CN101654668B - Hybridoma cell line and monoclonal antibody for producing angiostrongylus cantonensis V-stage larvae 55KD antigen - Google Patents
Hybridoma cell line and monoclonal antibody for producing angiostrongylus cantonensis V-stage larvae 55KD antigen Download PDFInfo
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Abstract
The invention discloses a hybridoma cell line ACV1 and a monoclonal antibody ACV McAb for producing angiostrongylus cantonensis V-stage larvae 55KD antigen. The antibody and the angiostrongylus cantonensis V-stage larvae 55KD antigen can be in specific binding. The preparation method of the antibody is simple, and the antibody can be obtained by direct secretion of the hybridoma cell line ACV1. The antibody has higher binding specificity and affinity with the angiostrongylus cantonensis V-stage larvae 55KD antigen, and the monoclonal antibody can be applied to detect the angiostrongylus cantonensis circulating antigen levels, thereby providing a method of early diagnosis of the angiostrongylus cantonensis.
Description
Technical field
The present invention relates to the method for the angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody of a kind of hybridoma that produces angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody and generation thereof.
Background technology
Angiostrongyliasis cantonensis (Angiostrongyliasis cantonensis) is meant by the angiostrongylus cantonensis larva at the human body internal migration; Invade that cns causes with acute severe headache serve as eosinophilia's property meningitis of mainly showing or meningoencephalitis (Eosinophilicmeningitis or Meningoencephalitis, EM).To the first routine case of Taiwan in 1945 report, this disease is successively at the China mainland report and be ascendant trend year by year, subsequently respectively in Wenzhou, collective's outbreak of epidemic among a small circle appears in Fujian, sickness rate reaches 44.8%.Angiostrongyliasis cantonensis was classified as the New Development transmissible disease by the Ministry of Health in 2003, but did not cause enough attention, lacked the understanding of this disease system, the diagnosis and treatment measure of detection means and standard efficiently.More than the local outbreak of epidemic in June, 2006 Pekinese, number of the infected, coverage is wide, beaten alarm bell to masses, caused personages of various circles of society's common concern.Along with people's food habits changes, and angiostrongyliasis cantonensis is soaring year by year, China's public health and people ' s health is caused serious threat, makes department of the Chinese government and medical institutions bear enormous economic loss and public opinion pressure.
The misdiagnosis rate of angiostrongyliasis cantonensis is high, mainly leans on the polypide that from patient's cerebrospinal fluid, detects children adult in age to make a definite diagnosis at present.Because this sick clinical symptom is very similar with many nervus centralis disorder diseases, and angiostrongyliasis cantonensis etiology diagnosis rate lower again (＜10%), brings very big difficulty to clinical diagnosis.So immunological method has important value as the auxiliary diagnosis means in the diagnosis of angiostrongyliasis cantonensis.But domesticly so far the diagnosis of this disease is also rested on the antibody test stage; Because of there being following point: the appearance of 1. infecting back antibody is later; 2. cure back antibody fully and still can have the long period in vivo; Can't carry out existing disease Infect And Diagnose and efficacy assessment, 3. higher cross reaction etc. arranged, limit the safety of these immune diagnostic methods with other helminthic infection.
Summary of the invention
The objective of the invention is to provides a kind of hybridoma cell strain and produces angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody for the deficiency that overcomes existing early diagnosis angiostrongyliasis cantonensis technology.
Be the realization above-mentioned purpose, the hybridoma cell strain that produces angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody provided by the present invention, name is called ACV1 (Angiostrongylus cantonensis V stage larva 1).This cell strain is stored in Chinese typical culture collection center on April 29th, 2009, and deposit number is CCTCC-C200930.
Hybridoma cell strain ACV1 produces the antigenic method for preparing monoclonal antibody of angiostrongylus cantonensis V-stage worm 55KD and may further comprise the steps:
(1) electroosmotic drainage is separated angiostrongylus cantonensis V phase larva 55KD albumen;
(2) to separate the albumen obtain from step (1) as the immunogen immune animal;
(3) get the immune animal splenocyte, itself and myeloma cell are merged, obtain hybridoma;
(4) from the animal ascites fluid of cell culture fluid or inoculation hybridoma, separate and the required monoclonal antibody of purifying.
Hybridoma cell strain ACV1 can produce the antigenic monoclonal antibody of angiostrongylus cantonensis V-stage worm 55KD; This antibody can have higher binding specificity and avidity with angiostrongylus cantonensis V phase larva 55KD antigen; This monoclonal antibody is applied to the detection of angiostrongylus cantonensis CAg level, the method for early diagnosis angiostrongyliasis cantonensis is provided.
Angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody called after ACV McAb by above-mentioned hybridoma ACV1 generation.
Below in conjunction with specific embodiment the present invention is done further explain.
Description of drawings
Fig. 1 is the karyotype observations of hybridoma.
Fig. 2 is the SDS-PAGE detected result of the monoclonal antibody that produces of purified hybridoma ACV1.
Fig. 3 is the Western Blot detected result of binding specificity of monoclonal antibody and the angiostrongylus cantonensis V phase larval antigens of purifying.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The acquisition of the angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody of hybridoma cell strain ACV1 and generation thereof
1, antigen prepd: in (Ampullaria gigas) Fushou spiral shell body, isolate angiostrongylus cantonensis III phase larva; Peroral infection SD rat is seized angiostrongylus cantonensis V phase larva from rat cerebral tissue after 21 days, with saline water rinsing 5 times; Extract angiostrongylus cantonensis V phase larval antigens by ordinary method; Run 12%PAGE glue (not adding SDS), 55KD size protein band was cut behind the glue under voltage 300V condition the electric osmose purifying 1 hour, collect PBS dialysis 24h behind the supernatant.Measure antigenic protein contnt (4.0mg/ml) with the Lowry method, put-20 ℃ of preservations.In addition, mouse serum is placed-20 ℃ of preservations.
2, immune animal
Choose 6～8 week female BALB/c mouses in age (immune animal can also be Mammalss such as mouse, rat, rabbit, goat, sheep, pig, donkey, horse), with 200 μ g antigens/only immune more than 2 months.Immunity for the first time adds Fu Shi Freund's complete adjuvant (0.1ml/ only), adds later freund 's incomplete adjuvant for the second time, and be 3 weeks immune pitch time.Preceding 3 days of cytogamy is got caudal vein blood, measures with indirect elisa method and tires, and the mouse tail vein booster immunization that selection is tired the highest once.
3, cytogamy
(1) preparation of feeder cell: a normal BALB/c mouse is put to death, take out spleen under the sterile state, remove surface-coating and fat, shred, place plate to grind, add serum-free RPMI RPMI-1640 and process single cell suspension, the meter viable count is about 5 * 10
5/ mL.
(2) preparation of immune spleen cell: the BALB/c mouse of step 2 booster immunization after three days put to death, take out spleen under the sterile state, remove surface-coating and fat; Shred, place plate to grind, add serum-free RPMI RPMI-1640 and process single cell suspension; The meter viable count is about 1 * 10
8Individual/mL.
(3) Sp2/0 myeloma cell's processing: get the Sp2/0 cell of exponential phase of growth, centrifugal, to wash once and be suspended in wherein with serum-free RPMI RPMI-1640, the meter viable count is about 2 * 10
7/ mL.
(4) immune spleen cell and Sp2/0 myeloma cell's fusion: the immune spleen cell of the Sp2/0 myeloma cell of step (3) and step (2) is even, centrifugal by 1: 5 mixed, evacuation supernatant as far as possible; Under 37 ℃ of water bath condition, add 50% polyoxyethylene glycol (MW1450) and carry out cytogamy then; Add the washing of serum-free RPMI RPMI-1640 again; Select nutrient solution (in the RPMI that contains 10% foetal calf serum 1640 substratum, to add 100 * aminopterin-induced syndrome storage liquid (HAT) with HAT; Sigma company) mix with the feeder cell of step (1) resuspended back; Join in the 96 porocyte culture plates, at 5%CO
2Cultivate in the incubator.Began to observe the hybridoma growing state from the 7th day, but grow to the naked eyes apparent time when cell, at microscopically HAT is selected nutrient solution to change into to contain HT to select nutrient solution (RPMI 1640 substratum that contain 2%HT), at CO
2Continue in the incubator to cultivate.Available ELISA method detects the height of antibody activity in the culture supernatant after about 3 days, and hybridoma is screened.
4, hybridoma screening
Adopt the hybridoma of ELISA method screening angiostrongylus cantonensis V-stage worm 55KD antigen-antibody, concrete grammar is:
(1), joins in the 96 hole enzyme plates and encapsulate every hole 100 μ l with antigen to the 5 μ g/L of 50mM carbonate buffer solution (pH 9.5) dilution step 1; 4 ℃ 12～24 hours; With PBS-T damping fluid (dissolving 8g NaCl in the 800mL zero(ppm) water, 0.2g KCl, 1.44g Na
2HPO
4, 0.24g KH
2HPO
4, regulate pH to 7.4 with HCl, add 1mLTween-20 then, the water constant volume is to 1L, room temperature preservation) give a baby a bath on the third day after its birth time;
(2) seal with 10% calf serum, every hole 100 μ l, 37 ℃ 30 minutes, give a baby a bath on the third day after its birth time with the PBS-T damping fluid;
(3) in 96 orifice plates, add the hybridoma supernatant of step 3, every hole 100 μ l, 37 ℃ 1 hour, give a baby a bath on the third day after its birth time with the PBS-T damping fluid;
(4) in 96 orifice plates, add the sheep anti mouse SA of horseradish peroxidase-labeled, every hole 50 μ l, 37 ℃ 30 minutes, give a baby a bath on the third day after its birth time with the PBS-T damping fluid;
(5) add alkaline phosphatase substrate solution (0.2M Tris liquid 50ml, 0.1N hydrochloric acid 40mL, MgCl
26H
2O 0.2g, LEVAMISOLE HCL 2mg, 1% sodium azide 5mL transfers pH to 8.3, adds water to 100mL), 37 ℃ were developed the color 15 minutes, and added 2N H
2SO
4, termination reaction is measured OD
450Value, OD
450The positive hybridoma that is worth 2.1 times of negative contrasts.
5, the foundation of the cloning of positive hybridoma cell and cell bank
(1) cloning of positive hybridoma cell: with limiting dilution assay (
G and C.Milstein.; 1975; The hybridoma in the positive hole that Nature 256:495) step 4 is filtered out carries out repeatedly subclone; Make the hybrid tumor cell monoclonalization of secrete monoclonal antibody; Thereby make its monoclonal antibody that can secrete homogeneous, simultaneously also avoided the hybridoma hypertrophy of secretory antibody not and the hybridoma of secretory antibody is lost.Cloning to all single clones' positive rate is 100% repeatedly, chooses strong positive clone enlarged culturing, and is a large amount of frozen as master cell bank.The continuous passage of part cell was cultivated more than 3 months, detected the stability of hybridoma secretory antibody with the method for following step 6.
(2) foundation of cell bank: positive hybridoma cell is being carried out in the cloning process, by fused cell, subclone, secondary subclone and an in-vitro cultivation more than 3 months three subclone cells of stably excreting antibody form master cell bank.Get the hybridoma cell strain complete detection of three subclones in the master cell bank, cultivate frozenly in a large number, set up master cell bank.Get the cell of master cell bank and cultivate in a large number, frozen after the check of antibody activity and mycoplasma is qualified, every batch is no less than 20 pipes, builds up the working cardial cell storehouse.Get 1 pipe recovery during each production, be used to prepare ascites antibody.
6, the evaluation of hybridoma
(1) antibody-secreting Detection of Stability: enlarged culturing is carried out in the wherein strain strong positive monoclonal cell strain (called after ACV1) to obtaining through screening, and getting part cell use saline water adjustment concentration is 2 * 10
6Individual/mL, it is inoculated in mouse peritoneal, the inoculating cell number is 1 * 10
6Individual/only.Formed ascites in about 7～10 days, increase to ascites, during the special bulge of belly, put to death mouse, get ascites, get supernatant after centrifugal and tire with ELISA method survey ascites.Other gets the part cell, and continuous passage was cultivated after 3 months, uses with above-mentioned same condition mouse is carried out the abdominal cavity inoculation, gets ascites, gets supernatant after centrifugal and surveys ascites and tire, and secondary ELISA measures the result and all reaches 1: 10 as a result
5, show that this strong positive monoclonal cell strain ACV1 has antibody-secreting stability preferably.
(2) hybridoma chromosome analysis: pair cell strain ACV1 goes down to posterity to cultivate after 36～48 hours and adds NST-757; Continue to cultivate collecting cell after 4～6 hours; Add the warm in advance 0.075M KCl solution suspension cell to 37 ℃, hypotonic processing was done in 37 ℃ of water-baths in 15～20 minutes; With cell with methyl alcohol/ice vinegar stationary liquid three times fixing after, be suspended in the stationary liquid, 4 ℃ 12～24 hours, centrifugal then; Remove supernatant, stay a little stationary liquid, drop in behind the mixing on the slide glass that just from frozen water, takes out, dispel cell suspension; Through flame for several times, seasoning is with 10%Giemsa staining fluid dyeing 10～20 minutes, flush away dye liquor; The karyotype of hybridoma, photomicrograph are observed in seasoning, mirror down.The karyotype observations of hybridoma is seen Fig. 1; The average chromosome number of hybridoma ACV1 is about 102; Near Sp2/0 cell and splenocyte chromosome number sum (BALB/c mouse splenocyte chromosome number is 40, and the average chromosome number of Sp2/0 cell is about 64), prove that this hybridoma cell strain is merged by Sp2/0 cell and mouse boosting cell; And the hybridoma after going down to posterity presents identical karyomit(e) detected result; The proof hybridoma carries the antibody staining body and produces gene in the process of going down to posterity, do not lose the antibody-secreting ability, and can give cell with this inheritance of ability.
(3) antibody subtype is identified: adopt monoclonal antibody hypotype inspection testing cassete (ImmunoType TMKit, Sigma) agent is carried out the evaluation of Tegeline hypotype to the Hybridoma Cell Culture supernatant, concrete grammar is: with PBS with 1: 1000 each antibody-like of dilution proportion (the anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and the IgM that prepare by ordinary method); With dilution antibody sandwich 96 hole enzyme plates (every hole 100 μ l, two holes of every antibody-like), 37 ℃ of incubations are after 1 hour then; Abandon coating buffer, wash 3 times, add the culture supernatant of monoclonal cell strain ACV1 by the amount in 100 μ l/ holes; 1 hour after scouring of room temperature incubation 3 times; The amount of pressing the 0.1mL/ hole adds the sheep anti-mouse igg (the green skies, Jiangsu company) of horseradish peroxidase-labeled, and the room temperature incubation washed 3 times after 30 minutes; Amount by 100 μ l/ holes adds horseradish peroxidase substrate reactions liquid (1mg/ml TMB); Room temperature 10～15 minutes blueness occurs and is positive findings, and the amount by 50 μ l/ holes adds 3N NaOH termination reaction at last.As a result, hybridoma ACV1 excretory antibody is the IgG2a subclass.
7, a large amount of preparations of the antibody of monoclonal cell strain ACV1 and specificity are identified
(1) obtain antibody ascites: choose 20～22g BALB/c mouse (Wenzhou Medical College's Experimental Animal Center provides), female, abdominal injection pristane, 0.5ml/ are only; After one week, the antibody positive hybridoma suspension of the logarithmic phase of cultivating is centrifugal, and sedimentation cell is suspended from serum-free RPMI 1640, and repeated centrifugation washing 2 times is resuspended in serum-free RPMI 1640, every mouse peritoneal injection hybridoma 1 * 10
7/ only, simultaneously offside reinject pristane and incomplete freund adjuvant equal-volume mixture 0.5ml/ only.After treating the remarkable bulge of mouse web portion after 7～10 days, collect ascites; With the ascites of collecting, centrifugal 30 seconds of 12000rpm removes bottom settlings and upper strata grease, collects stage casing liquid, surveys ascites with the ELISA method and tires, and the result meets the requirements, packing, and-20 ℃ are frozen subsequent use.
(2) affinitive layer purification of antibody: the AKTAFPLC protein chromatographic appearance with Pharmacia Biotech. company carries out affinity purification to the monoclonal antibody through preliminary purification; Concrete grammar is: the 0.02M PBS (pH 7.0) with 3 times of column volumes washes HiTrap Protein G chromatography column (1mL earlier; Amersham company) ethanol in the filler is used 0.02M PBS (pH 7.0) the balance chromatography column of 5 times of column volumes then, and 2mL is added chromatography column through the antibody-solutions of ammonium sulfate preliminary purification; 0.02M PBS (pH 7.0) flushing chromatography column with 5 times of column volumes; Collect effluent, when the IgG elution peak appears in ultraviolet rays detector, collect with the centrifuge tube that adds 60～100 μ l 0.05M Tris-HCl damping fluids (pH9.5) rapidly; Use 0.02M PBS (pH7.0) the flushing balance chromatography column of 5 times of volumes at last; Collect effluent, with the antibody purified packing ,-20 ℃ of preservations.
(3) antibody purity and concentration determination: respectively monoclonal antibody ascites and the antibody behind Protein G-Sepharose affinitive layer purification are carried out the SDS-PAGE electrophoresis detection by ordinary method; Detected result is seen Fig. 2 (swimming lane M: LMWP standard; Swimming lane ACV: purified monoclonal antibody); The electrophoretogram of the antibody of result behind affinitive layer purification presents Er Tiao district band, is the light chain and the heavy chain of antibody, and molecular weight is about 24KD and 53KD.Through sweep measuring, reach more than 98% through the antibody purity after the affinity chromatography, obtained the higher monoclonal antibody of purity.After 20mL mono-clonal ascites antibody purified with aforesaid method, being adjusted to original volume with PBS, is blank with PBS, measures the OD of antibody-solutions
450Value calculates AC and is about 10mg/mL.
(4) the monoclonal antibody specificity is identified: the monoclonal antibody and the antigenic binding specificity of angiostrongylus cantonensis V phase larva 55KD that detect above-mentioned purifying with immunoblotting reaction (Western Blot).After angiostrongylus cantonensis V phase larval antigens, schistosoma japonice ovum antigen, cysticercus cellulosae antigen, Paragonismus westermani adult antigen and Trichinella spiralis antigen carried out conventional SDS-PAGE, transferring on the nitrocellulose filter, is that a sheep anti-mouse igg anti-, the HRP mark is the two anti-immunoblotting reactions that carry out with the monoclonal antibody of above-mentioned purifying; (but Sambrook, J. wait the people to the concrete grammar reference; Molecular cloning: experiment guide, Cold Spring HarborLaboratory Press, second edition; 888 pages); The result sees Fig. 3 (swimming lane 1: angiostrongylus cantonensis V phase larval antigens, swimming lane 2: schistosoma japonice ovum antigen, swimming lane 3: cysticercus cellulosae antigen; Swimming lane 4: Paragonismus westermani adult antigen; Swimming lane 5: Trichinella spiralis antigen), show that prepared monoclonal antibody only combines with angiostrongylus cantonensis V phase larva 55KD antigen-specific, and do not combine with Japan schistosome antigen, Paragonismus westermani antigen, cysticercus cellulosae antigen and Trichinella spiralis antigen.
Above-mentioned experimental result shows that the monoclonal antibody that monoclonal cell strain ACV1 of the present invention produces has binding specificity preferably, with this antibody called after ACV McAb.
8, the antigenic avidity of ACV McAb and angiostrongylus cantonensis V phase larva 55KD is measured
Adopt noncompetitive ELISA method to measure ACV McAb and the antigenic avidity of angiostrongylus cantonensis V phase larva 55KD; Concrete grammar is: with 0.05mol/L, pH 9.6 carbonate buffer solutions with angiostrongylus cantonensis V phase larva 55KD antigen diluent to concentration be respectively 1000,500,250,125ng/mL; Add enzyme plate then; 100 μ l/ holes, 4 ℃ encapsulated 12 ~ 24 hours; Discard antigenic solution, wash plate 3 times with PBS (containing 0.05%Tween 20); Every hole adds the PBS that 150 μ l contain 1% bovine serum albumin, 37 ℃ of sealings 0.5 hour, and the monoclonal antibody purification ACV McAb that adds serial dilution is (since 1: 1000 by 10
2Multiple proportions is diluted), hatched 1 hour for 37 ℃ in 100 μ l/ holes, washes plate 3 times; Add two anti-sheep anti-mouse iggs (1: 2000) of HRP mark, hatched 0.5 hour for 37 ℃ in 100 μ l/ holes, washes plate 3 times; Add substrate 1mg/mL TMB, with substrate buffer solution (0.01%H
2O
2) dilution, 100 μ l/ holes, the room temperature lucifuge was placed 5 ~ 10 minutes; Use 2mol/L H at last
2SO
4Termination reaction is measured the A value of each hole 450nm, draws and measures 4 of curves; Read maximum absorbance (A value) the 1/2 pairing AC of each envelope antigen; Utilize Beatty calculation formula (Beatty JD, Beatty BG, Vlahos WG; Et al.Measurement ofmonoelonal antibody by non-competitive enzymeimmunoassay [J] .J Immunol Med; 1987,100 (1-2): 173-178.) confirm the affinity costant K value of the ACV McAb behind the purifying, the K value is 10.26 * 10 as a result
9Mol
-1, also have higher avidity with angiostrongylus cantonensis V phase larva 55KD antigen.
Detected result in the foregoing description shows that monoclonal antibody ACV McAb and angiostrongylus cantonensis V phase larva 55KD antigen that hybridoma cell strain ACV1 of the present invention produces have higher binding specificity and avidity.
The detection of angiostrongylus cantonensis CAg level
Angiostrongylus cantonensis CAg level is detected with the monoclonal antibody ACV McAb that hybridoma cell strain ACV1 produces with the ELISA method at present, may further comprise the steps:
(1) coated elisa plate: will use the angiostrongylus cantonensis V-stage worm 55KD antigen polyvalent antibody of ordinary method preparation to be diluted to concentration with 50mM carbonate buffer solution (pH 9.5) and be 1mg/mL; Every hole 100 μ l; Placed 12 ~ 24 hours for 4 ℃, use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then;
(2) seal the enzyme plate that has encapsulated: the elisa plate of calf serum closures quilt with 10%, 100 μ l/ holes, 37 ℃ are incubated 30 minutes;
(3) on enzyme plate, add patients serum or angiostrongylus cantonensis infecting mouse serum 100 μ l/ holes respectively, 37 ℃ of insulations 1 hour use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then;
(4) on enzyme plate, adding concentration is 0.2mg/L monoclonal antibody ACV McAb, 100 μ l/ holes, and 37 ℃ of insulations 1 hour use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then;
(5) add the sheep anti-mouse igg of horseradish peroxidase-labeled, 50 μ l/ holes, 37 ℃ of insulations 30 minutes use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then;
(6) add horseradish peroxidase substrate solution (1mg/mL TMB), 50 μ l/ holes, 37 ℃ were developed the color 15 minutes;
(7) add 2N H
2SO
450 μ l/ hole termination reactions;
(8) measure OD
450Value.
In the step (5) two is anti-also can be the anti-mouse or the anti-rabbit anti-antibody of px or alkaline phosphate ester enzyme labelling.
The result is through statistical analysis: (1) angiostrongylus cantonensis infecting mouse serum is all positive, compares with normal group mouse serum to have significant difference (P＜0.01); (2) detect the patients serum; Through comparing with etiological examination, clinical symptom and morbidity history; Find the cause of disease and learn the positive or have the angiostrongylus cantonensis CAg level among the typical angiostrongyliasis cantonensis clinical symptom patients serum all to want high than normal group, difference has statistical significance (P＜0.01).Show that the monoclonal antibody ACV McAb of hybridoma cell strain ACV1 generation of the present invention can be used for the detection of human serum angiostrongylus cantonensis CAg level.
The immunologic detection method of angiostrongylus cantonensis CAg also can be radioimmunology, enzyme-linked immunosorbent assay, sandwich immunization, immunoprecipitation or immunofluorescence etc.
In the above-mentioned detection method, the selection of testing sample can be from patient's serum and cerebrospinal fluid.
Interactional condition takes place in said monoclonal antibody and sample, can confirm according to detection method.
Those skilled in the art know, and for solid state reaction, can monoclonal antibody according to the invention be fixed in solid phase carrier, and reaction is at room temperature carried out usually, need in the testing process washing not with the step of monoclonal antibody bonded sample according to the invention.
For liquid phase reaction, can in the testing sample in being in specific buffer system, directly add monoclonal antibody of the present invention usually, then (like room temperature) takes place under the interactional temperature react being suitable for antigen-antibody.
The source of monoclonal antibody of the present invention is depended in the selection of SA in the said detection method.Those skilled in the art know how to select corresponding SA according to the source of monoclonal antibody.Said SA can be a labelled with radioisotope, includes but not limited to use be selected from:
32P,
125I, S,
2H etc.; Can be non-labelled with radioisotope also, include but not limited to use be selected from: marks such as horseradish peroxidase, SEAP, vitamin H, Streptavidin.The detection agent that uses in the detection method according to the invention depends on the affinity tag of the SA that testing process is used, and those skilled in the art know how to select suitable detection reagent.
Reaction conditions in the above-mentioned detection method and reagent all can be selected according to ordinary method.
The present invention also provides a kind of test kit of the people's of detection angiostrongylus cantonensis CAg level.
The test kit of detection people angiostrongylus cantonensis CAg level provided by the present invention can comprise the antigenic monoclonal antibody of angiostrongylus cantonensis V-stage worm 55KD that is produced by above-mentioned hybridoma cell strain ACV1.
Said test kit can comprise that also interactional SA takes place the proteic monoclonal antibody of angiostrongylus cantonensis V-stage worm 55KD that produces with above-mentioned hybridoma cell strain ACV1.
Said SA can be sheep anti-mouse igg or rabbit anti-mouse igg etc.
Said SA can be marker enzyme marks such as process horseradish peroxidase or Ostase, is preferably horseradish peroxidase, and horseradish peroxidase can be crosslinked on antibody through glutaraldehyde method or periodic acid method.
Also can comprise colour developing liquid A liquid and colour developing liquid B liquid in the test kit, said colour developing liquid A liquid is hydrogen peroxide or urea peroxide solution, and said colour developing liquid B liquid is O-Phenylene Diamine or tetramethyl biphenyl amine aqueous solution.
Use for convenient, said test kit also can comprise the washings that detects usefulness, like conventional washing reagents such as PBST; Confining liquid is like 10% calf serum etc.; Antibody diluent is like 50mM carbonate buffer solution (pH 9.5) etc.
Claims (4)
1. hybridoma cell strain that can produce angiostrongylus cantonensis V-stage worm 55KD antigen monoclonal antibody, it is characterized in that: said hybridoma cell strain is ACV1, its deposit number is CCTCC-C200930.
2. test kit that detects angiostrongylus cantonensis CAg level; Interactional SA takes place in the proteic monoclonal antibody of angiostrongylus cantonensis V-stage worm 55KD that it is characterized in that comprising the antigenic monoclonal antibody of angiostrongylus cantonensis V-stage worm 55KD that produced by hybridoma cell strain ACV1, produces with hybridoma cell strain ACV1; Said SA is selected from sheep anti-mouse igg or rabbit anti-mouse igg; Also comprise colour developing liquid A liquid and colour developing liquid B liquid in the test kit; Wherein, said hybridoma cell strain ACV1 deposit number is CCTCC-C200930.
3. test kit according to claim 2 is characterized in that: said SA is to pass through the horseradish peroxidase-labeled enzyme labelling, and horseradish peroxidase is crosslinked on antibody through glutaraldehyde method or periodic acid method; Said colour developing liquid A liquid is hydrogen peroxide or urea peroxide solution, and said colour developing liquid B liquid is O-Phenylene Diamine or tetramethyl biphenyl amine aqueous solution.
4. test kit according to claim 2 is characterized in that: said SA is to pass through Ostase marker enzyme mark; Said colour developing liquid A liquid is hydrogen peroxide or urea peroxide solution, and said colour developing liquid B liquid is O-Phenylene Diamine or tetramethyl biphenyl amine aqueous solution.
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