Liquid chromatography-mass spectrometry method for quantitatively detecting denosumab in serum
A technology for quantitative detection of denosumab, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of time-consuming, labor-intensive and expensive specific binding reagents, endogenous antibody interference detection results, and insufficient sensitivity, so as to improve the sensitivity and selectivity, standardization of operation, and increased sensitivity
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[0058] The present invention uses magnetic beads that can specifically bind to denosumab to immunocapture denosumab in serum samples, and then uses ProteinWorks Auto-eXpress Low kit to enzymolyze the immunocaptured serum samples, and the samples after enzymolysis Then purified and enriched by ProteinWorks μElution SPE Clean-up Kit. The invention uses the magnetic beads to immunocapture the denosumab, which can improve the sensitivity. In the present invention, the preparation method of the magnetic beads capable of specifically binding to denosumab preferably includes the following steps:
[0059] a) Give mice a large dose of denosumab injection subcutaneously, once a week, for 5 consecutive weeks, extract anti-denosumab antibody from the ascites of the mouse, and use Protein A magnetic beads to resist denosumab The monoclonal antibody was subjected to affinity purification to obtain the purified anti-denosumab antibody;
[0060] b) covalently cross-linking the activated bio...
Embodiment 1
[0093] Method validation for the quantitative determination of denosumab in human serum by UPLC-MS / MS
[0094] 1. Selection of characteristic peptides
[0095] Specific peptides in human serum were obtained by NCBI BLAST search. There are 2 specific peptides in the heavy chain, namely EVQLLESGGGLVQPGGSLR (SEQ ID NO.1) and GLEWVSGITGSGGSTYYADSVK (SEQ ID NO.2); 3 specific peptides in the light chain The specific peptides are respectively FSGSGSGTDFLTISR (SEQ ID NO.3), EIVLTQSPGTLSLSPGER (SEQ ID NO.4), LEPEDFAVFYCQQYGSSPR (SEQ ID NO.5). Denosumab was hydrolyzed with the standard procedure using ProteinWorks Kits, and the hydrolyzed samples were detected using Waters UPLCI-Class Vion MS / MS Qtof to confirm the charge state and main fragment ions of the candidate peptides. It was detected that there was interference in the EVQLLESGGGLVQPGGSLR (SEQ ID NO.1) peptide segment. After comparing the final 4 peptide segments, it was found that GLEWVSGITGSGGSTYYADSVK (SEQ ID NO.2) had the h...
Embodiment 2
[0188] Validation of the method for the quantitative determination of denosumab in monkey serum by LC-MS / MS
[0189] 1. Selection of characteristic peptides
[0190] The specific peptides in monkey serum were obtained by NCBI BLAST search. There are 2 specific peptides in the heavy chain, namely EVQLLESGGGLVQPGGSLR (SEQ ID NO.1) and GLEWVSGITGSGGSTYYADSVK (SEQ ID NO.2); there are 3 specific peptides in the light chain The specific peptides are respectively FSGSGSGTDFLTISR (SEQ ID NO.3), EIVLTQSPGTLSLSPGER (SEQ ID NO.4), LEPEDFAVFYCQQYGSSPR (SEQ ID NO.5). Denosumab was hydrolyzed using the standard procedure using ProteinWorks Kits, and the samples after enzymatic hydrolysis were detected using Waters UPLC I-Class Vion MS / MS Qtof to confirm the charge state and main fragment ions of the candidate peptides. It was detected that there was interference in the peptide EVQLLESGGGLVQPGGSLR (SEQ ID NO.1). After comparing the final 4 peptides, it was found that LEPEDFAVFYCQQYGSSPR (SE...
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