CN101921337B - Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application - Google Patents
Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application Download PDFInfo
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Abstract
The invention provides an antibody against lactate dehydrogenase of plasmodium vivax, which is a monoclonal antibody or polyclonal antibody specific to proteins shown in SEQ ID NO: 2. More preferably, the monoclonal antibody or polyclonal antibody is obtained by using a fusion protein formed by the protein shown in SEQ ID NO: 2 and the known label as an antigen immune animal; the monoclonal antibody is produced by a hybridoma cell strain of which the collection number is CGMCC No.3897 or No.3898. The invention also provides a hybridoma cell strain producing the antibody against the lactate dehydrogenase of plasmodium vivax, a preparation method and use of the antibody against the lactate dehydrogenase of plasmodium vivax, a related immunoassay method, a kit prepared from the antibody, etc. The antibody against the lactate dehydrogenase of plasmodium vivax can specifically detect the lactate dehydrogenase of plasmodium vivax, can be applied to specificity detection of vivax infection, has the advantages of high specificity, sensitive response and low cost, and is applicable to high-throughput detection.
Description
Technical field
The present invention relates to genetically engineered and immunological technique field, more specifically, relate to monoclonal antibody and polyclonal antibody technical field, refer to especially a kind of Plasmodium vivax Lactate Dehydrogenase (Pv-LDH) antibody, related manufacturing processes, hybridoma cell strain and application.
Background technology
Malaria (malaria) is the serious parasitosis of harm that is caused by Infected With Plasmodium.The plasmodium that infects the mankind has four kinds: plasmodium falciparum (Plasmodium falciparum, P.f), Plasmodium vivax (P.vivax, P.v), malariae (P.malariae, P.m) and Plasmodium ovale (P.ovale, P.o).According to the WHO statistics, there is the popular of malaria countries and regions, 107, the whole world and propagates, and 3,200,000,000 people are threatened by malaria.3～500,000,000 people's morbidities are arranged every year, and death toll reaches more than 100 ten thousand.
Being the key of effectively carrying out malaria control with diagnosing exactly fast, is also one of main intervening measure of global malaria control strategy.But the clinical diagnosis method of at present widespread use is based on the clinical symptom of malaria and diagnoses, because malaria symptom specificity is not strong and unreliable; Microscopy has that susceptibility is high, acquired information amount many (can determine worm kind, worm phase, metamorphosis and quantitatively) but, low, the blood sheet persistence of expense is convenient to " gold standard " that the advantage such as quality control is considered to present Infected With Plasmodium inspection and evaluation, but microscopy time and effort consuming, its accuracy depend on that to a great extent microscopy equipment, blood sheet are made and microscopy personnel's technology and experience.Unfortunately, these conditions are not being met at the scene or in the health organ of basic unit usually, cause the microscopy result insincere.
In recent years, the fast diagnosis method of malaria has had significant progress, using the test that immune chromatography method detects plasmodium specific antigens in the finger tip drop of blood can complete in 15 minutes, and operator only need the training of short period of time, need not electric power and special plant and instrument.Quick diagnosis is tested in the side area that lacks the microscopy condition and in emergency circumstances, is undoubtedly the important supplement to the microscopy diagnostic method.
Yet the commercialization detection kit of having developed at present both at home and abroad all can only detect plasmodium falciparum and non-malignant Infected With Plasmodium, for most of Endemic Area take the vivax malaria infection as main China, still lacks species specificity.Therefore, be badly in need of providing the species specificity that vivax malaria infects to detect, make up the deficiency that there is no at present the vivax malaria method for detecting specificity.
Summary of the invention
Main purpose of the present invention is exactly the problems and shortcomings for above existence, and a kind of antibody against lactate dehydrogenase of plasmodium vivax, related manufacturing processes, hybridoma cell strain and application are provided.Antibody against lactate dehydrogenase of plasmodium vivax energy specific detection Plasmodium vivax Lactate Dehydrogenase can be used for the specific detection that vivax malaria infects.Specificity is high, is quick on the draw, and cost is low, is suitable for high throughput testing.
In order to solve above-mentioned purpose, in a first aspect of the present invention, a kind of antibody against lactate dehydrogenase of plasmodium vivax is provided, has been characterized in, described antibody against lactate dehydrogenase of plasmodium vivax is that the albumen shown in SEQ ID NO:2 is had specific monoclonal antibody or polyclonal antibody.
" specificity " described in the present invention refers to that monoclonal antibody is to the recognition capability of corresponding antigens or approximate antigenic substance.Specificity is high, and the recognition capability of antigen is just strong.Therefore, above-mentioned antibody against lactate dehydrogenase of plasmodium vivax can specific recognition and in conjunction with the albumen shown in SEQ ID NO:2.Albumen shown in SEQ ID NO:2 can directly synthesize, and also can prepare by gene engineering method.In a specific embodiment of the present invention, the albumen shown in SEQ ID NO:2 can be to be produced by the 1st to 951 nucleotide coding of SEQ ID NO:1.
Preparing monoclonal antibody of the present invention antigen used is to obtain by engineered method, utilizes and express the polynucleotide sequence SEQ ID NO:1 that contains the antigen of the present invention of encoding in prokaryotic organism.Those skilled in the art knows the carrier that is applicable to express antigen of the present invention usually.Can select suitable carrier according to selected suitable promotor and goal gene sequence to be expressed.Can use any suitable host cell expression antigen of the present invention.Suitably host's example comprises: prokaryotic cell prokaryocyte such as intestinal bacteria, genus bacillus, streptomycete etc.The method of transduction, conversion or transfection is known in the art, includes, but not limited to virus infection, calcium chloride infection protocol, liposome transfection method, electroporation or microprojectile bombardment methods etc.In order to activate promotor, to select transformant or the required gene that increases, can cultivate by the host cell of transduction, transfection or conversion in the conventional nutritional medium of suitably modifying.The culture condition such as the temperature of using in cultivation, pH value are all generally to be determined by the host cell of selected expression specified protein, and these conditions are all well known to those skilled in the art.For a large amount of recombinant proteins that obtain, can adopt inducible promoter, and utilize the expression of inductor induced gene.In fact, " inductor " can be any material that can induce genetic expression in the host, can be chemical substance or environmental stimulus.In a preferred specific embodiment of the present invention, the inductor of employing is IPTG (isopropyl ss-D thiogalactoside).
In a specific embodiment of the present invention, use special primer and carry out the full length sequence that nucleotide sequence restructuring on pcr amplification and plasmid level has obtained Pv-LDH gene reading frame; And be cloned in prokaryotic expression carrier pET28a by the 951bp nucleotide sequence that EcoRI is connected with the XhoI enzyme, the step such as connection will comprise the whole opening code-reading frames of Pv-LDH gene, be built into prokaryotic expression plasmid, transform intestinal bacteria, under the inducing of IPTG, be expressed as the approximately fusion rotein rePv-LDH of Mr41000 of molecular weight.
Preferably, described monoclonal antibody or polyclonal antibody are that the fusion rotein that adopts the albumen shown in SEQ ID NO:2 and known label to form obtains as antigen-immunized animal.
More preferably, described known label is, but is not limited to histidine-tagged (His label), glutathione S-transferase (GST label), Trx label, S-tag label, HA label, HSV label, Myc label or VSV-G label.In a specific embodiment of the present invention, described known label is the His label, forms the HisPv-LDH fusion rotein with the albumen shown in SEQ ID NO:2.
More preferably, described monoclonal antibody adopts described fusion rotein to obtain as the antigen immune mouse, and described polyclonal antibody adopts described fusion rotein to obtain as the antigen immune rabbit.
Further, described monoclonal antibody has specificity to the HisPv-LDH fusion rotein, and the HisPf-LDH fusion rotein is not had specificity.
Preferably, described monoclonal antibody is the hybridoma cell strain generation of CGMCC No.3897 or No.3898 by preserving number.
In a specific embodiment of the present invention, the Subclass of antibody of the monoclonal antibody of the hybridoma cell strain Pv3 that the present invention obtains and Pv24 secretion is IgG1, and relative compatible constant is respectively 9.02 * 10
9Lmol
-1With 6.01 * 10
8Lmol
-1, the HisPv-LDH fusion rotein is had specificity, to the HisPf-LDH fusion rotein without specificity (Pv24) or weak reactivity (Pv3) is only arranged.And the identification antigen site of two monoclonal antibodies is not identical.
In a second aspect of the present invention, a kind of hybridoma cell strain is provided, be characterized in, described hybridoma cell strain is for generation of above-mentioned Plasmodium vivax Lactate Dehydrogenase monoclonal antibody, and the preserving number of described hybridoma cell strain is CGMCC No.3897 or No.3898.
Above-mentioned hybridoma cell strain has been deposited in one of international depositary institution, and " (China General Microbiological Culture Collection Center; CGMCC; address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; postcode: 100101); preservation date: on June 12nd, 2010, Classification And Nomenclature is mouse hybridoma cell (Mouse hybridoma) to China Committee for Culture Collection of Microorganisms's common micro-organisms " center ".
In a third aspect of the present invention, a kind of preparation method of above-mentioned antibody against lactate dehydrogenase of plasmodium vivax is provided, be characterized in, adopt the fusion rotein of the albumen shown in SEQ ID NO:2 and known label formation to prepare as antigen.
Preferably, described known label is, but is not limited to histidine-tagged (His label), glutathione S-transferase (GST label), Trx label, S-tag label, HA label, HSV label, Myc label or VSV-G label.In a specific embodiment of the present invention, described known label is the His label, forms the HisPv-LDH fusion rotein with the albumen shown in SEQ ID NO:2.
Preferably, described fusion protein immunization mouse, the splenocyte and the myeloma cell that get mouse are merged, filter out and to secrete the hybridoma that the albumen shown in SEQ ID NO:2 is had the monoclonal antibody of specific reaction, obtain described monoclonal antibody the animal ascites from the culture supernatant of described hybridoma or after injecting described hybridoma.
More preferably, the preserving number of described hybridoma cell strain is CGMCC No.3897 or No.3898.
In of the present invention one preferred specific embodiment,, get the splenocyte of mouse and the myeloma cell of syngeneic animal and merge as the antigen immune mouse with the rePv-LDH fusion rotein.Screening can produce the hybridoma of purpose antibody, carries out cloning and cultivates, and set up the hybrid cell strain.Aforesaid method is only exemplary, for example can use the same method the immunity other Mammalss, with its splenocyte as immunocyte.Can also select suitable myeloma cell be used for to merge, for example be used for myeloma cell from rat, mouse or hamster.Immunocyte and myeloma cell's fusion can be carried out according to ordinary method.
The hybridoma that screening produces purpose antibody carries out mono-clonal.The hybridoma cell strain of the generation monoclonal antibody of the present invention that obtains can go down to posterity in ordinary culture medium and cultivate or preserve for a long time in liquid nitrogen.When collecting monoclonal antibody of the present invention from hybridoma, can obtain antibody from hybridoma vitro culture supernatant liquor, perhaps hybridoma injected suitable Mammals and obtain antibody from animal ascites.A kind of front method is suitable for obtaining highly purified antibody, and a kind of rear method is suitable for obtaining in a large number antibody.By the antibody that aforesaid method obtains, can use the ordinary method purifying, such as saltout, the method such as gel-filtration, affinity chromatography.
In a fourth aspect of the present invention, provide the above-mentioned application of antibody against lactate dehydrogenase of plasmodium vivax in detection and/or auxiliary diagnosis plasmodium vivax infection disease.
In a specific embodiment of the present invention, provide the application in the Pv-LDH antigen protein that described monoclonal antibody exists in detecting human blood.Monoclonal antibody of the present invention has the antigen-binding specificity of height, the detection system of the present invention's foundation is used for the detection of patient's blood sample, all negative with the reaction of normal people's blood sample, with the subtertian malaria infected patient blood sample with comm on antigen structure also no cross reaction (Pv24 specificity 100%) or extremely low (the Pv3 cross reaction is only 15.38%, specificity is 90.47%), therefore, can be used for the specific detection that vivax malaria infects.
In a fifth aspect of the present invention, provide the above-mentioned application of antibody against lactate dehydrogenase of plasmodium vivax in the diagnostic kit of preparation detection and/or auxiliary diagnosis plasmodium vivax infection.
In a sixth aspect of the present invention, a kind of immunoassay is provided, be characterized in, described immunoassay adopts above-mentioned antibody against lactate dehydrogenase of plasmodium vivax.
Preferably, described immunoassay is ELISA immunoassay or immunochormatography.
In a specific embodiment of the present invention, by detection method being carried out preferably, set up many anti-detection systems that add monoclonal antibody, can reach ng level level to the detection sensitivity of rePv-LDH standard antigen.The linearity range that detects is larger, highly sensitive.Wherein, the sensitivity of Pv3 monoclonal antibody examination criteria antigen is 3.13ng/ml, and the Pv24 monoclonal antibody is 6.25ng/ml.In the detection of patient's blood sample, the susceptibility of Pv3 monoclonal antibody is that 93.51%, Pv24 monoclonal antibody is 85.71%.
In a seventh aspect of the present invention, a kind of test kit is provided, be characterized in, described test kit comprises above-mentioned antibody against lactate dehydrogenase of plasmodium vivax.
In a eighth aspect of the present invention, a kind of standard antigen of above-mentioned antibody against lactate dehydrogenase of plasmodium vivax is provided, be characterized in, described standard antigen is the rePv-LDH recombinant protein.
The double-antibody sandwich elisa detection system that the present invention sets up, the marker (marker can be the materials such as radio isotope, fluorescent chemicals, vitamin H and enzyme) that contains monoclonal antibody of the present invention, polyclonal antibody and be used for being combined with monoclonal antibody, and for detection of other reagent and damping fluid.Standard antigen provided by the invention can be used for whole detection system is demarcated.Therefore, detection system of the present invention can directly or further be converted into the ELISA detection kit after optimization fully.For a person skilled in the art, according to content of the present invention, utilize said monoclonal antibody, prepare corresponding testing product, such as Kit and/or Rapid detection test strip etc. is apparent.Therefore, be also the present invention's content required for protection.
Beneficial effect of the present invention is:
1, antibody against lactate dehydrogenase of plasmodium vivax of the present invention is that the albumen shown in SEQ ID NO:2 is had specific monoclonal antibody or polyclonal antibody, can be specifically in conjunction with the albumen shown in SEQ ID NO:2, thereby can be used for the specific detection that vivax malaria infects, specificity is high.
2, antibody against lactate dehydrogenase of plasmodium vivax of the present invention has specificity to the HisPv-LDH fusion rotein, the HisPf-LDH fusion rotein do not had specificity, thereby can carry out the species specificity detection that vivax malaria infects, make up the deficiency that there is no at present the vivax malaria method for detecting specificity, specificity is high, is quick on the draw.
3, immunoassay of the present invention and test kit can be used for the species specificity detection that vivax malaria infects, and make up the deficiency that there is no at present the vivax malaria method for detecting specificity, and specificity is high, is quick on the draw, and cost is low, is suitable for high throughput testing and large-scale promotion application.
Description of drawings
Fig. 1 is the pcr amplification product electrophorogram of pLDH gene.M:DNA marker, Pv: Plasmodium vivax, Pf: plasmodium falciparum.
Fig. 2 is that the digestion with restriction enzyme of Pv-LDH recombinant plasmid is identified.M:DNA marker, 1:pET28a empty plasmid, 2: recombinant plasmid double digestion, the PCR product of 3:Pv-LDH.
Fig. 3 is soluble analysis and the purifying of rePv-LDH recombinant antigen.M: albumen marker, 1: before inducing, 2: after inducing, 3: cellular lysate liquid supernatant, 4: cellular lysate liquid precipitate, 5: purification column effluent liquid, 6: purifying protein.
Fig. 4 is rePv-LDH recombinant antigen protein matter engram analysis.M: albumen marker, 1:rePv-LDH rabbit immune serum, 2: Healthy Rabbits serum, 3: patients infected with Plasmodium vivax pooled serum, 4: subtertian malaria patient's pooled serum, 5: the Healthy People pooled serum.
Fig. 5 is the different dilution association reaction curves of Pv3 monoclonal antibody.
Fig. 6 is the different dilution association reaction curves of Pv24 monoclonal antibody.
Fig. 7 is typical curve and the detection sensitivity that Pv3 and Pv24 monoclonal antibody detect the rePv-LDH recombinant antigen.
Fig. 8 is that the Pv3 monoclonal antibody detects diluting blood sample titre curve.
Fig. 9 is that the Pv24 monoclonal antibody detects diluting blood sample titre curve.
Figure 10 is that the light absorption value of the different classes of blood sample of monoclonal antibody detection compares.
Embodiment
The inventor obtains a kind of antibody against lactate dehydrogenase of plasmodium vivax through extensive and deep research, and this antibody against lactate dehydrogenase of plasmodium vivax has excellent specific binding effect to Plasmodium vivax Lactate Dehydrogenase.Completed on this basis the present invention.
In order more clearly to understand technology contents of the present invention, be described with reference to the accompanying drawings especially exemplified by following examples.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.In embodiment, various chemical reagent commonly used used, be the commercially available prod.
The preparation of embodiment 1 antigen (aminoacid sequence shown in SEQ ID NO:2)
1.1 the extraction of plasmodium gene group DNA
Adopt DNA extraction test kit (Omega) to extract plasmodial genomic dna from the blood sample that derives from the District of Shanghai patients infected with Plasmodium vivax, consult and use specification sheets and carry out, the DNA of extraction is in-20 ℃ of preservations.
1.2 design of primers and pcr amplification
According to the target gene sequence in GenBank (accession number is DQ060151) design Auele Specific Primer.The sequence of upstream primer is the nucleotide sequence shown in SEQ ID NO:3, and the sequence of downstream primer is the nucleotide sequence shown in SEQ ID NO:4.Introduce respectively EcoR I and Xho I restriction enzyme site at 5 of two primers ' end.Primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.Take the genomic dna that extracts as template, reaction conditions is 95 ℃ of 5min; 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations; 72 ℃ of 10min.Detect amplified production with 1% agarose gel electrophoresis, result as shown in Figure 1.The sequencing result demonstration, the nucleotide sequence of amplified fragments is as shown in SEQ ID NO:1, and its corresponding aminoacid sequence is as shown in SEQ ID NO:2.According to the above results, obtained required target fragment through pcr amplification.
1.3 the structure of recombinant plasmid
Pcr amplification product is after DNA gel purification kit (Axygen) purifying, with EcoR I/Xho I double digestion (New England Biolabs), 1% agarose electrophoresis is separated and purifying again, and the DNA fragmentation of recovery is connected with the pET28a expression vector of same process double digestion processing by 5: 1 ratios and transforms the JM109 bacterial strain.Through kalamycin resistance screening, from the bacterium colony that grows random choose several increase bacterium and cultivate, extract plasmid and do double digestion analysis (Fig. 2), select to have the cloning and sequencing (Invitrogen) of correct Insert Fragment.
1.4rePv-LDH the expression of recombinant antigen and purifying
Expression plasmid Transformed E .coli BL21 (DE3) Host Strains that will contain correct Insert Fragment, after cultivating through spending the night, the random choose bacterium colony is cultured to absorbancy (A in the LB substratum that contains kantlex
600Value)=0.6 o'clock, add isopropyl ss-D thiogalactoside (IPTG, 1mmol/L) to induce 4～5h.Bacteria suspension after inducing is centrifugal, collect thalline, be resuspended in PBS damping fluid (0.01mol/L, pH 7.4), after ultrasonic degradation, the centrifugal 30min of 12000 * g collects respectively upper cleer and peaceful precipitation and makes soluble analysis.The polyhistidyl (6 * His-tag) labels, the use NI-NTA agarose resin affinity purification recombinant protein that utilize the pET28a expression vector to carry.Elutriant is concentrated through Ultrafree-15 centrifugal ultrafiltration (Millipore) device, the purity (Fig. 3) of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (separation gel 15%, concentrated glue 5%) purification Identification recombinant antigen.
1.5 the preparation of polyclonal antibody and purifying
The fully emulsified water-in-oil antigen emulsion of making of the rePv-LDH recombinant antigen of purifying and isopyknic Fu Shi Freund's complete adjuvant, subcutaneous multi-point injection immunize rabbit after neck (300 μ g/ are only).Use identical antigen emulsion immunity 1 time after 4 weeks, after this every 2 weeks repeat immunity 1 time, amount to immunity 5 times again.Collect rabbit ear vein blood every 2 weeks before immunity and after immunity respectively, ELISA measures serum antibody titer.The heart blood sampling, collect immune serum after immune programme for children finishes, and conventional ELISA surveys immune serum and tires.The purifying of polyclonal antibody adopts salting-out process, gets antibody 2ml and adds in clean centrifuge tube, adds 2ml physiological saline mixing; Dropwise add the 4ml saturated ammonium sulphate, 4 ℃ of standing 2h or spend the night; The centrifugal 30min of 5000g, precipitation is again with 50% saturated ammonium sulphate washing 2 times; Make resolution of precipitate with 2ml PBS, this protein soln added in dialysis tubing, to 4 ℃ of dialysed overnight of PBS damping fluid, during change liquid 2-3 time.
1.6Western Blot detects
Recombinant antigen rePv-LDH is at about M
r41000 places and rabbit immune serum polyclonal antibody have strong reaction, with vivax malaria and subtertian malaria patients serum, the specific reaction band are arranged also, as shown in Figure 4.Show that recombinant antigen can be good at simulating the structure of natural antigen.
The preparation of embodiment 2, monoclonal antibody
2.1 animal immune
2.1.1 animal: female, 5 of 6～8W Balb/c in age mouse (available from the 2007-0005 of Shanghai Si Laike laboratory animal limited liability company (Shanghai)).
The purifying rePv-LDH recombinant antigen of preparation in 2.1.2 antigen: embodiment 1,50 μ g//times.
2.1.3 immunization route and program: initial immunity: rePv-LDH antigen and the abundant mixing of equal-volume complete Freund's adjuvant form water-in-oil, in mouse back intracutaneous multi-point injection.Immunity for the second time after 3 weeks, rePv-LDH antigen and incomplete Freund's adjuvant equal-volume mixing, back intracutaneous multi-point injection.Afterwards every 3 all immunity once (intraperitoneal does not contain adjuvant), and get tail blood 20 μ l in 5-7 days after immunity, survey antibody titer with the ELISA method.When antibody titer reaches requirement, tail vein booster immunization (not containing adjuvant)), get spleen after Yu Santian, carry out cytogamy.
2.2 cytogamy
2.2.1 the preparation of feeder cell: the Balb/c mouse draws neck to put to death, soaking disinfection 5min in 75% alcohol.Cut the abdomen outer skin open, inject mouse peritoneal with 5ml serum-free RPMI-1640, rock gently rear extraction peritoneal fluid, centrifugal counting.Adjust cell concn to 0.5～2 * 10 with 20% calf serum 1640
5/ ml plants immediately in 96 well culture plates, the 0.1ml/ hole.
2.2.2 the preparation of splenocyte: after last booster immunization the 3rd day, the aseptic spleen of getting mouse was placed in the RPMI-1640 of serum-free, with bolt in syringe, spleen was driven away brokenly, by nylon net filter, prepared cell suspension.The centrifugal 5min of 1000rpm is resuspended in 5ml serum-free RPMI-1640 counting cells concentration after abandoning supernatant.
2.2.3 myeloma cell's preparation: select mouse myeloma strain SP 2/0 (available from Shanghai cell biological institute of the Chinese Academy of Sciences), collect the myeloma cell of logarithmic phase, the washing numeration, adjusting concentration is 4～5 * 10
5/ ml is suspended in the serum-free RPMI-1640 standby.
2.2.4 cytogamy: splenocyte is mixed with 10: 1 (cell count ratio) with SP 2/0 cell, the centrifugal 5min of 1000rpm, the supernatant that inclines flicks the cell mass that settles down, and makes it fully loose.50%PEG-4000 with 1ml pipette, extract 0.7ml adds in 1min while stirring, adds rear standing 90s.Stop merging with the serum-free RPMI-1640 afterwards, must slowly add (1ml/min) during beginning, then accelerate gradually, the limit edged stirs, and adds altogether the 30ml stop buffer.Whole fusion process must be carried out under 37 ℃ of water-baths.The centrifugal 5min of 800rpm removes supernatant, continues next step operation.
2.2.5 cell cultures: bullet is loose gently with the cell mass after centrifugal.Fully loose rear resuspended with the HAT substratum until cell, join in 96 orifice plates of completing with feeder cell every hole 200 μ l.Be placed in 37 ℃, 5% (volume percent) CO
2Incubator in cultivate.After one week, observe the clonal growth situation and record every hole clone's number under inverted microscope.Can draw the nutrient solution supernatant at the bottom of account for culture hole during 1/3 area carries out ELISA and detects until Growth of Cells.
2.3 the screening of positive colony and subclone
2.3.1ELISA detect:, be placed in 4 ℃ and spend the night (50ng/ml) every hole 100 μ l coated elisa plates of detectable antigens rePv-LDH and re-Pf-LDH (subtertian malaria serum lactic dehydrogenase recombinant antigen) with coating buffer; The enzyme plate that will be coated with washings washs 3 times, each 3min, and then with the 1%BSA-PBS sealing, every hole 200 μ l, room temperature is placed 2h; The deblocking liquid that inclines, the same washing, every hole adds 100 μ l Hybridoma Cell Culture supernatants to be checked, hatches 1h in 37 ℃; The culture supernatant of inclining, after washing, every hole adds sheep anti-mouse igg-HRP (horseradish peroxidase) binding substances 100 μ l of 1: 3000 (working concentration) dilution, hatches 1h in 37 ℃.Unnecessary enzyme conjugates is discarded the same washing.Every hole adds the tmb substrate reaction solution of 100 μ l, and after color development at room temperature 10-30min, every hole adds stop buffer (the 2mol/L H of 50 μ l
2SO
4) termination reaction.Survey light absorption value with microplate reader 450nm, light absorption value greater than negative hole more than 2 times the person positive, select the rePv-LDH antigen-reactive positive, and the negative cell hole of re-Pf-LDH antigen-reactive carries out subclone.
2.3.2 the subclone of hybridoma (Single cell culture): adopt limiting dilution assay to carry out cloning.The same 2.2.1 of the preparation of feeder cell detects cell in positive hole wash-out counting gently in the culture plate with ELISA.Become 30 cell/ml to be seeded in 96 orifice plates that are covered with feeder cell with nutrient solution serial dilution cell suspension, the 0.1ml/ hole.Cultivated about 10 days, and when the hybridoma colony grows at the bottom of the hole 1/3 area, began to measure antibody activity in supernatant.The positive cell hole that antibody activity is arranged is cloned again, carry out altogether 3 times.
Through above-mentioned ELISA screening and the subclone of repeating, filter out at last the hybridoma cell strain of the monoclonal antibody of the 2 anti-rePv-LDH antigens of strains energy stably excreting, respectively called after Pv3 and Pv24.Preserving number is respectively CGMCCNo.3897 or No.3898.
2.4 a large amount of preparations of monoclonal antibody and preliminary purification
2.4.1 the preparation of mouse ascites and collection: with the hybridoma enlarged culturing, choose 20～22g Balb/c female mice, abdominal injection hybridoma 2 * 10
7/ only, after mouse web portion obviously expands, collect ascites after 10～14d.The centrifugal 5min of ascites 3000g with collecting gets supernatant ,-20 ℃ of storages.
2.4.2 the preliminary purification of ascites: adopt salting-out process, get ascites 2ml and add in clean centrifuge tube, add 2ml physiological saline mixing; Dropwise add the 4ml saturated ammonium sulphate, 4 ℃ of standing 2h or spend the night; The centrifugal 30min of 5000g, precipitation is again with 50% saturated ammonium sulphate washing 2 times; Make resolution of precipitate with 2ml PBS, this protein soln added in dialysis tubing, to 4 ℃ of dialysed overnight of PBS damping fluid, during change liquid 2-3 time.
The evaluation of embodiment 3, monoclonal antibody
3.1 Subclass of antibody
Use mouse monoclonal antibody subclass detection kit (Sigma) to measure.With sheep anti-mouse igg 1, IgG2a, IgG2b, IgG3, IgA and IgM use respectively coating buffer 1: 2000 dilution, and 100 μ l/ holes are coated, 4 ℃ spend the night or 37 ℃ hatch 2h; With washings PBST washing 3 times, 3 minutes/time, every hole added 100 μ l monoclonal antibody cells and supernatant to be detected, hatched 1 hour for 37 ℃; The same washing 3 times adds sheep anti mouse multivalent immunoglobulin (G, A, the M) antibody of horseradish peroxidase-labeled of dilution in 1: 5000, and every hole 100 μ l are hatched 1h for 37 ℃; The same washing three times, every hole add the tmb substrate solution 100 μ l of fresh configuration, and 37 ℃ of lucifuges were hatched 20 minutes; Every hole adds 50 μ l 2M H
2SO
4Termination reaction.Read OD on microplate reader
450Light absorption value, coated subclass classification is the antibody isotype of supernatant to be measured take the positive reaction hole.Qualification result shows, the Subclass of antibody of the Pv-LDH cell strain of monoclonal antibody Pv3 of the present invention's preparation and Pv24 secretion is IgG1.
3.2 the recognition site of antibody
3.2.1 site competition ELISA: with the dilution in 1: 200 of Pv3 and Pv24 monoclonal antibody, get 100 μ l/ hole coated elisa plates, 4 ℃ are spent the night; Wash plate 3 times with PBS (containing 0.05%Tween-20), 1%BSA seals 1h; The same washing adds respectively each the 100 μ l of mixed solution after Pv-LDH recombinant antigen (antigen final concentration 1: 1000) diluted Pv3 and 37 ℃ of effect 1h of Pv24 monoclonal antibody with 1: 500 respectively.Simultaneously, the contrast take the Pv-LDH recombinant antigen (1: 1000) that do not add monoclonal antibody as basic point, 37 ℃ of reaction 1h; The samely wash plate 3 times, add Pv-LDH rabbit polyclonal antibody (1: 500), 37 ℃ of reaction 1h; The samely wash plate 3 times, add goat anti-rabbit igg-HRP, 37 ℃ of reaction 1h; Wash plate 3 times, the tmb substrate colour developing.Calculate antigen after the different monoclonal antibody competing reactions of saturation capacity, the inhibiting rate of residue site and coated antibody reaction.Inhibiting rate calculation formula: inhibiting rate=(absorbancy after basic point contrast absorbancy-competition)/basic point contrast absorbancy * 100%.Take inhibiting rate＞50% as criterion.
The demonstration of site competition ELISA result, Pv3 and Pv24 monoclonal antibody are respectively 75.58% and 58.18% to the competition inhibiting rate of self, all greater than 50%; And the inhibiting rate that both vie each other is respectively 12.79% and 45.45%, all less than 50%, shows that the epitope of two monoclonal antibody identifications is different.
Table 1. competitive ELISA is measured the binding site of monoclonal antibody
3.2.2 site blocking-up ELISA: with the Pv-LDH recombinant antigen 100 μ l/ hole coated elisa plates of dilution in 1: 1000,4 ℃ are spent the night; Wash plate 3 times with PBS (containing 0.05%Tween-20), 1%BSA seals 1h; The same washing adds respectively and dilutes Pv3 and Pv24 monoclonal antibody at 1: 100, establishes simultaneously normal mouse serum and PBS contrast, 37 ℃ of effect 1h; The samely wash plate 3 times, add the HRP marker (working concentration 1: 3000) of Pv3 monoclonal antibody, 37 ℃ of reaction 1h; Wash plate 3 times, the tmb substrate colour developing.The contrast take normal mouse serum and PBS as basic point is respectively calculated envelope antigen respectively after the blocking effect of Pv3 and Pv24 monoclonal antibody, to the inhibiting rate of the Pv3 monoclonal antibody reaction of mark.Inhibiting rate calculation formula: inhibiting rate=(absorbancy after basic point contrast absorbancy-blocking-up)/basic point contrast absorbancy * 100%.Take inhibiting rate＞50% as criterion.
The Pv3 monoclonal antibody is respectively 77.85% (contrast take normal mouse serum as basic point) and 80.12% (contrast take PBS as basic point) to the inhibiting rate of the Pv3 monoclonal antibody of mark, all greater than 50%; And the Pv24 monoclonal antibody is respectively 8.72% and 18.07% to the inhibiting rate of the Pv3 monoclonal antibody of mark, and all less than 50%, namely Pv24 can not block the combination of Pv3 monoclonal antibody and antigen.The site is blocked the ELISA result and is also shown, the epitope of two monoclonal antibody identifications is different.
Table 2. blocking-up ELISA measures the binding site of monoclonal antibody
3.3 the relative affinity of antibody is measured
With reference to (Journal of Immunological Methods, 1987, the non-competing ELISA method mensuration of 100:173-179) setting up such as Beatty.At first determine the suitableeest package amount of Pv-LDH recombinant antigen, draw with 4 concentration (concentration ratio is 1: 2: 4: when Pv-LDH recombinant antigen 8) is coated with, different extent of dilution and the A of monoclonal antibody
450The association reaction curve of nm (Fig. 5, Fig. 6), half (is A to obtain respectively its maximum absorbance A value
50%) locate corresponding antibody concentration.By formula (n Ab '-Ab) calculates affinity costant to Kaff=(n-1)/2.In formula Ab and Ab ' represent respectively the antigen coated concentration of any 2 curves be Ag and Ag ' time A
50%Antibody concentration, guarantee Ag＞Ag ' when note calculating, n=Ag/Ag ', each antigen coated amount just can calculate 1 Kaff value, then compares in twos, when n=2, can get 3 K values; During n=4, can get 2 K values; During n=8, can get 1 K value, obtain the mean of 6 K values as net result.The demonstration of table 3 result, the relative compatible constant of 2 monoclonal antibodies that the present invention obtains is respectively 9.02 * 10
9Lmol-1 and 6.01 * 10
8Lmol
-1
The non-competing ELISA method of table 3. is measured the relative compatible constant of monoclonal antibody
3.4 the specificity of antibody
RePv-LDH antigen and rePf-LDH antigen are diluted to the concentration 100 μ l/ hole coated elisa plates of 1 μ g/ml with coating buffer, 4 ℃ spend the night or 37 ℃ hatch 2h; With washings PBST washing 3 times, 3 minutes/time; Pat dry the PBST sealing that rear use contains 1%BSA, 37 ℃ of wet box effect 1h; The same washing 3 times adds the odd contradictive hydroperitoneum of doubling dilution, and extent of dilution is 1: 1000,2000,4000,8000,16000,32000,64000,128000,37 ℃ and hatches 1h; The same washing 3 times adds the sheep anti-mouse igg antibody of peroxidase labelling of dilution in 1: 3000, hatches 1h for 37 ℃; The same washing 3 times adds the tmb substrate colour developing, with the negative contrast of the serum of normal Balb/c mouse.
Table 4 result shows, odd contradictive hydroperitoneum and the rePf-LDH antigen anergy (Pv24) of the present invention preparation or weak reactivity (Pv3) is only arranged, negative with the reaction of normal Balb/c mouse serum, and with rePv-LDH antigen, strong reactivity is arranged, cross over 4 extent of dilution between the platform area of antibody dilution, show that the specificity of antibody and affinity are better.
Table 4. indirect ELISA detected result
The sensitivity of embodiment 4, detection rePv-LDH standard substance
4.1 the foundation of double-antibody sandwich elisa
The rePv-LDH antigen of concentration known is preserved the liquid doubling dilution, with Pv-LDH rabbit source polyclonal antibody and Pv3 and two mono-clonals of Pv24 totally 3 antibody independent assortments carry out the double-antibody sandwich elisa test, the antigen amount that can detect with the double-antibodies sandwich ELISA of the monoclonal antibody of measuring homophyletic not and how anti-pairing or combination.Selection detects the most responsive a kind of pairing of antigen amount or combination and sets up double-antibodies sandwich ELISA and detect Pv-LDH antigen in malaria patient and normal people's blood sample.Criterion: the many parts of average light absorption value (A of normal human serum
450) negative reference value, patient's blood sample detected value/positive threshold value of negative reference value (P/N) 〉=2.0.Result shows, and is the highest with the sensitivity that many anti-double fastener heart indirect ELISA methods that add monoclonal antibody detect the rePv-LDH recombinant antigen.Therefore, we select the method to set up detection system.
4.2 Specification Curve of Increasing method
With Pv-LDH rabbit source polyclonal antibody as coated antibody (concentration 10 μ g/L)), monoclonal antibody Pv3 and Pv24 are respectively as detecting antibody (concentration is 10 μ g/L).Concrete operations are as follows: with Pv-LDH rabbit source polyclonal antibody 100 μ l/ hole wrapper sheets, 4 ℃ spend the night or 37 ℃ hatch 2h; PH7.4PBST washes plate 3 times, and every hole adds 2%BSA, seals 1h at 37 ℃ of temperature, and PBST washes plate 1 time; Every hole adds 100 μ l with the standard rePv-LDH antigen serial dilution solution of normal people's whole blood (1: 5) dilution, hatches 1h for 37 ℃; After using PBST to wash plate 6 times, every hole adds 100 μ l Pv3 or Pv24 antibody, hatches 1h for 37 ℃; The samely wash plate 3 times, add the sheep anti-mouse igg antibody of the peroxidase labelling of dilution in 1: 3000, hatch 1h for 37 ℃; The samely wash plate 3 times, add tmb substrate liquid, 37 ℃ of lucifuges colour developings 15-20 minute; Every hole adds 50 μ l, and 2mol/L sulfuric acid termination reaction detects A
450Value is according to the A that obtains
450Value is made rePv-LDH antigen concentration typical curve.
4.3rePv-LDH recombinant antigen typical curve and detection sensitivity
Take normal people's whole blood sample of 1: 5 as diluent, with rePv-LDH recombinant antigen (400 μ g/ml) from 1: 500 doubling dilution to 1: 512000, not contain the negative contrast of whole blood diluent of antigen; Then adopt 4.2 method to measure respectively A
450Value according to measurement result, is drawn the rePv-LDH recombinant antigen to A
450The typical curve (Fig. 7) of value.According to the result of Fig. 7, it is linear that recombinant antigen standard reaction curve is between 3.13～800ng/ml, and the linear relationship in 6.25～200ng/ml scope is best, and with the absorbance ratio of negative control greater than 2.0.Therefore, typical curve can be used for the demarcation of recombinant protein concentration in 6.25～200ng/ml scope.With 2.0 times of positive threshold values of negative control light absorption value, the detection sensitivity of two monoclonal antibodies is respectively that Pv3 is that 3.13ng/ml and Pv24 are 6.25ng/ml.
4.4 diluting blood sample titre curve plotting
Make strong positive, the positive and weak positive mixed blood sample with the malaria disease human blood sample of many parts of different infection intensities respectively, with mixing sample since 1: 1 doubling dilution to 1: 512, then adopt 4.2 method to measure respectively the A of patient's blood sample of different extension rates
450Value.According to measurement result, in the drafting blood sample, antigen is to A
450The titre curve (Fig. 8 and Fig. 9) of value.The result of Fig. 8 and Fig. 9, the pooled serum of different infection intensities is good in 1: 2～8 scope internal linear relations, and with the ratio of negative control absorbancy greater than 2.0.Therefore, blood sample can dilute in 1: 2～8 scopes, be used for the detection of blood sample Pv-LDH antigen, but the extent of dilution of blood sample is less, and the susceptibility of detection reaction is higher.
With the double-antibodies sandwich ELISA of above-mentioned foundation, 161 parts of whole blood samples (wherein, 32 parts of 77 parts of vivax malarias, 52 parts of subtertian malarias and normal peoples) are detected, detected result sees Table 5.Result shows, the susceptibility of Pv3 monoclonal antibody and specificity are respectively 93.51% and 90.47%, and the susceptibility that is 15.38%, Pv24 monoclonal antibody with the cross reaction of subtertian malaria is 85.71% a little less than Pv3, but specificity is 100%, with subtertian malaria blood sample no cross reaction.Two monoclonal antibodies all can be distinguished different classes of blood sample (Figure 10) preferably.In the double-antibody sandwich elisa detection system that the present embodiment uses, polyclonal antibody and monoclonal antibody only are the antibody with the salting-out process preliminary purification, if above-mentioned antibody by being further purified as methods such as affinity chromatographys, may be obtained more preferably susceptibility and specificity.
Table 5. double-antibody sandwich elisa detects patient's blood sample result
PLDH (Plasmodium lactate dehydrogenase, pLDH) only produced by living worm body, in blood, the level of pLDH is parallel relevant to parasitemia, detect the existing state that pLDH can differentiate polypide, be used for the curative effect of monitoring and judgement medicine, thereby can become the comparatively desirable target antigen of malaria detection.
The present invention is used for by the monoclonal antibody specific of a kind of Plasmodium vivax Lactate Dehydrogenase (Pv-LDH) is provided the specific detection that vivax malaria infects, and makes up the deficiency that there is no at present the vivax malaria method for detecting specificity.
In sum, antibody against lactate dehydrogenase of plasmodium vivax energy specific detection Plasmodium vivax Lactate Dehydrogenase of the present invention can be used for the specific detection that vivax malaria infects, and specificity is high, is quick on the draw, and cost is low, and is suitable for high throughput testing.
In this specification sheets, the present invention is described with reference to its specific embodiment.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Claims (5)
1. antibody against lactate dehydrogenase of plasmodium vivax, it is characterized in that, described antibody against lactate dehydrogenase of plasmodium vivax is that the albumen shown in SEQ ID NO:2 is had specific monoclonal antibody, and the hybridoma cell strain that described monoclonal antibody is CGMCC No.3897 by preserving number produces.
2. a hybridoma cell strain, is characterized in that, described hybridoma cell strain is for generation of antibody against lactate dehydrogenase of plasmodium vivax according to claim 1, and the preserving number of described hybridoma cell strain is CGMCC No.3897.
3. the preparation method of an antibody against lactate dehydrogenase of plasmodium vivax according to claim 1, it is characterized in that, obtain described monoclonal antibody animal ascites from the culture supernatant of hybridoma or after injecting described hybridoma, the preserving number of described hybridoma cell strain is CGMCC No.3897.
4. the application of antibody against lactate dehydrogenase of plasmodium vivax according to claim 1 in the diagnostic kit of preparation detection and/or auxiliary diagnosis plasmodium vivax infection.
5. a test kit, is characterized in that, described test kit comprises antibody against lactate dehydrogenase of plasmodium vivax according to claim 1.
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| CN110951703B (en) * | 2019-12-23 | 2023-04-07 | 杭州贤至生物科技有限公司 | Plasmodium vivax lactate dehydrogenase recombinant protein and preparation of monoclonal antibody thereof |
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| CN1222936A (en) * | 1996-02-14 | 1999-07-14 | 巴斯德研究所 | Recombinant protein containing c-terminal fragment of plasmodium MSP-1 |
| CN1414018A (en) * | 1993-09-10 | 2003-04-30 | 美国国有健康与人类服务部 | Binding region of erythrocyte binding protein of plasmodium vivax and plasmodium falciform |
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| CN1414018A (en) * | 1993-09-10 | 2003-04-30 | 美国国有健康与人类服务部 | Binding region of erythrocyte binding protein of plasmodium vivax and plasmodium falciform |
| CN1222936A (en) * | 1996-02-14 | 1999-07-14 | 巴斯德研究所 | Recombinant protein containing c-terminal fragment of plasmodium MSP-1 |
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| Title |
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| 江莉 等.间日疟原虫和恶性疟原虫乳酸脱氢酶基因的序列和重组抗原表位分析.《中国寄生虫学与寄生虫病杂志》.2010,第28卷(第2期), * |
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