CN102043053A - Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen - Google Patents
Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen Download PDFInfo
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Abstract
The invention provides a nano magnetic bead labeled by chicken yolk immune globulin IgY and used for resisting the soluble antigen of schistosoma japonica ovums and a chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen. The method comprises the steps of: immunizing a hen with specific antigens of schistosoma japonica with a method of subcutaneous multi-point injection, and extracting, purifying and identifying specific IgY from an egg yolk; and coupling the polyclone IgY on a magnetic bead, and detecting the circulating antigen of the schistosoma by using the magnetic bead-IgY polyclone antibody as a capture antibody and using the specific monoclone IgY antibody as a detecting antibody. The method not only can be used for capturing more antigens by using the magnetic bead and the IgY to increase the sensibility, but also benefits the increase of detecting specificity by using the monoclonal antibody. Thus, the high combination and coordination of sensibility and specificity, required in immunology diagnosis, are realized.
Description
Technical field
The present invention relates to the detection technique of Schistosoma japonicum circulating antigen, be specifically related to detect the chicken yolk antibody one magnetic bead ELISA diagnostic techniques of Schistosoma japonicum circulating antigen.
Background technology
Blood fluke is a kind of worm that colonizes in the vein blood vessel, but domestic animals such as infected person and ox cause snail fever interior multiple mammal.Snail fever patient can cause the advanced schistosomiasis based on cirrhosis and ascites if can not get diagnosing timely and treating, and patient completely loses labour capacity and self care ability, and finally because of hepatic coma or the death of uncontrollable upper gastrointestinal bleeding.At present, snail fever is still the parasitic disease 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 reaches 200,000,000 more than, China popular be Japanese schistosomiasis.Snail fever China Yangtze river basin 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, oncomelania area is 14,300,000,000 m2, and 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 mammals such as people and ox, pig, mouse are bilharzial definitive hosts, when contacts such as people or ox contain the epidemic disease water of blood fluke cercaria, cercaria pierces host's skin and takes off afterbody and is transformed into virgin worm, after host's hypodermis 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 and enter vena portae hepatica.Virgin worm grows sexual organ in vena portae hepatica and tentatively breaks up the female male worm in back and fill 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 one 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.Under suitable condition, 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, is in light, grade and moderate infection (5-10%).In the diagnosis of snail fever, the excrement inspection finds that worm's ovum is a foundation of making a definite diagnosis infection by Schistosoma, 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 the specific antibody that detects anti-schistosome antigen in patient's serum.Antibody detection method commonly used has intracutaneous test (intradermal test, IDT), circum oval precipitating test (circumoval precipitin test, COPT), indirect hemagglutination test (HA test) (indirect haemagglutination test, IHA), enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), IFA (indirect fluorescent antibody method, IFA) etc.But the antibody life period is long behind infection by Schistosoma, 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 blood fluke CIC ELISA, so CAg content is less.So CAg detects for the early diagnosis of snail fever 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 all require higher to the sensitivity and the specificity of detection method.
At home and abroad under researcher's the joint efforts, the immunology diagnosis of snail fever has been obtained obvious improvement, but still has some critical technical problems.
(1) efficacy assessment of existing snail fever detection system is worth all undesirable, main cause is that existing a large amount of detectable 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 diagnostic system of antigen.
(2) main difficulty of circulating antigen detection method is the patient particularly the circulating antigen content in the chronic phase patient body is considerably less, the susceptibility of present detection architecture is lower, does not especially also have very efficient ways in the assessment in the slight popular district a large amount of to China.
(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; Diagnostic criteria objectifies inadequately, has personal error etc.
Though used schistosome antibody diagnostic method reaches its maturity at present, 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 drug therapy, the snail fever 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 not reporting at present has the high schistosome antigen detectable of susceptibility, seeks responsive and special circulating antigen detection method and also pushes the main direction that vast popular district is the research and development of schistosomiasis diagnosis reagent to.
Chicken yolk immune globulin (Egg yolk immunoglobulin, IgY) be that IgG antibody selectivity in the chicken blood is transferred in the yolk and formed, be the typical low-molecular-weight antibody that is prevalent in birds, Reptilia and amphibian animal 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 mammal IgG; Be equivalent to mammal IgG on function, and there is bigger difference in the antigenicity aspect, the IgG than mammal property aspect immunodiagnosis has more advantage.
IgY is made up of two heavy chains (H) and light chain (L), does not have hinge arrangement.Molecular weight is 180KDa (7.8S), and heavy chain is 67-70KDa, and light chain is 25Kda.A small size 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 extron (being arranged in the introne between C2 and C3 constant region extron), 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 5.7S IgY (Δ Fc) can coexist as with a kind of animal or individualism.
IgY antibody is only found in vertebrate, 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 vertebrate such as grade is replied the antibody that is produced to antigen and presents narrower, restricted diversity.Antibody L of chicken and H gene 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 gene conversion 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 agglutinating 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 agglutinating reaction.The antibody of duck often can not show precipitation or agglutination effectively, even those antibody that aggegation does not take place still can not obtain the ability of aggegation antigen under the concentration that improves salt.This is because the distance of two Fab fragments is too near, owing to sterically hindered reason, has hindered they and combining of molecular antigen greatly.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 factor 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 following advantage is 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 immune 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 factor 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.
IgY antibody begins widespread use since the eighties in 20th century, and Dr.C.Staak at first uses this term of IgY technology in nineteen ninety-five.1996, the production of IgY antibody and application were defined as the IgY technology.Nowadays, it has been a global standardized technique.Ever-increasing document number shows that the IgY technology is just becoming the new focus of field of medicaments.In recent years, IgY is being widely used aspect the diagnosis of disease, the infantile diarrhea that causes as enteropathogenic E (EPEC), people's palilate knurl 16 types infect, even can diagnose anoxic specifically, but in the immunodiagnosis of snail fever, do not appear in the newspapers as yet, do not see correlation technique and product yet about the IgY of preparation anti-schistosome antigen.
Immunomagnetic bead technique (immunomagnetic bead techniques) is that the magnetic microsphere with high homogeneity is a solid support, and pan coating immunity aglucon such as antibody etc. utilize specific immune response separation detection target material from mixed liquor.(Immunomagnetic beads Enzyme-linked Immunosorbent Assay IMB-ELISA) is a kind of novel solid-phase immunity detection technique of setting up, more conventional ELISA sensitivity to magnetic bead enzyme-linked immuno assay technology on the basis of ELISA principle.IMB-ELISA compares with common ELISA has following main advantage: among (1) common ELISA, the antibody of bag quilt has only the Fab section of minority Ig molecule to combine with the corresponding antigens specificity in the liquid phase, most Fab sections are difficult to combine with antigen because of towards sulidus face or parallel with antigen; And magnetic bead is spherical in shape, and particle is small, and quantity is many, and total surface area increases, and the chance that the antibody molecule of its pan coating is combined with antigen increases greatly.(2) antibody of bag quilt is when capture antigen, and association reaction also mainly occurs on the interface of solid phase.And antigen-antibody reaction can be carried out in liquid phase among the IMB-ELISA, has improved the accessibility of antibody and epitope, and this reaction pattern has improved the efficient of reaction greatly, thereby more traditional solid phase ELISA is highly sensitive.(3) immunomagnetic beads can carry out biochemical again.Immunomagnetic beads after the regeneration can be realized recycling.(4) entire reaction course of immunomagnetic bead technique can be finished in 1 hour, and is easy and simple to handle quick, thereby great application prospect is also arranged in immune detection.Have not yet to see the report and the ripe kit that immunomagnetic bead technique are applied to the snail fever immunology diagnosis.
Summary of the invention
The present invention's method immune hen of the subcutaneous multi-point injection of specific antigen of Schistosoma japonicum, extraction, purifying and identify specific IgY from egg yolk, again polyclone IgY coupling is associated on the magnetic bead, with magnetic bead one IgY polyclonal antibody is capture antibody, specific monoclonal IgG antibody carries out the detection of circulating antigen of schistosome for detecting antibody, can utilize magnetic bead and IgY to catch 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.This novel circulating antigen of schistosome detection method---IgY-IMB-ELISA can be schistosomiasis diagnosis a kind of detection kit with high sensitivity and high specific is provided, also start new technology and means simultaneously for the diagnosis of the detection of other numerous pathogen and infectious diseases.
Task of the present invention provides a kind of anti schistosoma worm's ovum soluble antigen (soluble egg antigens, the nanometer magnetic bead of chicken yolk immune globulin IgY mark SEA).
Another task of the present invention provides a kind of IgY-magnetic bead ELISA method of vitro detection schistosome antigen.
Another task of the present invention provides a kind of kit that detects schistosome antigen.
Realize that technical scheme of the present invention is:
Anti schistosoma worm's ovum soluble antigen (soluble egg antigens provided by the invention, the nanometer magnetic bead of chicken yolk immune globulin IgY mark SEA), be with behind the method immune hen of bilharzial specific antigen with injection, from being extracted specific IgY the egg yolk of immune hen, again this specific IgY is marked at and forms on the nanometer magnetic bead.
The preparation method of the chicken yolk immune globulin IgY of anti schistosoma worm's ovum soluble antigen SEA may further comprise the steps:
Step 1, preparation schistosoma japonice ovum soluble antigen SEA;
Step 3, from extracted the egg yolk of immune hen, purification specificity IgY.
The concrete preparation method of above-mentioned steps one 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 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, 4 ℃ of precipitation 48h, the centrifugal 1h of 10000 * g draws supernatant, collect packing, be worm's ovum soluble antigen SEA.
Above-mentioned steps two is described with the schistosoma japonice ovum soluble antigen SEA immune hen and the method for collecting egg to be: 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 is 30 μ g SEA/, each 10d at interval, 7d collection egg after immunity is first carried out and is marked on 4 ℃ of refrigerators preservations.
Above-mentioned steps three is described from being extracted the egg yolk of immune hen, the method of purification specificity IgY is: the egg of getting 60d behind the initial immunity, remove eggshell, egg white with deionized water flush away yolk outside, remove yolk clothing film, phosphate buffer (Phosphate Buffer Solution with the 0.01mol/L pH 7.6 of yolk and two volumes, PBS) mix, and fully stir, adding polyglycol 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, the PBS that former vitelline bodies with 1/6 amass is dissolution precipitation again, dialysed 2 hours for 4 ℃ in the distilled water, change water therebetween 3~4 times, be concentrated into 1/10 of original volume among the most rearmounted PEG, collect the IgY after concentrating, packing postposition-20 ℃ preservation is standby.
The present invention with the method that specific IgY is marked on the nanometer magnetic bead is: with the magnetic bead of diameter 800nm with the distilled water washing after, 0.1M PBS (pH7.4 contains 1%BSA) suspends again and adjusts magnetic bead concentration to 10mg/ml; Getting 5ml magnetic bead suspension adds the 50mg carbodiimide and shakes 15min for 37 ℃, after PBS washes 2 times, make the 2.5m1 suspension, add anti schistosoma worm's ovum soluble antigen (soluble egg antigens, SEA) chicken yolk immune globulin IgY 0.5ml puts 37 ℃ of shaking table vibrations and hatches 90min; Adding the sealing of 0.01M glycocoll (pH8.2 contains 1%BSA) damping fluid spends the night; PBS solution 5ml is outstanding again, and 4 ℃ store for future use.
The IgY-magnetic bead ELISA method of vitro detection schistosome antigen provided by the invention may further comprise the steps:
(1) the IgY bag is added the decrement reaction tube by magnetic bead (magnetic bead concentration 1mg/ml, antibodies concentration is 10 μ g IgY/mg magnetic beads) 200 μ l/ pipe; Described IgY bag can be the nanometer magnetic bead of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen by magnetic bead;
The serum to be checked 200 μ l/ pipe of (2) dilutions in 1: 2 adds in the decrement reaction tube; Negative control is a normal serum; Blank is the PBST (containing 0.05%Tween-20) of 0.1M pH 7.4, and 60min is hatched in 37 ℃ of vibrations;
(3) reaction tube is put on the magnet absorption magnetic bead to supernatant and is become clarification after, remove supernatant, with the 0.1M pH 7.4Tris-HCl lavation buffer solution washing that contains 0.05%Tween-20 3 times;
(4) every pipe adds the monoclonal antibody of dilution in 1: 1000 of horseradish peroxidase-labeled, and 90min is hatched in 37 ℃ of vibrations
(5) by the described method washing of above-mentioned steps (3);
(6) add substrate solution 3,3 ', 5 by 200 μ L/ pipe, 5 '-tetramethyl benzidine, 37 ℃ of lucifuge reaction 25min;
(7) every pipe adds the 2M concentrated sulphuric acid cessation reaction of 30 μ l;
(8) after reaction tube was put on the magnet and to be become clarification to supernatant, every pipe was drawn 100 μ l reaction supernatant and is drawn in the 96 hole ELISA Plate, puts the ELISA readout instrument and reads A
450Value;
(9) result judges: value/negative A value (S/N) 〉=2.1 is promptly positive to measure A.
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 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 items 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 items hole of 96 hole ELISA Plate, 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 10d at interval.7d collection egg after immunity is first carried out and is marked on 4 ℃ of refrigerators preservations.
3. the polyglycol method is extracted and purifying IgY
Get the egg of 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 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, the PBS that the former vitelline bodies with 1/6 amass is dissolution precipitation again.To putting in the bag filter that conventional processing crosses, 4 ℃ of dialysis 2h change water therebetween 3~4 times in the distilled water.The most rearmounted Macrogol 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 skimmed milk power with 1% are spent the night.-resisting to be the IgY (working concentration is 1: 1000) after the immunity, non-immune IgY is in contrast; Two anti-are the goat-anti chicken IgG of horseradish peroxidase-labeled (working 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 be two master tapes and five bands of 66000 and 35000, sees 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 50 μ g, and 4 ℃ of sealings of 5% bovine serum albumin(BSA) (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 absorbance.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 20 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 cultured 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 IgGl, 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-IMB-ELISA detection method provided by the invention
7.1 animal grouping, infection and 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 blood fluke.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 infection by Schistosoma is successful.The control group mice of Gan Raning had not both seen then that worm's ovum do not see adult yet.
7.2IgY in conjunction with magnetic bead: the magnetic bead of diameter 800nm (Beijing Bo Mai company) with the distilled water washing after, 0.1M PBS (pH7.4 contains 1%BSA) suspends again and adjusts magnetic bead concentration to 10mg/ml; Get 5ml magnetic bead suspension and add 50mg carbodiimide (EDC, Sigma company) and shake 15min for 37 ℃, after PBS washes 2 times, make the 2.5ml suspension, add 0.5ml IgY antibody, put 37 ℃ of shaking tables vibrations and hatch 90min; Adding the sealing of 0.01M glycocoll (pH8.2 contains 1%BSA) damping fluid spends the night; PBS solution 5ml is outstanding again, 4 ℃ of storages.
7.3 the foundation of IgY-IMB-ELISA method of the present invention
(1) the IgY bag is by magnetic bead (magnetic bead concentration 1mg/ml, antibodies concentration is 10 μ g IgY/mg magnetic beads) 200 μ l/ pipe adding decrement reaction tube, described IgY bag is the nanometer magnetic bead of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen by magnetic bead;
The serum to be checked 200 μ l/ pipe of (2) dilutions in 1: 2 adds in the decrement reaction tube; Negative control is a normal serum; Blank is the PBST (containing 0.05%Tween-20) of 0.1M pH 7.4.60min is hatched in 37 ℃ of vibrations.
(3) reaction tube is put on the magnet absorption magnetic bead to supernatant and is become clarification after, remove supernatant.With the 0.1M pH 7.4Tris-HCl lavation buffer solution washing that contains 0.05%Tween-20 3 times.
(4) every pipe adds the HRP-NP28 (working concentration 1: 6000) of 200 μ l, and 90min is hatched in 37 ℃ of vibrations.
(5) washing is with 3.
(6) add substrate solution 3,3 ', 5 by 200 μ L/ pipe, 5 '-tetramethyl benzidine (TMB), 37 ℃ of lucifuge reaction 25min.
(7) every pipe adds the 2M concentrated sulphuric acid cessation reaction of 30 μ l.
(8) after reaction tube was put on the magnet and to be become clarification to supernatant, every pipe was drawn 100 μ l reaction supernatant and is drawn in the 96 hole ELISA Plate, puts ELISA readout instrument (Multiskan Ascent 354, Finland Labsystems company) and reads A
450Value.
(9) result judges: value/negative A value (S/N) 〉=2.1 is positive to measure A.
7.4IgY-IMB-ELISA clinical small sample testing result
Annotate: the chronic schistosomiasis patients serum is from Wuhan City CDC snail fever research institute; The acute schistosomiasis patients serum is from parasitic disease inspection center of Tongji Medical College, Huazhong Science and Technology Univ.; Be the excrement inspection japonice ovum positive.Lung fluke patient and liver fluke patients serum are respectively from the popular district in Guangxi and Guangzhou; Normal human serum is from the healthy person in Shandong, the popular district of non-blood fluke.
Result of the present invention shows: the present invention has prepared the IgY antibody of stable, special anti schistosoma SEA, and with the nano level magnetic bead of this antibody sandwich, has set up the immunomagnetic beads ELISA diagnostic method based on this IgY.This method can be used for detecting circulating antigen of schistosome, and its clinical diagnosis specificity reaches 100%, and susceptibility reaches more than 90%.
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 drug therapy, the snail fever 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 detectable of susceptibility is arranged.The immunomagnetic beads elisa technique that the present invention is based on IgY uses the IgY that is coated on the nanometer magnetic bead as capture antibody, combines the common detection hypersensitivity of magnetic bead and IgY; Simultaneously, guaranteed the specificity that detects with monoclonal antibody again as detecting antibody.These all are the unexistent advantages of detection means commonly used at present.
Description of drawings
Fig. 1 is IgY and IgG tactic pattern figure.
Fig. 2 is that the SDS-PAGE of IgY analyzes, and A figure is that the non-reduced type SDS-PAGE of IgY analyzes, and 1 place is the IgY through purifying after the immunity among the figure, and 2 places are the IgY that does not purify after the immunity among the figure; B figure is that the reduced form SDS-PAGE of IgY analyzes, and 1 place is the IgY that not immune yolk is purified among the figure, and 2 places are the IgY that immunity back yolk is purified among the figure.
Fig. 3 is that the Western blotting of IgY analyzes, and 1 place is the IgY reaction of purifying with not immune yolk among the figure; Among the figure 2 places be with immunity after the yolk IgY reaction of purifying.
Embodiment
Embodiment 1 is marked at method on the nanometer magnetic bead with specific IgY
The present invention with the method that specific IgY is marked on the nanometer magnetic bead is: with the magnetic bead of diameter 800nm with the distilled water washing after, 0.1M PBS (pH7.4 contains 1%BSA) suspends again and adjusts magnetic bead concentration to 10mg/ml; Getting 5ml magnetic bead suspension adds the 50mg carbodiimide and shakes 15min for 37 ℃, after PBS washes 2 times, make the 2.5ml suspension, add anti schistosoma worm's ovum soluble antigen (soluble egg antigens, SEA) chicken yolk immune globulin IgY 0.5ml puts 37 ℃ of shaking table vibrations and hatches 90min; Adding the sealing of 0.01M glycocoll (pH8.2 contains 1%BSA) damping fluid spends the night; PBS solution 5ml is outstanding again, and 4 ℃ store for future use.
The clinical small sample of IgY-IMB-ELISA method of the present invention detects to be used and the result
Annotate: the chronic schistosomiasis patients serum is from Wuhan City CDC snail fever research institute; The acute schistosomiasis patients serum is from parasitic disease inspection center of Tongji Medical College, Huazhong Science and Technology Univ.; Be the excrement inspection japonice ovum positive.Lung fluke patient and liver fluke patients serum are respectively from the popular district in Guangxi and Guangzhou; Normal human serum is from the healthy person in Shandong, the popular district of non-blood fluke.
Embodiment 3
The kit of detection schistosome antigen provided by the invention comprises:
(1) the nanometer magnetic bead 25ml of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen;
(2) standard items (dried frozen aquatic products) are 1 bottle; Face with preceding and be diluted to 1ml, face with preparation in preceding 15 minutes with distilled water;
(3) sample diluting liquid is 1 bottle: 25ml;
(4) detect with 1 bottle of antibody: 50ml;
(5) cleansing solution is 1 bottle: 500ml;
(6) substrate solution is 1 bottle: 20ml;
(7) stop buffer is 1 bottle: 20ml;
(8) operation instructions portion.
The operation instruction of the kit of detection schistosome antigen provided by the invention: each reagent before use balance to room temperature.Each reaction all should be established standard pipe 2 pipe and testing sample pipes.IgY bag is added the decrement reaction tube by magnetic bead 200 μ l/ pipe, adds the standard items or the testing sample 200 μ l of dilution more respectively, mixing gently, and 37 ℃ of vibrations were hatched 60 minutes.After reaction tube put on the magnet absorption magnetic bead to supernatant and become clarification, discard liquid, cleansing solution is washed 3 times.Every hole adds to be detected with antibody 200 μ l, and 90min is hatched in 37 ℃ of vibrations.The same method washing 3 times.Every pipe adds substrate solution 200 μ l, 37 ℃ of lucifuge colour developing 25min.Every pipe adds stop bath 30 μ l, cessation reaction.After reaction tube was put on the magnet and to be become clarification to supernatant, every pipe was drawn 100 μ l reaction supernatant and is drawn in the 96 hole ELISA Plate, puts the ELISA readout instrument and reads the A450 value.
Claims (9)
1. the nanometer magnetic bead of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen, it is characterized in that, it is with behind the method immune hen of bilharzial specific antigen with injection, from being extracted specific IgY the egg yolk of immune hen, again this specific IgY is marked at and forms on the nanometer magnetic bead.
2. the nanometer magnetic bead of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen according to claim 1, it is characterized in that, the preparation method of the chicken yolk immune globulin IgY of anti schistosoma worm's ovum soluble antigen SEA may further comprise the steps:
Step 1, preparation schistosoma japonice ovum soluble antigen SEA;
Step 2, usefulness schistosoma japonice ovum soluble antigen SEA immune hen, and collect egg;
Step 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, it is characterized in that, the preparation method of 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 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, 4 ℃ of precipitation 48h, the centrifugal 1h of 10000 * g draws supernatant, collect packing, be 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, describedly be: the SEA 0.5ml of 60 μ g/ml is mixed with isopyknic complete freund adjuvant with the schistosoma japonice ovum soluble antigen SEA immune hen and the method for collecting egg, 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 is 30 μ g SEA/, 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 being extracted the egg yolk of immune hen, the method of purification specificity IgY is: the egg of getting 60d behind the initial immunity, remove eggshell, egg white with deionized water flush away yolk outside, remove yolk clothing film, phosphate buffer (Phosphate Buffer Solution with the 0.01mol/L pH 7.6 of yolk and two volumes, PBS) mix, and fully stir, adding polyglycol 6000 (PEG 6000) to ultimate density is 5%, and be stirred to PEG and dissolve fully, the centrifugal 20min of 4420 * g room temperature, with supernatant liquid filtering, continue to add PEG to its concentration be 20%, fully after the stirring and dissolving 12, the centrifugal 10min of 000 * g room temperature, remove supernatant, the PBS that former vitelline bodies with 1/6 amass is dissolution precipitation again, dialysed 2 hours for 4 ℃ in the distilled water, change water therebetween 3~4 times, 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. anti schistosoma worm's ovum soluble antigen (soluble egg antigens according to claim 1, the nanometer magnetic bead of chicken yolk immune globulin IgY mark SEA), it is characterized in that, the method that specific IgY is marked on the nanometer magnetic bead is: with the magnetic bead of diameter 800nm with the distilled water washing after, 0.1M PBS (pH7.4 contains 1%BSA) suspends again and adjusts magnetic bead concentration to 10mg/ml; Getting 5ml magnetic bead suspension adds the 50mg carbodiimide and shakes 15min for 37 ℃, after PBS washes 2 times, make the 2.5ml suspension, add anti schistosoma worm's ovum soluble antigen (soluble egg antigens, SEA) chicken yolk immune globulin IgY 0.5ml puts 37 ℃ of shaking table vibrations and hatches 90min; Adding the sealing of 0.01M glycocoll (pH8.2 contains 1%BSA) damping fluid spends the night; PBS solution 5ml is outstanding again, and 4 ℃ store for future use.
7. the method for a vitro detection schistosome antigen may further comprise the steps:
(1) the IgY bag is added the decrement reaction tube by magnetic bead (magnetic bead concentration 1mg/ml, antibodies concentration is 10 μ g IgY/mg magnetic beads) 200 μ l/ pipe;
The serum to be checked 200 μ l/ pipe of (2) dilutions in 1: 2 adds in the decrement reaction tube; Negative control is a normal serum; Blank is the PBST (containing 0.05%Tween-20) of 0.1M pH 7.4, and 60min is hatched in 37 ℃ of vibrations;
(3) reaction tube is put on the magnet absorption magnetic bead to supernatant and is become clarification after, remove supernatant, with the 0.1M pH 7.4Tris-HCl lavation buffer solution washing that contains 0.05%Tween-20 3 times;
(4) every pipe adds the monoclonal antibody of dilution in 1: 1000 of horseradish peroxidase-labeled, and 90min is hatched in 37 ℃ of vibrations
(5) by the described method washing of above-mentioned steps (3);
(6) add substrate solution 3,3 ', 5 by 200 μ L/ pipe, 5 '-tetramethyl benzidine, 37 ℃ of lucifuge reaction 25min;
(7) every pipe adds the 2M concentrated sulphuric acid cessation reaction of 30 μ l;
(8) after reaction tube was put on the magnet and to be become clarification to supernatant, every pipe was drawn 100 μ l reaction supernatant and is drawn in the 96 hole ELISA Plate, puts the ELISA readout instrument and reads A
450Value;
(9) result judges: value/negative A value (S/N) 〉=2.1 is promptly positive to measure A.
8. the method for a kind of vitro detection schistosome antigen according to claim 7 is characterized in that described IgY bag is the nanometer magnetic bead of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen by magnetic bead.
9. a kit that detects schistosome antigen is characterized in that, it includes the nanometer magnetic bead of the chicken yolk immune globulin IgY mark of anti schistosoma worm's ovum soluble antigen.
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