-IgG level of the human serum is prepared, is used for immediately detecting the MOG-IgG level in the serum of MOGAD patients and healthy people, and accurately quantifies the MOG-IgG level of the serum by calculating the MOG-IgG positive cell percentage of the Q2 quadrant. FIGS. 2A and 2B are illustrations of results of detecting the MOG-IgG level in human serum using the GFP-MOG-HEK293 cell model (non-cryopreserved GFP-MOG-HEK293 cells, corresponding to example 3) prepared on the same day, 2A being a negative result and 2B being a positive result; FIGS. 2C and 2D are graphs showing the results of MOG-IgG level in human serum using the GFP-MOG-HEK293 cell model recovered after freezing for 136 days (corresponding to example 2), with 2C being a negative result and 2D being a positive result.
The final test results of examples 2 and 3, both of which had a higher agreement, are shown in fig. 3. Wherein, the abscissa: detection results (positive cells%) based on recovered GFP-MOG-HEK293 cells after 136 days of cryopreservation; ordinate: detection results based on non-cryopreserved GFP-MOG-HEK293 cells (% positive cells); r20.9611, with a higher degree of fit.
The inventor also inspects that the detection material GFP-MOG-HEK293 cell model prepared by the invention can still accurately and quantitatively detect the MOG-IgG level in the serum after the cryopreservation time reaches 5 months.
Claims (10)
1. A method for constructing a cryopreserved cell model for immediately detecting MOG-IgG level in human serum comprises the following steps:
step 1, constructing a recombinant GFP-MOG plasmid;
step 2, transfecting the GFP-MOG plasmid into an HEK293 cell to establish the GFP-MOG-HEK293 cell;
and 3, subpackaging and freezing the GFP-MOG-HEK293 cells to obtain a frozen cell model capable of detecting the serum MOG-IgG level immediately.
2. The method for constructing a frozen cell model according to claim 1, wherein the step 1 comprises the steps of: adding EcoRI/NheI enzyme cutting sites at two ends of an MOG target gene; inserting EcoRI/NheI enzyme cutting sites into the pIRES2-AcGFP1 vector; inserting MOG target gene into pIRES2-AcGFP1 vector by DNA Ligation Kit to obtain GFP-MOG plasmid; and fourthly, identifying the recombinant plasmid by agarose gel electrophoresis by using restriction enzyme Hind III.
3. The method for constructing a frozen cell model according to claim 1, wherein the transfection in step 2 is selected from the group consisting of transfecting a GFP-MOG plasmid into a HEK293 cell using a Neon electrotransfection system, and establishing a GFP-MOG-HEK293 cell; preferred Neon electrotransfection system conditions are pulse voltage 1100V, pulse time 20ms, pulse number 2 pulses.
4. The method for constructing a frozen cell model according to claim 1, wherein the step 3 comprises the steps of: suspending GFP-MOG-HEK293 cells in a cell freezing medium, placing the cell freezing medium in a container, and freezing; and secondly, the container in the step one is transferred to liquid nitrogen for long-term storage, and a frozen cell model capable of detecting the MOG-IgG level of the serum in real time is obtained.
5. The method for constructing a frozen cell model according to claim 4, wherein the GFP-MOG-HEK293 cells are cultured at (1-5). times.106The cells/mL density is re-suspended in the cell freezing solution, preferably the container in the (i) is a freezing box with controllable cooling rate, the cooling rate in the box of the freezing box is-1 ℃/minute, and the freezing condition in the (i) is refrigerator for 12 hours at-80 ℃.
6. A method for detecting MOG-IgG level in human serum immediately by using the cryopreserved cell model of any one of claims 1 to 5, comprising the following steps: after the frozen cell model is recovered, the frozen cell model is mixed with a sample to be tested for primary incubation, and after washing, an APC-labeled anti-human IgG fluorescent antibody Miltenyi Biotec, Order No. 130-.
7. The detection method according to claim 6, wherein the recovery is performed by thawing the cryopreserved GFP-MOG-HEK293 cells, rapidly adding the thawed cells to a test tube, adding a buffer solution, centrifuging, and washing.
8. The detection method according to claim 7, wherein the recovery is performed by thawing the cryopreserved GFP-MOG-HEK293 cells by a water bath constant temperature constant speed oscillator (37 ℃, 5 rpm/s), rapidly adding the thawed GFP-MOG-HEK293 cells into a 15mL test tube, adding 10mL of a 37 ℃ preheated 0.01M phosphate buffered saline (0.01M PBS) buffer, centrifuging (1200rpm/8min), and completing one washing and cell recovery after three washing.
9. The assay of claim 6 wherein said first incubation is performed with a test serum sample at a ratio of 1: 5, diluting with PBS-2 buffer solution, placing the test tube containing 50 μ L of diluted serum sample and GFP-MOG-HEK293 cells in a rotary incubator for incubation at 4 ℃, 300rpm and 30 min.
10. The detection method according to claim 6, wherein the second incubation is performed with a fluorescent antibody in a ratio of 1: 20, diluted with PBS-2 buffer, the tube containing 100. mu.L of diluted fluorescent antibody and GFP-MOG-HEK293 cells was incubated in a rotary incubator at 4 ℃ and 300rpm for 30 min.
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