CN112961237B - Humanized IgM monoclonal antibody standard substance and preparation method thereof - Google Patents
Humanized IgM monoclonal antibody standard substance and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a humanized IgM monoclonal antibody standard substance and a preparation method thereof, wherein the method comprises the following steps: screening a phage antibody library aiming at a virus antigen to obtain an IgG prototype antibody meeting the requirement of an affinity threshold; after the affinity of the prototype IgG antibody is matured, the optimized heavy chain variable region of the antibody is grafted to the constant region of the human IgM antibody to form a chimeric heavy chain, and the Kappa light chain can be unchanged. Expressing and purifying to obtain humanized IgM monoclonal antibody standard substance raw material; performing purity characterization, protein activity characterization and structure characterization on humanized IgM monoclonal antibody standard substance raw materials; on the basis of fully characterizing the purity and confirming the consistency with the target antibody sequence, the humanized IgM monoclonal antibody standard substance is subjected to the value determination by adopting an isotope dilution mass spectrometry based on amino acid analysis through the uniformity and stability inspection.
Description
Technical Field
The invention relates to the technical field of IgM antibody detection standard substances, in particular to a preparation method of a humanized IgM monoclonal antibody standard substance.
Background
The immunological detection is one of the important methods for clinical detection, is a specific diagnostic method, is widely used for clinical detection, confirmed diagnosis and epidemiological investigation, and can be used for detecting unknown antibodies by using known antigens or detecting unknown antigens by using known antibodies (such as serological detection). The immunological detection method is to detect a target substance by utilizing the specificity and reversible spatial binding between antigen and antibody. The immunodetection method can be mainly divided into virus antigen detection and serum IgM/IgG antibody detection according to different detection targets; according to different detection principles, the method can be divided into colloidal gold method, immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence method and the like. The immunological detection is used as an important component of diagnosis and treatment work in hospitals, provides a basis for clinical diagnosis, treatment medication and the like, and has the most direct influence on the clinical diagnosis result and the treatment effect due to the quality of the immunological detection.
IgM is the largest molecular weight immunoglobulin, distributed mainly in serum. IgM is the most primitive antibody. In ontogeny, the body first synthesizes antibodies, also IgM, before IgG and IgA appear in succession. In the early stages of infection, IgM plays a role in pioneer immunity. The IgM antibody can be clinically detected to be one of the indexes for early diagnosis of some infectious diseases.
A Reference Material (RM) is a material or substance with one or more sufficiently uniform and well-defined characteristic values for calibrating equipment, evaluating measurement methods or assigning values to the material. The standard substance is a main tool for realizing accurate, consistent and comparable detection results, and is also a measuring physical standard for ensuring effective transmission of quantity values. With the increase of the supervision and management force of the in vitro diagnostic reagent and the continuous improvement of the requirements on the detection technology in China, the demand and the dependence on standard substances are stronger and stronger. The serum IgM antibody detection also needs standard substances for quality control and evaluation of the detection method and the kit.
However, for human clinical standards, many standards are extracted from the blood of patients, which greatly limits the development of clinical standards. If the patient is difficult to find or the blood content is low, in vitro expression and purification by recombinant protein technology is considered. Aiming at the research and development of serum IgM antibody standard substances of difficult-to-obtain raw materials, if recombinant protein technology design and preparation are not applied, it is difficult to imagine that sufficient and consistent serum source IgM antibody standard substances extracted from human sources are distributed in time and applied to the whole world, especially applied to the emergency epidemic crisis like the Xinguan. The reason for this is that the number of the existing clinical diagnosis antibody standard substances is only limited, namely, the antibodies need to be extracted from the blood of patients and the sources of raw materials are limited; secondly, the characterization and value fixing difficulty for the extracted polyclonal antibody is too large, and a reference value fixing method applicable to the extracted polyclonal antibody needs to be established.
Specifically, taking an acute infectious disease as an example, the development cycle of a detection reagent is generally short, and the development and production of a standardized process are difficult in a short time, so that the problems of inconsistent performance and poor quality are inevitable. The evaluation of the kit performance and the quality control are also very urgent. The standard substance is used as a carrier of an accurate value, is a 'weight' for evaluating the quality of the reagent, and can be used for developing and verifying a detection method, performing quality control and evaluation on the development and evaluation of the kit, and the like.
At the present stage, reagent companies mostly directly adopt positive serum as a quality control product for IgM detection research and development. Positive sera were pooled patient-inactivated sera, not purified by extraction, of very low purity and of polyclonal origin (because of the presence of very high abundance of background antibodies against (viral) antigens in addition to serum matrix and antibodies against (viral) antigens). The defect of the quality control product is that the quality control product has biological safety risk and batch source difference, cannot be accurately copied and also cannot be accurately characterized, and the standard substance with extremely high requirements on purity, stability, uniformity and fixed value accuracy is developed on the basis of the quality control product. Many manufacturers and research and development organizations cannot obtain positive serum in time, and only can rely on the function of partially replacing quality control products by substances which are automatically developed by enterprises in the research and development process, so that the development of virus IgM antibody standard substances which have no biological safety risk, can be accurately copied, and can be accurately characterized and quantified is necessary.
In conclusion, IgM in patient serum, particularly in acute infectious disease serum caused by unknown viruses belongs to a polyclonal antibody, and IgM raw materials cannot be obtained and applied to preparation of standard substances. The IgM monoclonal antibody standard substance can be designed and developed by utilizing the characteristics of safety, easy production, reproducibility, accurate representation and accurate value determination by applying a reference method, and can be used for the development of an immunodetection method of serum antibodies in infectious diseases, the quality control and evaluation of a detection kit and the quality control of a laboratory, thereby breaking through the bottleneck limitation of the development and application of the serum antibody standard substance in clinical diseases at present.
However, because the IgM antibody is a polymer and comprises a plurality of subunits, the molecular weight is more than 1000KDa, the heavy chain of each subunit comprises a plurality of glycosylation sites, and the IgM antibody has a complex structure and a large molecular weight. At present, no standard substance preparation method aiming at the immunoglobulin with the complex structure and the maximum molecular weight exists. Therefore, it is necessary to establish a preparation method of the humanized IgM monoclonal antibody standard substance.
Disclosure of Invention
The invention aims to provide a humanized IgM monoclonal antibody standard substance and a preparation method thereof. The monoclonal antibody standard substance prepared by the invention can simulate an antiviral polyclonal antibody in patient serum and react with an antigen and a secondary antibody in an antibody detection kit, so that the performance and reliability of the antibody detection kit and the method are evaluated, and quality control is provided for detection of a sudden acute infectious disease serum antibody. The method has good practicability, accuracy, reliability and traceability.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of a humanized IgM monoclonal antibody standard substance comprises the following steps:
(1) screening a phage antibody library aiming at a virus antigen to obtain an IgG prototype antibody meeting the requirement of an affinity threshold; the antibody library comprises a normal human natural library, a patient antibody library, a synthetic human antibody library and an antibody library obtained by immunizing animals, and the total library capacity is 109The above;
(2) grafting the heavy chain variable region of the IgG antibody to a constant region framework of a human IgM heavy chain, wherein a Kappa light chain can be unchanged, and constructing a humanized IgM monoclonal chimeric antibody matched with the original IgG antibody;
(3) performing cell suspension culture, performing transient transfection to express humanized IgM antibody, centrifuging to remove cells, collecting supernatant, and performing separation and purification by adopting Protein L affinity purification, ion exchange and molecular sieving to obtain a standard substance raw material of the humanized IgM monoclonal antibody;
(4) carrying out purity characterization on the humanized IgM monoclonal antibody;
(5) carrying out protein activity characterization on the humanized IgM monoclonal antibody;
(6) carrying out structural characterization on the humanized IgM monoclonal antibody, wherein structural characterization information comprises molecular weight measurement after cutting off sugar chains of the IgM monoclonal antibody, molecular weight measurement after reducing the cut off sugar chains and peptide spectrum characterization;
(7) the method comprises four steps of oligosaccharide chain release, marking, liquid chromatography-mass spectrometry analysis and data processing, and the relative content abundance and the molecular weight of each sugar type are obtained through analysis. Calculating to obtain the sugar chain weighted average molecular weight of the single glycosylation site of the humanized IgM monoclonal antibody protein according to the relative content abundance and the molecular weight of each sugar type;
(8) calculating the complete molecular weight of the humanized IgM monoclonal antibody based on the determinations of (6) and (7), and calculating the complete molecular weight of the humanized IgM monoclonal antibody according to IgM heavy chain and light chain molecular weights and sugar chain weighted average molecular weights of single glycosylation sites;
(9) on the basis of fully characterizing the purity and confirming the consistency with the target antibody sequence through structure, after uniformity and stability inspection, the standard substance is determined by adopting an isotope dilution mass spectrometry method based on amino acid analysis.
Wherein, in the step (1), an IgG prototype antibody is prepared, 2 or more IgG prototype antibodies meeting the requirement of an affinity threshold are screened against the virus antigen, and the prototype antibodies need to be directed against different epitopes of the antigen; the affinity threshold Kd<10-7。
Before the step (2), the obtained IgG prototype antibody can be subjected to in vitro affinity maturation according to the requirement, so that the affinity is improved;
in the step (2), the heavy chain variable region of the IgG antibody after affinity maturation is grafted and cloned to the heavy chain constant region of the human IgM antibody, and the Kappa light chain can be unchanged.
In the step (3), cells are subjected to suspension culture, the humanized IgM antibody is transiently transfected and expressed, the cells are removed by centrifugation, supernatant is collected, Protein L affinity purification is adopted, and an eluent adopts an acetate buffer solution with the pH value of 3.0; adjusting the pH value of the collected protein to 7.4, performing ion exchange purification by using a hydrophobic mixed mechanism filler captoMMC, and performing linear gradient elution by using a low-salt buffer solution; and after the eluted target protein is subjected to ultrafiltration concentration, continuously separating and purifying by using a molecular sieve Superdex200 according to the molecular weight to obtain the purified IgM protein.
In the step (4), the purity characterization method comprises polyacrylamide gel electrophoresis and high performance gel exclusion chromatography; the purity of the standard substance raw material of the monoclonal antibody is more than or equal to 95 percent; and (4) if the purity is less than 95%, continuing to purify the IgM monoclonal antibody according to the method described in the step (4), and increasing the purity to more than 95%.
In the step (5), the activity characterization method comprises an enzyme-linked immunosorbent assay, a surface plasmon resonance titer determination method and a biofilm interference method.
And (6) in the step (6), mass spectrometry is carried out to determine the molecular weight of the light chain of the reduced IgM monoclonal antibody, the molecular weight of the heavy chain of the reduced IgM monoclonal antibody is cut off, and whether the molecular weight is consistent with the theoretical molecular weight is confirmed.
Performing peptide spectrum analysis on the monoclonal antibody, performing enzyme digestion treatment on the monoclonal antibody, detecting the monoclonal antibody by a mass spectrometer, and performing database search through software to process an MS spectrum to obtain a peptide map sequence coverage rate;
improving the sequence coverage rate of a peptide spectrum by various enzyme cutting modes of trypsin and chymotrypsin or multiple mass spectrometry; the sequence coverage of the peptide map needs to be more than 90%.
In the step (7), peptide-N-glycosidase F (PNGase F) is adopted to release oligosaccharide chains, commercial reagents can be adopted to mark the oligosaccharide chains, qualitative and quantitative analysis is carried out to obtain the relative content abundance and molecular weight of each sugar type, and the sugar chain weighted average molecular weight of the single glycosylation site of the humanized IgM monoclonal antibody protein is calculated.
In the step (8), the calculation of the complete molecular weight of the humanized IgM monoclonal antibody is based on the determinations of (7) and (8), and the complete molecular weight of the humanized IgM monoclonal antibody is calculated from the following formula according to the IgM heavy chain and light chain molecular weights and the sugar chain weighted average molecular weight of the single glycosylation site.
Molecular weight of IgM subunit (sugar free): (MW)Heavy load+MWLight and lightweight)×2sIn-chain×2＝MWSubunit (sugar-free)Da；
Molecular weight of IgM antibody (no sugar): MWSubunit (sugar-free)×n-sChain chamber×2＝MWIgM (sugar free)Da；
Sugar chain molecular weight of IgM antibody: (MW)Sugar chain-weighted average molecular weight-18)×m×n＝MWIgM sugar chainDa；
Complete molecular weight (sugar content) of humanized IgM monoclonal antibody protein of N protein: MWIgM (sugar free)+MWIgM sugar chain＝
MWIgM integrityDa；
Wherein:
MWheavy load-cleaving the IgM heavy chain molecular weight (Da) after sugar chain reduction;
MWlight and lightweight-cutting off the molecular weight (Da) of the IgM light chain after reduction of the sugar chain;
sin-chain-the logarithm of intrachain disulfide bonds contained in the IgM subunit;
MWsubunit (sugar-free)IgM subunit molecular weight (Da) without sugar chains
n is the number of IgM subunits contained by the IgM antibody;
schain chamber-the number of pairs of disulfide bonds between the subunit chains comprised by the IgM antibody;
MWIgM (sugar free)-IgM molecular weight (Da) without sugar chains;
MWsugar chain-weighted average molecular weight-a sugar chain weighted average molecular weight (Da) of a single glycosylation site of the IgM antibody;
m is the number of glycosylation sites contained in the IgM subunit;
MWIgM sugar chain-the sugar chain molecular weight of the IgM antibody;
MWIgM integrity-the complete molecular weight of the IgM antibody.
In the step (9), the humanized IgM monoclonal antibody standard substance is subjected to homogeneity test, an isotope dilution mass spectrometry method is adopted for determination, and whether the standard substance is homogeneous or not is determined through analysis by a statistical method; carrying out stability inspection on the humanized IgM monoclonal antibody standard substance, wherein the stability inspection comprises short-term stability and long-term stability, the short-term stability is stability inspection of one week under a simulated transportation condition, and the long-term stability is stability of 6 months or more under a storage condition at-70 ℃;
and (3) taking leucine, valine, isoleucine and phenylalanine standard substances as standards, taking isotope-labeled leucine, valine, isoleucine and phenylalanine as internal standards, and adopting an isotope dilution mass spectrometry to carry out value determination on the humanized IgM monoclonal antibody standard substance, wherein the molecular weight used in the value determination is obtained in the step (8).
Compared with the prior art, the invention has the outstanding effects that:
(1) at present, no preparation method aiming at an IgM antibody standard substance exists in the prior art, and no available IgM antibody standard substance exists.
(2) The preparation method of the IgM antibody standard substance for detecting the infectious disease serum antibody, which is established by the invention, is a completely innovative method, and a new idea for developing the antibody standard substance for detecting the serum IgM antibody is created, and the preparation method mainly comprises two aspects of design and preparation of IgM antibody standard substance raw materials, characterization of antibody proteins and value determination.
Two bottlenecks resulting in a limited number of standard substances of the existing clinical diagnostic antibodies: firstly, the raw materials need to be extracted from the blood of a patient and are limited in source; secondly, the characterization and value fixing difficulty for the extracted polyclonal antibody is too large, and a reference value fixing method applicable to the extracted polyclonal antibody needs to be established. In the past, for clinical standard substances of human origin, many substances need to be extracted from patient blood. If the patient is difficult to find or the blood content is low, in vitro expression and purification by recombinant protein technology is considered. Aiming at the research and development of serum IgM antibody standard substances of difficult-to-obtain raw materials, if recombinant protein technology design and preparation are not applied, it is difficult to imagine that sufficient and consistent serum source IgM antibody standard substances extracted from human sources are distributed in time and applied to the whole world, and particularly applied to the emergency epidemic crisis like Xinguan.
(3) The humanized IgM antibody standard substance developed by the method established by the invention can simulate the antivirus IgM polyclonal antibody in human serum, and compared with the existing positive serum used as a detection quality control product, the humanized IgM antibody standard substance overcomes the infectious risk defect of infectious disease positive serum and the defect that the IgM polyclonal antibody in the positive serum cannot be accurately characterized, quantified and copied.
At the present stage, reagent companies mostly directly adopt positive serum as a quality control product for IgM detection research and development. The positive serum is a mixed patient inactivated serum, which is only subjected to inactivation treatment and is not subjected to extraction and purification, and is of very low purity and polyclonal origin (except for a serum matrix and antibodies against (viral) antigens, and also contains extremely high abundance of background antibodies against (viral) antigens). The defect of the quality control product is that the quality control product has biological safety risk and batch source difference and cannot be accurately copied; meanwhile, due to the high complexity of components, accurate characterization and quantification cannot be achieved in the prior art of human beings, and positive serum cannot be used for value determination in a reference method and cannot realize value tracing. The standard substance is required to have high purity and good stability, so the positive serum is not suitable to be used as a raw material of the standard substance, and the problem can be effectively solved by developing the (virus) antigen IgM monoclonal antibody standard substance which has no biological safety risk, can be accurately copied and can be accurately characterized and quantified.
The humanized IgM antibody standard substance developed by the method established by the present invention can be accurately characterized, quantified and duplicated, and can be used for the quality control and evaluation of infectious disease serum antibody detection kits and the verification and development of detection methods.
(4) The method established by the invention can be used for developing IgM antibody standard substances required by a series of IgM antibody detection, and can develop a plurality of IgM monoclonal antibody standard substances aiming at different antigen epitopes to form an IgM antibody standard substance library with a plurality of epitopes so as to better simulate polyclonal antibodies in serum.
(5) The IgM antibody protein valuing method established by the invention has the advantages that the standard substance has the value traceability, and the content measurement result can be directly traced to SI unit.
The humanized IgM monoclonal antibody standard substance and the preparation method thereof according to the present invention will be further described with reference to the accompanying drawings and specific examples.
Drawings
FIG. 1 is a graph showing the results of mass spectrometry and deconvolution of heavy chains of N-protein human IgM monoclonal antibody after desugaring reduction;
FIG. 2 is a graph showing the results of mass spectrometry and deconvolution of light chains of the N protein human IgM mabs after desugaring reduction;
FIG. 3 is the traceability of the N protein human IgM monoclonal antibody solution standard substance fixed value result.
Detailed Description
Example 1
For acute infectious diseases caused by the novel suddenly-outbreak virus, the research process of the 2019-nCoV novel coronavirus N protein humanized IgM monoclonal antibody solution standard substance is shown in a new coronavirus mainstream behavior example of 2019-nCoV 2020, and the specific design and experimental process are as follows:
1. screening of 2019-nCoV novel coronavirus N protein human IgM monoclonal antibody and protein expression
Based on the novel coronavirus N gene (28274-29533 site) sequence published in NCBI GenBank, expresses N complete protein, and utilizes phage display library (the library capacity is about 10) constructed after N protein is used to immunize animals9) Screening the N antigen to obtain IgG prototype antibody with certain affinity (affinity threshold Kd)<10-7). And performing affinity maturation on the IgG prototype antibody, and cloning the optimized IgG antibody variable region to the constant region of the human IgM antibody. HEK293F cells were cultured in suspension, transiently transfected to express IgM, and cell supernatants were collected after culture.
2. Purification and purity characterization of 2019-nCoV novel coronavirus N protein human IgM monoclonal antibody
Purifying the cell supernatant to obtain a standard substance raw material of the N protein human IgM monoclonal antibody, wherein the purification steps comprise: centrifuging the cell supernatant at 8000rpm for 20min x2 times, and filtering the supernatant with 0.45um membrane; adjusting the pH value to 7.4, loading the protein affinity column at 4 ℃, washing the column by PBS, eluting by using acetate buffer with the pH value of 3.0, and collecting target protein; the pH of the collected protein is quickly adjusted to 7.4 and is diluted by 10 times, and then the protein is loaded with an ion exchange and hydrophobic mixed mechanism filler captoMMC; after washing by the low-salt buffer, carrying out linear gradient elution by using a salt concentration gradient; ultrafiltering and concentrating the eluted target protein; and continuing to separate and purify by using a molecular sieve Superdex200 according to the molecular weight, and discarding IgM antibodies which are collected by monomers and dimers and have the same natural state.
After the standard substance raw material is obtained, the obtained N protein humanized IgM antibody is firstly subjected to SDS-PAGE electrophoresis purity characterization, and the electrophoresis conditions are as follows: 20 μ g N of protein material was mixed with an equal volume of 1 Xelectrophoresis loading buffer and boiled in a boiling water bath for 5min, loading 10 μ g N of protein per lane. Separating with Mini-PROTEAN precast gel of Bolete company at electrophoresis voltage of 120V for 45 min. After the electrophoresis is finished, dyeing is carried out by using Coomassie brilliant blue, and after the dyeing is finished, decoloring and imaging are carried out by using a methanol-acetic acid solution.
The purity of the obtained N protein human IgM monoclonal antibody raw material is characterized by adopting a high performance gel exclusion chromatography, and the liquid phase chromatography conditions are as follows:
a Wyatt WTC-030S5 high performance liquid phase size exclusion chromatography column (5 μm, 7.8 mm. times.30 cm) was used, the column temperature was 25 ℃, the mobile phase was phosphate buffer (Corning 21-040-CV PBS buffer), isocratic elution was performed at a flow rate of 0.8ml/min, the operating instrument was Agilent1200, and the detector included: the UV ultraviolet detector (detection wavelength 280nm), the RI differential refraction detector and the MALS multi-angle light scattering detector are used for collecting for 20min, the concentration of the diluted sample is about 1mg/mL, the sample injection amount is 30 mu L, parallel measurement is carried out for 3 times, and 100mM citric acid buffer solution is used as blank control.
The MALS multi-angle light scattering detector can directly measure the molecular weight distribution, the molecular weight of the protein shown in the figure is 1128.28kD, the calculated deviation of the molecular weight is +/-3.85%, and the IgM antibody is confirmed to be a hexamer.
The purity of the standard substance of the N protein human IgM monoclonal antibody solution was measured 3 times, and the average purity of the standard substance of the N protein human IgM monoclonal antibody solution was 95.76%, the relative standard deviation was 0.4%, and the content of the monomer and polymer forms was 4.24%, which were counted according to the area normalization method (as shown in Table 1).
TABLE 1 characterization result of purity of standard substance SEC of N protein human IgM monoclonal antibody solution
3. Activity characterization of N protein human IgM monoclonal antibody
And (3) evaluating the affinity of the N protein human IgM monoclonal antibody and the N protein antigen by using a biomacromolecule interaction analyzer Biacore. N protein human IgM mab (20. mu.g/. mu.l, pH4.5 sodium acetate solution) was immobilized on the chip by amino coupling at a fixed value of about 2000RU, 3 channels. The mobile phase is the novel coronavirus N recombinant protein antigen, and the mobile phase is diluted in a gradient way, the flow rate is 30 mu l/min, 30 mu l of sample injection is carried out, the dissociation is carried out for 120s, and 10 mu l of Gly (pH 1.5) is used for regeneration. The N antigen is 47.08kD in length, and the affinity test is repeated 4 times. As shown in Table 2, the affinity between the N protein human IgM mab and the N protein antigen was about 1.7e to 8M.
TABLE 2 affinity evaluation results of N protein human IgM monoclonal antibody and N protein antigen
4. Structure characterization and molecular weight determination of IgM monoclonal antibody
The N protein human IgM monoclonal antibody protein is composed of subunits in which two heavy chains (mu) are covalently paired with two light chains (L) and exists in the form of hexamer (mu 2L2)6, and after IgM subunits are assembled, the heavy chains are covalently linked by interchain disulfide bonds. There are 17 disulfide bonds within the subunits and 12 disulfide bonds between the chains.
Detecting by using a Waters Xevo G2-XS Qtof mass spectrometer; the analysis software is Waters UNIFI, and the software is used for deconvoluting MS spectra to obtain corresponding molecular weight information.
The sample treatment method is as follows:
the molecular weight of the protein sample is determined after reducing and sugar cutting. The method for determining the reducing molecular weight after desugaring is to take 50 mu g of a protein desugarized sample, add DTT to make the final concentration 20mM, react for 60min at 56 ℃, add solution A (0.1% formic acid + 2% acetonitrile) to dilute to 1mg/mL, and inject 0.1ug for analysis.
The chromatographic conditions used by the instrument were as follows:
a chromatographic column: waters BEH Protein C4 chromatography column; 1.7 μm; ID 2.1 mm; length 50 mm.
Mobile phase: solution A, 0.1% formic acid water; liquid B, 0.1% formic acid acetonitrile.
Flow rate: 0.2 mL/min.
Elution conditions:
the mass spectrum conditions are as follows: the data acquisition time is 5 minutes; MS Range: 1000-9000;
and (3) carrying out data analysis by adopting Waters UNIFI software to carry out deconvolution processing on the MS spectrum to obtain molecular weight information.
After the N protein human IgM monoclonal antibody sample is cut with sugar and reduced, a heavy chain molecular weight mass spectrogram and a deconvolution result are shown in figure 1, and the measured heavy chain molecular weight after sugar cutting is consistent with the theoretical cut sugar heavy chain molecular weight. FIG. 2 shows the mass spectrum and deconvolution results of the light chain after reduction of the N protein human IgM monoclonal antibody, the light chain does not contain sugar chains, and the measured molecular weight of the light chain is consistent with the theoretical molecular weight.
The sugar type and content of the N-glycosylation modification of the N protein human IgM monoclonal antibody protein are determined. And after the IgM monoclonal antibody is cut off, the N-sugar is marked by a Waters Rapi Fluor MS marking reagent and then the detection is carried out. Detection was performed using a Waters UPLC with FLR detector in tandem with a Waters Xevo G2-XS Qtof mass spectrometer. The analysis software was Waters UNIFI.
The conditions used for the instrument are as follows:
a chromatographic column: waters BEH Glycan chromatography column; 1.7 μm; ID 2.1 mm; length 150 mm.
Mobile phase: solution A, 50mM formic acid (pH 4.4); liquid B and acetonitrile.
Elution conditions:
the mass spectrum conditions are as follows: the data acquisition time is 55 minutes; MS Range: 500-;
the structural analysis of the N-sugar chain (oligosaccharide is connected by asparagine) comprises four steps of oligosaccharide chain release, labeling, liquid chromatography-mass spectrometry analysis and data processing. Then, mass spectrometry and structural analysis were performed on the oligosaccharide fragments cleaved from IgM to obtain fine structural information of the sugar chains contained in the IgM monoclonal antibody protein, as shown in table 3. And calculating to obtain the sugar chain weighted average molecular weight 1704.72 of the single glycosylation site of the N protein human IgM monoclonal antibody protein according to the relative content abundance and theoretical molecular weight of each sugar type.
TABLE 3 Structure and content information of N protein human IgM monoclonal antibody protein sugar chain
Based on the above characterization of the structure and molecular weight of the N protein human IgM monoclonal antibody protein, we calculate according to the molecular weight of IgM heavy chain and light chain, and the sugar chain weighted average molecular weight of a single glycosylation site to obtain:
molecular weight of IgM subunit (sugar free): (62616.5+23259.82) × 2-17 × 2 ═ 171718.64 Da;
molecular weight of IgM antibody (no sugar): 171718.64 × 6-12 × 2 ═ 1030287.84 Da;
sugar chain molecular weight of IgM antibody: (1704.72-18) × 10 × 6 ═ 101203.3161 Da;
complete molecular weight (containing sugar) of N protein human IgM monoclonal antibody protein: 1030287.84+101203.3161 ═ 1131491.156 Da;
the sugar content of the IgM monoclonal antibody protein was 8.9%.
5. Peptide profile of IgM mAbs
To further confirm the primary structure of IgM mab, we performed peptide profiling. The IgM monoclonal antibody is separated by a nano-liter liquid capillary chromatographic column after enzyme digestion treatment and then is treated by Thermo ScientificTMAnd detecting by a Q active Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer, and processing an MS map by database search through pFind software to obtain the coverage rate of a peptide map sequence.
The experimental method is that the protein sample is added into 50mM ammonium bicarbonate solution until the total volume is 100 mu L, and the mixture is shaken, vortexed and uniformly mixed. And (3) carrying out DTT reduction and IAM alkylation on the solution to be subjected to enzymolysis. The protein samples are subjected to enzyme digestion by two enzymes, namely Chymotrypsin and Trypsin. Taking 2 μ L of the enzymolysis solution, adding 0.1% FA 8 μ L, centrifuging at 15000g speed for 15min, taking 5 μ L of supernatant, and performing LC-MS/MS analysis.
The liquid phase parameters of the apparatus were as follows:
the mass spectrum parameters of the instrument are as follows:
full scan resolution: 70000; AGC Target: 1e 6; maximum IT: 20 ms; scanning range: 200-2000 m/z. dd-MS2 resolution: 35000; AGC Target: 1e 5; maximum IT: 60 ms; loop count: 20; (N) CE/tapped: 30.
data analysis database retrieval of peptide map maps was performed using pFind software, variable modification: carbammidopropyl (C), Deamidination (NQ), oxygenation (M) leakcut: tolerance deviation: MS 20ppm, MS/MS 50 ppm. The database matching adopts a local database constructed by C-kappa region and C _ H constant region sequences of two monoclonal antibodies.
The peptide profile assay results were: after the N protein human IgM monoclonal antibody trypsin and Chymotrypsin are subjected to enzyme digestion, the sequence coverage rate of the light chain constant region is 100%, and the measurement result shows that the N protein human IgM monoclonal antibody sample can be matched with a theoretical sequence.
6. Uniformity test
A certain number of minimum packing units (which can be sampled according to the method shown in a random number table) are randomly extracted, and the extracted samples are measured by adopting a high-precision test method under the same control of test conditions, so that the difference among the samples is completely reflected by the non-uniformity of the samples. Analysis of variance was used to perform statistical tests for homogeneity.
According to the number of units dispensed, a certain number of units are randomly extracted from the initial stage, the middle stage and the final stage of standard substance dispensing respectively for uniformity analysis. The number of units for subpackaging the IgM antibody standard substances is 180, 11 tubes are extracted during uniformity test, and the uniformity test is carried out by adopting an isotope dilution mass spectrometry method based on amino acid analysis to the N protein human IgM monoclonal antibody solution standard substances.
The uniformity test data of the N protein human IgM monoclonal antibody solution standard substance are shown in Table 4, and the uniformity statistics shows that the F value is less than F0.05The standard substance showed good homogeneity.
TABLE 4 homogeneity test data of N protein human IgM monoclonal antibody solution standard substance
7. Stability test
And (3) adopting an amino acid hydrolysis-isotope dilution mass spectrometry method to investigate the stability of the prepared standard substance. Short term stability experiments focus on the stability of standard substances under specific transport conditions. According to the current transportation conditions of China, goods can be transported to main cities of China within 7 days by using express transportation. And (3) extracting a proper amount of standard substance samples, marking, respectively placing in a specified environment, and checking according to a specified period. And (3) respectively inspecting the short-term stability of the N protein human IgM monoclonal antibody solution standard substance for 0, 1, 3, 5 and 7 days at three different temperatures of 4 ℃, room temperature and 37 ℃.
The short-term stability test result of the standard substance is as follows: the N protein human IgM monoclonal antibody solution standard substance can be stably stored for 7 days at three different temperatures of 4 ℃, room temperature and 37 ℃. The storage condition is that the mixture is stored in a refrigerator at the temperature of-70 ℃. The transportation condition is dry ice transportation.
The long-term stability experiments concern the stability profile of the standard substances under specific storage conditions. Storage conditions for this study were-70 ℃. Table 5 shows the long-term stability of the N protein human IgM monoclonal antibody solution standard substance at-70 ℃.
TABLE 5 Long-term stability test data of N protein human IgM monoclonal antibody solution standard substance
8. Standard substance constant value
(I) Experimental method
And (3) carrying out value determination on the N protein human IgM monoclonal antibody solution standard substance by two operators by adopting the established isotope dilution mass spectrometry method based on amino acid analysis.
HPLC-IDMS analysis was performed in Agilent1200 liquid phase system tandem AB5500 triple quadrupole mass spectrometry, using multiple reaction monitoring mode (MRM). By optimizing the chromatographic conditions, the following chromatographic separation conditions were selected: the column was a spanetex C18 column (2.6 μm 150 × 2.1mm) from philips corporation; elution conditions: mobile phase a (water) containing 0.1% Trifluoroacetic acid (TFA) was eluted with mobile phase B (100% acetonitrile) in equal gradient at 91:9 volume ratio for 10min at flow rate of 200 μ l/min.
Optimizing mass spectrum conditions: since the experiment employs the MRM mode, the ionization condition, Declustering Potential (DP) and Entrance Potential (EP) of the parent ion scan, and Collision Energy (CE) generated by the daughter ions are mainly optimized, and the acquisition condition and the monitored ion pair (Q1> Q3) are shown in table 6.
TABLE 6 Mass Spectrometry Collection conditions and ion pairs monitored during HPLC-IDMS analysis
Calculation formula according to bracket method:
wherein C is the concentration of the amino acid after the sample is hydrolyzed; m is the mass of the sample in the hydrolysis solution; r1 is the peak area ratio of amino acid to labeled amino acid in the high-standard solution; r2 is the peak area ratio of amino acid to labeled amino acid in the low standard solution; p is the purity of the amino acid; m is marked as the mass of the labeled amino acid in the hydrolyzed sample; the R sample is the peak area ratio of the hydrolyzed amino acid and the marked amino acid of the sample; w1 is the mass ratio of amino acid to labeled amino acid in the high-standard solution; r2 is the mass ratio of amino acid to labeled amino acid in the low standard solution.
In the fixed value research of the monoclonal antibody protein solution standard substance, four amino acids of Val, Leu, Ile and Phe are adopted to quantify the protein. The concentrations of Val, Leu, Ile and Phe amino acids after hydrolysis are firstly obtained, then the protein content is calculated according to the concentration of each amino acid, and finally the average value of the protein contents determined by the four amino acids is the accurate content of the protein.
(II) results of the experiment
The N protein human IgM monoclonal antibody solution standard substances were individually quantified by isotope dilution mass spectrometry, and the results are shown in table 7.
TABLE 7 protein content results (μ g/g) of four amino acids of the N-protein humanized IgM antibody
9. Measurement traceability
The standard substances are all subjected to value determination by adopting an isotope dilution mass spectrometry method, and the national amino acid primary standard substances are used as traceability standards, namely Val national primary standard substance (GBW 09236), Leu national primary standard substance (GBW 09237) and Ile national primary standard substance (GBW 09238) Phe national primary standard substance (GBW 09235). The amino acid standard substance is subjected to value determination by adopting a quantitative nuclear magnetism and mass balance method, and the final result can be traced to SI unit, wherein the tracing graph is shown in figure 3.
10. Verification and trial of standard substance
And (3) evaluating a commercially available colloidal gold immunochromatographic test strip by using the developed N protein human IgM monoclonal antibody solution standard substance and determining a positive reference value. Because no standard substance exists, the colloidal gold immunochromatographic test strip cannot provide parameters such as detection limit and the like, the developed N protein humanized IgM monoclonal antibody solution standard substance can provide a detection limit reference value for the test strip, and the comparability and quality control of a detection result are realized.
And evaluating according to the operation steps of the colloidal gold immunochromatographic test strip product specification. The concentrations of the standard substances of the N protein humanized IgM antibody are respectively set as follows: 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 5000ng/mL, 10000ng/mL, 50000 ng/mL.
The detection limit of the 4-model colloidal gold immunochromatographic test strip is shown in Table 8 according to the fixed value of the developed standard substance.
TABLE 8 summary of the evaluation results of the colloidal gold immunochromatographic test strip
From the above results, it can be seen that: the developed N protein humanized IgM monoclonal antibody solution standard substance can be used for carrying out quality control and evaluation on the performance of the antibody detection kit and the method.
Example 2
For common important infectious diseases, such as AIDS, IgM monoclonal antibody standard substance can be developed according to the technical scheme of the invention:
at present, there is no evidence standard substance for AIDS IgM antibody in China, and there are two main reasons:
1. the human serum of the AIDS patient can not be obtained and processed under the daily condition as a standard substance;
2. even if tens of human AIDS patient serum can be obtained, the components in the human AIDS patient serum are very complex, and only the anti-AIDS virus polyclonal IgM antibody is contained in 1 percent or even less than 0.1 percent, so that the human AIDS patient serum is difficult to accurately characterize and quantify so as to become a certified standard substance.
Therefore, the scheme in the embodiment 1 is adopted to screen IgG antibodies aiming at AIDS antigens, humanized transformation and variable region grafting are carried out to obtain humanized IgM antibodies, the humanized IgM antibodies are used as raw materials after expression and purification in mammalian cells, sufficient purity and structure representation are carried out in sequence, and finally, the pure IgM antibodies resisting the AIDS antigens are subjected to value determination according to the isotope dilution mass spectrometry established in the embodiment 1, so that the AIDS IgM monoclonal antibody standard substance is obtained and is used for development, verification, quality control and unification of clinical diagnosis methods.
In order to highlight the advantageous effects of the present invention, the following comparative examples are exemplified.
Comparative example
When the antibody obtained from the immune animal is directly used as a standard substance raw material and is not subjected to humanized modification:
the obtained IgM is from immunized animal species and theoretically should not react with reagents in a kit, so the IgM loses the function of serving as a reference standard and cannot be used for development of an immunodetection method for IgM antibodies in patient serum, quality control and evaluation of a detection kit, quality control in a laboratory and consistency of measurement results.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (8)
1. A preparation method of a humanized IgM monoclonal antibody standard substance is characterized by comprising the following steps:
(1) screening a phage antibody library aiming at a virus antigen to obtain an IgG prototype antibody meeting the requirement of an affinity threshold; the antibody library comprises a normal human natural library, a patient antibody library, a synthetic human antibody library and an antibody library obtained by immunizing animals, and the total library capacity is 109The above;
(2) grafting the heavy chain variable region of the IgG antibody to a human IgM heavy chain constant region framework, and constructing a humanized IgM monoclonal chimeric antibody matched with the original IgG antibody, wherein the Kappa light chain is unchanged;
(3) performing cell suspension culture, performing transient transfection to express humanized IgM monoclonal chimeric antibody, centrifuging to remove cells, collecting supernatant, and performing separation and purification by adopting Protein L affinity purification, ion exchange and molecular sieving to obtain a standard substance raw material of the humanized IgM monoclonal antibody;
(4) carrying out purity characterization on the humanized IgM monoclonal antibody;
(5) carrying out protein activity characterization on the humanized IgM monoclonal antibody;
(6) carrying out structural characterization on the humanized IgM monoclonal antibody, wherein structural characterization information comprises molecular weight measurement after cutting off sugar chains of the IgM monoclonal antibody, molecular weight measurement after reducing the cut off sugar chains and peptide spectrum characterization;
(7) the method comprises the steps of (1) determining the sugar type and content of humanized IgM monoclonal antibody protein N-glycosylation modification, including four steps of oligosaccharide chain release, labeling, liquid chromatography-mass spectrometry analysis and data processing, and analyzing to obtain the relative content abundance and molecular weight of each sugar type; calculating to obtain the sugar chain weighted average molecular weight of the single glycosylation site of the human IgM monoclonal antibody protein according to the relative content abundance and the molecular weight of each sugar type;
(8) calculating the complete molecular weight of the humanized IgM monoclonal antibody based on the assay results of (6) and (7): based on the humanized IgM heavy and light chain molecular weights and the sugar chain weighted average molecular weights of the individual glycosylation sites,
calculating the complete molecular weight of the humanized IgM monoclonal antibody by the following formula;
molecular weight of IgM subunit (sugar free):
；
molecular weight of IgM antibody (no sugar):
；
sugar chain molecular weight of IgM antibody:
Da；
complete molecular weight (sugar content) of humanized IgM monoclonal antibody protein of N protein:
；
wherein:
n is the number of IgM subunits contained by the IgM antibody;
m is the number of glycosylation sites contained in the IgM subunit;
(9) on the basis of fully characterizing the purity and confirming the consistency with the target antibody sequence through structure, after uniformity and stability inspection, the standard substance is determined by adopting an isotope dilution mass spectrometry method based on amino acid analysis.
2. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 1, characterized in that: in the step (1), firstly preparing IgG prototype antibodies, and screening 2 or more IgG prototype antibodies meeting the requirement of an affinity threshold against the virus antigens, wherein the prototype antibodies need to be directed against different epitopes of the antigens; the affinity threshold Kd<10-7。
3. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 2, characterized in that: before the step (2), performing in-vitro affinity maturation on the obtained IgG prototype antibody according to requirements to improve the affinity;
in the step (2), the heavy chain variable region of the IgG antibody after affinity maturation is grafted and cloned to the heavy chain constant region of the human IgM antibody, and the Kappa light chain is unchanged.
4. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 3, characterized in that: in the step (3), cells are subjected to suspension culture, the human IgM antibody is transiently transfected and expressed, the cells are removed by centrifugation, supernatant is collected, Protein L affinity purification is adopted, and an eluent adopts an acetate buffer solution with the pH value of 3.0; adjusting the pH value of the collected protein to 7.4, performing ion exchange purification by using a hydrophobic mixed mechanism filler captoMMC, and performing linear gradient elution by using a low-salt buffer solution; and after the eluted target protein is subjected to ultrafiltration concentration, continuously separating and purifying by using a molecular sieve Superdex200 according to the molecular weight to obtain the purified IgM protein.
5. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 4, characterized in that: in the step (4), the purity characterization method comprises polyacrylamide gel electrophoresis and high performance gel exclusion chromatography; the purity of the standard substance raw material of the monoclonal antibody is more than or equal to 95 percent; if the purity is less than 95%, continuing to purify the IgM monoclonal antibody according to the method described in the step (4), and increasing the purity to more than 95%;
in the step (5), the activity characterization method comprises an enzyme-linked immunosorbent assay, a surface plasmon resonance titer determination method and a biofilm interference method.
6. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 5, characterized in that: in the step (6), mass spectrometry is carried out to determine the molecular weight of the light chain of the reduced IgM monoclonal antibody, the molecular weight of the heavy chain of the reduced IgM monoclonal antibody is cut off, and whether the molecular weight is consistent with the theoretical molecular weight is confirmed;
performing peptide spectrum analysis on the monoclonal antibody, performing enzyme digestion treatment on the monoclonal antibody, detecting the monoclonal antibody by a mass spectrometer, and performing database search through software to process an MS spectrum to obtain a peptide map sequence coverage rate;
improving the sequence coverage rate of a peptide spectrum by various enzyme cutting modes of trypsin and chymotrypsin or multiple mass spectrometry; the sequence coverage of the peptide map needs to be more than 90%.
7. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 6, characterized in that: in the step (7), the peptide-N-glycosidase F is adopted to release the oligosaccharide chain, a commercial reagent is adopted to mark the oligosaccharide chain, qualitative and quantitative analysis is carried out to obtain the relative content abundance and the molecular weight of each sugar type, and the sugar chain weighted average molecular weight of the single glycosylation site of the human IgM monoclonal antibody protein is obtained through calculation.
8. The method for producing a humanized IgM monoclonal antibody standard substance according to claim 7, characterized in that: in the step (9), the humanized IgM monoclonal antibody standard substance is subjected to homogeneity test, an isotope dilution mass spectrometry method is adopted for determination, and whether the standard substance is homogeneous or not is determined through analysis by a statistical method; carrying out stability inspection on the humanized IgM monoclonal antibody standard substance, wherein the stability inspection comprises short-term stability and long-term stability, the short-term stability is stability inspection of one week under a simulated transportation condition, and the long-term stability is stability of 6 months or more under a storage condition at-70 ℃;
and (3) taking leucine, valine, isoleucine and phenylalanine standard substances as standards, taking isotope-labeled leucine, valine, isoleucine and phenylalanine as internal standards, and adopting an isotope dilution mass spectrometry to carry out value determination on the humanized IgM monoclonal antibody standard substance, wherein the molecular weight used in the value determination is obtained in the step (8).
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