A kit for individualized diagnosis of M protein in multiple myeloma patients and application thereof

Abstract

The application discloses a kit for individualized diagnosis of M protein of a multiple myeloma patient and application thereof, relates to the technical field of multiple myeloma related M protein detection, and the detection reagent comprises Kunitz type soybean trypsin inhibitor and 1% formic acid solution. The Kunitz type soybean trypsin inhibitor is used as a standard substance, mass spectrometry technology is used to individualize detection of the accurate molecular weight and peak intensity of M protein complete light chains in blood, and the content of the M protein complete light chains in the patient is accurately quantified according to the amount of the added standard substance, so that a new technical method and support are provided for detection of multiple myeloma M protein microresiduals in a treatment process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of multiple myeloma related M protein detection, and more particularly to a kit for individualized diagnosis of M protein of multiple myeloma patients and application thereof. BACKGROUND

[0002] Multiple myeloma (MM) is a common hematological malignancy characterized by the abnormal proliferation of monoclonal plasma cells in the bone marrow. M protein is an abnormal protein found in the serum or urine of MM patients, which is secreted by monoclonal plasma cells. Its detection is of great significance for the diagnosis, treatment monitoring and prognosis evaluation of MM. Therefore, it is of great value to develop an accurate and efficient M protein detection method.

[0003] Although there are currently various methods for detecting M protein, such as serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), serum free light chain (sFLC), etc., these methods have certain limitations, such as low sensitivity, weak specificity, and complicated operation, etc. With the development of molecular biology and immunology technology, M protein mass spectrometry detection methods based on standard protein quantification have gradually shown their unique advantages.

[0004] Therefore, it is a technical problem that needs to be solved by those skilled in the art to develop a new M protein quantitative detection method based on mass spectrometry technology. SUMMARY

[0005] Therefore, the present application is proposed.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The first aspect of the embodiment of the present application is the application of Kunitz Trypsin Inhibitor (KTI) in the preparation of a kit for detecting multiple myeloma related M protein.

[0008] The second aspect of the embodiment of the present application provides a detection reagent for multiple myeloma related M protein, which comprises Kunitz Trypsin Inhibitor and 1% formic acid solution.

[0009] In a preferred embodiment, the Kunitz Trypsin Inhibitor contains 216 amino acids, and the amino acid sequence is shown in SEQ ID NO. 1.

[0010] The third aspect of the embodiment of the present application provides application of the detection reagent for the M protein related to multiple myeloma in preparation of a kit for detecting the M protein related to multiple myeloma.

[0011] The fourth aspect of the embodiment of the present application provides a kit for detecting the M protein related to multiple myeloma, comprising Kunitz type soybean trypsin inhibitor and 1% formic acid solution.

[0012] The fifth aspect of the embodiment of the present application provides a system for detecting the M protein related to multiple myeloma, comprising the kit and a MALDI-TOF mass spectrometer.

[0013] The sixth aspect of the embodiment of the present application provides use of the system for detecting the M protein related to multiple myeloma, which is used for quantitative residual detection of the M protein related to multiple myeloma for non-diagnostic treatment purposes.

[0014] In a preferred embodiment, the process comprises:

[0015] S1: capturing the M protein intact light chain in the plasma by an immunization affinity microsphere;

[0016] S2: obtaining a working solution by adding a certain amount of Kunitz type soybean trypsin inhibitor standard in the kit to the sample after mixing;

[0017] S3: detecting the working solution by a MALDI-TOF mass spectrometer;

[0018] S4: obtaining the accurate molecular weight of the M protein intact light chain by correcting the mass spectrum after the accurate molecular weight of the Kunitz type soybean trypsin inhibitor molecule;

[0019] S5: reading the Kunitz type soybean trypsin inhibitor mass spectrum peak intensity (S1) and IMLC peak intensity (S2) in the MALDI TOF MS result by felxAnalysis software, and calculating the concentration C2 of IMLC = 40*(S2 / S1) / C1 mg / mL.

[0020] The immunization affinity microsphere comprises Capture Select Kappa XL and Capture Select LC-Lambda capture microsphere

[0021] Compared with the prior art, the detection reagent can be used for diagnosing multiple myeloma patients, improving the accuracy and efficiency of diagnosis, and helping patients to obtain better treatment opportunities. Secondly, it can be used for treatment monitoring of multiple myeloma patients, helping clinicians to understand the treatment effect in time and adjusting the treatment plan. In addition, it can also be used for prognosis evaluation of multiple myeloma patients, providing a scientific basis for the rehabilitation and prevention of recurrence of patients. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0023] Figure 1 The figure is the mass spectrum result graph of the accurate molecular weight of KTI molecules measured by high-resolution Q-Exactive MS.

[0024] Figure 2 The figure is the accurate molecular weight and quantitative mass spectrum graph of IMLC molecules detected by MALDI TOF MS and corrected by the accurate molecular weight of KTI molecules. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0026] The present application uses the detection reagent to prepare a M protein detection kit based on standard protein quantification and MALDI-TOF MS technology, which can realize rapid and accurate diagnosis of multiple myeloma patients, and provide strong support for clinical diagnosis and treatment. The research and application of the kit will help to improve the diagnosis and treatment level of multiple myeloma and improve the quality of life of patients.

[0027] The Kunitz type soybean trypsin inhibitor contains 216 amino acids, and the amino acid sequence is shown as SEQ ID NO. 1.

[0028] MKSTIFFLFL FCAFTTSYLP SAIADFVLDN EGNPLENGGT YYILSDITAF GGIRAAPTGNERCPLTVVQS RNELDKGIGT IISSPYRIRF IAEGHPLSLK FDSFAVIMLC VGIPTEWSVV EDLPEGPAVKIGENKDAMDG WFRLERVSDD EFNNYKLVFC PQQAEDDKCG DIGISIDHDD GTRRLVVSKN KPLVVQFQKLDKESLAKKNH GLSRSE.

[0029] The isolation, detection and accurate molecular weight identification of M protein in the embodiment are achieved by the following steps:

[0030] 1) Mix 20 μL of plasma with 160 μL of 10 mM PBS buffer uniformly, and make sure that the plasma and the buffer are fully mixed.

[0031] 2) Add 20 μL of Capture Select Kappa XL and Capture Select LC-Lambda capture microspheres (mixed at a ratio of 1:1) to the mixture, and then perform oscillation reaction for 45 minutes to ensure that the microspheres can effectively capture target molecules.

[0032] 3) After the reaction is completed, remove the supernatant by low-speed centrifugation. Then, use 200 μL of 10 mM PBS buffer to wash three times, and then use pure water to wash three times. After each washing, retain the microsphere precipitate by low-speed centrifugation to remove non-specifically bound impurities.

[0033] 4) Add 20 μL of 50 mM TECP solution (prepared by 1% formic acid) to the washed microsphere precipitate, and perform oscillation elution for 10 minutes to release the captured target molecules.

[0034] 5) After elution is completed, perform high-speed centrifugation at 15000 g for 5 minutes. After centrifugation, take 20 μL of IMLC eluent and place it in an EP tube.

[0035] 6) Dissolve KTI powder in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg / mL.

[0036] 7) Add 20 μL of C1 mg / mL KTI standard working solution to the IMLC eluent to mix thoroughly to obtain a spiked working solution.

[0037] 8) Take 1 μL of the working solution after adding the standard to perform MALDI-TOF MS detection.

[0038] 9) Obtain the KTI mass spectrum peak intensity (S1) and IMLC peak intensity (S2) by felxAnalysis software, then the concentration C2 of IMLC = 40 * (S2 / S1) / C1 mg / mL.

[0039] Example 1 Qualitative and quantitative mass spectrometry analysis of M protein using a kit

[0040] 1. M protein separation method in plasma

[0041] 1) Take 20 uL of plasma from 10 multiple myeloma patients in a 0.6 mL centrifuge tube, add 160 uL of 10 mM PBS and vortex to dilute the plasma.

[0042] 2) After adding 20 μL of Capture Select Kappa XL and Capture Select LC-Lambda 1:1 mixed microspheres, mix well by oscillation for 30 min to capture the M protein light chain in the plasma.

[0043] 3) After low-speed centrifugation, discard the supernatant, add 200 μL of 10 mM PBS buffer and wash 3 times, then add 200 μL of pure water and wash 3 times.

[0044] 4) Add 50 mM TECP solution (1% formic acid) to elute the M protein light chain bound to the microspheres, and obtain 30 μL of M protein light chain solution.

[0045] 2. Mass spectrometry detection of individualized M protein complete light chain

[0046] 1) Dissolve KTI powder in 1% formic acid solution to prepare KTI standard working solution with a concentration of C1 mg / mL, add 20 μL of C1 mg / mL KTI standard working solution to the IMLC eluate and mix well to obtain the working solution after adding the standard.

[0047] 2) Mix 1 μL of the above working solution with 1 μL of 20 mg / mL α-cyano-4-hydroxycinnamic acid solution and spot on MTP AnchorChip™ target, then dry at room temperature and perform mass spectrometry analysis.

[0048] 3) Detection instrument: MALDI-TOF MS, equipped with matrix-assisted laser desorption ionization source, flexControl 3.0.0 data acquisition software and flexAnalysis 3.3 (Bruker Daltonics) data processing software.

[0049] 4) Mass spectrometry condition: SmartBeam-II laser system, positive ion mode.

[0050] 3. Qualitative and quantitative analysis of M protein intact light chain

[0051] 1) Qualitative analysis: The accurate molecular weight of IMLC molecule was obtained after correcting the mass spectrum by the accurate molecular weight of KTI molecule (19965.0630 Da, determined by high-resolution Q-Exactive MS, as shown in Figure 1 ). Figure 2

[0052] 2) Quantitative analysis: The KTI mass spectrum peak intensity (S1) and IMLC peak intensity (S2) in the MALDI TOF MS result were read by felxAnalysis software, then the concentration C2 of IMLC was calculated as C2 = 40*(S2 / S1) / C1 mg / mL. In this experiment, the concentration C1 was 10 mg / mL, and the quantitative results are shown in Table 1.

[0053] Table 1 Mass spectrometry quantitative results of M protein intact light chain

[0054]

[0055] The clinical information of 10 patients is shown in Table 2:

[0056] Table 2

[0057]

[0058]

[0059] Result analysis: In this case, patients 1, 2, 3, 4, 5, 6, 7 and 8 were in stage 1 according to Mayo 2012 staging, and the M protein quantitative results were all less than 2.5 mg / mL. Patient 9 was in stage 3 according to Mayo 2012 staging, and the M protein intact light chain quantitative result was 9.82 mg / mL. Patient 10 was in stage 4 according to Mayo 2012 staging, and the M protein intact light chain quantitative result was greater than 15 mg / mL. It can be seen that the M protein quantitative results are basically consistent with the clinical stages of the patients, and the quantitative results well reflect the disease status of the patients.

[0060] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0061] ​The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and, while certain modifications are discussed, it is desired to be protected in accordance with the spirit and scope of the application. Therefore, the application is not limited to the specific embodiments shown and described, but only by the scope of the appended claims, unless otherwise specified.

Claims

1. Application of Kunitz-type soybean trypsin inhibitor as an internal standard in the preparation of a multiple myeloma-related M protein detection kit; The kit quantifies the complete light chain of the M protein using mass spectrometry; the Kunitz-type soybean trypsin inhibitor contains 216 amino acids, the amino acid sequence of which is shown in SEQ ID NO.1, and its molecular weight is 19965.063 Da, and it is stable in 1% formic acid solution; The isolation, detection, and precise molecular weight identification of the M protein are achieved through the following steps: 1) Mix 20 μL of plasma with 160 μL of 10 mM PBS buffer until well combined, ensuring that the plasma and buffer are fully mixed; 2) Add 20 μL of CaptureSelect Kappa XL and CaptureSelect LC-Lambda microspheres to the mixture at a 1:1 ratio, and then perform a shaking reaction for 45 minutes to ensure that the microspheres can effectively capture the target molecules; 3) After the reaction is complete, remove the supernatant by low-speed centrifugation; Subsequently, the sample was washed three times with 200 μL of 10 mM PBS buffer, followed by three more washes with pure water. After each wash, the microsphere precipitate is retained by low-speed centrifugation to remove non-specifically bound impurities; 4) Add 20 μL of 50 mM TECP solution, prepared with 1% formic acid, to the washed microsphere precipitate and elute by shaking for 10 minutes to release the captured target molecules. 5) After elution, centrifuge at 15000g for 5 minutes; after centrifugation, take 20μL of IMLC eluent and place it in an EP tube; 6) Dissolve KTI powder in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg / mL; 7) Add 20 μL of 1 mg / mL KTI standard working solution to the IMLC eluent and mix thoroughly to obtain the spiked working solution; 8) Take 1 μL of the spiked working solution and perform MALDI-TOFMS analysis; 9) Obtain the KTI mass spectrometry peak intensity (S1) and IMLC peak intensity (S2) using felxAnalysis software. Then, the concentration of IMLC, C2, is 40*(S2 / S1) / C1 mg / mL.

2. The application of a multiple myeloma-associated M protein detection reagent in the preparation of a multiple myeloma-associated M protein detection kit, characterized in that, The detection reagent includes a Kunitz-type soybean trypsin inhibitor and a 1% formic acid solution; the multiple myeloma-associated M protein is the complete light chain of the M protein; the Kunitz-type soybean trypsin inhibitor contains 216 amino acids, the amino acid sequence is shown in SEQ ID NO.1, its molecular weight is 19965.063 Da, and it is stable in a 1% formic acid solution; The isolation, detection, and precise molecular weight identification of the M protein are achieved through the following steps: 1) Mix 20 μL of plasma with 160 μL of 10 mM PBS buffer until well combined, ensuring that the plasma and buffer are fully mixed; 2) Add 20 μL of CaptureSelect Kappa XL and CaptureSelect LC-Lambda microspheres to the mixture at a 1:1 ratio, and then perform a shaking reaction for 45 minutes to ensure that the microspheres can effectively capture the target molecules; 3) After the reaction is complete, remove the supernatant by low-speed centrifugation; Subsequently, the sample was washed three times with 200 μL of 10 mM PBS buffer, followed by three more washes with pure water. After each wash, the microsphere precipitate is retained by low-speed centrifugation to remove non-specifically bound impurities; 4) Add 20 μL of 50 mM TECP solution, prepared with 1% formic acid, to the washed microsphere precipitate and elute by shaking for 10 minutes to release the captured target molecules. 5) After elution, centrifuge at 15000g for 5 minutes; after centrifugation, take 20μL of IMLC eluent and place it in an EP tube; 6) Dissolve KTI powder in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg / mL; 7) Add 20 μL of 1 mg / mL KTI standard working solution to the IMLC eluent and mix thoroughly to obtain the spiked working solution; 8) Take 1 μL of the spiked working solution and perform MALDI-TOFMS analysis; 9) Obtain the KTI mass spectrometry peak intensity (S1) and IMLC peak intensity (S2) using felxAnalysis software. Then, the concentration of IMLC, C2, is 40*(S2 / S1) / C1 mg / mL. The Kunitz-type soybean trypsin inhibitor was used as an internal standard.

3. The application of the detection reagent for multiple myeloma-associated M protein according to claim 2 in the preparation of a multiple myeloma-associated M protein detection kit, characterized in that, The detection kit includes a Kunitz-type soybean trypsin inhibitor and a 1% formic acid solution; the kit quantifies the intact light chain of the M protein using mass spectrometry; the Kunitz-type soybean trypsin inhibitor contains 216 amino acids, the amino acid sequence of which is shown in SEQ ID NO.1, and its molecular weight is 19965.063 Da, and it is stable in a 1% formic acid solution.

4. The use of a multiple myeloma-associated M protein detection system, characterized in that, The purpose is for quantitative residual detection of multiple myeloma-related M protein for non-diagnostic and therapeutic purposes, and the detection system includes the detection kit and MALDI-TOF mass spectrometer as described in claim 3. The isolation, detection, and precise molecular weight identification of the M protein are achieved through the following steps: 1) Mix 20 μL of plasma with 160 μL of 10 mM PBS buffer until well combined, ensuring that the plasma and buffer are fully mixed; 2) Add 20 μL of CaptureSelect Kappa XL and CaptureSelect LC-Lambda microspheres to the mixture at a 1:1 ratio, and then perform a shaking reaction for 45 minutes to ensure that the microspheres can effectively capture the target molecules; 3) After the reaction is complete, remove the supernatant by low-speed centrifugation; Subsequently, the sample was washed three times with 200 μL of 10 mM PBS buffer, followed by three more washes with pure water. After each wash, the microsphere precipitate is retained by low-speed centrifugation to remove non-specifically bound impurities; 4) Add 20 μL of 50 mM TECP solution, prepared with 1% formic acid, to the washed microsphere precipitate and elute by shaking for 10 minutes to release the captured target molecules. 5) After elution, centrifuge at 15000g for 5 minutes; after centrifugation, take 20μL of IMLC eluent and place it in an EP tube; 6) Dissolve KTI powder in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg / mL; 7) Add 20 μL of 1 mg / mL KTI standard working solution to the IMLC eluent and mix thoroughly to obtain the spiked working solution; 8) Take 1 μL of the spiked working solution and perform MALDI-TOFMS analysis; 9) Obtain the KTI mass spectrometry peak intensity (S1) and IMLC peak intensity (S2) using felxAnalysis software. Then, the concentration of IMLC, C2, is 40*(S2 / S1) / C1 mg / mL. The Kunitz-type soybean trypsin inhibitor was used as an internal standard.

5. The use according to claim 4, characterized in that the process include: S1: Captures the intact light chain of M protein in plasma via immunoaffinity microspheres; S2: The working solution is obtained by adding a certain amount of Kunitz-type soybean trypsin inhibitor standard from the kit to the sample mixture; S3: The working solution was detected by MALDI-TOF mass spectrometry; S4: The accurate molecular weight of the complete light chain of the M protein was obtained by correcting the mass spectrum using the precise molecular weight of the Kunitz-type soybean trypsin inhibitor molecule. S5: Read the mass spectrometry peak intensity (S1) and IMLC peak intensity (S2) of Kunitz-type soybean trypsin inhibitor from the MALDITOFMS results using felxAnalysis software, and calculate the concentration of IMLC C2 = 40*(S2 / S1) / C1 mg / mL.

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