CN117288952A - Antibody composition for detecting multiple myeloma based on full spectrum flow cytometry and application thereof - Google Patents
Antibody composition for detecting multiple myeloma based on full spectrum flow cytometry and application thereof Download PDFInfo
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides an antibody composition for detecting multiple myeloma based on full spectrum flow cytometry and application thereof, belonging to the technical field of multiple myeloma detection. The antibody composition includes a CD3 antibody, a CD20 antibody, an HLA-DR antibody, a CD8 antibody, a CD11b antibody, a CD10 antibody, a CD71 antibody, a CD28 antibody, a CD14 antibody, a CD13 antibody, an igκ antibody, a CD15 antibody, an igλ antibody, a CD117 antibody, a CD56 antibody, a CD45 antibody, a CD19 antibody, a CD34 antibody, a CD138 antibody, a CD4 antibody, a CD38 antibody, a CD22 antibody, and a CD27 antibody. The kit containing the antibody composition can be applied to a full spectrum flow cytometer, and can accurately perform primary screening on patients with multiple myeloma.
Description
Technical Field
The invention belongs to the technical field of disease detection, in particular to the technical field of multiple myeloma detection, and particularly relates to an antibody composition for detecting multiple myeloma based on full spectrum flow cytometry and application thereof.
Background
Multiple Myeloma (MM) is a serious hematological malignancy, the malignant cells originating from abnormal plasma cells in bone marrow tissue. The main symptoms of the disease include hypercalcemia, bone diseases, renal insufficiency, anemia and the like. Disease progression and subsequent relapse are characterized by subcloning and drug resistance, and almost all patients eventually develop a relapse and refractory type. In recent years, laboratory detection is carried out on patients with multiple myeloma by using a plurality of high-sensitivity detection technologies, so that basis is provided for early diagnosis of multiple myeloma, and the method is a research hotspot of clinical test subjects.
Chinese patent application publication CN111912983a discloses an antibody composition for detecting minimal residual lesions of multiple myeloma, a kit and application thereof. The antibody composition is composed of antibody CD319 or antibody CD269, and antibodies CD138, CD38, CD184, CD27, CD19, CD56, ckappa and clamda. The kit containing the antibody composition can be used for detecting tiny residual lesions after conventional chemotherapy drug treatment. However, this protocol is only suitable for detecting minimal residual lesions after treatment in patients with established diagnosis, and cannot be used for the initial diagnosis of the disease.
Disclosure of Invention
In order to solve the above problems, a first aspect of the present invention is to provide an antibody composition for detecting multiple myeloma based on full spectrum flow cytometry. The antibody compositions of the invention can be used for making a preliminary diagnosis in cases where a patient is suspected of having multiple myeloma, and are useful for clinically classifying diseases.
Specifically, the technical scheme for achieving the purpose of the invention is as follows:
an antibody composition for detecting multiple myeloma based on whole-spectrum flow cytometry, the antibody composition comprising a CD3 antibody, a CD20 antibody, an HLA-DR antibody, a CD8 antibody, a CD11b antibody, a CD10 antibody, a CD71 antibody, a CD28 antibody, a CD14 antibody, a CD13 antibody, an igκ antibody, a CD15 antibody, an igλ antibody, a CD117 antibody, a CD56 antibody, a CD45 antibody, a CD19 antibody, a CD34 antibody, a CD138 antibody, a CD4 antibody, a CD38 antibody, a CD22 antibody, and a CD27 antibody.
In a preferred embodiment, each antibody is a fluorescein-labeled antibody.
In a further preferred embodiment, the fluorescein label of the CD3 antibody is cflur V420, the fluorescein label of the CD20 antibody is cflur V450, the fluorescein label of the HLA-DR antibody is BV510, the fluorescein label of the CD8 antibody is cflur V547, the fluorescein label of the CD11B antibody is BV570, the fluorescein label of the CD10 antibody is BV605, the fluorescein label of the CD71 antibody is BV650, the fluorescein label of the CD28 antibody is BV711, the fluorescein label of the CD14 antibody is BV750, the fluorescein label of the CD13 antibody is BV785, the fluorescein label of the Ig kappa antibody is AF488, the fluorescein label of the CD15 antibody is cflur B548, the fluorescein label of the Ig lambda antibody is PE, the fluorescein label of the CD117 antibody is cflur 610, the fluorescein label of the CD56 antibody is PE-5, the fluorescein label of the CD45 is Cy 45, the fluorescein label of the CD 19-CD 7 is cfrcf 27, the fluorescein label of the APC is CD38, the fluorescein label of the CD27 is Fire 9, and the fluorescein label of the CD38 is Fire antibody is firrb 720.
In a second aspect, the invention provides the use of the antibody composition in the manufacture of a product for detecting multiple myeloma based on full spectral flow cytometry.
In a preferred embodiment, the product is a kit.
In a third aspect the invention provides a kit for detecting multiple myeloma based on full spectrum flow cytometry, said kit comprising said antibody composition.
In a preferred embodiment, the kit comprises a erythropoietin, a membrane breaker and a fluorescent agent.
In preferred embodiments, the volume ratio of the HLA-DR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the igkappa antibody, the iglambda antibody, the CD117 antibody, the CD34 antibody, the CD22 antibody, the CD27 antibody, the CD45 antibody, the CD3 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD19 antibody, the CD38 antibody, the CD20 antibody, the CD138 antibody, the CD4 antibody, the CD56 antibody, the CD15 antibody is 5:5:5:5:5:5:5:5:3:2.5:2.5:2.5:2.5:2.5:2.5:2.5:2:2:2.2:2:2:2:2.1.25:1.
In a fourth aspect, the invention provides the use of the antibody composition in the preparation of a flow cytometric sample for detecting multiple myeloma based on full spectrum flow cytometry.
In a preferred embodiment, the step of preparing a flow cytometry-based on-machine sample for detecting multiple myeloma comprises:
the CD3 antibody, the CD20 antibody, the HL-ADR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD15 anti-antibodyThe body, the CD117 antibody, the CD56 antibody, the CD45 antibody, the CD19 antibody, the CD34 antibody, the CD138 antibody, the CD4 antibody, the CD38 antibody, the CD22 antibody, the CD27 antibody are added to a flow tube in a volume ratio of 2.5:2:5:5:5:5:2.5:2.5:2.5:1:1.25:3:2.5:5:2:2:2:2.5:5:5:5, and a test sample is added to the flow tube such that the number of cells in the flow tube is 2 x 10 5 Mixing uniformly, and incubating in a dark place; adding erythrocyte lysate, mixing, and incubating in dark; centrifuging, removing supernatant, adding buffer solution, washing, centrifuging again, and removing supernatant; adding a membrane breaker, uniformly mixing, incubating in a dark place, centrifuging, and removing the supernatant; adding the Ig kappa antibody and the Ig lambda antibody, uniformly mixing, incubating in a dark place, adding the buffer solution for washing, centrifuging, removing the supernatant, adding the buffer solution for resuspension, and adding a fluorescence stabilizer to obtain a flow cell on-machine sample; wherein the sample to be tested is bone marrow, peripheral blood or body cavity effusion (e.g. hydrothorax).
According to the invention, a patient sample is prepared into a cell suspension containing the antibody composition, and the cell suspension is detected by a full spectrum flow cytometry, so that the obtained data is analyzed, and whether abnormal expression exists in cells is judged. Plasma cells were judged for abnormalities by igκ, igλ, CD117, CD56, CD45, CD19, CD138, CD27, CD38 antibodies. Typically, the phenotype of the plasma cell is CD38+/CD56+/CD19-/CD138+, and after finding out this population, analysis of Igkappa/Iglambda is performed to determine if Igkappa/Iglambda <0.5 or Igkappa/Iglambda >3 is an abnormal plasma cell. And then judging whether other cell line abnormalities besides plasma cell abnormalities exist or not through judging the residual antibodies, wherein the T lymphocyte line-related antigens are as follows: CD3, CD8, CD4; the B lymphocyte line related antigens are: CD20, CD19, CD22; the myeloid-related antigens are: CD11b, CD14, CD13, CD15, CD117, CD56; stem progenitor cell line antigens are: HLA-DR, CD10, CD117, CD34, CD38; the erythrocyte line antigens are: CD71. If the series of antigens are abnormal, other kits are selected according to different series for further diagnosis.
In a preferred embodiment, each antibody is a fluorescein-labeled antibody.
In a further preferred embodiment, the fluorescein label of the CD3 antibody is cflur V420, the fluorescein label of the CD20 antibody is cflur V450, the fluorescein label of the HLA-DR antibody is BV510, the fluorescein label of the CD8 antibody is cflur V547, the fluorescein label of the CD11B antibody is BV570, the fluorescein label of the CD10 antibody is BV605, the fluorescein label of the CD71 antibody is BV650, the fluorescein label of the CD28 antibody is BV711, the fluorescein label of the CD14 antibody is BV750, the fluorescein label of the CD13 antibody is BV785, the fluorescein label of the Ig kappa antibody is AF488, the fluorescein label of the CD15 antibody is cflur B548, the fluorescein label of the Ig lambda antibody is PE, the fluorescein label of the CD117 antibody is cflur 610, the fluorescein label of the CD56 antibody is PE-5, the fluorescein label of the CD45 is Cy 45, the fluorescein label of the CD 19-CD 7 is cfrcf 27, the fluorescein label of the APC is CD38, the fluorescein label of the CD27 is Fire 9, and the fluorescein label of the CD38 is Fire antibody is firrb 720.
Compared with the prior art, the invention has the following beneficial effects: the antibody composition is special for a full-spectrum flow cytometry, can be used for primary screening of patients with multiple myeloma by full-spectrum flow cytometry, can distinguish normal plasma cells from abnormal plasma cells by 1 tube, and can save a great deal of samples. And the detection indexes of the 1-pipe are rich, so that the detection rate can be effectively improved. Meanwhile, the repeated application of the framework antibody is reduced, and the cost is greatly saved. The malignant tumor cells can be accurately found out by using the kit through data statistics of 362 times of multiple myeloma patient detection, combining clinical data and comparison with a traditional flow cytometer. Comparing with the detection result of the traditional flow cytometer, the diagnosis accuracy is found to be 100%, namely all samples can detect abnormal cells. The differences were statistically not significant (P > 0.05) compared to the proportion of abnormal plasma cells in nucleated cells from patients with multiple myeloma. The antigen types and the expression intensities are consistent, and the difference of the expression rates of the positive antigens has no statistical significance (P is more than 0.05). Meanwhile, the kit can make up for the technical deficiency of the full spectrum flow cytometry for diagnosing the multiple myeloma at the present stage.
Drawings
FIGS. 1 to 6 are test results of the kit of example 2 for detecting a patient sample with multiple myeloma using full-spectrum flow cytometry;
FIG. 7 is a graph comparing the results of the test of multiple myeloma patient samples using the kit of comparative example 1 and the kit of example 2.
Detailed Description
The following description sets forth a clear and complete description of the present invention, in connection with embodiments, so that those skilled in the art will fully understand the present invention. It will be apparent that the described embodiments are only some, but not all, of the preferred embodiments of the present invention. Any equivalent alterations or substitutions for the following embodiments without any inventive effort by those of ordinary skill in the art are intended to be within the scope of the present invention.
The sources of fluorescein-labeled antibodies used in the examples below are shown in table 1.
Table 1 sources of fluorescein-labeled antibodies in the examples
| Antibody name | Fluorescein (Lu) | Purchasing source | Goods number | |
| 1 | CD3 | cFluor V420 | Cytek Biosciences | R7-10053 |
| 2 | CD20 | cFluor V450 | Cytek Biosciences | R7-20015 |
| 3 | HLADR | BV510 | BioLegend | 307646 |
| 4 | CD8 | cFluor V547 | Cytek Biosciences | R7-10063 |
| 5 | CD11b | BV570 | BioLegend | 301325 |
| 6 | CD10 | BV605 | BioLegend | 312222 |
| 7 | CD71 | BV650 | BioLegend | 334116 |
| 8 | CD28 | BV711 | BioLegend | 302948 |
| 9 | CD14 | BV750 | BioLegend | 367136 |
| 10 | CD13 | BV785 | BioLegend | 301726 |
| 11 | Igκ | AF488 | BioLegend | 316512 |
| 12 | CD15 | cFluor B548 | Cytek Biosciences | R7-10025 |
| 13 | Igλ | PE | BioLegend | 316608 |
| 14 | CD117 | cFluor BYG610 | Cytek Biosciences | R7-10023 |
| 15 | CD56 | PE-Cy5 | BioLegend | 362516 |
| 16 | CD45 | PerCP-Cy5.5 | Cytek Biosciences | R7-10006 |
| 17 | CD19 | cFluor BYG710 | Cytek Biosciences | R7-20009 |
| 18 | CD34 | PE-Cy7 | BioLegend | 343516 |
| 19 | CD138 | BV-421 | BD Biosciences | 562935 |
| 20 | CD4 | cFluor R668 | Cytek Biosciences | R7-10049 |
| 21 | CD38 | cFluor R720 | Cytek Biosciences | R7-20061 |
| 22 | CD22 | APC-Fire750 | BioLegend | 363522 |
| 23 | CD27 | APC-Fire810 | BioLegend | 393214 |
The phosphate concentration of the PBS buffer in the examples was 0.02mol/L, and pH=7.2 to 7.6.
Example 1
The present example provides a kit for detecting multiple myeloma using full spectrum flow cytometry. The kit comprises an antibody composition as shown in table 2.
Table 2 antibody compositions for detection of multiple myeloma based on full spectral flow cytometry and markers and amounts thereof
Further, each of the antibodies in table 2 is a fluorescein-labeled antibody. The fluorescein label for each antibody is shown in table 2, with fluorescein in one-to-one correspondence with the antibody.
Further, the kit also comprises erythrocyte lysate, a membrane breaker and a fluorescent stabilizer.
Specifically, the erythrocyte lysate is one of BD lysis Buffer 10×Conntrate (product number: 555899, manufactured by BD Co., ltd.) and Kaplan hemolysin (manufactured by Henan Kaplan Biotechnology Co., ltd.). The membrane breaker is one of BD Cytofix/Cytoperm Fixation and Permeabilization Solution (product number: 554722), beckman PerFix-nc and BD IntraSure. The fluorescent stabilizer is BD Brilliant Stain Buffer.
Example 2
The present embodiment uses the kit in embodiment 1 to detect a sample to be detected, including the following steps:
preparing a sample to be tested: the concentration of the bone marrow sample is adjusted to 5X 10 6 ～5×10 7 Individual cells/mL.
The amounts in Table 2 were followed for the cFluor V420-labeled CD3 antibody, the cFluor V450-labeled CD20 antibody, the BV 510-labeled HL-ADR antibody, the cFluor V547-labeled CD8 antibody, the BV 570-labeled CD11B antibody, the BV 605-labeled CD10 antibody, the BV 650-labeled CD71 antibody, the BV 711-labeled CD28 antibody, the BV 750-labeled CD14 antibody, the BV 785-labeled CD13 antibody, the cFluor B548-labeled CD15 antibody, the cFluor BYG 610-labeled CD117 antibody, and the PE-Cy 5-labeled CD117 antibodyCD56 antibody, perCP-Cy5.5 labeled CD45 antibody, cFluor BYG710 labeled CD19 antibody, PE-Cy7 labeled CD34 antibody, BV421 labeled CD138 antibody, cFluor R668 labeled CD4 antibody, cFluor R720 labeled CD38 antibody, APC-Fire750 labeled CD22 antibody, APC-Fire810 labeled CD27 antibody are sequentially added to a flow tube, and a sample to be tested is added to give a cell number of 2X 10 in the flow tube 5 Separately, the mixture was incubated for 15min at room temperature under dark conditions, 1mL of 1 XBD lysis Buffer 10 Xconcentration red blood cell lysate was added, gently mixed, and incubated for 15min at room temperature under dark conditions.
1mL of a 1 XBD lysis Buffer 10 XConntrate erythrocyte lysate was added to the flow tube, and after mixing, incubated at room temperature for 8min in the absence of light. The flow tube was then placed on a low speed centrifuge and centrifuged at 300rcf/min for 5 minutes to remove the supernatant.
1mL of PBS buffer solution is added into a flow tube, uniformly mixed, and centrifuged at 150rcf/min for 5 minutes, and the supernatant is removed; then 0.5mL BD Cytofix/Cytoperm Fixation and Permeabilization Solution membrane breaker is added, the mixture is gently mixed, and incubated for 10min at room temperature in a dark place. Centrifuging at 300rcf/min for 5min in a low-speed centrifuge, and removing the supernatant; AF 488-labeled Ig kappa antibody and PE-labeled Ig lambda antibody were sequentially added according to the amounts shown in Table 2, gently mixed, and incubated for 30min in the dark. Then adding 1mL of PBS, uniformly mixing, centrifuging at a speed of 150rcf/min for 5 minutes, and removing the supernatant; add 150 μl PBS buffer for resuspension; then 50 mu L BD Brilliant Stain Buffer of a fluorescent light stabilizer was added.
And (3) placing the flow tube on a Cytek NL-CLC flow cytometer for detection, adjusting instrument conditions according to each quality control, acquiring 5 ten thousand signals, exporting data into an fcs format, and analyzing by using Beckman kaluza software, wherein the analysis results are shown in figures 1-6. In FIGS. 1 to 6, the MM population (red color in the drawing) is an abnormal cell population, and positive expression of CD38, CD138, CD56, igkappa, and the phenotype is an abnormal plasma cell population, so that multiple myeloma can be diagnosed.
It should be noted that, in addition to the bone marrow sample, the sample to be tested may also be a peripheral blood sample or a body cavity effusion sample (for example, hydrothorax) according to practical situations. The concentration of the sample to be measured is adjusted to 5 multiplied by 10 6 ～5×10 7 Individual cells/mL, and then used for detection.
Example 3
This example demonstrates the results of the assay in example 2 using a conventional flow cytometer (Wei Shuangyu. Diagnostic value analysis of multiple myeloma by flow cytometry immunophenotyping [ J ]. J. Of clinical medicine literature, 2020.). The sample to be measured is a bone marrow sample. 362 samples tested showed 100% diagnostic accuracy using the method of example 2 compared to the conventional flow cytometer, i.e., all samples detected abnormal cells. The proportion of abnormal plasma cells in nucleated cells of the patients with multiple myeloma in the two groups of experiments was compared, and the difference was not statistically significant (P > 0.05). The antigen types and the expression intensities are consistent, and the difference of the expression rates of the positive antigens has no statistical significance (P is more than 0.05).
Comparative example 1
In this comparative example, the BV 421-labeled CD138 antibody of example 2 was replaced with the APC-labeled CD138 antibody, and the test sample (bone marrow sample) was examined in the same manner as in example 2, and the results of the examination are shown in FIG. 7. FIG. 7 (a) shows the results of CD138 antibody clustering in example 2, and (b) shows the results of CD138 antibody clustering in this comparative example. As can be seen from fig. 7, the detection result of the CD138 antibody of example 2 was positive, whereas the detection result of the CD138 antibody of the present comparative example was negative, affecting the judgment of the detection result.
The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Various modifications and alterations of this invention will occur to those skilled in the art. Any and all such simple and equivalent variations and modifications are intended to be included within the scope of this invention.
Claims (10)
1. An antibody composition for detecting multiple myeloma based on whole-spectrum flow cytometry, wherein the antibody composition comprises a CD3 antibody, a CD20 antibody, an HLA-DR antibody, a CD8 antibody, a CD11b antibody, a CD10 antibody, a CD71 antibody, a CD28 antibody, a CD14 antibody, a CD13 antibody, an igκ antibody, a CD15 antibody, an igλ antibody, a CD117 antibody, a CD56 antibody, a CD45 antibody, a CD19 antibody, a CD34 antibody, a CD138 antibody, a CD4 antibody, a CD38 antibody, a CD22 antibody, and a CD27 antibody.
2. The antibody composition of claim 1, wherein each antibody is a fluorescein-labeled antibody.
3. The antibody composition of claim 2, wherein the CD3 antibody has a fluorescein label of cflur V420, the CD20 antibody has a fluorescein label of cflur V450, the HLA-DR antibody has a fluorescein label of BV510, the CD8 antibody has a fluorescein label of cflur V547, the CD11B antibody has a fluorescein label of BV570, the CD10 antibody has a fluorescein label of BV605, the CD71 antibody has a fluorescein label of BV650, the CD28 antibody has a fluorescein label of BV711, the CD14 antibody has a fluorescein label of BV750, the CD13 antibody has a fluorescein label of BV785, the Ig kappa antibody has a fluorescein label of AF488, the CD15 antibody has a fluorescein label of cflur B548, the CD117 antibody has a fluorescein label of cflur g610, the CD56 antibody has a fluorescein label of BV5, the CD45 has a fluorescein label of cfrce 45, the CD27 has a fluorescein label of CD27, the CD19 has a fluorescein label of cfrce 138, and the CD 19-B antibody has a fluorescein label of cfrce.g 9.
4. Use of an antibody composition according to any one of claims 1 to 3 for the preparation of a product based on full spectral flow cytometry for the detection of multiple myeloma.
5. The use according to claim 4, wherein the product is a kit.
6. A kit for detecting multiple myeloma based on full spectrum flow cytometry, comprising the antibody composition of any one of claims 1-3.
7. The kit of claim 6, wherein the kit comprises a erythrocyte lysate, a membrane breaker, and a fluorescent agent.
8. The kit of claim 6, wherein the volume ratio of the HLA-DR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the igκ antibody, the igλ antibody, the CD117 antibody, the CD34 antibody, the CD22 antibody, the CD27 antibody, the CD45 antibody, the CD3 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD19 antibody, the CD38 antibody, the CD20 antibody, the CD138 antibody, the CD4 antibody, the CD56 antibody, the CD15 antibody is 5:5:5:5:5:5:5:5:3:2.5:2.5:2.5:2.5:2.5:2.2:2:2.1.25:1.
9. Use of an antibody composition according to any one of claims 1 to 3 for the preparation of a flow cytometric sample for the detection of multiple myeloma based on full spectrum flow cytometry.
10. The use of claim 9, wherein the step of preparing a flow cytometric sample for detecting multiple myeloma based on full spectrum flow cytometry comprises:
the CD3 antibody, the CD20 antibody, the HL-ADR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the CD28 antibody, the CD14 antibody the CD13 antibody, the CD15 antibody, the CD117 antibody, the CD56 antibody, the CD45 antibody, the CD19 antibody, the CD34 antibody, the CD138 antibody, the CD4 antibody, the CD38 anti-antibodyThe body, the CD22 antibody and the CD27 antibody are added into a flow tube according to the volume ratio of 2.5:2:5:5:5:5:5:2.5:2.5:2.5:1.25:3:2.5:5:2:2:2.5:5:2:2.5:5, and a sample to be tested is added, so that the number of cells in the flow tube is 2X 10 5 Mixing uniformly, and incubating in a dark place; adding erythrocyte lysate, mixing, and incubating in dark; centrifuging, removing supernatant, adding buffer solution, washing, centrifuging again, and removing supernatant; adding a membrane breaker, uniformly mixing, incubating in a dark place, centrifuging, and removing the supernatant; adding the Ig kappa antibody and the Ig lambda antibody, uniformly mixing, incubating in a dark place, adding the buffer solution for washing, centrifuging, removing the supernatant, and then
Then adding the buffer solution for resuspension, and adding a fluorescence stabilizer to obtain a flow cell loading sample; wherein,
the sample to be tested is marrow, peripheral blood or body cavity effusion.
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