CN108659126A - Anti-human ApoA1 monoclonal antibodies and its preparation method and application - Google Patents

Anti-human ApoA1 monoclonal antibodies and its preparation method and application Download PDF

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CN108659126A
CN108659126A CN201710216853.2A CN201710216853A CN108659126A CN 108659126 A CN108659126 A CN 108659126A CN 201710216853 A CN201710216853 A CN 201710216853A CN 108659126 A CN108659126 A CN 108659126A
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apoa1
monoclonal antibodies
cardiovascular
cerebrovascular disease
disease
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CN108659126B (en
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张�杰
张艳
王荣芳
钱震斌
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Diasys Diagnostic Systems Shanghai Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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Abstract

The invention discloses a kind of anti-human Apolipoprotein A1 (ApoA1) mouse resource monoclonal antibody D11 10, the heavy chain subgroup of the ApoA1 monoclonal antibodies D11 10 is IgG2b, light chain subtype κ, heavy chain, chain variable region amino acid sequence are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4.Compared to existing based on the ApoA1 detection methods that ApoA1 is mostly anti-and establishes, the present invention is based on the ApoA1 detection methods that ApoA1 monoclonal antibodies D11 10 is established, the recall rate of coronary heart disease (CHD) can be significantly improved, particularly with HDL C in the CHD patient of normal range (NR), recall rate is significantly increased.Therefore, the present invention is based on the completely new detection method that ApoA1 monoclonal antibodies D11 10 is established, correlation of the detected value with cardiovascular and cerebrovascular disease of ApoA1 can be more preferably embodied, there is important clinical meaning for the diagnosis of cardiovascular and cerebrovascular disease, prevention and management.

Description

Anti-human ApoA1 monoclonal antibodies and its preparation method and application
Technical field
The invention belongs to field of biological detection, and in particular to a kind of anti-human Apolipoprotein A1 (ApoA1) mouse resource monoclonal is anti- Body D11-10 and the hybridoma cell strain for secreting the monoclonal antibody, and using the monoclonal antibody in clinical detection people periphery In blood the content of ApoA1 albumen with predict occur cardiovascular and cerebrovascular disease risk and its to the diagnosis of the disease.
Background technology
Apolipoprotein A1 (ApoA1) is the major structural protein of high-density lipoprotein (HDL), and physiological action is to help HDL is helped to capture free cholesterol.In addition, ApoA1, which also has, stablizes prostacyclin, promotes vasodilation and inhibit blood platelet solidifying It is poly-, resist the effects that vascular plaque is formed.
Traditionally, the detection of Clinical practice HDL-C and ApoA1 albumen carrys out the level of HDL metabolism in antimer.Serum/blood The reduction of ApoA1 and HDL-C levels in slurry prompt the dangerous of cardiovascular and cerebrovascular disease (CHD, cerebral apoplexy etc.) to increase, are heart and brain One of the sensitive indexes of vascular diseases hazard assessment.
However having studies have shown that in more than 130,000 CHD patients at present, the HDL-C levels for exceeding 4 one-tenth patients are in normal model It encloses, so HDL-C can not completely effectively reflect disease risks.Fibrates, niacin and cholesterol ester transport protein (CETP) Retarding agent etc. promoted HDL-C level drug cannot all reduce endpoints (entirely because or CHD caused by the death rate), and can not drop Low palsy relative risk further prompts HDL-C to be unable to the metabolic condition of HDL in actual response body.HDL by new life to ripe, then To the dynamic process for by liver recycling degradation being a series of complex.It can will be internal by the method for ultracentrifugation and dielectrophoresis HDL points in metabolic process are different hypotypes, and clinical correlation can more be reacted researches show that the hypotype of HDL changes compared with HDL-C With the correlation of cardiovascular and cerebrovascular disease.Therefore, monitoring specifically seems particularly heavy with the relevant functionality HDL hypotypes of disease in vivo It wants.
The HDL hypotypes separation that laboratory uses at present and identification method operation difficulty are big, technology requires high, experimental period It is long, it is difficult to meet clinical testing requirements quickly, high-throughput, be not suitable for clinical application.Although detecting the main egg of HDL in serum Bai Chengfen ApoA1, to a certain degree can in antimer HDL metabolism state.But detection method is to be based on ApoA1 mostly The Immunological Method of polyclonal antibody cannot detect the variation of HDL hypotypes.Therefore, develop it is a kind of it is completely new, clinic can be met Quickly and it is high-throughput require, and can detect and the method for the relevant HDL hypotypes of cardiovascular and cerebrovascular disease, for cardiovascular and cerebrovascular disease Prevent and management has important clinical meaning.
Invention content
The present invention is prepared for a kind of ApoA1 monoclonal antibodies D11-10, the heavy chain of the ApoA1 monoclonal antibodies D11-10 Hypotype is IgG2b, light chain subtype κ, secreted by BALB/c mouse hybridoma cell strain D11-10, the BALB/c mouse is miscellaneous Tumor cell strain D11-10 is handed over to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation Date is on March 9th, 2017, deposit number CGMCC No.13807.Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3.
The present invention provides a kind of ApoA1 monoclonal antibodies D11-10, the ApoA1 monoclonal antibodies D11-10 can know Other ApoA1 albumen specific antigen epitope, and can screen accordingly and the relevant HDL specific subtypes of the cardiovascular and cerebrovascular disease (Asias D11-10 Type).
In the present invention, the heavy chain variable region of the ApoA1 monoclonal antibodies D11-10, the amino acid sequence of light chain variable region Respectively as shown in SEQ ID NO.3 and SEQ ID NO.4；Encode the nucleotide sequence of the heavy chain variable region, light chain variable region Respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
With the people ApoA1 of recombination reacting for specificity, the ApoA1 monoclonal antibodies can occur for the ApoA1 monoclonal antibodies D11-10 D11-10 and people's ApoA1 binding forces of recombination are 1.73 × 10-10M, not with the recombinant protein blood that is obtained in identical expression system Clear amyloid A 1 (SAA1) and β2Microglobulin (β2M) react, also not with commercially available natural bovine serum albumin(BSA) (BSA) it reacts.
In the present invention, the preparation process of the ApoA1 monoclonal antibodies D11-10 includes：Mouse peritoneal is tied up to using hybridoma The ascites for preparing the monoclonal antibody containing D11-10 obtains monoclonal antibody through Protein A/G column affinitive layer purifications.It is tried using Enzyme-linked Immunosorbent Assay Test hypotype and affinity that (ELISA) measures antibody.
In the present invention, the ApoA1 monoclonal antibodies D11-10 can screen the ApoA1 in specific conformation HDL particles, the energy quilt The specific HDL conformations that the monoclonal antibody is screened are referred to as HDL D11-10 hypotypes.
Present invention finds the high correlations of HDL D11-10 hypotypes and CHD, it is proposed that ApoA1 monoclonal antibodies D11-10 is making The application being ready for use in the product of diagnosis cardiovascular and cerebrovascular disease.Wherein, the cardiovascular and cerebrovascular disease in-vitro diagnosis product includes examination Agent box, test paper, solid support immune detection tool；The solid support includes array, microarray or protein array.
The present invention also provides a kind of diagnostic products of cardiovascular and cerebrovascular disease, including ApoA1 monoclonal antibodies D11- as described above 10；Wherein, the diagnostic products of the cardiovascular and cerebrovascular disease include the immune detections tool such as kit, test paper, solid support；Institute It includes array, microarray or protein array to state solid support.
The present invention also provides a kind of methods that the risk of cardiovascular and cerebrovascular disease occurs for prediction object to be measured, pass through the heart The diagnostic products of cranial vascular disease measure the ApoA1 protein contents of object to be measured, and cardiovascular and cerebrovascular disease occurs to object to be measured Risk is predicted that, when the ApoA1 protein contents of object to be measured are less than the reference value of normal population, cardiovascular and cerebrovascular occurs for prediction The risk of disease is higher.
The present invention also provides a kind of methods of diagnosis cardiovascular and cerebrovascular disease, are produced by the diagnosis of the cardiovascular and cerebrovascular disease Product measure the content of the ApoA1 albumen of object to be measured, are diagnosed to cardiovascular and cerebrovascular disease.
The present invention also provides a kind of kits comprising ApoA1 monoclonal antibodies D11-10 as described above.
The present invention also provides the application of the diagnostic products of the cardiovascular and cerebrovascular disease, the diagnosis of the cardiovascular and cerebrovascular disease Product can be by ApoA1 monoclonal antibodies D11-10 identifications, quantitative specific ApoA1 epitopes, to reflect HDL D11- in peripheral blood The level of 10 hypotypes, to monitor cardiovascular and cerebrovascular disease risk and for the diagnosis of cardiovascular and cerebrovascular disease.
In the present invention, the diagnostic products of the cardiovascular and cerebrovascular disease can be used in whole blood, serum or blood plasma and organize The detection of ApoA1 in extract.
In the present invention, the cardiovascular and cerebrovascular disease includes mainly forming caused disease by vascular plaque.
In the present invention, the cardiovascular and cerebrovascular disease is CHD, cerebral apoplexy or atherosclerosis.
The beneficial effects of the present invention are ApoA1 monoclonal antibodies D11-10 of the present invention can be by identifying that specific ApoA1 is anti- Former epitope screens HDL D11-10 hypotypes, and the binding force of the monoclonal antibody and recombined human ApoA1 albumen is 1.73 × 10-10M has Higher affinity and specificity；And in the albumen that is measured of the present invention, the monoclonal antibody only with recombination and in blood it is natural ApoA1 occur specific reaction, do not reacted with the unrelated proteins such as such as SAA1, β 2M and BSA.D11-10 pairs of the ApoA1 monoclonal antibodies The test result of group's difference sample is in discrete distribution, prompts the HDL that it can be between reflected sample heterogeneous, can pass through Detection to ApoA1 carrys out the level of HDL D11-10 hypotypes in reflected sample.Compared to existing HDL-C detection techniques and ApoA1 The testing result of Immunoturbidimetric kit (based on ApoA1 is mostly anti-), HDL D11-10 hypotypes of the present invention have more with CHD diseases Correlation can significantly distinguish CHD and control group, be improved particularly underwent coronaries of the HDL-C in normal reference range Radiography makes a definite diagnosis the concordance rate of the CHD sufferers of (golden standard).Therefore, the inspection of ApoA1 monoclonal antibodies D11-10 development is utilized Survey method can improve the sensitivity of the prior art, improve the recall rate to cardiovascular and cerebrovascular disease.ApoA1 using the present invention is mono- Anti- D11-10, development are suitable for the kit of clinical high-throughput immunoturbidimetry automatic detection, it can be achieved that cardiovascular and cerebrovascular disease Risk profile and clinical monitoring, while further being to illustrate the occurrence and development mechanism of disease to lay the foundation.
Description of the drawings
The binding force that Fig. 1 is ApoA1 monoclonal antibodies D11-10 detects.It is coated with plate with recombined human ApoA1 albumen, is serially diluted ApoA1 monoclonal antibody D11-10 are detected, the combination of ApoA1 monoclonal antibodies D11-10 and recombined human ApoA1 albumen using Salmonella method Power is 1.73 × 10-10M。
Fig. 2 is the amino acid sequence of the heavy chain variable region and light chain variable region of ApoA1 monoclonal antibodies D11-10, and encodes its heavy chain The nucleotide sequence of variable region and light chain variable region.
Fig. 3 is ApoA1 monoclonal antibodies D11-10 and resists the testing result to clinical sample more and compare.A：It is utilized respectively ApoA1 The content of ApoA1 in control group and CHD group sample that monoclonal antibody D11-10 and ApoA1 Immunoturbidimetric kit measures；B：Respectively Utilize the sample of control group and CHD group HDL-C >=1mM that ApoA1 monoclonal antibody D11-10 and ApoA1 Immunoturbidimetric kits measure The content of ApoA1 in this.For 1.37g/L, (90% confidence interval of control group that ApoA1 monoclonal antibodies measure is as cut- Off values)；For 1.00g/L (《Clinical examination regulation》The cut-off values for ApoA1 detections recorded).
Fig. 4 is compared with the ApoA1 tertiles in the detection CHD and control group of ApoA1 monoclonal antibodies D11-10.
Specific implementation mode
In conjunction with following specific examples and attached drawing, the present invention is described in further detail.The process of the implementation present invention, Condition, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to the following content specially referred to It is bright that content is not particularly limited.
Experimental method used in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1：The preparation and purification of ApoA1 monoclonal antibodies D11-10
1. material：6 × His-ApoA1 of fusion protein, 8 week old female BAl BIcs/c mouse
2. method and result
It is prepared by 2.1 antigens
2.1.1 the structure of people ApoA1 recombinant proteins expression plasmid
Gene order (the Gene ID for obtaining ApoA1 are inquired in GenBank:335), according to sequence design polymerase chain React (PCR) primer.Go out the DNA fragmentation of people's ApoA1 genes using people source cDNA as template amplification, PCR product is through 1.0% agarose Detected through gel electrophoresis blend compounds QIAquick Gel Extraction Kit recycles respective segments, and the ApoA1 genetic fragment sizes of PCR amplification are 875bp. PCR product is connected into expression vector pColdTMII (6 × His labels that the carrier contains are for purifying), conversion to TaKaRa DH5 α host strains, picking monoclonal carry out plasmid extraction and sequence verification, and sequencing result is consistent with GenBank retrievals sequence.
2.1.2 the expression and purification of people ApoA1 recombinant proteins
The pCold of people's ApoA1 albumen will be expressedTMII recombinant plasmid transformed e. coli bl21s.With IPTG induced expressions, warp (according to specification operating instruction) after Qiagen nickel affinity chromatography column purifications, 6 × His-ApoA1 of people's ApoA1 recombinant proteins is obtained (6 × His-ApoA1 fusion proteins).
2.2 mouse immune
According to《Current Protocols in Immunology》Middle operating process, user ApoA1 recombinant proteins with Isometric Freund's complete adjuvant emulsification, is immunized the BALB/c mouse of 8 week old, then uses incomplete Freund's adjuvant breast Change, be immunized four times altogether, each inoculation amount is 200 μ g albumen.
Preparation, screening and the monoclonal of 2.3 hybridomas
The splenocyte of the BALB/c mouse of separating immune is merged with murine myeloma cell Sp2/0.By ELISA method, Hybridoma is detected.It is the positive to measure 6 × His-ApoA1 results, and measures the clone that His-SAA results are negative It is retained.By limiting dilution culture, until obtaining stable hybridoma monoclonal cell.Culture supernatant containing antibody is used Protein A affinity columns carry out after purification, being used for subsequent detection.
2.4ApoA1 monoclonal antibody D11-10 hypotypes, specificity and affinity detection
2.4.1ApoA1 monoclonal antibody D11-10 subtype identifications
Use Southern Biotech companies SBA ClonotypingTMSystem/HRP monoclonal antibodies light chain and again Chain parting kit is identified of the present invention according to manufacturers instruction using enzyme-linked immunosorbent assay (ELISA) method The heavy chain subgroup of ApoA1 monoclonal antibodies D11-10 is IgG2b, light chain subtype κ.
2.4.2ApoA1 the specificity of monoclonal antibody D11-10 and affinity detection
Using the specificity of Salmonella detection ApoA1 monoclonal antibodies D11-10, it is coated on solid-phase media respectively commercially available natural ApoA1 albumen and BSA or with 6 × His labels different recombinant proteins such as：ApoA1, SAA1 and β2M, detection monoclonal antibody is to it Identification situation.As a result it is anti-to show that only with recombination and natural people's ApoA1 albumen specificity occurs for ApoA1 monoclonal antibodies D11-10 Answer, not with the recombinant protein SAA1 and β that are obtained in identical expression system2M reacts, and does not also occur with commercially available natural B SA Reaction, shows good specificity.
Affinity (binding force) of the monoclonal antibody to its antigen is detected using Salmonella method.By 96 hole elisa plate coatings ApoA1 albumen, coating buffer are the Tris.HCl of 50mM pH8.5.It is positioned in wet box, 4 DEG C are overnight, after PBST is washed 4 times, 2% 37 DEG C of BSA closing 2h, wash 2 times.A concentration of ApoA1 monoclonal antibodies D11-10 being serially diluted is added, 37 DEG C are incubated 1 hour； PBST is washed 4 times.To the goat anti-mouse antibody of horseradish peroxidase (Horseradish Peroxidase, HRP) label 1: 2000 dilution is carried out, 50 μ L are added per hole, is incubated at room temperature 1.5 hours；PBST is washed 4 times.Then 50 bottoms μ L HRP are added per hole Object (H2O2+ TMB), room temperature is protected from light incubation about 0.5 hour, and 50 μ L 2N H are then added per hole2SO4, survey A450nmValue.Absorption value is big It is judged as the positive in twice of negative control hole or more.The light absorption value that positive hole is diluted by concentration gradient calculates ApoA1 monoclonal antibodies As a result the binding force of D11-10 shows the specific reaction that ApoA1 monoclonal antibodies D11-10 is relied on ApoA1 albumen generation concentration gradients (Fig. 1), the binding force that the antibody and people's ApoA1 albumen is calculated is 1.73 × 10-10M。
In conclusion ApoA1 monoclonal antibodies D11-10 is IgG2bThe binding force of subclass, antibody is 1.73 × 10-10M, only and people ApoA1 native proteins and recombinant protein generation specific reaction, and nothing to do with Protein S AA1, β2M and BSA does not react (table 1)。
The feature of table 1ApoA1 monoclonal antibodies D11-10
+：It reacts；：It does not react.
Embodiment 2：The measurement of the variable region sequences of ApoA1 monoclonal antibodies D11-10
1. material：Trizol (Invitrogen), primer are synthesized by Sheng Gong bio-engineering corporations, reverse transcription and PCR reagent It purchases from TaKaRa companies.
2. method and result：
The first chain of 2.1 Total RNAs extractions and cDNA synthesize
The hybridoma in exponential phase is collected, total serum IgE is extracted according to Trizol specification operating processes.It uses Spectrophotometer and agarose gel electrophoresis identify total serum IgE qualitative, quantitative.
According to TaKaRa companies PrimeScriptTMII 1st Strand cDNA Synthesis Kit's illustrates to synthesize cDNA。
The gene fragment amplification and sequencing of 2.2ApoA1 monoclonal antibody D11-10 heavy chain variable regions (VH) and light chain variable region (VL)
According to TaKaRa company's T aq enzyme specifications, using the reaction system of 50 μ LPCR.
The article Universal PCR amplification of mouse delivered according to Wang etc. immunoglobulin gene variable regions:the design of degenerate primers and an 3 ' to of assessment of the effect of DNA polymerase, 5 ' exonuclease activity, by raw work Bio-engineering corporation's synthetic primer.
Amplification condition：94 DEG C of pre-degenerations 5 minutes, 94 DEG C are denaturalized 30 seconds；57 DEG C are annealed 30 seconds；72 DEG C extend 30 seconds, and totally 35 A cycle, 72 DEG C 10 minutes.
PCR product is summarized as follows using Axygen companies glue recovery catalyst box (operation is according to manufacturers instruction)：By ApoA1 The agarose gel electrophoresis band of monoclonal antibody D11-10 heavy chain variable regions (VH) and light chain variable region (VL) gene PCR product is cut glue and is returned After receipts, quantitative determination and electrophoretic analysis are carried out.Target fragment is connected into T-A cloning vectors, after inverted, screening, picking sun Property clone transfer to Sheng Gong bio-engineering corporations be sequenced.The DNA sequence dna that sequencing is obtained passes through online software IMGT/V-QUEST points Analysis, the nucleotide sequence for encoding its heavy chain variable region (VH) and light chain variable region (VL) is respectively SEQ ID NO.1 and SEQ ID The amino acid sequence of No.2, heavy chain variable region (VH) and light chain variable region (VL) is respectively SEQ ID NO.3 and SEQ ID NO.4 (Fig. 2).
Embodiment 3：The detection method established based on ApoA1 monoclonal antibodies D11-10 cardiovascular and cerebrovascular disease risk profile and examine Disconnected clinical application
1. material：
ApoA1 monoclonal antibodies D11-10；The serum sample of people taking physical examination (no History of Coronary Heart Disease) and coronary heart disease (CHD) patient.
2. method and result：
2.1 prepare pooled serum calibration object and assignment
Using the serum sample of 20 people taking physical examinations, every takes in isometric serum to centrifuge tube (each 100 μ L), sets The packing of 200 μ L/ pipes, freezes in -80 DEG C of refrigerators after being mixed well in 4 DEG C.It takes out one within second day, is placed in 4 DEG C of defrostings, balance is extremely After room temperature, using the ApoA1 Immunoturbidimetric kits of desai company to pooled serum sample assignment.Pooled serum sample ApoA1 A concentration of 1.66g/L, and in this, as the calibration object for subsequently establishing ELISA detection method.
The detection of 2.2 clinical samples and cardiovascular and cerebrovascular disease correlation analysis
2.2.1 the collection of clinical sample and relevant clinical information：
Control group serum sample 38 is collected respectively and coronary heart disease (CHD) organizes serum sample 52.Control group is strong for physical examination Kang Renqun, triglycerides, cholesterol, high density and low-density lipoprotein are in normal physiologic values range, and no inflammation reaction, Without coronary heart disease and liver and kidney disease history；CHD group is the coronary heart disease crowd that coronary angiography is made a definite diagnosis.
2.2.2 double-antibody sandwich elisa is detected clinical sample and interpretation of result
2.2.2.1 the detection of the drafting of standard curve and clinical sample
Rabbit-anti ApoA1 antibody is coated with to 96 hole elisa plates, a concentration of 1 μ g/mL, coating buffer is 50mM pH8.5's Tris.HCl.Elisa plate is positioned in wet box, 4 DEG C overnight.It is washed 4 times with cleaning solution (PBS solution of 2%BSA) within second day Afterwards, the PBS solution of 200 μ L 2%BSA, 37 DEG C of closing 2h are added per hole.It is washed 4 times with cleaning solution, calibration is added in plate respectively Product and clinical serum sample.Pooled serum prepared by step 2.1 is with 1/20,1/40,1/80,1/160,1/320,1/640,1/ 1280 ratios are diluted, and are separately added into plate, 50 holes μ L/, and as calibration object, dilution is that the PBS containing 2%BSA is molten Liquid.To add the hole of dilution as blank control.The clinical serum sample being collected into carries out 1/80 times dilute with dilution respectively It releases, to ensure that concentration of specimens is located in the calibration curve concentration range drawn.Each clinical sample is surveyed using two multiple holes Examination.It is incubated 1 hour at 37 DEG C after sample is added in elisa plate, cleaning solution washs 4 times.Using dilution by ApoA monoclonal antibodies D11- 10 are diluted to 1 μ g/mL, and 50 μ L are added per hole, after being incubated at room temperature 1.5 hours, wash 4 times.It is dilute with 1: 2000 ratio using dilution The goat anti-mouse secondary antibody for releasing horseradish peroxidase (Horseradish Peroxidase, HRP) label adds 50 μ L per hole, After 37 DEG C are incubated 1 hour, cleaning solution washs 4 times.Then 50 μ LHRP substrates (H are added per hole2O2+ TMB), incubation at room temperature about 0.5 Hour, it is last that 50 μ L 2N H are added per hole2SO4, survey A450nmValue.It is dense according to the ApoA1 theoretical proteins of various concentration pooled serum Angle value and actual measurement A450nmValue fits calibration curve.Each clinical sample can be according to its A450nmValue, calculates in calibration curve The measured concentration value of ApoA1.
2.2.2.2ApoA1 the analysis of monoclonal antibody D11-10 testing results and the comparison with how anti-testing result
Sample is divided into control group and CHD groups according to clinical diagnosis information, and (underwent coronary radiography is made a definite diagnosis, i.e., clinic is examined The CHD sufferers group of disconnected goldstandard diagnosis), by each sample using the measured value of ApoA1 monoclonal antibodies D11-10 of the present invention and The measured value of ApoA1 Immunoturbidimetric kits (how anti-) is analyzed with GraphPad Prism 6, makes a distribution map (figure 3A)：The ordinate each put represents the ApoA1 contents for surveying the sample, and abscissa represents affiliated grouping.
Fig. 3 A show that control group and each sample of CHD groups pass through ApoA1 monoclonal antibody D11-10 and ApoA1 Immunoturbidimetric kits The testing result of (how anti-).The distribution that monoclonal antibody measures ApoA1 in CHD groups is low compared with the totality of control group；It is resist also in figure 3 a more It shows similar as a result, still obviously difference is that the distribution of how anti-test result is concentrated very much, cannot embody and there emerged a Body is heterogeneous.The test result for reviewing monoclonal antibody is screened since ApoA1 monoclonal antibodies have in specific conformation HDL (D11-10 hypotypes) particle ApoA1 epitopes ability, so ApoA1 content distributions be in discrete type, prompt D11-10 hypotypes HDL Different Individual point Cloth is different.
Due to monoclonal antibody only identifies on a HDL particle ApoA1 protein antigenic determinants of exposure and mostly it is anti-can identify it is more The difference of a antigenic determinant causes the normal reference range based on mostly anti-ApoA1 Immunoturbidimetric kits not to be suitable for base In the detection method of D11-10 monoclonal antibodies development.Therefore, the judgement of monoclonal antibody D11-10 measurement results needs to establish independent with reference to model It encloses.It is in Non-Gaussian Distribution after being summarized due to testing result, therefore uses median (25% percentile, 75% percentile) mode Statement：The control group A poA1 contents that ApoA1 monoclonal antibodies D11-10 is measured are 4.05 (2.43,6.00) g/L, control group A poA1 monoclonal antibodies The cut-off values of the confidence interval of D11-10 measurement results 90% are 1.37g/L (in Fig. 3Line)；And ApoA1 is immune Normal reference range cut-off values than turbid kit (how anti-) are 1.00g/L (in Fig. 3Line) (《Clinical examination is advised Journey》).The content that monoclonal antibody measures CHD groups ApoA1 is 1.64 (0.77,3.14) g/L, and it is small that statistics monoclonal antibody detects measured value in CHD groups In the number and proportion of cut-off values be respectively 22 and 42% (22/52).It is more anti-to measure CHD group ApoA1 contents and be 1.18 (0.99,1.32) g/L.Measured value is less than the number and proportion difference of cut-off values in the how anti-detection CHD groups of statistics For 13 and 25% (13/52).Illustrate more CHD cases that monoclonal antibody can detect, is more kissed with clinical CHD diagnosis goldstandards It closes, illustrates the high sensitivity of the ApoA1 detection methods developed based on monoclonal antibody in existing ApoA1 Immunoturbidimetric kits.
It further carries out analysis to the level of the HDL-C of CHD patient to find, the HDL-C in 38 control groups is in just Normal reference interval range (HDL-C >=1mM), but 52 CHD patients in collection also have 32 HDL-C to be in nominal reference area Between range (HDL-C >=1mM), illustrate only to detect again HDL-C can not completely in antimer the heterogeneity of HDL and with disease Correlation, therefore HDL-C has significant limitations as CHD risks and assumptions.Further to patient CHD of 32 HDL-C >=1mM Subgroup and 38 control groups are analyzed with GraphPad Prism 6, make a distribution map (Fig. 3 B).Same monoclonal antibody detection As a result it is still in a discrete distribution, it is 1.63 (0.86,2.91) g/L that monoclonal antibody, which measures ApoA1 contents in CHD groups, and under cut-off values There are 13 samples (13/32,41%) in side；Mostly anti-test result is reviewed, distribution is still concentrated, and prompt cannot distinguish between individual The heterogeneity of HDL.ApoA1 contents 1.27 (1.19,1.40) g/L in mostly anti-measurement CHD groups, all in range of normal value, i.e., There is no sample distribution (0/32) (Fig. 3 B, table 2) below the cut-off values of how anti-measurement result, i.e., in 32 HDL-C >=1mM CHD patient in, monoclonal antibody can increase by 41% recall rate compared to existing ApoA1 Immunoturbidimetric kits.Monoclonal antibody is prompted to survey Examination can improve the deficiency in how anti-detection and HDL-C detections, illustrate the inspection of the ApoA1 monoclonal antibodies D11-10 development using the present invention Survey method can improve the accuracy and sensitivity of ApoA1 and HDL-C in prediction CHD risks.
To sum up, compared to existing ApoA1 Immunoturbidimetric kits (how anti-), ApoA1 monoclonal antibodies D11-10 of the present invention is utilized The detection method of foundation has higher consistency with clinical diagnosis result, more especially in the CHD crowd of HDL-C >=1mM None is consistent for anti-testing result and clinical diagnosis result, and the recall rate of monoclonal antibody is 41%, substantially increases ApoA1 and HDL-C Accuracy in prediction CHD risks and sensitivity.
The ApoA1 comparision contents of table 2 CHD group ApoA1 monoclonal antibodies D11-10 and how anti-detection
§Monoclonal antibody test result cut-off values are that 1.37g/L (refers to 90% confidence area of control group monoclonal antibody test result Between).
※Mostly anti-test result cut-off values are 1.00g/L (referring to clinical examination operating instruction).
ApoA1 content representations are：Median (25% percentile, 75% percentile).
Since monoclonal antibody identifies HDL D11-10 hypotypes in crowd, it is in a discrete distribution in crowd, and in control group and CHD Between distribution it is variant, i.e., below cut-off values that CHD groups were more be distributed in.Knot is measured with CHD group ApoA1 monoclonal antibodies D11-10 Control group and CHD groups are grouped analysis (Fig. 4) by the tertile of fruit.As shown in Fig. 4 A and 4C, in minimum point of position t1 of ApoA1 Only have 2 in (0.10~0.90g/L) group, in control group and fall within the group (2/38,5.3%), both control group was most of not low In cut-off values, antithesis with CHD groups.And divide in hyte t2 (0.91~2.80g/L) group, control group has 11 (11/ 38,28.9%), wherein only 2 are less than cut-off values；High score hyte t3 (>In 2.90g/L), 66% control group sample (25/38) it is respectively positioned on this.Therefore control group proportion in the tertile group rises to 66% from 5.3%, 28.9% respectively, Chi-square Test its with significant difference (p<0.01)；Correlation analysis discovery further is carried out to the tertile group data, The result that ApoA1 monoclonal antibodies measure and disease negatively correlated (r=-0.377, p<0.01)；Due in patient CHD, having suitable one The HDL of some patientss is there is no reducing, still in normal range (NR), therefore, for subgroups of the part HDL in normal range (NR) (HDL >=1mM) is grouped analysis also according to above-mentioned tertile, as shown in Fig. 4 B and 4D, control group proportion difference For 7.9% (3/38), 15.8% (6/38), 76.3% (29/38), the control group crowd more than 3/4 is in high score hyte, the inspection of card side Testing display has significant difference (p<0.01)；Further correlation analysis is found, in the subgroup of the HDL >=1mM, The result that ApoA1 monoclonal antibodies D11-10 is measured (is increased to r=-0.435p with disease associated higher from r=-0.377<0.01)； Illustrate sensitiveer than existing HDL-C and ApoA1 immunoturbidimetries method using the detection method of ApoA1 monoclonal antibodies D11-10 development With it is accurate, the correlation for the CHD sufferer crowds being especially in nominal reference section with HDL-C is significantly increased.
In conclusion the ApoA1 monoclonal antibodies D11-10 of the present invention and its detection method and kit of development can screen and CHD Negatively correlated specific HDL hypotypes, i.e. HDL D11-10 hypotypes.The hypotype is distributed in patient CHD and its control group all in discrete Type prompts the HDL of group heterogeneous.In CHD, the testing result of monoclonal antibody is inhomogenous and overall relatively low, prompts HDL D11-10 hypotypes are reduced such as the CHD courses of disease, may be related to the severity of disease；And hypotype phase in control group To function that is more, prompting the hypotype that may there is anti-plaque to be formed.In the detection to clinical CHD patient, ApoA1 monoclonal antibodies are relied on The detection method of D11-10 can significantly improve the recall rate of CHD, especially the CHD patient in HDL-C normal (HDL-C >=1mM) In, in the case where existing ApoA1 immunoturbidimetries reagent is completely negative, can still there be 41% recall rate, substantially increase With the consistency of clinical coronary artery angiographic diagnosis (golden standard).Therefore the ApoA1 monoclonal antibodies D11-10 of the present invention is based on The detection method and kit of monoclonal antibody development can improve the defect of existing ApoA1 detection kits and method, to heart and brain blood The clinical monitoring of pipe disease is of great significance.
The protection content of the present invention is not limited to above example.Without departing from the spirit and scope of the invention, originally Field technology personnel it is conceivable that variation and advantage be all included in the present invention, and with appended claims be protect Protect range.
<110>Desai diagnostic system（Shanghai）Co., Ltd
<120>Anti-human ApoA1 monoclonal antibodies and its preparation method and application
<160> 4
<210> 1
<211> 352
<212> DNA
<213>Artificial sequence
<400> 1
CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTT
CTGGCTACAGTTTTACAAGCTACTGGATGCATTGGATAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGG
ATACATTAATCCTAGCAGTGGTTATACTGAATACAGTCAGAAGTTCAAGGACAAGGCCACAATGACTGCTGAC
AAATATTCCACAACAGCCTACATACAACTAAGCAGTGTGACATCTACGGACTCTGCAGTCTATTACTGTGCAT
CCTATGGTAACTACAAATTTGTTTACTGGGGCCAAGGGAGTCTGGTCACGGTCTCTGCAA
<210> 2
<211> 322
<212> DNA
<213>Artificial sequence
<400> 2
GATATTGTGATCACACAGTCTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGG
CAAGTCAGGACATTAACAATTATTTACACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTA
CTACACGTCCAGATTACAGTCAGGAGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTC
ATCATTACCAACCTGGAGCAAGAAGATTTCGCCACTTACTTTTGCCAACAGGGTGATACGCTTCCAATCACGT
TCGGCTCGGGGACAAATTTGGAAATAAAGC
<210> 3
<211> 117
<212>Amino acid
<213>Artificial sequence
<400> 3
QVQLQQSGAELAKPGASVKMSCKASGYSFTSYWMHWIKQRPGQGLEWIGYINPSSGYTEYSQKFKDKATMTAD
KYSTTAYIQLSSVTSTDSAVYYCASYGNYKFVYWGQGSLVTVSA
<210> 4
<211> 107
<212>Amino acid
<213>Artificial sequence
<400> 4
DIVITQSTSSLSASLGDRVTISCRASQDINNYLHWYQQKPDGTVKLLIYYTSRLQSGVPSRFSGSGSGTDYSL
IITNLEQEDFATYFCQQGDTLPITFGSGTNLEIK

Claims (12)

1. a kind of ApoA1 monoclonal antibodies D11-10, which is characterized in that the ApoA1 monoclonal antibodies D11-10 can be special Property identification ApoA1 albumen specific antigen epitopes.
2. ApoA1 monoclonal antibodies D11-10 as described in claim 1, which is characterized in that the ApoA1 monoclonal antibodies D11-10 is secreted by BALB/c mouse hybridoma cell strain D11-10, the BALB/c mouse hybridoma cell strain D11-10 preservations In China Committee for Culture Collection of Microorganisms's common micro-organisms center, the deposit date is on March 9th, 2017, deposit numbers For CGMCC No.13807.
3. ApoA1 monoclonal antibodies D11-10 as described in claim 1, which is characterized in that the ApoA1 monoclonal antibodies The heavy chain subgroup of D11-10 is IgG2b, light chain subtype κ, the amino acid sequence of heavy chain variable region and light chain variable region is respectively such as Shown in SEQ ID NO.3 and SEQ ID NO.4；Encode the nucleotide sequence of the heavy chain variable region and light chain variable region respectively such as Shown in SEQ ID NO.1 and SEQ ID NO.2.
4. as any one of them ApoA1 monoclonal antibodies D11-10 of claims 1 to 3 is being prepared for diagnosing cardiovascular and cerebrovascular Application in disease product.
5. a kind of diagnostic products of cardiovascular and cerebrovascular disease, which is characterized in that include any one of them of such as claims 1 to 3 ApoA1 monoclonal antibodies D11-10.
6. the diagnostic products of cardiovascular and cerebrovascular disease as claimed in claim 5, which is characterized in that the cardiovascular and cerebrovascular disease is examined Stopping pregnancy product include kit, test paper, solid support；The solid support includes array, microarray or protein array.
7. a kind of kit, which is characterized in that any one of them ApoA1 monoclonal antibodies including such as claims 1 to 3 D11-10。
8. the application of the diagnostic products such as cardiovascular and cerebrovascular disease described in claim 5 or 6, which is characterized in that the heart and brain blood The diagnostic products of pipe disease are used to detect the level of ApoA1 in human body, or are used for Division identification HDL-C hypotypes, or for monitoring Cardiovascular and cerebrovascular disease risk and diagnosis for cardiovascular and cerebrovascular disease.
9. application as claimed in claim 8, which is characterized in that the cardiovascular and cerebrovascular disease includes being formed to cause by vascular plaque Disease.
10. application as claimed in claim 9, which is characterized in that the cardiovascular and cerebrovascular disease is coronary heart disease, cerebral apoplexy or artery Atherosis.
11. a kind of detection method of ApoA1, which is characterized in that the method includes：Utilize any one institute of claims 1 to 33 The characteristics of ApoA1 monoclonal antibodies D11-10 stated can identify ApoA1 specific antigen epitopes carries out immune detection to ApoA1.
12. a kind of BALB/c mouse hybridoma cell strain D11-10, which is characterized in that the BALB/c mouse hybridoma cell strain D11-10 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the deposit date is March 9 in 2017 Day, deposit number CGMCC No.13807.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265002A (en) * 2021-05-25 2021-08-17 德赛诊断系统(上海)有限公司 Anti-human ApoA1 monoclonal for identifying HDL subclass positively correlated with coronary heart disease and preparation and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265002A (en) * 2021-05-25 2021-08-17 德赛诊断系统(上海)有限公司 Anti-human ApoA1 monoclonal for identifying HDL subclass positively correlated with coronary heart disease and preparation and application thereof
CN113265002B (en) * 2021-05-25 2023-10-31 德赛诊断系统(上海)有限公司 Antihuman ApoA1 monoclonal for identifying HDL subclass positively correlated with coronary heart disease, and preparation and application thereof

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