Use of a recombinant vector for identifying human IgG transfected cells

Abstract

This invention provides an application for identifying human IgG recombinant vector transfected cells. The cells carry a peptide that binds to the Fc-III fragment of human IgG and a fluorescent protein gene sequence. These cells can be used as a control or quality control for autoantibody detection reagents. When using autoantibody detection reagents to detect autoantibodies in samples, this invention can prevent false negatives caused by errors in detection procedures, invalid body fluid samples, etc.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of recognizing human IgG recombinant vector transfected cells. Background Technology

[0002] Cell-based indirect immunofluorescence is a method for detecting autoantibodies in body fluids, used for the diagnosis of autoimmune diseases. This method involves co-expressing the antigen specifically recognized by the autoantibody to be detected with a labeled fluorescent protein in viable cells and immobilizing it on a carrier plate to create a detection reagent for that specific autoantibody. The specific autoantibody in body fluids binds to this antigen, and the autoantibody is labeled using indirect immunofluorescence to develop color. The presence of the specific autoantibody in body fluids is determined by observing the overlap between the antigen reporter fluorescence and the immunofluorescence using a fluorescence microscope.

[0003] Cell-based indirect immunofluorescence uses mammalian cells to express antigens, whose antigen structures are closer to human proteins than other expression systems. Furthermore, the antigen and fluorescent protein are co-expressed, and two excitation lights can be used to observe and confirm that the spatial distribution of the antigen and antibody is consistent. Therefore, this method has very high accuracy.

[0004] Several cell-based indirect immunofluorescence assay reagents have been developed and applied to various autoantibody detection methods. However, this method currently lacks suitable positive controls or quality control materials, making it impossible to confirm the validity of the test when a negative result is obtained. False negatives due to operational errors, expired body fluid samples, or other reasons cannot be ruled out. Summary of the Invention

[0005] To address the shortcomings of existing methods and solve the problem of lacking controls or quality control materials, this invention proposes an application for identifying human IgG recombinant vector transfected cells. When preparing autoantibody detection reagents, these cells are used as controls or quality control materials. When using autoantibody detection reagents to detect autoantibodies in body fluid samples, if the cells of this invention produce a positive result, it indicates that the detection procedure is correct and the body fluid sample is valid, thereby preventing false negatives caused by detection errors, invalid body fluid samples, etc.

[0006] An application of cells transfected with a recombinant human IgG vector, wherein the cells transfected with the recombinant human IgG vector carry a peptide that binds to the Fc-III fragment of human IgG and a fluorescent protein gene sequence, and wherein the cells transfected with the recombinant human IgG vector are used as a control or quality control for an autoantibody detection reagent.

[0007] Furthermore, the fluorescent protein gene sequence includes GFP, RFP, YFP, mCherry, mKate2, DsRed, EGFP, EYFP, TurboGFP, Venus, Citrin, CFP, mOrange, mPlum, or mRuby.

[0008] A method for preparing cells transfected with a recombinant human IgG vector includes the following steps:

[0009] Step 1: Obtain the Fc-III-Linker-E sequence through artificial synthesis or other means, and add two restriction enzyme sites, BamHI and PmeI, to both ends of the target gene sequence; insert the target gene with restriction enzyme sites into the pcDNA3.1(+) vector, with the insertion sites being BamHI / PmeI, to obtain the recombinant vector, which is named pcDNA3.1(+) / Fc-III-Linker-E;

[0010] Step 2: Transfect the recombinant plasmid vector pcDNA3.1(+) / Fc-III-Linker-E into 293T cells to obtain pcDNA3.1(+) / Fc-III-Linker-E-293T cells;

[0011] Step 3: Fix pcDNA3.1(+) / Fc-III-Linker-E-293T cells with a fixative and block the cells with a blocking agent;

[0012] E: Fluorescent protein gene sequence.

[0013] Furthermore, in step 2, pcDNA3.1(+) / Fc-III-Linker-E is transfected into 293T cells using lentivirus and non-liposome transfection reagent, and stable expression cell lines are obtained by screening with G418, puromycin or hygromycin.

[0014] Furthermore, the plasmid vector pcDNA3.1(+) can be replaced with pLV, pCMV, pEGFP-N1, pEGFP-C1 or pVAX1.

[0015] Furthermore, the concentration of G418 is 100µg / ml-1000µg / ml, the concentration of puromycin is 1µg / ml-10µg / ml, and the concentration of hygromycin is 50µg / ml-500µg / ml.

[0016] Furthermore, the fixative is selected from one or more of aldehydes, methanol, ethanol, acetone, osmium tetroxide, picric acid, chloroform, chromic acid, mercuric chloride, and dichromic acid to prepare a solution.

[0017] Furthermore, the blocking agent includes BSA, goat serum, or casein.

[0018] An autoantibody detection reagent includes a slide, a climbing plate, and a fluorescently labeled antibody, characterized in that the climbing plate is fixed on the slide, and the climbing plate is attached to cells transfected with overexpressing target antigen and cells transfected with recombinant vector overexpressing human IgG recognition.

[0019] Further, the target antigens include PLA2R1, THSD7A, NELL-1, AQP4, MOG, GFAP, MBP, NMDAR, AMPAR, GABABR, LGI1, CASPR2, IgLON5, GAD65, mGluR1, mGluR5, Neurexin3α, GABAARα1, GABAARβ3, gAchR, AchR, Titin, Musk, LRP4, SOX1, Hu, Yo, Ri, Amphiphysin, CV2, Ma1, Ma2, Tr, NF155, NF186, CNTN1, CNTN2, CASPR1, Jo-1, or MDA5.

[0020] Furthermore, the fluorescently labeled antibodies include goat anti-human secondary antibodies, rabbit anti-human secondary antibodies, mouse anti-human secondary antibodies, or sheep anti-human secondary antibodies.

[0021] Furthermore, the fluorescein includes FITC, Alexa Fluorescent 488, Alexa Fluorescent 594, Dylight 488, Dylight 594, Cyanine 3, Pacific Blue, or TRITC.

[0022] Furthermore, when using the fluorescent protein gene sequences and fluoresceins to prepare detection reagents, combinations of fluorescent protein gene sequences and fluoresceins with different color systems are selected for easy differentiation and observation.

[0023] This invention also provides an application of recognizing human IgG recombinant vector transfected cells in the preparation of autoantibody detection reagents.

[0024] Technical effect

[0025] 1) The human IgG recombinant vector transfected cells described in this invention can be used as a control or quality control for indirect immunofluorescence diagnostic reagents to prevent false negatives caused by incorrect detection operations, invalid body fluid samples, etc.

[0026] 2) Utilizing recombinant human IgG vectors overexpressed in eukaryotic cells to detect antibodies in human body fluids eliminates the need for antigen extraction and purification, shortens the reagent preparation cycle, and maintains the native conformation of the antigen, thereby increasing the sensitivity and specificity of the detection reagent;

[0027] 3) An indirect immunofluorescence diagnostic reagent that recognizes recombinant human IgG vector transfected cells was used. As long as the sample contains human IgG, it will show a positive result. Attached Figure Description

[0028] Figure 1 Fluorescence patterns of serum and PBS samples, respectively.

[0029] Figure 2 The fluorescent secondary antibodies are Dylight 488-labeled IgG and Dylight 488-labeled IgM fluorescence images, respectively.

[0030] Figure 3 Fluorescence images of cerebrospinal fluid samples at different concentrations

[0031] Figure 4 Fluorescence image of anti-PLA2R antibody negative sample when measured by anti-PLA2R antibody detection reagent

[0032] Figure 5 Fluorescence image when anti-PLA2R antibody positive samples are detected by anti-PLA2R antibody detection reagent Detailed Implementation

[0033] The present invention will now be described in more detail with reference to the embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional reagent stores.

[0034] Example 1

[0035] Preparation of cells transfected with a recombinant human IgG vector.

[0036] Step 1: Construction of the recombinant vector plasmid:

[0037] The Fc-III-Linker-mCherry sequence was obtained through artificial synthesis, and two restriction enzyme sites, BamHI and PmeI, were added to both ends of the target gene sequence. The target gene with restriction enzyme sites was inserted into the pcDNA3.1(+) vector at the BamHI / PmeI insertion sites to obtain the recombinant vector, which was named pcDNA3.1(+) / Fc-III-Linker-mCherry.

[0038] Step 2: Obtain stable expression cell lines:

[0039] (1) Cell culture: Eukaryotic cells were cultured in 10% FBS+DMEM high glucose medium at 37°C in a 5% CO2 incubator;

[0040] (2) Cell transfection: When the cell density reaches 50%-60%, the recombinant plasmid vector obtained in step 1 is transfected into the cells using lipo8000. Replace with fresh culture medium 24 hours after transfection.

[0041] (3) Antibiotic screening: 48 hours after transfection, the medium was replaced with antibiotic G418. The medium was replaced with antibiotic every 2-3 days for 7-10 days to obtain stable expression cell lines. The stable transfected cell lines were tested with positive samples of different concentrations.

[0042] Step 3: Cell fixation

[0043] (1) Take the stable expression cell line and untransfected cells obtained in step 2 and culture them separately until the cell density reaches 80%-90%;

[0044] (2) Fixation: Cells were fixed at room temperature for 15 minutes using 4% paraformaldehyde;

[0045] (3) Cleaning: Clean 3 times with 200ul of detergent;

[0046] (4) Blocking: Block with 5% BSA at room temperature for 30 minutes.

[0047] Example 2

[0048] A reagent for detecting anti-human IgG antibodies includes a slide, a cell spreader containing cells transfected with a recombinant vector overexpressing human IgG, and a Dylight 488-labeled IgG fluorescent secondary antibody. The cell spreader is fixed on the slide. Serum samples are tested using this reagent, with PBS as a control. The cell spreader containing cells transfected with the recombinant vector overexpressing human IgG is prepared in Example 1, with cells attached to the spreader.

[0049] (1) Dilute serum samples or PBS at a ratio of 1:10 or higher;

[0050] (2) Add the sample to the detection well at a rate of 50~200µL / well;

[0051] (3) Incubate at 37°C for 1 hour or at 4°C overnight in the dark;

[0052] (4) Wash three times with 1XPBST, five minutes each time;

[0053] (5) Add Dylight 488-labeled IgG fluorescent secondary antibody to the reagent wells at a dosage of 50 µL / well;

[0054] (6) Incubate at 37°C in the dark for 0.5 hours;

[0055] (7) Wash three times with 1XPBST, five minutes each time;

[0056] (8) Add 100µL / well of PBS;

[0057] (9) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0058] Test results as follows Figure 1 As shown, the immunofluorescence signal of serum can overlap with the fluorescence image of antigen reporter signal. Since the serum sample contains human IgG, Human-IgG is stained with green fluorescence, and the result is positive. In contrast, the sample used in the control is PBS, which does not contain human IgG, so Human-IgG is not stained with green fluorescence, and the result is negative.

[0059] Example 3

[0060] A reagent for detecting anti-human IgG antibodies includes a slide, a cell spreader containing cells transfected with a recombinant vector overexpressing human IgG, a Dylight 488-labeled IgG fluorescent secondary antibody, and a Dylight 488-labeled IgM fluorescent secondary antibody. The cell spreader is fixed on the slide. Serum samples are tested using this reagent. The cell spreader containing cells transfected with the recombinant vector overexpressing human IgG is prepared in Example 1, with cells attached to the spreader.

[0061] (1) Dilute serum samples at a ratio of 1:10 or higher;

[0062] (2) Add the sample to the detection well at a rate of 50~200µL / well;

[0063] (3) Incubate at 37°C for 1 hour or at 4°C overnight in the dark;

[0064] (4) Wash three times with 1XPBST, five minutes each time;

[0065] (5) Add Dylight 488-labeled anti-human IgG fluorescent secondary antibody to the first reagent well at a dosage of 50µL / well, and add Dylight 488-labeled anti-human IgM fluorescent secondary antibody to the second reagent well;

[0066] (6) Incubate at 37°C in the dark for 0.5 hours;

[0067] (7) Wash three times with 1XPBST, five minutes each time;

[0068] (8) Add 100µL / well of PBS;

[0069] (9) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0070] Test results as follows Figure 2 As shown, the sample contained human IgG. Cells in the first reagent well adsorbed human IgG, hence Human-IgG showed green fluorescence staining, resulting in a positive result. Cells in the second reagent well did not adsorb human IgM, hence Human-IgM did not show green fluorescence staining, resulting in a negative result. The results demonstrate that cell slides transfected with the recombinant vector overexpressing human IgG have specific binding to human IgG and do not bind to human IgM.

[0071] Example 4

[0072] A reagent for detecting anti-human IgG antibodies includes a slide, a cell spreader plate containing cells transfected with a recombinant vector overexpressing human IgG, and a Dylight 488-labeled IgG fluorescent secondary antibody. The cell spreader plate is fixed on the slide. This reagent is used to test cerebrospinal fluid samples. The cell spreader plate containing cells transfected with the recombinant vector overexpressing human IgG is prepared in Example 1, with cells attached to the spreader plate.

[0073] (1) Dilute cerebrospinal fluid samples at a ratio of 1:10 or higher;

[0074] (2) Add the sample to the detection well at a rate of 50~200µL / well;

[0075] (3) Incubate at 37°C for 1 hour or at 4°C overnight in the dark;

[0076] (4) Wash three times with 1XPBST, five minutes each time;

[0077] (5) Add an appropriate amount of Dylight 488-labeled anti-human IgG fluorescent secondary antibody to the reagent well;

[0078] (6) Incubate at 37°C in the dark for 0.5 hours;

[0079] (7) Wash three times with 1XPBST, five minutes each time;

[0080] (8) Add 100µL / well of PBS;

[0081] (9) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0082] Test results as follows Figure 3As shown, the cerebrospinal fluid (CSF) samples contained human IgG. The CSF in the first well was diluted 20-fold, and the CSF in the second well was diluted 200-fold. Therefore, Human-IgG showed green fluorescence staining, and the results were positive in both wells. The CSF samples with lower dilutions showed stronger green fluorescence staining of Human-IgG, while those with higher dilutions showed weaker green fluorescence staining. These results demonstrate that fluorescence intensity reflects the level of human IgG in the CSF sample.

[0083] Example 5

[0084] A detection reagent for anti-human PLA2R antibody includes a slide, a cell slide transfected with cells overexpressing PLA2R vector, a cell slide transfected with cells overexpressing recombinant vector recognizing human IgG, a cell slide transfected with cells overexpressing mCherry, and a Dylight 488-labeled IgG fluorescent secondary antibody. The three cell slides are simultaneously immobilized on the same slide to form a single detection reagent.

[0085] Cell slides of cells transfected with the recombinant vector that overexpresses human IgG recognition were used as positive controls, while cell slides of cells transfected with mCherry overexpression were used as negative controls.

[0086] I. Preparation Method

[0087] Step 1: Construction of the recombinant vector plasmid:

[0088] The CDS sequences of PLA2R, human IgG binding peptide, and mCherrry were obtained as target genes through PCR or artificial synthesis. The target genes with restriction enzyme sites were inserted into a eukaryotic cell expression vector containing the fluorescent protein mCherry to obtain a recombinant plasmid. The recombinant plasmid vector was then extracted and prepared for use.

[0089] Step 2: Obtain stable expression cell lines:

[0090] (1) Cell culture: Eukaryotic cells were cultured in 10% FBS+DMEM high glucose medium at 37°C in a 5% CO2 incubator;

[0091] (2) Cell transfection: When the cell density reaches 50%-60%, the recombinant plasmid vector obtained in step 1 is transfected into the cells using lipo8000. Replace with fresh culture medium 24 hours after transfection.

[0092] (3) Antibiotic screening: 48 hours after transfection, the medium was replaced with antibiotic G418. The antibiotic-containing medium was changed every 2-3 days for 7-10 days to obtain stable expression cell lines. The stable transfected cell lines were then tested using different concentrations of positive samples. The results are as follows: Figure 1 As shown;

[0093] Step 3: Cell fixation

[0094] (1) Take the stable expression cell line and untransfected cells obtained in step 2 and culture them separately until the cell density reaches 80%-90%;

[0095] (2) Fixation: Cells were fixed at room temperature for 15 minutes using 4% paraformaldehyde;

[0096] (3) Cleaning: Clean 3 times with 200ul of detergent;

[0097] (4) Blocking: Block with 5% BSA at room temperature for 30 minutes.

[0098] II. Detection Methods

[0099] (1) Dilute serum samples at a ratio of 1:10 or higher;

[0100] (2) Add the sample to the detection well at a rate of 50~200µL / well;

[0101] (3) Incubate at 37°C for 1 hour or at 4°C overnight in the dark;

[0102] (4) Wash three times with 1XPBST, five minutes each time;

[0103] (5) Add an appropriate amount of Dylight 488-labeled anti-human IgG fluorescent secondary antibody to the reagent reaction area;

[0104] (6) Incubate at 37°C in the dark for 0.5 hours;

[0105] (7) Wash three times with 1XPBST, five minutes each time;

[0106] (8) Add 100µL / well of PBS;

[0107] (9) Observe and photograph fluorescence under a 20X objective lens of a fluorescence microscope.

[0108] Test results as follows Figure 4 , Figure 5As shown, negative serum samples do not contain anti-PLA2R specific autoantibodies, therefore Human-IgG does not show green fluorescence staining, resulting in a negative result. However, cell slides of cells transfected with the recombinant vector overexpressing human IgG show green fluorescence staining, while mCherry cell slides do not, indicating that the sample testing is normal and the results are reliable. Conversely, positive serum samples contain anti-PLA2R specific autoantibodies, therefore Human-IgG shows green fluorescence staining, resulting in a positive result. Additionally, cell slides of cells transfected with the recombinant vector overexpressing human IgG show green fluorescence staining, while mCherry cell slides do not, indicating that the sample testing is normal and the results are reliable.

Claims

1. An application of cells transfected with a recombinant human IgG vector, wherein the cells transfected with the recombinant human IgG vector carry a peptide fragment that binds to the Fc-III fragment of human IgG and a fluorescent protein gene sequence; the application of the cells transfected with the recombinant human IgG vector as a control or quality control for an autoantibody detection reagent, wherein the reagent comprises a slide, a climbing slide, and a fluorescently labeled antibody, characterized in that... The climbing slide is fixed on the slide, and the climbing slide is attached to cells transfected with the target antigen and cells transfected with the recombinant vector that recognizes human IgG.

2. The application of the method for identifying human IgG recombinant vector-transfected cells according to claim 1, characterized in that, The fluorescent protein gene sequences include GFP, RFP, YFP, mCherry, mKate2, DsRed, EGFP, EYFP, TurboGFP, Venus, Citrin, CFP, mOrange, mPlum, or mRuby.

3. The application of the method for recognizing human IgG recombinant vector-transfected cells according to claim 1, characterized in that... The method for preparing the transfected cells includes the following steps: Step 1: Obtain the Fc-III-Linker-E sequence through artificial synthesis or other means, and add two restriction enzyme sites, BamHI and PmeI, to both ends of the target gene sequence; insert the target gene with restriction enzyme sites into the pcDNA3.1(+) vector, with the insertion sites being BamHI / PmeI, to obtain the recombinant vector, which is named pcDNA3.1(+) / Fc-III-Linker-E; Step 2: Transfect the recombinant plasmid vector pcDNA3.1(+) / Fc-III-Linker-E into 293T cells to obtain pcDNA3.1(+) / Fc-III-Linker-E-293T cells; Step 3: Fix pcDNA3.1(+) / Fc-III-Linker-E-293T cells with a fixative and block the cells with a blocking agent; E: Fluorescent protein gene sequence.

4. The method for preparing cells transfected with a recombinant vector for recognizing human IgG according to claim 3, characterized in that, The plasmid vector pcDNA3.1(+) can be replaced with pLV, pCMV, pEGFP-N1, pEGFP-C1 or pVAX1.

5. The method for preparing cells transfected with a recombinant vector for recognizing human IgG according to claim 3, characterized in that, In step 2, pcDNA3.1(+) / Fc-III-Linker-E was transfected into 293T cells using lentivirus and non-liposome transfection reagent, and stable expression cell lines were obtained by screening with G418, puromycin or hygromycin.

6. The method for preparing cells transfected with a recombinant vector for recognizing human IgG according to claim 5, characterized in that, The concentration of G418 is 100µg / ml-1000µg / ml, the concentration of puromycin is 1µg / ml-10µg / ml, and the concentration of hygromycin is 50µg / ml-500µg / ml.

7. The method for preparing cells transfected with a recombinant vector for recognizing human IgG according to claim 3, characterized in that, The fixative is selected from one or more of aldehydes, methanol, ethanol, acetone, osmium tetroxide, picric acid, chloroform, chromic acid, mercuric chloride, and dichromic acid to prepare a solution; the blocking agent includes BSA, goat serum, or casein.

8. An autoantibody detection reagent, comprising a slide, a climbing slide, and a fluorescently labeled antibody, characterized in that, The climbing slide is fixed on a slide, on which are attached cells transfected with the target antigen and cells transfected with the human IgG recombinant vector. The human IgG recombinant vector transfected cells carry a peptide fragment that binds to the Fc-III fragment of human IgG and a fluorescent protein gene sequence. The human IgG recombinant vector transfected cells are used as a control or quality control for autoantibody detection reagents.

9. The autoantibody detection reagent according to claim 8, characterized in that, The target antigens include PLA2R1, THSD7A, NELL-1, AQP4, MOG, GFAP, MBP, NMDAR, AMPAR, GABABR, LGI1, CASPR2, IgLON5, GAD65, mGluR1, mGluR5, Neurexin3α, GABAARα1, GABAARβ3, gAchR, AchR, Titin, Musk, LRP4, SOX1, Hu, Yo, Ri, Amphiphysin, CV2, Ma1, Ma2, Tr, NF155, NF186, CNTN1, CNTN2, CASPR1, Jo-1, or MDA5.

10. The autoantibody detection reagent according to claim 8, characterized in that, The fluorescently labeled antibodies include goat anti-human secondary antibodies, rabbit anti-human secondary antibodies, mouse anti-human secondary antibodies, or sheep anti-human secondary antibodies; the fluorescent dyes include FITC, Alexa Fluorescent 488, Alexa Fluorescent 594, Dylight 488, Dylight 594, Cyanine 3, Pacific Blue, or TRITC.

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