Method for detecting mass spectrometry flow cytometry antibodies and its applications

Abstract

The present invention provides a method for detecting antibodies by mass cytometry and its application, which relates to the technical field of antibody detection. In the present invention, the amino group at the Fc end of the antibody is modified to form a structure that can be reduced to a thiol group, and then reduced to a thiol group, which binds to the maleimide on the FluoReporter product X8-polymer, greatly increasing the number of polymers linked to one antibody, meaning that more metals are linked, and greatly enhancing the detected metal signal. The present invention solves the technical problems that when using the labeling reagent X8-polymer in mass cytometry to detect weakly expressed antigen proteins, the signal is not obvious, and when detecting a large number of markers, using low-sensitivity metal tags results in low metal channel signals.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody detection, and particularly to a method for detecting antibodies by mass spectrometry flow cytometry and its application. Background Art

[0002] In recent years, mass spectrometry flow cytometry technology has been widely used in single-cell level research due to its advantages of high throughput, low background, and high sensitivity. When detecting cell markers (surface or intracellular proteins) by mass spectrometry flow cytometry, almost all commercially available polymer-based labeling reagents - X8 polymer produced by Fluidigm are used. It belongs to bifunctional chelating agents (BFCAs) and can chelate metal isotopes and antibodies simultaneously. The polymer consists of a main chain, DTPA ligands, and a linker (maleimide) for connecting antibodies; each polymer can bind 20 - 50 metal ions, and each antibody contains approximately 100 - 250 metal ions. Although the current metal content is sufficient for detecting and quantifying biomolecules with high content, there are still the following technical problems: (1) When detecting weakly expressed antigen proteins, the signal is not obvious and needs to be improved; (2) When detecting a large number of markers, it is sometimes difficult to avoid using metal tags with low sensitivity, resulting in low signals in metal channels.

[0003] In view of this, the present invention is particularly proposed. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a method for detecting antibodies by mass spectrometry flow cytometry, which can improve the detection signal of weakly expressed antigen proteins and enhance the signal of low-sensitivity metal channels.

[0005] Another purpose of the present invention is to provide an application of the method for detecting antibodies by mass spectrometry flow cytometry.

[0006] In order to achieve the above purposes of the present invention, the following technical solutions are specifically adopted:

[0007] In a first aspect, a method for detecting antibodies by mass spectrometry flow cytometry includes the following steps:

[0008] (a) Reacting the amino group at the Fc end of the antibody with compound a to obtain a modified antibody;

[0009] Wherein, compound a has the structure represented by general formula (1) or general formula (2):

[0010]

[0011] Wherein, R1 is selected from groups that react with amino groups;

[0012]

[0013] Among them, R2 and / or R3 are selected from groups that react with amino groups;

[0014] (b) The modified antibody in step (a) is reduced to form sulfhydryl groups, obtaining a reduced antibody;

[0015] (c) The reduced antibody in step (b) reacts with the labeling reagent X8-polymer to obtain a sample;

[0016] The X8-polymer is linked with metal ions;

[0017] (d) The sample in step (c) is processed and then detected by mass cytometry.

[0018] Furthermore, the group that reacts with amino groups is selected from one of ester groups, acid anhydrides, isothiocyanates, and carboxyl groups.

[0019] Furthermore, the compound a includes at least one of thioacetyl succinic anhydride, thioacetyl diester, thioacetyl diacid, dithio anhydride, dithio acid, and dithio ester.

[0020] Furthermore, the reducing reagent for reduction includes at least one of TCEP, hydroxylamine, sodium borohydride, DTT, DTE, and THPP.

[0021] Furthermore, the conditions for reduction include water bath incubation at a temperature of 30 - 40 °C for 25 - 35 min.

[0022] Furthermore, in step (c), the conditions for the reaction include water bath incubation at a temperature of 30 - 40 °C for 80 - 100 min.

[0023] Furthermore, in step (d), the device for detection by mass cytometry includes a mass cytometer.

[0024] In a second aspect, an application of the method according to any one of the above in the detection of low-abundance proteins.

[0025] Furthermore, the protein includes weakly expressed antigen proteins.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] The method for detecting antibodies by mass cytometry provided by the present invention can improve the detection signal of weakly expressed antigen proteins and enhance the signal of metal channels with low sensitivity. Specifically, in the method of the present invention, the amino group at the Fc end of the antibody is modified to form a structure that can be reduced to a thiol group, and then the thiol group is formed through reduction and combined with the maleimide on the FluoReporter product X8-polymer. Thus, not only the thiol group after the reduction of the disulfide bond on an antibody is connected to the polymer, but also the thiol group generated by the reaction and reduction of the amino group is connected to the polymer, thereby greatly increasing the number of polymers connected to an antibody. The increase in the number of polymers connected to the antibody means an increase in the metals it binds, and also greatly enhances the metal signal detected by mass cytometry technology.

[0028] The application of the method for detecting antibodies by mass cytometry provided by the present invention has the same advantages as the above method and will not be elaborated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 It is a schematic diagram of the modification and reduction of the amino group at the Fc end of the antibody provided by one embodiment of the present invention;

[0031] Figure 2 It is a schematic diagram of the modification and reduction of the amino group at the Fc end of the antibody provided by one embodiment of the present invention;

[0032] Figure 3 It is a comparison chart of metal ion signal detection provided by Example 1 and Comparative Example 1 of the present invention;

[0033] Figure 4 It is a flow cytometry diagram obtained from the test example of the present invention (the detected antibody is CD25);

[0034] Figure 5 It is a flow cytometry diagram obtained from the test example of the present invention (the detected antibody is CD14);

[0035] Figure 6 It is a flow cytometry diagram obtained from the test example of the present invention (the detected antibody is TCRγδ). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] According to a first aspect of the present invention, there is provided a method for detecting an antibody by mass cytometry, comprising the following steps:

[0038] (a) Reacting the amino group at the Fc end of the antibody with compound a to obtain a modified antibody;

[0039] Among them, compound a has a structure represented by general formula (1) or general formula (2):

[0040]

[0041] Among them, R1 is selected from groups that react with amino groups;

[0042]

[0043] Among them, R2 and / or R3 are selected from groups that react with amino groups;

[0044] (b) The modified antibody in step (a) is reduced to form a thiol group to obtain a reduced antibody;

[0045] (c) Reacting the reduced antibody in step (b) with the labeling reagent X8-polymer to obtain a sample, wherein X8-polymer is linked with metal ions;

[0046] (d) The sample in step (c) is processed and then detected by mass cytometry technology.

[0047] The method for detecting an antibody by mass cytometry provided by the present invention can improve the detection signal of weakly expressed antigen proteins and enhance the signal of metal channels with low sensitivity; specifically, the method of the present invention modifies the amino group at the Fc end of the antibody to make the amino group into a structure that can be reduced to a thiol group, and then forms a thiol group through reduction, and binds to the maleimide on the X8-polymer polymer of the FluoProbe product. In this way, not only the thiol group after the reduction of the disulfide bond on an antibody is linked to the polymer, but also the thiol group generated by the reaction and reduction of the amino group is linked to the polymer, so that the number of polymers linked to an antibody is greatly increased. The increase in the number of polymers linked to the antibody means that the metal linked to it increases, which also makes the metal signal detected by mass cytometry technology greatly enhanced.

[0048] In a preferred embodiment, the group reacting with the amino group in the present invention is selected from, but not limited to, an ester group, an acid anhydride, isothiocyanate, and a carboxyl group.

[0049] The amino group at the Fc terminus of the antibody reacts with compound a to convert the amino group into a structure that can be reduced to a mercapto group, and then is reduced to form a mercapto group, as shown in Figure 1 , where R is a structure that can react with the amino group.

[0050] In a preferred embodiment, compound a in the present invention includes, but is not limited to, at least one of thioacetyl succinic anhydride, thioacetyl diester, thioacetyl diacid, dithioanhydride, dithioacid, and dithioester.

[0051] The amino group at the Fc terminus of the antibody is modified by a specific compound to become a structure that can be reduced to a mercapto group, and then is reduced to form a mercapto group, as shown in Figure 2 .

[0052] In a preferred embodiment, the reducing reagent for reduction in the present invention includes, but is not limited to, at least one of TCEP, hydroxylamine, sodium borohydride, DTT, DTE, and THPP, which is more conducive to reducing the modified antibody to form a mercapto group.

[0053] In the present invention, the reduction conditions include, but are not limited to, water bath incubation at a temperature of 30 - 40 °C for 25 - 35 min, which is more conducive to the progress of the reduction reaction and improves the reduction effect.

[0054] In a preferred embodiment, in step (c), the reaction conditions include water bath incubation at a temperature of 30 - 40 °C for 80 - 100 min, which is more conducive to improving the ligation effect between the labeling reagent X8-polymer and the antibody.

[0055] In the present invention, the device for detection using the mass cytometry technology includes a mass cytometer.

[0056] Compared with the prior art, the detection method provided by the present invention can enhance the overall detection signal. Especially for the detection of low-abundance and weakly expressed antigen proteins, it can greatly enhance their detection signals. The detection method of the present invention has an obvious enhancing effect on the metal channel signals with low sensitivity. Even when there are many detection markers, good metal signal detection results can be obtained in each channel, thereby greatly improving the utilization rate of the metal channels, greatly increasing the number of indicators that can be detected in one tube of sample, saving more samples, being more likely to obtain more information, and even when the cell number in the sample is slightly less, relatively stronger signals can still be detected.

[0057] According to the second aspect of the present invention, there is provided an application of the method according to any one of the above in the detection of low-abundance proteins.

[0058] In the present invention, the protein includes but is not limited to weakly expressed antigen proteins.

[0059] Although the content of some weakly expressed antigen proteins in the human body is low, they are essential, especially for the detection, diagnosis, and prevention of some diseases. Weakly expressed antigen proteins play a crucial role. When the method of the present invention is applied to the detection of low-abundance weakly expressed antigen proteins, the detection signal can be greatly enhanced.

[0060] The present invention will be further described below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0061] Example 1

[0062] A method for detecting antibodies by mass spectrometry flow cytometry, comprising the following steps:

[0063] A. Antibody modification: In a 0.1 M PBS solution of 0.15 M NaCl, 5 μL of thioacetyl succinic anhydride solution and 50 μL of antibody (1 mg / mL) are added, such that the molar ratio of thioacetyl succinic anhydride to the antibody is 20:1; then, incubated at room temperature for 30 min, and then transferred to a 10 kDa ultrafiltration tube, centrifuged at room temperature for 10 min, and washed 2 times with PBS to obtain the modified antibody;

[0064] Among them, the antibody used is the naked antibody of Biolegend: CD3;

[0065] B. Antibody reduction: Transfer 100 μg of the modified antibody to a 50 kDa ultrafiltration tube, and add TBS buffer and a reducing agent (the reducing agent is TCEP, and its final concentration is 4 mM), incubate in a water bath at 37 °C for 30 min. After the incubation, add TBS buffer and wash repeatedly 2 - 3 times;

[0066] C. Connecting antibody: The X8-polymer conjugated with metal ions is poured into a 50 kDa column containing the reduced antibody, incubated in a water bath at 37 °C for 90 min. After the incubation, wash repeatedly 3 - 5 times with TBS buffer;

[0067] D. Collect the product of step C and dilute it with TBS to 0.5 mg / mL;

[0068] E. For 1 mL of peripheral blood sample, add 1× lysin in a ratio of 1:20, mix well by shaking, incubate at room temperature for 10 min, centrifuge at 350 g at room temperature for 8 min, resuspend with PBS and centrifuge again;

[0069] F. Add a Pt solution with a final concentration of 0.5 μM;

[0070] G. Add 2 mL of Cell Staining Buffer to terminate the reaction, centrifuge at 300 g for 5 min at room temperature, discard the supernatant, and leave approximately 50 μL of residual liquid;

[0071] H. Add 5 μL of Fc receptor blocker to each tube of sample, resuspend the cells, and incubate at room temperature for 10 min;

[0072] I. Add 50 μL of antibody mixture (the amount of each antibody is 0.5 μg and buffer), and incubate at room temperature for 30 min;

[0073] J. Add 2 mL of Cell Stain Buffer, centrifuge at 300 g for 5 min at room temperature, discard the supernatant; repeat once;

[0074] K. Add Ir at a final concentration of 125 nM, incubate at room temperature for 1 h, and then centrifuge at 800 g for 5 min at room temperature;

[0075] L. Count the cells, and according to the counting result, dilute the sample with 2% dilute nitric acid to make the cell concentration between 0.5 - 1×10^6 cells / mL;

[0076] M. Perform mass spectrometry flow cytometry ICP-MS on the machine for testing, and the signal value of metal ions (115In) is shown in Figure 3 。

[0077] Example 2

[0078] The difference between this example and Example 1 is that the antibody used in this example is the naked anti-CD25 from Biolegend, and the rest are the same as in Example 1. Detection is performed using the mass spectrometry flow cytometer helios, and the FCS data after detection is analyzed using Flowjo.

[0079] Example 3

[0080] The difference between this example and Example 1 is that the compound for modifying the antibody in this example is thioacetyl diester, and the rest are the same as in Example 1, and the same detection effect as in Example 1 can be achieved.

[0081] Example 4

[0082] The difference between this example and Example 1 is that the compound for modifying the antibody in this example is dithioanhydride, and the rest are the same as in Example 1, and the same detection effect as in Example 1 can be achieved.

[0083] Example 5

[0084] The difference between this example and Example 1 is that the compound for modifying the antibody in this example is dithioester, and the rest are the same as in Example 1, and the same detection effect as in Example 1 can be achieved.

[0085] Comparative Example 1

[0086] The difference between this comparative example and Example 1 is that in step A of this comparative example, the antibody was not modified, and the rest were the same as in Example 1. The signal value of the metal ion (115In) is shown in Figure 3 , and the results show that the signal value of 115In measured in Example 1 is about 3 times that of Comparative Example 1.

[0087] Comparative Example 2

[0088] The difference between this comparative example and Example 2 is that in step A of this comparative example, the antibody was not modified, and the rest were the same as in Example 1. The results show that for the low-abundance antigen CD25, the content of CD25 detected by the method of Example 2 increased significantly, about 2 times that detected by Comparative Example 2.

[0089] Test Example

[0090] The antibodies CD25, CD14, and TCRgd were detected by the method of mass spectrometry flow cytometry for detecting antibodies in Example 1 as the test group, and the detection method with the antibody not modified was used as the control group to obtain flow cytometry diagrams. The results are shown in Figure 4 , Figure 5 and Figure 6 .

[0091] Flow cytometry diagram: Look at the stability of the data from time and event length. By removing the quality control EQ beads, circle the cells, remove the adhesion and circle the mononuclear cells, then circle the live cells, circle the lymphocytes, and then gradually circle the target cells through surface markers.

[0092] From Figure 4 , Figure 5 and Figure 6 it can be seen that the metal signal intensity detected for CD25 in the test group is about 2 times that of the control group, the metal signal intensity detected for CD14 in the test group is about 4 times that of the control group, and the metal signal intensity detected for TCRgd in the test group is about 2 times that of the control group.

[0093] In summary, the method of the present invention modifies the amino group at the Fc end of the antibody to make the amino group into a structure that can be reduced to a mercapto group, and then forms a mercapto group through reduction, which binds to the maleimide on the Fludara product X8-polymer. Thus, not only the mercapto group after the reduction of the disulfide bond on an antibody is connected to the polymer, but also the mercapto group generated by the reaction and reduction of the amino group is connected to the polymer, so that the number of polymers connected to an antibody is greatly increased. The increase in the number of polymers connected to the antibody means that the metal connected to it increases, which also makes the metal signal detected by the mass spectrometry flow cytometry technology greatly enhanced.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting antibodies by mass spectrometry flow cytometry, characterized in that, It includes the following steps: (a) The amino group at the Fc end of the antibody reacts with compound a to obtain a modified antibody; Among them, the compound a has the structure represented by general formula (1) or general formula (2): （1）； Among them, R1 is selected from the groups reactive with amino groups; （2）； Among them, R2 and / or R3 are selected from the groups reactive with amino groups; (b) The modified antibody in step (a) is reduced to form sulfhydryl groups to obtain a reduced antibody; (c) The reduced antibody in step (b) reacts with the labeling reagent X8-polymer to obtain a sample; The X8-polymer is connected with metal ions; (d) The sample in step (c) is processed and then detected by mass cytometry.

2. The method according to claim 1, wherein The group reactive with amino groups is selected from one of ester groups, acid anhydrides, isothiocyanates, and carboxyl groups.

3. The method according to claim 1, wherein The compound a includes at least one of thioacetyl succinic anhydride, thioacetyl diester, thioacetyl diacid, dithio acid anhydride, dithio acid, and dithio ester.

4. The method according to claim 1, characterized in that The reducing reagent for reduction includes at least one of TCEP, hydroxylamine, sodium borohydride, DTT, DTE, and THPP.

5. The method according to claim 4, wherein The conditions for reduction include water bath incubation at a temperature of 30-40 °C for 25-35 min.

6. The method according to claim 1, characterized in that, In step (c), the conditions for the reaction include water bath incubation at a temperature of 30-40 °C for 80-100 min.

7. The method according to claim 1, wherein In step (d), the equipment for detection by mass cytometry includes a mass cytometer.

8. Use of the method according to any one of claims 1-7 in the detection of low-abundance proteins.

9. The application according to claim 8, wherein The protein includes weakly expressed antigen proteins.

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