CN101643507A - Egg-yolk antibody for resisting soluble egg antigen of Japanese blood flukes, preparation method thereof and application thereof - Google Patents
Egg-yolk antibody for resisting soluble egg antigen of Japanese blood flukes, preparation method thereof and application thereof Download PDFInfo
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Abstract
In the invention, a method of hypodermic multi-point injection of specific antigens of blood flukes is used to immunize a hen, the specific IgY is extracted, purified and identified from egg yolks, and an ELISA diagnosis system IgY-ELISA is established by combining the monoclonal antibody technique. The invention establishes a method for detecting circulating antigens of blood flukes, which is novel, highly efficient, sensitive and specific and also has a curative effect evaluation action. Moreover, the invention also initially provides a novel technique and means for detecting other pathogensand diagnosing infectious diseases.
Description
Technical field
The present invention relates to parasitic diagnostic techniques, particularly Schistosoma japonicum
Background technology
Schistosomicide is a kind of worm that colonizes in the vein blood vessel, but domestic animals such as infected person and ox cause schistosomicide interior multiple Mammals.Schistosomicide patient can cause the advanced schistosomiasis based on liver cirrhosis and ascites if can not get diagnosing timely and treating, and patient completely loses work capacity and self care ability, and finally because of hepatic coma or the death of uncontrollable upper gastrointestinal hemorrhage.At present, schistosomicide is still the parasitosis of a kind of serious harm human health and Economic development, and there are 74 popular these diseases in countries and regions in the whole world, and annual number of the infected still reaches 200,000,000 more than, is schistosomiasis japanica at China's popular.Schistosomicide China Yangtze valley and on the south 370 counties and cities of 13 provinces such as Hunan, Hubei, Jiangxi, Anhui, Jiangsu, Yunnan, Sichuan, Zhejiang, Guangdong, Guangxi, Shanghai, Fujian popular, accumulative total the infected reaches 1,160 ten thousand people, and oncomelania area is 14,300,000,000 m
2, compromised population is more than 100,000,000.Therefore, the situation that faced of prevention and cure of snail fever work is still very severe.
Multiple Mammalss such as people and ox, pig, mouse are bilharzial final hosts, when contacts such as people or ox contain the epidemic disease water of schistosomulum, cercaria pierces host's skin and takes off afterbody and is transformed into virgin worm, after host's subcutis is done short stay, intravasation or lymphatic vessel, with blood flow through the right heart to lung, enter systemic circulation by the left heart again, the virgin worm that arrives mesenteric artery can pass capillary vessel and enter hepatic vein.Virgin worm is after hepatic vein is grown the preliminary differentiation of sexual organ, and promptly female, hero is filled the span of a man's arms, and divides a word with a hyphen at the end of a line that mesenteric vein and hemorrhoidal veins are lived away from home again, mating, lays eggs.Pierce skin from cercaria and reach maturity and lay eggs to polypide, Schistosoma japonicum needs 24 days approximately.Adult parasitizes portal vein-mesenteric vein system, and female worm lays eggs in intestinal submucosa vein tip.Part worm's ovum is deposited in liver or the colon intestinal tissue with venous blood flow, and another part worm's ovum can fall into enteric cavity with the tissue of ulceration, and excretes with host's ight soil.The essential entry of the worm's ovum that excretes is hatched miracidium under conditions such as suitable temperature, osmotic pressure and illumination, pierce in the oncomelania body during host's oncomelania in the middle of miracidium runs into it, develops into cercaria through the asexual reproductive phase of mother sporocyst, daughter sporocyst.Cercaria can infect new host after breechblock is overflowed, the breeding of beginning a new generation.
In early days, correct diagnosis is the important step of control schistosomiasis endemic.China is through the prevention and cure of schistosomiasis work of over half a century, and the schistosomiasis infection rate in present most epidemic-stricken areas and patient's infectiosity all have obvious decline, are in light grade and moderate infection (5-10%).In the diagnosis of schistosomicide, the excrement inspection finds that worm's ovum is a foundation of making a definite diagnosis schistosomicide, but because all multifactor influences such as infected degree and infective stage are measured in ovulation, susceptibility in moderate or the inspection of low schistosomiasis endemic district excrement can not be satisfactory, the loss height, particularly, rely on the excrement inspection can not satisfy the needs of schistosomiasis diagnosis merely, so immunologic test has become the important means of schistosomiasis diagnosis in China.Traditional immunology diagnosis mainly is to detect the antigenic specific antibody of schistosomicide in patient's serum.Antibody detection method commonly used has intradermal test (intradermal test, IDT), circum oval precipitating test (circumoval precipitin test, COPT), hemagglutination test (HA test) (indirect haemagglutination test.IHA) indirectly, enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), indirect fluorescent antibody test (indirect fluorescent antibody method, IFA) etc.But the antibody lifetime is long, can't distinguish existing disease and previous infection, and is little to the directive significance of patient.And circulating antigen (circulating antigens, CAg) be the excrete when people's endobiosis of the polypide that lives, it is generally acknowledged, CAg content height in the acute schistosomiasis patients serum, and in the chronic schistosomiasis patients serum, because CAg constantly combines with serum antibody and forms the schistosomicide circulating immune complex, so CAg content is less.So CAg detects for the early diagnosis of schistosomicide and distinguishes that existing disease is infected and previous infection is valuable especially, and can be as the index of efficacy assessment, but because of content is few, so higher to the susceptibility requirement of detection method.
At home and abroad under investigator's the joint efforts, the immunology diagnosis of schistosomicide has been obtained obvious improvement, but still has some critical technical problems.
(1) efficacy assessment of existing schistosomicide detection system is worth all undesirable, major cause is that existing a large amount of detection reagent all is to detect antibody, and antibody still can detect in serum at the 1-3 after the patient even after the more than ten years, can't distinguish existing disease and previous infection, therefore be required to be efficacy assessment and provide a kind of effectively to detect the diagnositc system of antigen.
(2) main difficulty of circulating antigen detection method is the patient particularly the intravital circulating antigen content of chronic phase patient is considerably less, the susceptibility of present detection architecture is lower, does not especially also have very efficient ways on to the assessment in a large amount of slight popular districts.
(3) the schistosomiasis diagnosis reagent type is a lot, and level is uneven, difference and problem of unstable that the reagent quality existence is produced between criticizing; Case definition objectifies inadequately, easily causes personal errors etc.
Current, seek responsive and special circulating antigen detection method and push the main direction that vast popular district is the research and development of schistosomiasis diagnosis reagent to.
Immunoglobulin of Yolk (Egg yolk immunoglobulin, IgY) be that IgG antibody selectivity in the chicken blood is transferred in the yolk and formed, be the typical lower molecular weight antibody that is prevalent in birds, Reptilia and batrachians serum and the yolk, do not have the 50kDa heavy chain γ that is similar to IgG, and the heavy chain of IgY is obviously greater than the heavy chain of Mammals IgG; Be equivalent to Mammals IgG on function, and there is bigger difference in the antigenicity aspect, the IgG than mammalian source property aspect immunodiagnosis has more advantage.
IgY forms (see figure 1) by two heavy chains (H) and light chain (L), does not have hinge arrangement, and molecular weight is 180KDa (7.8S), and heavy chain is 67-70KDa, and light chain is 25Kda, and a small volume copy is arranged, and 120KDa (5.7S) is present in wild duck and some Chelonian.The H chain of less IgY lacks C3, C4 district, owing to used special not end exon (being arranged in the intron between C2 and C3 constant region exon), 5.7S IgY and F (ab) 2 fragments are quite similar, so correct name is IgY (Δ Fc), its H chain is υ (Δ Fc).7.8S IgY and 5.7SIgY (Δ Fc) can coexist as with a kind of animal or Individual existence.IgY antibody is only found in vertebrates, there is diversity in its molecular structure, relevant with antigen binding site formation component, its encoding gene is variable gene (V), diversified gene (D) and in conjunction with gene (J), and the reorganization of selection at random by V, D and J gene produces all diversity primary antibodies.Low vertebrates such as grade is replied the antibody that is produced to antigen and presents narrower, restricted diversity.Antibody L of chicken and H locus only have a V gene and a J gene, have only D gene cluster to have (16 genes).Therefore the Ig gene diversity of chicken is that form with genetic modification produces by homologous recombination, lacks the mechanism of the somatic mutation of selecting high-affinity.
IgY and IgY (Δ Fc) contain 2 antigen binding sites, in principle, can precipitation or agglutination reaction phenomenon take place with a plurality of antigens, but true really not so.The antibody capable of most of chickens conjugated antigen securely but has only under high salt concn (as 1.5M NaCl) just can cause precipitation or agglutination reaction.The antibody of duck often can not show precipitation or agglutination effectively, still can not obtain the antigenic ability of aggegation even agglutinative antibody takes place for those under the concentration that improves salt.This is because two segmental distances of Fab are too near, owing to sterically hindered reason, has hindered them and has combined with macromole is antigenic.Under high salt or low pH situation, the distance between IgY release Fab is to allow their conjugated antigen molecules independently of one another.Many effector functions of Ig are by Fc district mediation, so IgY (Δ Fc) can not activate mammiferous complement system, not combine with the RF factor, Rheumatoid factors, polyclonal and function such as SPA, SPG combination.From the phyletic evolution angle, the existence of Δ Fc Igs is to have certain selective advantage.
As the source of antibody a lot of advantages are arranged as immune animal with egg with hen.(1) because kind is that distance is big between taking place, hen can be to the stronger immunne response of mammiferous antigen generation.(2) antibody of the hen generation high-affinity that can continue.The mammalian antigen of the high conservative of 20-30mg can cause that the IgY that height is tired secretes for a long time.(3) undamaged antibody is collected.The egg that only needs to collect immune hen can obtain IgY.(4) high yield.A hen can give birth to about 280 eggs in 1 year, and every egg contains the IgY of 100-150mg, and every chicken can produce the IgY of 28-42g every year like this.(5) discord Rheumatoid factors, polyclonal combination.In detection, use IgY and can avoid false positive results.(6) do not react with complement.But mammiferous IgG activating complement causes false-negative generation.IgY discord complement reacts, and when detecting mammiferous serum, can reduce the interference that is caused by complement.
Summary of the invention
Task of the present invention provide a kind of anti schistosoma worm's ovum soluble antigen (soluble egg antigens, SEA)
ChickenYolk immunoglobulin (Egg yolk immunoglobulin, IgY).
Another task of the present invention provides a kind of method of vitro detection circulating antigen of schistosome.
Another task of the present invention provides a kind of test kit that detects schistosome antigen.
The technical scheme that realizes first task of the present invention is:
Anti schistosoma worm's ovum soluble antigen (soluble egg antigens provided by the invention, SEA) Immunoglobulin of Yolk (Egg yolk immunoglobulin, IgY), be with behind the method immune hen of bilharzial specific antigens with injection, from by the specific IgY that extracts the egg yolk of immune hen, the method of the Immunoglobulin of Yolk of preparation anti schistosoma worm's ovum soluble antigen SEA may further comprise the steps:
(1) preparation schistosoma japonice ovum soluble antigen SEA;
(2), and collect egg with schistosoma japonice ovum soluble antigen SEA immune hen;
(3) from extracted the egg yolk of immune hen, purification specificity IgY.
The concrete grammar of the described preparation schistosoma japonice ovum of above-mentioned steps (1) soluble antigen SEA is: the oncomelania that will infect Schistosoma japonicum is at 25 ℃, the cercaria of overflowing under the sufficient situation of illumination, the cercaria that at least 20 oncomelanias are overflowed mixes, percutaneous infection cleaning level new zealand white rabbit, infective dose is 800 cercaria/rabbits, infects the rabbit that anesthesia is infected after 45 days, the physiological saline heart perfusion, cut open then and kill and take out liver, separate and collection liver worm's ovum.With the worm's ovum collected in ice bath repeatedly after the homogenate, 4 ℃ of precipitation 48h, the centrifugal 1h of 10000 * g draws supernatant, collects packing, is worm's ovum soluble antigen SEA.
The described concrete grammar with schistosoma japonice ovum soluble antigen SEA immune hen and collection egg of above-mentioned steps (2) is: the SEA 0.5ml of 60 μ g/ml mixed with isopyknic complete freund adjuvant, and fully emulsified after vein immunity under the wing.10d after the first immunisation, use instead antigen add incomplete freund adjuvant through the chicken back subcutaneous booster immunization 3 times, immunizing dose be 30 μ g SEA/ only, each 10 days at interval,, carry out and be marked on 4 ℃ of refrigerators and preserve from immune back 7 days first collection eggs.
Above-mentioned steps (3) is described from being extracted the egg yolk of immune hen, the concrete grammar of purification specificity IgY is: get behind the initial immunity 60 days egg, remove eggshell, egg white with deionized water flush away yolk outside, remove yolk clothing film, phosphate buffered saline buffer (Phosphate Buffer Solution with the 0.01mol/LpH 7.6 of yolk and two volumes, PBS) mix, and fully stir, adding polyoxyethylene glycol 6000 (PEG 6000) to ultimate density is 5%, and be stirred to PEG and dissolve fully, the centrifugal 20min of 4420 * g room temperature is with supernatant liquid filtering, continue to add PEG to its concentration be 20%, fully the centrifugal 10min of 12,000 * g room temperature after the stirring and dissolving removes supernatant liquor, the PBS that former vitellus with 1/6 amass is dissolution precipitation again, 4 ℃ of dialysis 2h change water therebetween 3～4 times in the distilled water, are concentrated into 1/10 of original volume among the most rearmounted PEG, collect the IgY after concentrating, packing postposition-20 ℃ preservation is standby.
The technical scheme that realizes second task vitro detection of the present invention circulating antigen of schistosome is: with the chicken yolk antibody contact measured serum of anti schistosoma soluble egg antigen.
The technical scheme that realizes the 3rd task of the present invention is: the test kit of detection schistosome antigen provided by the invention comprises:
(1) with two of 96 hole elisa plate of the IgY of anti-SEA bag quilt;
(2) freeze-drying standard substance are the healthy human serum in non-schistosomicide epidemic-stricken area, 1 bottle: 0.5mg, and face with preceding and be diluted to 1ml with distilled water, face in preceding 15 minutes and prepare;
(3) sample diluting liquid is the phosphate buffered saline buffer of 0.01mol/l pH 7.4,1 bottle: 15ml;
(4) antibody is used in detection, is the anti-SEA monoclonal antibody of horseradish peroxidase mark, 1 bottle: 25ml;
(5) washings is phosphate buffered saline buffer-polysorbas20 of 0.05%pH 7.4,1 bottle: 500ml;
(6) substrate solution is tetramethyl benzidine, 1 bottle: 20ml;
(7) stop buffer is 2mol/l sulfuric acid, 1 bottle: 20ml;
(8) process specifications: every plate is all established standard orifice 2 holes and testing sample hole, adds the standard substance or the testing sample 100 μ l of dilution respectively, mixing gently, and 37 ℃, 120 minutes, discard liquid, dry, washings is washed plate 5 times, and the every hole of 350 μ l/ dries; Every hole adds to be detected with antibody 100 μ l, and 37 ℃, 60 minutes, washings was washed plate 5 times, and the every hole of 350 μ l/ dries; Every hole adds substrate solution 90 μ l, and 37 ℃ of lucifuges developed the color 30 minutes; Every hole adds stop bath 50 μ l, termination reaction.With the absorbance of ELISA readout instrument in each hole of 450nm wavelength measurement, the ratio of absorbance of being fond of the absorbance of sample and standard substance is promptly positive greater than 2.1.Operation notice: 1. each reagent answers balance to room temperature before use; 2. each washing time should not be less than 1 minute; 3. 4 ℃ of preservations of test kit were please finished using in 12 months after the opening as far as possible.
The present invention utilizes the method immune hen of the subcutaneous multi-point injection of bilharzial specific antigens, extraction, purifying and identify specific IgY from egg yolk, and in conjunction with monoclonal antibody technique, set up ELISA diagnosis system (IgY-ELISA), set up a kind of new efficient, sensitivity and special, had the method for the detection circulating antigen of schistosome of efficacy assessment effect simultaneously.Also start new technology and means simultaneously for the diagnosis of the detection of other numerous pathogenic agent and infectious diseases.
Experimental data
1. the preparation of Schistosoma japonicum SEA
The oncomelania hupensis of the infection Schistosoma japonicum of going down to posterity in this laboratory (continent strain) is at 25 ℃, the cercaria of overflowing under the sufficient situation of illumination, the cercaria that at least 20 oncomelanias are overflowed mixes, percutaneous infection new zealand white rabbit (Tongji Medical Institute's Experimental Animal Center provides, the cleaning level), infective dose is 800 cercaria/rabbits, infect the rabbit that anesthesia is infected after 45 days, the physiological saline heart perfusion cuts open then and kills and take out liver, separates and collection liver worm's ovum.With the worm's ovum collected in ice bath repeatedly after the homogenate, behind 4 ℃ of precipitation 48h, the centrifugal 1h of 10000 * g.Draw supernatant, collect packing, be the worm's ovum soluble antigen (soluble egg antigens, SEA).
The mensuration of SEA protein content adopts BCA method (BCA Protein Assay Kit, U.S. BIPEC).With concentration be 0, the standard substance 20 μ l of 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml, 1mg/ml are added to respectively in the standard substance hole of 96 hole enzyme plates, again sample 20 μ l are added in the sample well, each hole adds the BCA working fluid of 200 μ l, 37 ℃ of reaction 30min, 562nm reads absorbance in the place.The drawing standard curve, the protein content of calculation sample.The BCA method records protein content 〉=5mg/ml of SEA.
2. immunity and egg are collected
The SEA 0.5ml of 60 μ g/ml is mixed with isopyknic complete freund adjuvant, fully emulsified after vein immunity under the wing.10d after the first immunisation, use instead antigen add incomplete freund adjuvant through the chicken back subcutaneous booster immunization 3 times, immunizing dose be 30 μ g SEA/ only, each 10 days at interval.7d collection egg after immunity is first carried out and is marked on 4 ℃ of refrigerators preservations.
3. the polyoxyethylene glycol method is extracted and purifying IgY
Get behind the initial immunity 60 days egg, remove eggshell, with the egg white of deionized water flush away yolk outside.Remove yolk clothing film, (Phosphate Buffer Solution PBS) mixes, and fully stirs with the phosphate buffered saline buffer of the 0.01mol/L pH 7.6 of yolk and two volumes.Add 5% PEG and be stirred to PEG and dissolve fully.Room temperature centrifugal (4420 * g, 20min).With supernatant liquid filtering, continue to add PEG to 20%, fully after the stirring and dissolving room temperature centrifugal (12,000 * g, 10min).Remove supernatant liquor, the PBS that the former vitellus with 1/6 amass is dissolution precipitation again.Put in the dialysis tubing that conventional processing crosses, 4 ℃ of dialysis are 2 hours in the distilled water, change water therebetween 3～4 times.The most rearmounted polyethylene glycol 6000 is concentrated into 1/10 of original volume in (PEG 6000), and ℃ preservation of collection packing postposition-20 is standby.The protein concentration of IgY is about 6mg/ml after purifying.Every egg can obtain the IgY of 61mg after saltouing, dialyse, concentrating.
4.IgY sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (Westernblotting) analyze
Non-reduced type SDS-PAGE detects relative molecular mass (Mr) and degree of purification, and its concentrated gum concentration is 5%, and resolving gel concentration is 7%.Reduced form SDS-PSGE identifies specificity, and its concentrated gum concentration is 5%, and resolving gel concentration is 15%; The applied sample amount in every hole is 7 μ g.Western blotting after SEA is carried out the SDS-PAGE electrophoresis, transfers on the nitrocellulose filter, and 4 ℃ of sealings of the skim-milk with 1% are spent the night.One anti-is the IgY (concentration is 1: 1000) after the immunity, and non-immune IgY in contrast; Two anti-are the goat-anti chicken IgY of horseradish peroxidase-labeled (concentration is 1: 100000).4-chloro-1-naphthols+3%H
2O
2Colour developing.
Complete IgY behind the purifying has a master tape, and its relative molecular mass Mr is about 130000.The result of reduced form SDS-PAGE shows: the IgY after the cracking is totally seven bands, wherein contains Mr and is two master tapes and five bands of 66000 and 35000 and see Fig. 2.Western blotting result shows that SEA can be discerned combination by the IgY after the immunity, and does not react with non-immune IgY, sees Fig. 3.
5.IgY the detection of susceptibility
The every hole of elisa plate bag is by the IgY of 30 μ g, and 4 ℃ of sealings of 10% bovine serum albumin (BSA) are spent the night, PBS-tween (Tween) 20 (PBST) washing of 0.05%pH 7.4 3 times; The 37 ℃ of incubation 1h of SEA that add serial dilution; The same IgG that adds anti-SEA after 3 times that washs with PBST, behind 37 ℃ of incubation 1h, the same method washing, the 37 ℃ of incubation 1h of goat anti-rabbit igg (working concentration is 1: 100000) that add horseradish peroxidase (HRP) mark, add substrate OPD colour developing 15min, on microplate reader (Multiskan Ascent 354, Finland Labsystems company), read absorbancy.The double-antibody sandwich elisa method records the result and shows: SEA is diluted to 2.4ng/ml with antigen, and its A value is 0.4546, and the A value of negative control is 0.2132, and S/N is greater than 2.1, and the result is still positive.
6. preparation and the mark of the monoclonal antibody NP28-5B of anti schistosoma SEA
4 the week age BALB/C mice, infect 8 schistosoma japonicum cercariaes, infect and got mouse boosting cell as the hybridoma donorcells in back 4 months.Donorcells is mixed with homology murine myeloma cell SP2/0, add PEG4000 and make its fusion.Cells and supernatant and Schistosoma japonicum SEA are carried out ELISA, and positive porocyte is through subclone repeatedly, till the culture supernatant in all hybridomas growth holes all can produce positive reaction with SEA.This moment, confirmation form clonal antibody clone was set up.
Go down to posterity and cultivate back collection culturing cell supernatant, behind 50% saturated ammonium sulphate, be dissolved in 0.01M, the PBS of pH7.4, it is standby that-70 ℃ of refrigerators are placed in the back of fully dialysing.Identify that through immune double diffusion the homotype of NP28-5B is IgG1, its target antigen molecular weight is 140KD.Adopt simple and easy sodium periodate method, horseradish peroxidase (HRP, RZ 〉=3.0) is bonded on the NP28/5B, determine its working concentration through titration.
7.IgY-sandwich ELISA (Sandwich-ELISA) diagnostic techniques
7.1 the animal grouping is infected and the serum collection
40 of the female BALB/C mice of 20-25g are divided into 2 groups at random, and one group is infected 40/mouse of schistosoma japonicum cercariae (the conventional infection method in this area) through skin, and another group is done normal control, does not infect schistosomicide.It is all identical that two groups of mouse are raised condition, diet drinking-water etc.
Infect after 45 days, 2 groups of mouse are all gathered peripheric venous blood and separation of serum, and-20 ℃ of preservations are standby.Mouse blood sampling back anesthesia, THPV physiological saline pours into towards worm, and makees the hepatic tissue compressing tablet, and the microscopy worm's ovum is to confirm the mouse infection success.Behind worm, every mouse of infected group all can be gone out adult, and the visible a large amount of ova nodules of hepatic tissue compressing tablet prove that schistosomicide is successful.The control group mice of Gan Raning had not both seen then that worm's ovum do not see adult yet.
7.2?IgY-sandwich-ELISA
(1) get the every hole of IgY30 μ g/ml bag that above-mentioned preparation identifies by 96 hole elisa plates, 4 ℃ spend the night after, with the PBST washing of 0.1%pH7.4 3 times.
The 37 ℃ of sealings in (2) 10% bovine serum BSA200 μ l/ holes 1h, the same method is washed 3 times.
(3) add the bilharzial mouse serum of infection to be checked that dilutes at 1: 10,37 ℃ of incubation 2h replace serum to be checked to make blank with PBST, and health of the same race not infected mice serum is made negative control.The same method is washed 3 times behind the incubation.
(4) NP28 of dilution in 1: 6000 of adding HRP mark, 37 ℃ of incubation 30min, the same method is washed 3 times
(5) the 37 ℃ of colour developings in substrate TMB100 μ l/ hole 25min.
(6) 2mol/l sulfuric acid 50 μ l/ hole termination reactions read the absorbance at 450nm place on microplate reader
The result shows: serum A value of all infection schistosomicide mouse of infected group and the ratio of negative control mouse are all greater than 2.1, the average A value is 1.48, negative control group average A value is 0.36, and S/N ratio reaches 4.0, exceeds far that ELISA is positive in detecting need to reach 2.1 requirement with negative ratio.Every part of serum sample of this experiment repeats 2 times at least, good stability.
In sum, result of study of the present invention shows, the technology of the present invention can prepare the IgY antibody of stable, special anti schistosoma SEA, and the technology based on the sandwich-ELISA diagnosis schistosomicide of this IgY is provided, and the susceptibility that infection animal is detected in the laboratory reaches 100%.
Though current schistosome antibody diagnostic method reaches its maturity, but critical problem is an antibody positive, and not distinguish the examinee be that existing disease is infected or previous infection, because antibody is cured 3-4 the patient, even still can exist in the serum after the longer time, so can't provide valuable guidance to pharmacological agent, the schistosomicide patient an innocent person that can allow some cure takes medicine.The Detection of antigen positive means that then the patient is acute infection, because polypide existence alive, just meeting released antigen material in blood are arranged in the body.Also do not report at present the high schistosome antigen detection reagent of susceptibility is arranged.The sandwich elisa technique that the present invention is based on IgY obtains antibody with IgY as pouncing on, monoclonal antibody NP28 is for detecting antibody, can pounce on I gY and obtain more antigens, thereby raising susceptibility, the specificity that can improve detect with monoclonal antibody again combines and coordination with special height thereby reach desired sensitivity in the immunology diagnosis.And IgY antibody is to extract from egg non-invasively, and the source is abundant, the yield height, and homogeneity is good, with Mammals cross reaction does not take place, and these all are that routine is done the unexistent advantage that detects with Mammals antibody.
Description of drawings
Fig. 1 is IgY and IgG structural pattern figure;
Fig. 2 is that the SDS-PAGE of IgY analyzes, and A figure is that non-reduced type SDS-PAGE analyzes, and 1 place is the IgY through purifying after the immunity, and 2 places are the IgY that does not purify after the immunity; B figure analyzes for reduced form SDS-PAGE, and 1 place is the IgY that not immune yolk is purified, and 2 places are the IgY that immunity back yolk is purified;
Fig. 3 is that the Western blotting of IgY analyzes, and 1 place be the IgY reaction of purifying with not immune yolk, 2 places be with immunity after the IgY of yolk purification react.
Embodiment
The preparation of Schistosoma japonicum SEA
The oncomelania hupensis of the infection Schistosoma japonicum of going down to posterity in this laboratory (continent strain) is at 25 ℃, the cercaria of overflowing under the sufficient situation of illumination, the cercaria that at least 20 oncomelanias are overflowed mixes, percutaneous infection new zealand white rabbit (Tongji Medical Institute's Experimental Animal Center provides, the cleaning level), infective dose is 800 cercaria/rabbits, infect the rabbit that anesthesia is infected after 45 days, the physiological saline heart perfusion cuts open then and kills and take out liver, separates and collection liver worm's ovum.With the worm's ovum collected in ice bath repeatedly after the homogenate, behind 4 ℃ of precipitation 48h, the centrifugal 1h of 10000 * g.Draw supernatant, collect packing, be the worm's ovum soluble antigen (soluble egg antigens, SEA).
The mensuration of SEA protein content adopts BCA method (BCA Protein Assay Kit, U.S. BIPEC).With concentration be 0, the standard substance 20 μ l of 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml, 1mg/ml are added to respectively in the standard substance hole of 96 hole enzyme plates, again sample 20 μ l are added in the sample well, each hole adds the BCA working fluid of 200 μ l, 37 ℃ of reaction 30min, 562nm reads absorbance in the place.The drawing standard curve, the protein content of calculation sample.The BCA method records protein content 〉=5mg/ml of SEA.
Immunity and egg are collected
The SEA 0.5ml of 60 μ g/ml is mixed with isopyknic complete freund adjuvant, fully emulsified after vein immunity under the wing.10d after the first immunisation, use instead antigen add incomplete freund adjuvant through the chicken back subcutaneous booster immunization 3 times, immunizing dose be 30 μ g SEA/ only, each 10 days at interval,, carry out and be marked on 4 ℃ of refrigerators and preserve from immunity back 7d collection egg first.
Embodiment 3
The polyoxyethylene glycol method is extracted and purifying IgY
Get behind the initial immunity 60 days egg, remove eggshell, with the egg white of deionized water flush away yolk outside, remove yolk clothing film, (Phosphate Buffer Solution PBS) mixes, and fully stirs with the phosphate buffered saline buffer of the 0.01mol/L pH 7.6 of yolk and two volumes.Add 5% PEG and be stirred to PEG and dissolve fully.Room temperature centrifugal (4420 * g, 20min).With supernatant liquid filtering, continue to add PEG to 20%, fully after the stirring and dissolving room temperature centrifugal (12,000 * g, 10min).Remove supernatant liquor, the PBS that the former vitellus with 1/6 amass is dissolution precipitation again.Put in the dialysis tubing that conventional processing crosses, 4 ℃ of dialysis are 2 hours in the distilled water, change water therebetween 3～4 times.The most rearmounted polyethylene glycol 6000 is concentrated into 1/10 of original volume in (PEG 6000), and ℃ preservation of collection packing postposition-20 is standby.The protein concentration of IgY is about 6mg/ml after purifying.Every egg can obtain the IgY of 61mg after saltouing, dialyse, concentrating.
Preparation and the mark of the monoclonal antibody NP28-5B of anti schistosoma SEA
4 the week age BALB/C mice, infect 8 schistosoma japonicum cercariaes, infect and got mouse boosting cell as the hybridoma donorcells in back 4 months.Donorcells is mixed with homology murine myeloma cell SP2/0, add PEG4000 and make its fusion.Cells and supernatant and Schistosoma japonicum SEA are carried out ELISA, and positive porocyte is through subclone repeatedly, till the culture supernatant in all hybridomas growth holes all can produce positive reaction with SEA.This moment, confirmation form clonal antibody clone was set up.
Go down to posterity and cultivate back collection culturing cell supernatant, behind 50% saturated ammonium sulphate, be dissolved in 0.01M, the PBS of pH7.4, it is standby that-70 ℃ of refrigerators are placed in the back of fully dialysing.Identify that through immune double diffusion the homotype of NP28-5B is IgG1, its target antigen molecular weight is 140KD.Adopt simple and easy sodium periodate method, horseradish peroxidase (HRP, RZ 〉=3.0) is bonded on the NP28/5B, determine its working concentration through titration.
Claims (7)
1. the chicken yolk antibody of an anti schistosoma worm's ovum soluble antigen SEA, it is characterized in that, it is with behind the method immune hen of bilharzial specific antigens with injection, from the specific immunoglobulin immune globulin IgY that is extracted the egg yolk of immune hen.
2. prepare the method for the chicken yolk immune globulin IgY of anti schistosoma worm's ovum soluble antigen SEA, may further comprise the steps:
(1) preparation schistosoma japonice ovum soluble antigen SEA;
(2), and collect egg with schistosoma japonice ovum soluble antigen SEA immune hen;
(3) from extracted the egg yolk of immune hen, purification specificity IgY.
3. the method for the chicken yolk immune globulin IgY of preparation schistosoma japonice ovum soluble antigen SEA according to claim 2, the preparation method who it is characterized in that described schistosoma japonice ovum soluble antigen SEA is: the oncomelania that will infect Schistosoma japonicum is at 25 ℃, the cercaria of overflowing under the sufficient situation of illumination, the cercaria that at least 20 oncomelanias are overflowed mixes, percutaneous infection cleaning level new zealand white rabbit, infective dose is 800 cercaria/rabbits, infect the rabbit that anesthesia is infected after 45 days, the physiological saline heart perfusion, cut open then and kill and take out liver, separate and the collection liver worm fourth of the twelve Earthly Branches.With the worm's ovum collected in ice bath repeatedly after the homogenate, 4 ℃ of precipitation 48h, the centrifugal 1h of 10000 * g draws supernatant, collects packing, is worm's ovum soluble antigen SEA.
4. the method for the chicken yolk immune globulin IgY of preparation schistosoma japonice ovum soluble antigen SEA according to claim 2, it is characterized in that described method with schistosoma japonice ovum soluble antigen SEA immune hen is: the SEA0.5ml of 60 μ g/ml is mixed with isopyknic complete freund adjuvant, fully emulsified after vein immunity under the wing.10d after the first immunisation, use instead antigen add incomplete freund adjuvant through the chicken back subcutaneous booster immunization 3 times, immunizing dose be 30 μ g SEA/ only, each 10d at interval.7d collection egg after immunity is first carried out and is marked on 4 ℃ of refrigerators preservations.
5. the method for the chicken yolk immune globulin IgY of preparation schistosoma japonice ovum soluble antigen SEA according to claim 2, it is characterized in that described from extracted the egg yolk of immune hen, the method for purification specificity IgY is: the egg of getting 60d behind the initial immunity, remove eggshell, with the egg white of deionized water flush away yolk outside.Remove yolk clothing film, (Phosphate Buffer Solution PBS) mixes, and fully stirs with the phosphate buffered saline buffer of the 0.01mol/L pH 7.6 of yolk and two volumes.Adding polyoxyethylene glycol 6000 (PEG 6000) to ultimate density is 5%, and is stirred to PEG and dissolves fully.The centrifugal 20min of 4420 * g room temperature, with supernatant liquid filtering, continuing to add PEG is 20% to its concentration, the centrifugal 10min of 12,000 * g room temperature after the abundant stirring and dissolving.Remove supernatant liquor, the PBS that the former vitellus with 1/6 amass is dissolution precipitation again, and 4 ℃ of dialysis 2h change water therebetween 3～4 times in the distilled water.Be concentrated into 1/10 of original volume among the most rearmounted PEG, collect the IgY after concentrating, packing postposition-20 ℃ preservation is standby.
6. the method for a vitro detection circulating antigen of schistosome is characterized in that, with the chicken yolk antibody contact measured serum of the described anti schistosoma soluble egg antigen of claim 1.
7. test kit that detects schistosome antigen comprises:
(1) with two of 96 hole elisa plate of the IgY of anti-SEA bag quilt;
(2) 1 bottle of 0.5mg of freeze-drying standard substance for preparing with the healthy human serum in non-schistosomicide epidemic-stricken area;
(3) the phosphate buffer 1 bottle 15ml of sample diluting liquid 0.01mol/l pH 7.4;
(4) detect with 1 bottle of the anti-SEA monoclonal antibody of antibody horseradish peroxidase mark: 25ml
(5) phosphate buffered saline buffer-polysorbas20 of washings 0.05%pH 7.4 is 1 bottle: 500ml
(6) the substrate solution tetramethyl benzidine is 1 bottle: 20ml
(7) stop buffer 2mol/l sulfuric acid is 1 bottle: 20ml
(8) process specifications.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101968485A (en) * | 2010-04-09 | 2011-02-09 | 中国疾病预防控制中心寄生虫病预防控制所 | Method for detecting schistosome circulating antigen and enzyme-linked immune kit thereof |
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN104650224A (en) * | 2014-12-22 | 2015-05-27 | 浙江省医学科学院 | Nano-antibody aiming at SEA (Soluble Egg Antigen), and coding sequence and application thereof |
| CN106771197A (en) * | 2017-03-15 | 2017-05-31 | 河南农业大学 | A kind of sandwich ELISA detection method of Cryptosporidum parvum and its application |
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2008
- 2008-08-07 CN CN200810048728A patent/CN101643507A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101968485A (en) * | 2010-04-09 | 2011-02-09 | 中国疾病预防控制中心寄生虫病预防控制所 | Method for detecting schistosome circulating antigen and enzyme-linked immune kit thereof |
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN104650224A (en) * | 2014-12-22 | 2015-05-27 | 浙江省医学科学院 | Nano-antibody aiming at SEA (Soluble Egg Antigen), and coding sequence and application thereof |
| CN106771197A (en) * | 2017-03-15 | 2017-05-31 | 河南农业大学 | A kind of sandwich ELISA detection method of Cryptosporidum parvum and its application |
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