Antibody combination suitable for detecting DLBCL immune microenvironment by imaging mass spectrometer, kit and application

Abstract

The invention discloses an antibody combination suitable for detecting DLBCL immune microenvironment by an imaging mass spectrometer, a kit and application. The antibody combination is an antibody for resisting alpha SMA, an antibody for resisting HLADR, an antibody for resisting CD20, an antibody for resisting CD56, an antibody for resisting Vimentin, an antibody for resisting CD8a, an antibody for resisting CCL17, an antibody for resisting CCR6, an antibody for resisting IL3CD123, an antibody for resisting CXCL12, an antibody for resisting BCL2, an antibody for resisting PD1, an antibody for resisting CD163, an antibody for resisting CD45RO, an antibody for resisting ICOS, an antibody for resisting GranzymeB, an antibody for resisting CXCR5, an antibody for resisting Ki67, an antibody for resisting PDL1, an antibody for resisting Bcl6, an antibody for resisting CD31, an antibody for resisting CD163, an antibody for resisting CD45RO, an antibody for resisting CD27, an And all the antibodies are marked. The DLBCL immune microenvironment can be more comprehensively and efficiently identified by using the antibody combination disclosed by the invention.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to an antibody combination, a kit and uses thereof suitable for detecting the immune microenvironment of DLBCL by an imaging mass spectrometer. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. In gene expression profiling studies, the composition of the tumor microenvironment has emerged as an important predictor of prognosis in DLBCL. Other studies have emphasized the importance of individual immune cell populations, such as CD4+ T cells, CD8+ T cells, macrophages, dendritic cells, and conventional regulatory T cells (Tregs), but none of these studies have simultaneously and comprehensively characterized the tumor immune microenvironment.

[0003] Imaging mass cytometry (IMC) is a new molecular imaging technology that combines the advantages of high throughput and exploration of spatial distribution characteristics. Through high-dimensional analysis, IMC can show the localization and relative quantification of different cell subsets in the tumor microenvironment, thereby achieving an in-depth analysis of the number, spatial localization and interaction of each cell subset in the tumor immune microenvironment. The use of IMC can accurately present the composition and spatial distribution of each cell subset in the DLBCL immune microenvironment, which is of great significance for finding the key cell subsets that induce the immunosuppressive microenvironment, in-depth exploration of the molecular mechanism that induces the formation of the inhibitory immune microenvironment in DLBCL, and improving the prognosis of DLBCL. Summary of the invention

[0004] Therefore, a kit suitable for detecting the immune microenvironment of diffuse large B-cell lymphoma using imaging mass spectrometry technology can more comprehensively and efficiently identify the immune microenvironment of DLBCL, and the present invention is specially proposed.

[0005] To achieve the above-mentioned purpose, an antibody combination for detecting the immune microenvironment of DLBCL by imaging mass spectrometry flow cytometry is provided, characterized in that the antibody combination is anti-α_SMA, HLA_DR, CD20, CD56, Vimentin, CD8a, CCL17, CCR6, IL3_CD123, CXCL12, BCL2, PD1, CD163, CD45RO, ICOS, GranzymeB, CXCR5, Ki_67, PDL1, Bcl_6, CD31, CD3, CD45, CD27, LAG_3, PDL2, CXCL13, CD127, FoxP3, CD21, CD4, CD25, CCL21, IDO, CD68 antibodies; all antibodies are labeled.

[0006] Further, the antibody combination is an anti-α_SMA antibody labeled with 141Pr, recorded as 141Pr_α_SMA, and so on, recorded as 160Gd_HLA_DR, 142Nd_CD20, 161Dy_CD56, 143Nd_Vimentin, 162_Dy_CD8a, 144Nd_CCL17, 163Dy_CCR6, 145Nd_IL3_CD123, 164Dy_CXCL12, 146Nd_BCL2, 165Ho_PD1, 147Sm_CD163, 166Er_CD45RO, 148Nd_ICOS, 167Er_ GranzymeB, 149Sm_CXCR5, 168Er_Ki_67, 150Nd_PDL1, 169Tm_Bcl_6, 151Eu_CD31, 170Er_CD3, 152Sm_CD45, 171Yb_CD27, 153Eu_LAG_3, 172Yb_PDL2, 154Sm_CXCL13, 173Yb_CD127, 155Gd_FoxP3, 174Yb_CD21, 156Gd_CD4, 175Lu_CD25, 158Gd_CCL21, 176Yb_IDO, 159Tb_CD68.

[0007] Furthermore, the method for preparing the antibody in the antibody combination is:

[0008] A1. Preparation of lanthanide-loaded polymers:

[0009] Preloading: Resuspend the Maxpar polymer with Maxpar L-Buffer; then add the corresponding lanthanide metal solution, preferably, the final concentration of the lanthanide metal solution is 2.5mM; mix and incubate, preferably at 37°C for 30-40 minutes; Maxpar polymer is X8 Antibody Labeling Kit,142Nd—4Rxn, X8 AntibodyLabeling Kit,144Nd—4Rxn, X8 Antibody Labeling Kit,145Nd—4Rxn, X8 Antibody Labeling Kit,149Sm—4Rxn, X8 Antibody Labeling Kit,154Sm—4Rxn, X8 Antibody Labeling Kit,158Gd—4Rxn, X8 Antibody LabelingKit,160Gd—4Rxn, X8 Antibody Labeling Kit,161Dy—5Rxn, X8Antibody Labeling Kit,164Dy—4Rxn, X8 Antibody Labeling Kit,166Er—4Rxn, X8 Antibody Labeling Kit,169Tm—4Rxn, X8 Antibody LabelingKit,173Yb—4Rxn, X8 Antibody Labeling Kit,174Yb—4Rxn and X8Antibody Labeling Kit,176Yb—4Rxn;

[0010] Purification: adding the above-obtained product to a filter containing Maxpar L-Buffer, centrifuging, preferably, centrifuging at 12,000 x g for 25 minutes at room temperature; discarding the waste liquid in the collection tube; adding Maxpar C-Buffer to the filter, centrifuging, preferably centrifuging at 12,000 x g for 30 minutes at room temperature; discarding the waste liquid in the collection tube; obtaining the lanthanide-loaded polymer;

[0011] A2. Preparation of the corresponding partially reduced antibody:

[0012] Add Maxpar R-Buffer and antibody to a 50kDa filter, centrifuge at room temperature; preferably, centrifuge at 12,000xg for 10 minutes; discard the liquid after centrifugation; then add 4mM TCEP-R-buffer, mix thoroughly and incubate, preferably, incubate at 37°C for 30 minutes; add Maxpar C-Buffer, centrifuge, preferably, centrifuge at 12,000xg for 10 minutes at room temperature; discard the waste liquid, then add Maxpar C-Buffer; centrifuge at 12,000xg for 10 minutes at room temperature; discard the waste liquid; obtain the corresponding partially reduced antibody;

[0013] The order of the above steps A1 and A2 can be swapped or performed simultaneously, with no order requirement.

[0014] A3. Coupling of antibodies with lanthanide-loaded polymers:

[0015] Resuspend the product obtained in A1 in Maxpar C-Buffer and add it to the product obtained in A2, mix well and incubate, preferably, incubate at 37° C. for 60-120 minutes to obtain an antibody coupling mixture;

[0016] A4. Washing of metal-coupled antibodies:

[0017] Add Maxpar W-Buffer to the product obtained in A3, centrifuge, preferably, centrifuge at 12,000 x g for 10 minutes at room temperature; discard the waste liquid; and then rinse repeatedly with Maxpar W-Buffer for multiple times; preferably, repeat the rinsing 1-5 times;

[0018] A5. Recycling of metal-conjugated antibodies:

[0019] Add Maxpar W-Buffer to the 50kDa filter to rinse the filter walls; then invert the 50kDa filter into a new collection tube and centrifuge the inverted filter / collection tube assembly, preferably at 1000xg for 2 minutes; remove the inverted filter from the collection tube, rinse the filter walls with Maxpar W-Buffer, and place the inverted filter back into the collection tube; centrifuge the inverted filter / collection tube assembly to collect the precipitate, preferably at 1000xg for 2 minutes.

[0020] The present invention also provides a kit, characterized in that it contains the antibody combination.

[0021] The present invention also provides a method for detecting the immune microenvironment of diffuse large B-cell lymphoma, characterized in that the antibody combination or the kit is used.

[0022] The present invention also provides a method for detecting the immune microenvironment of DLBCL using an imaging mass spectrometer, which is characterized by comprising the following steps:

[0023] Air-dry the paraffin tissue sections to be tested at room temperature, and then bake them in an oven at 65±5℃ for 30-60min; take out the sections, immerse them in xylene twice at room temperature, and dewax them for 10±1min respectively. Move the sections up and down from time to time during the period to ensure sufficient dewaxing; immerse the sections in 100%, 100%, 95%, 95%, and 75% ethanol in turn, each time for 5±1min; slowly immerse the sections in a vertical staining bottle containing preheated 1xAntigen Retrieval buffer and incubate at 97±3℃ for 30±5min, then cool to 70±5℃ at room temperature, transfer to a vertical staining bottle containing ddH2O, wash for 10±2min, then transfer to a vertical staining bottle containing DPBS, wash for 10±2min, and use an immunohistochemistry pen to draw circles around the sections; use SuperBlock TMBlock with blocking buffer for 45±2 minutes; remove the blocking solution, drip the antibody onto the tissue section, incubate overnight at 4±2°C, wash several times with DPBS containing 0.2% Triton-X, and then wash several times with DPBS; drip the diluted dye onto the tissue section, stain at room temperature in a wet box, rinse, and dry for detection on an imaging mass spectrometer.

[0024] The detection reagent of the present invention can detect 36 indicators simultaneously, is suitable for detecting the immune microenvironment of diffuse large B-cell lymphoma by imaging mass spectrometry flow cytometer, and can identify the immune microenvironment of DLBCL more comprehensively and efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is one of the detection index diagrams of Example 1.

[0026] Figure 2 This is the second detection index diagram of Example 1.

[0027] Figure 3 This is the third detection index diagram of Example 1.

[0028] Figure 4 It is one of the detection index diagrams of Example 2.

[0029] Figure 5 This is the second detection index diagram of Example 2.

[0030] Figure 6 This is the third detection index diagram of Example 2. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0032] The antibody combination and kit for detecting the immune microenvironment of diffuse large B-cell lymphoma by imaging mass spectrometry flow cytometry of the present invention include commercial reagents, as shown in Table 1:

[0033] Table 1 Reagent List

[0034]

[0035]

[0036] And homemade antibodies:

[0037] 24002 and homemade antibodies:

[0038] 142Nd_CD20, 144Nd_CCL17, 145Nd_IL3_CD123, 149Sm_CXCR5, 154Sm_CXCL13, 158Gd_CCL21, 160Gd_HLA _DR, 161Dy_CD56, 164Dy_CXCL12, 166Er_CD45RO, 169Tm_Bcl_6, 173Yb_CD127, 174Yb_CD21, 176Yb_IDO. (The preparation methods of these antibodies are shown in the "Antibody Preparation Steps" below, except that the metals therein are selected to correspond to the antibodies. For example, in 142Nd_CD20, the antibody is CD20, which is labeled with 142Nd, and the lanthanide metal is 142Nd; in 144Nd_CCL17, the antibody is CCL17, which is labeled with 144Nd, and the lanthanide metal is 144Nd, ...).

[0039] The reagents needed to make your own antibodies are:

[0040] Table 2 List of reagents required for homemade antibodies

[0041]

[0042]

[0043] Maxpar polymers include those listed in Table 2: X8Antibody Labeling Kit,142Nd—4Rxn, X8Antibody Labeling Kit,144Nd—4Rxn, X8 Antibody Labeling Kit,145Nd—4Rxn, X8Antibody Labeling Kit,149Sm—4Rxn, X8 AntibodyLabeling Kit,154Sm—4Rxn, X8Antibody Labeling Kit,158Gd—4Rxn, X8 Antibody Labeling Kit,160Gd—4Rxn, X8 Antibody Labeling Kit,161Dy—5Rxn, X8Antibody Labeling Kit,164Dy—4Rxn, X8 Antibody LabelingKit,166Er—4Rxn, X8Antibody Labeling Kit,169Tm—4Rxn, X8Antibody Labeling Kit,173Yb—4Rxn, X8Antibody Labeling Kit,174Yb-4Rxn and X8 Antibody Labeling Kit,176Yb—4Rxn.

[0044] The steps for making antibodies (142Nd_CD20, 144Nd_CCL17, 145Nd_IL3_CD123, 149Sm_CXCR5, 154Sm_CXCL13, 158Gd_CCL21, 160Gd_HLA_DR, 161Dy_CD56, 164Dy_CXCL12, 166Er_CD45RO, 169Tm_Bcl_6, 173Yb_CD127, 174Yb_CD21, 176Yb_IDO) are as follows:

[0045] 1) Preloading of lanthanides in polymers:

[0046] 1. Spin the Maxpar polymer tube in a microcentrifuge for 10 seconds to ensure the reagent is at the bottom of the tube;

[0047] 2. Resuspend the Maxpar polymer in 95 μL Maxpar L-Buffer;

[0048] 3. Mix thoroughly by pipetting;

[0049] 4. Add 5 μL of the corresponding lanthanide metal solution (final concentration is 2.5 mM) to the test tube;

[0050] 5. Mix thoroughly by pipetting and incubate at 37°C for 30-40 minutes. During the incubation period, start the buffer exchange and partial reduction of the antibody step.

[0051] 2) Buffer exchange and partial reduction of antibodies:

[0052] 1. Add 300 μL Maxpar R-Buffer to the 50 kDa filter;

[0053] 2. Add up to 200 μL of antibody to the filter with R-Buffer added;

[0054] 3. Centrifuge at 12,000 x g for 10 minutes at room temperature;

[0055] 4. Mix 8 μL of 0.5M TCEP stock solution with 992 μL of Maxpar R-Buffer and dilute the 0.5M TCEP stock solution to 4mM in Maxpar R-Buffer. Each labeled antibody requires 100 μL of 4mM TCEP-R-buffer.

[0056] 5. Discard the liquid after centrifugation;

[0057] 6. Add 100 μL of 4 mM TCEP-R-buffer to each antibody and mix thoroughly with a pipette;

[0058] 7. Incubate at 37°C for 30 minutes (strict timing).

[0059] 3) Purification of polymers preloaded with lanthanides:

[0060] 1. Add 200 μL Maxpar L-Buffer to the 3 kDa filter;

[0061] 2. Add the lanthanide-loaded polymer mixture to the filter containing Maxpar L-Buffer;

[0062] 3. Centrifuge at 12,000 x g for 25 minutes at room temperature;

[0063] 4. Add 300 μL Maxpar C-Buffer to the filter and centrifuge at 12,000 x g for 30 minutes at room temperature;

[0064] 4) Purification of partially reduced antibodies:

[0065] 1. Remove the partially reduced antibody from the 37°C incubator;

[0066] 2. Add 300 μL Maxpar C-Buffer to each 50 kDa filter;

[0067] 3. Centrifuge at 12,000 x g for 10 minutes at room temperature. Another microcentrifuge can be used at this step to avoid time conflict with polymer washing.

[0068] 4. Discard the waste liquid and add 400 μL Maxpar C-Buffer to the filter;

[0069] 5. Centrifuge at 12,000 x g for 10 minutes at room temperature.

[0070] 5) removing the 3 kDa filter containing the lanthanide polymer from the centrifuge and discarding the waste liquid;

[0071] 6) Remove the 50 kDa filter containing the partially reduced antibody from the centrifuge and discard the waste liquid;

[0072] 7) Coupling of antibodies and lanthanide-loaded polymers:

[0073] 1. Using a pipette, resuspend the lanthanide-loaded polymer in 60 μL Maxpar C-Buffer;

[0074] 2. Transfer the resuspended liquid to the corresponding 50kDa filter of partially reduced antibody;

[0075] 3. Pipette to mix evenly;

[0076] 4. Incubate at 37°C for at least 60 minutes (maximum 2 hours);

[0077] 8) Washing metal-coupled antibodies:

[0078] 1. Add 300 μL of Maxpar W-Buffer to the antibody coupling mixture;

[0079] 2. Centrifuge at 12,000 x g for 10 minutes at room temperature and discard the waste liquid;

[0080] 3. Repeat three more times with 400 μL Maxpar W-Buffer (four washes in total);

[0081] 9) Recovering metal-coupled antibodies:

[0082] 1. Add 50 μL Maxpar W-Buffer to the 50 kDa filter and mix with a pipette to rinse the filter wall.

[0083] 2. Invert the 50 kDa filter into a new collection tube;

[0084] 3. Centrifuge the inverted filter / collection tube assembly at 1000 x g for 2 minutes;

[0085] 4. Remove the inverted filter from the collection tube, rinse the filter wall with an additional 50 μL Maxpar W-Buffer, and place the inverted filter back into the collection tube;

[0086] 5. Centrifuge the inverted filter / collection tube assembly at 1000 x g for 2 minutes;

[0087] 10) Yield determination and storage of metal-coupled antibodies:

[0088] 1. Using Maxpar W-Buffer as blank control, quantify the labeled antibody by measuring the absorbance at 280 nm;

[0089] 2. Dilute the antibody to a final concentration of 0.5 mg / mL using Antibody Stabilizer PBS and store at 4°C.

[0090] A method for using an antibody or a kit for detecting the immune microenvironment of diffuse large B-cell lymphoma using an imaging mass spectrometry flow cytometer comprises the following steps:

[0091] 1. Baking: restore the paraffin tissue sections to room temperature and dry them, then bake them in a 65°C oven for 30-60 minutes;

[0092] 2. Preheat 1x Antigen Retrieval Buffer: Dilute 100× Antigen Retrieval Buffer to 1× with ddH2O, pour the diluted buffer into a plastic vertical staining bottle, the height must be completely covered; set the water bath to 97℃, place the vertical staining bottle in it to preheat for later use;

[0093] 3. Dewaxing: Take out the paraffin sections (4um), immerse them in two cylinders of xylene at room temperature, and dewax for 10 minutes respectively. Move the sections up and down from time to time to ensure sufficient dewaxing;

[0094] 4. Rehydration: Soak the sections in a series of ethanol concentrations (100%, 100%, 95%, 95%, 75%) for 5 minutes each time;

[0095] 5. Rinse the slides with ddH2O for 5 minutes (place the staining bottle on a horizontal shaker);

[0096] 6. Place the sections in a vertical staining bottle containing preheated 1x Antigen Retrieval buffer and incubate at 97°C for 30 minutes. Do not tighten the lid to maintain internal and external pressure balance.

[0097] 7. After incubation, place the vertical staining bottle at room temperature and cool it naturally to 70 degrees Celsius in 10 minutes;

[0098] 8. Transfer the slices into a vertical staining bottle containing ddH2O and wash for 10 minutes;

[0099] 9. Transfer the slices into a vertical staining bottle containing DPBS and wash for 10 minutes;

[0100] 10. Use a PAP pen to draw a circle around the slice and use SuperBlock TM Block with blocking buffer for 45 min;

[0101] 11. Use SuperBlock TM Prepare antibody mix with blocking buffer and estimate the total volume of mix. 100ul system is enough to stain 1cm2 The antibody was centrifuged at 13000g for 2 minutes and the antibody was aspirated from the top of the liquid to avoid antibody aggregation. When preparing the mix, SuperBlock was first added to the EP tube. TM Blocking buffer, then add antibody, the antibody dilution ratio is shown in Table 3:

[0102] Table 3 Antibody dilution ratio table

[0103]

[0104]

[0105] 12. Aspirate the blocking solution, drop the antibody onto the tissue section (if the antibody cannot be spread, apply it horizontally with the tip of the gun, being careful not to scratch the section), and incubate overnight at 4°C;

[0106] 13. Wash twice with DPBS containing 0.2% Triton-X for 8 minutes, and shake slowly on a shaker;

[0107] 14. Wash with DPBS for 8 minutes × 2 times, and shake slowly on a shaker;

[0108] 15. Dilute Cell-ID with DPBS at room temperature TM Intercalator-Ir (1:400), drop the diluted dye onto the area of ​​the tissue section and stain it in a humidified chamber at room temperature for 30 min;

[0109] 16. Rinse with ddH2O for 5 minutes;

[0110] 17. Air-dry the slides at room temperature for at least 20 minutes before testing on an imaging mass spectrometer.

[0111] Embodiment 1:

[0112] Sample: Lymph node samples were collected from a patient with diffuse large B-cell lymphoma and prepared into paraffin tissue sections.

[0113] Detection method: Same as above.

[0114] Result analysis: The detection results were analyzed using MCDviewer software, with different pseudo colors given to the above markers. Figure 1-3 The method of the present invention can detect 36 indicators simultaneously.

[0115] Embodiment 2:

[0116] Sample: Lymph node samples were collected from a patient with diffuse large B-cell lymphoma and prepared into paraffin tissue sections.

[0117] Detection method: Same as above.

[0118] Result analysis: The detection results were analyzed using MCDviewer software, with different pseudo colors given to the above markers. Figure 4-6 The method of the present invention can detect 36 indicators simultaneously.

[0119] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. An antibody combination suitable for detecting the immune microenvironment of DLBCL by imaging mass spectrometry, characterized in that: The antibody combination was anti-α_SMA, HLA_DR, CD20, CD56, Vimentin, CD8a, CCL17, CCR6, IL3_CD123, CXCL12, BCL2, PD1, CD163, CD45RO, ICOS, GranzymeB, CXCR5, Ki_67, PDL1, Bcl_6, CD31, CD3, CD45, CD27, LAG_3, PDL2, CXCL13, CD127, FoxP3, CD21, CD4, CD25, CCL21, IDO, CD68 antibodies; all antibodies were labeled.

2. The antibody combination according to claim 1, characterized in that The antibody combination is an anti-α_SMA antibody labeled with 141Pr, recorded as 141Pr_α_SMA, and the like is recorded as 160Gd_HLA_DR, 142Nd_CD20, 161Dy_CD56, 143Nd_Vimentin, 162_Dy_CD8a, 144Nd_CCL17, 163Dy_CCR6, 145Nd_IL3_CD123, 164Dy_CXCL12, 146Nd_BCL2, 165Ho_PD1, 147Sm_CD163, 166Er_CD45RO, 148Nd_ICOS, 167Er_Gr anzymeB, 149Sm_CXCR5, 168Er_Ki_67, 150Nd_PDL1, 169Tm_Bcl_6, 151Eu_CD31, 170Er_CD3, 152Sm_CD45, 171Yb_CD27, 153Eu_LAG_3, 1 72Yb_PDL2, 154Sm_CXCL13, 173Yb_CD127, 155Gd_FoxP3, 174Yb_CD21, 156Gd_CD4, 175Lu_CD25, 158Gd_CCL21, 176Yb_IDO, 159Tb_CD68.

3. The antibody combination according to claim 1, characterized in that The method for preparing the antibody in the antibody combination is as follows: A1. Preparation of lanthanide-loaded polymers: Preloading: Resuspend the Maxpar polymer with Maxpar L-Buffer; then add the corresponding lanthanide metal solution, preferably, the final concentration of the lanthanide metal solution is 2.5mM; mix and incubate, preferably at 37°C for 30-40 minutes; Maxpar polymer is X8 Antibody Labeling Kit,142Nd—4Rxn, X8 Antibody LabelingKit,144Nd—4Rxn, X8 Antibody Labeling Kit,145Nd—4Rxn, X8Antibody Labeling Kit,149Sm—4Rxn, X8 Antibody Labeling Kit,154Sm—4Rxn, X8 Antibody Labeling Kit,158Gd—4Rxn, X8 Antibody LabelingKit,160Gd—4Rxn, X8 Antibody Labeling Kit,161Dy—5Rxn, X8Antibody Labeling Kit,164Dy—4Rxn, X8 Antibody Labeling Kit,166Er—4Rxn, X8 Antibody Labeling Kit,169Tm—4Rxn, X8 Antibody LabelingKit,173Yb—4Rxn, X8 Antibody Labeling Kit,174Yb—4Rxn and X8Antibody Labeling Kit,176Yb—4Rxn; Purification: adding the above-obtained product to a filter containing Maxpar L-Buffer, centrifuging, preferably, centrifuging at 12,000 x g for 25 minutes at room temperature; discarding the waste liquid in the collection tube; adding Maxpar C-Buffer to the filter, centrifuging, preferably centrifuging at 12,000 x g for 30 minutes at room temperature; discarding the waste liquid in the collection tube; obtaining the lanthanide-loaded polymer; A2. Preparation of the corresponding partially reduced antibody: Add Maxpar R-Buffer and antibody to a 50kDa filter, centrifuge at room temperature; preferably, centrifuge at 12,000xg for 10 minutes; discard the liquid after centrifugation; then add 4mM TCEP-R-buffer, mix thoroughly and incubate, preferably, incubate at 37°C for 30 minutes; add Maxpar C-Buffer, centrifuge, preferably, centrifuge at 12,000xg for 10 minutes at room temperature; discard the waste liquid, then add Maxpar C-Buffer; centrifuge at 12,000xg for 10 minutes at room temperature; discard the waste liquid; obtain the corresponding partially reduced antibody; The order of the above steps A1 and A2 can be swapped or performed simultaneously, with no order requirement. A3. Coupling of antibodies with lanthanide-loaded polymers: Resuspend the product obtained in A1 in Maxpar C-Buffer and add it to the product obtained in A2, mix well and incubate, preferably, incubate at 37° C. for 60-120 minutes to obtain an antibody coupling mixture; A4. Washing of metal-coupled antibodies: Add Maxpar W-Buffer to the product obtained in A3, centrifuge, preferably, centrifuge at 12,000 x g for 10 minutes at room temperature; discard the waste liquid; and then rinse repeatedly with Maxpar W-Buffer for multiple times; preferably, repeat the rinsing 1-5 times; A5. Recycling of metal-conjugated antibodies: Add Maxpar W-Buffer to the 50kDa filter to rinse the filter walls; then invert the 50kDa filter into a new collection tube and centrifuge the inverted filter / collection tube assembly, preferably at 1000xg for 2 minutes; remove the inverted filter from the collection tube, rinse the filter walls with Maxpar W-Buffer, and place the inverted filter back into the collection tube; centrifuge the inverted filter / collection tube assembly to collect the precipitate, preferably at 1000xg for 2 minutes.

4. A kit, characterized in that: A combination of antibodies comprising any one of claims 1 to 3.

5. A method for detecting the immune microenvironment of diffuse large B-cell lymphoma, characterized in that: The antibody combination according to any one of claims 1 to 3 or the kit according to claim 4 is used.

6. A method for detecting the immune microenvironment of DLBCL using imaging mass spectrometry, characterized in that: The following steps are included: Air-dry the paraffin tissue sections to be tested at room temperature, and then bake them in an oven at 65±5℃ for 30-60min; take out the sections, immerse them in xylene twice at room temperature, and dewax them for 10±1min respectively. Move the sections up and down from time to time during the period to ensure sufficient dewaxing; immerse the sections in 100%, 100%, 95%, 95%, and 75% ethanol in turn, each time for 5±1min; slowly immerse the sections in a vertical staining bottle containing preheated 1xAntigen Retrieval buffer and incubate at 97±3℃ for 30±5min, then cool to 70±5℃ at room temperature, transfer to a vertical staining bottle containing ddH2O, wash for 10±2min, then transfer to a vertical staining bottle containing DPBS, wash for 10±2min, and use an immunohistochemistry pen to draw circles around the sections; use SuperBlock TM Block with blocking buffer for 45±2 minutes; remove the blocking solution, drip the antibody onto the tissue section, incubate overnight at 4±2°C, wash several times with DPBS containing 0.2% Triton-X, and then wash several times with DPBS; drip the diluted dye onto the tissue section, stain at room temperature in a wet box, rinse, and dry for detection on an imaging mass spectrometer.

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