CN102331501A - Method for detecting trichinella circulating antigen by utilizing IgY-McAb sandwich ELISA (enzyme-linked immuno sorbent assay) - Google Patents
Method for detecting trichinella circulating antigen by utilizing IgY-McAb sandwich ELISA (enzyme-linked immuno sorbent assay) Download PDFInfo
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Abstract
The invention discloses a method for detecting a trichinella circulating antigen by utilizing IgY-McAb sandwich ELISA (enzyme-linked immnuo sorbent assay). With an anti-trichinella muscle larval ES antigen egg yolk antibody IgY as a capture antibody and an anti-trichinella ES antigen McAb as a detection antibody, the preparation method of the IgY comprises the following steps of: adding the trichinella muscle larval into a culture medium to carry out sterile culture, purifying and dialyzing supernate and then concentrating and drying to obtain the ES antiagen, determining protein concentration and then immunizing roman legehenne by utilizing the ES antigen, collecting the IgY from the produced egg yolk by adopting a saturate ammonium sulphate precipitation method, purifying and determining concentration, and sub-packaging for later use; and the preparation method of the McAb comprises the following steps of: establishing a hybridoma cell strain secreting the anti-trichinella muscle larval ES antigen, cloning by adopting a limiting dilution method, purifying McAb by applying an octanoic acid-ammonia sulphate method, determining the concentration and sub-packaging for later use. The invention has the advantage that a new method which has high sensitivity and specificity and has wide application prospect is provided for early detection of trichinella CAg in blood serum of an infected animal.
Description
Technical field
The present invention relates to biology, parasitology, serology and immunological technique; Especially relate to chicken yolk immune globulin (Egg Yolk Immunoglobulin; IgY)-monoclonal antibody (Monoclonal Antibody; McAb) sandwich ELISA (Enzyme Linked Immunosorbent Assay, ELISA) method of trichina CAg in the detection serum.
Background technology
Trichinosis (trichinellosis) is that a kind of people beast that is universal serious harm health suffers from parasitic disease altogether, and is main because of eating raw or eat half a lifetime due to the meat and goods thereof that contains trichinzation property larva.But known more than 150 kinds of animal natural infection trichinas (
Trichinella spiralis), be popular in 66 countries (or area).China is except that Hainan Province, and other provinces all have the report of zoogenetic infection trichina, and China a lot of human body trichinosises have taken place breaks out in recent years.Because the traditional detection method of trichinzation mainly relies on muscle biopsy; Low and the performance difficulty of this method recall rate; In recent years, Chinese scholars is attempted to realize the detection to trichinzation through the trichina antibody that detects in the serum, but can only after infection, 3 ~ 4 weeks just can be detected trichina antibody usually; And this moment, part severe infection person possibly can not be used for trichinous early diagnosis so detect trichina antibody because of acute stage complication death.Result of study shows; Drainage-secretion (Excretory-Secretory that trichinella larvae is produced in host internal migration and growth course; ES) thing and polypide the surface cast can get into blood and become CAg (Circulating Antigens, CAg), this type of antigenic content is directly proportional with infection intensity; And time of occurrence is early than specific antibody; Its half life period also is significantly shorter than specific antibody, thereby trichina CAg is expected to be used for discriminating and the efficacy assessment that early detection, the past and existing disease to trichinzation are infected in the detection serum; Yet the CAg content in the serum is lower usually, and recall rate is merely 19% ~ 54% when using conventional method, has greatly limited and has detected applying of CAg, therefore, needs the new method of the detection trichina CAg that sets up a kind of susceptibility height, high specificity at present badly.
Summary of the invention
The object of the present invention is to provide a kind of method with high susceptibility and specific IgY-McAb sandwich ELISA detection trichina CAg.
For realizing above-mentioned purpose, the present invention can take following technical proposals:
IgY-McAb sandwich ELISA of the present invention detects the method for trichina CAg, it be with anti-trichina muscle larvae ES antigen chicken yolk antibody IgY as capture antibody, with anti-trichina ES antigen McAb as detecting antibody; Said anti-trichina muscle larvae ES antigen chicken yolk antibody IgY and anti-trichina ES antigen McAb are prepared from through following method respectively:
(A) the anti-trichina muscle larvae ES antigen chicken yolk antibody IgY of preparation:
The trichina muscle larvae is added axenic cultivation in the nutrient culture media, and supernatant purifying dialysis back concentrate drying promptly gets ES antigen, and packing is for use behind the mensuration protein concentration; With above-mentioned trichina muscle larvae ES antigen immune Luo Man laying hen, the egg yolk that produces collect IgY with the saturated ammonium sulphate method, measure concentration behind the purifying, both must resist trichina muscle larvae ES antigen chicken yolk antibody IgY, subsequent use after the packing;
(B) the anti-trichina ES antigen McAb of preparation:
Set up the hybridoma cell strain of the anti-trichina muscle larvae ES antigen of secretion, use limiting dilution assay to carry out cloning, use sad-ammonium sulfate method purifying McAb, packing is subsequent use after the mensuration concentration.
During detection, adopt the chessboard titrimetry to confirm the righttest concentration that encapsulates of IgY, encapsulate damping fluid with carbonate and the IgY of optimum concentration is encapsulated in the reacting hole of ELISA Plate as capture antibody; The serum to be checked that adds the optimal proportion dilution; To add reacting hole successively as the McAb that detects antibody and the goat anti-mouse igg of horseradish peroxidase-labeled, and add the substrate colour developing at last and judge testing result.
The invention has the advantages that early detection for trichina CAg in the infection animal serum provides a kind of susceptibility higher and have a new method of wide application prospect; Overcome conventional method owing to the low shortcoming that is difficult to detect of CAg content in the serum, helped trichina CAg in the serum and detect applying in practice.The present invention collects trichina muscle larvae ES antigen through in vitro culture; And use anti-trichina muscle larvae ES antigen I gY and McAb respectively as capture antibody and detection antibody; Set up the IgY-McAb sandwich ELISA method that trichina CAg detects in the serum, can realize early detection, compared with existing method to trichinzation; The sensitivity and the specificity of early detection have been improved to a great extent, the present invention also can be used for previously and existing disease is infected examination and efficacy assessment.
IgY (chicken yolk immune globulin) is that the being selected property of IgG in the chicken blood is transferred in the yolk and formed; And be immunoglobulin class unique in the yolk; Though its structure is similar with IgG; But have more advantage in the serology context of detection than the IgG of mammal property, can avoid combining to take place cross reaction with rheumatoid factor etc., specificity is higher; In addition, IgY can combine with more epitope and play the signal amplification, can improve the susceptibility of detection; The report that IgY is used for the trichinosis diagnosis is not arranged at present both at home and abroad as yet.McAb is by the generation of B cell hybridoma cell line, only to the monospecific antibody of a kind of antigenic determinant, and the detection that can be trichinzation provides the instrument of high special, sensitivity.
Description of drawings
Fig. 1 is that the IgY-McAb sandwich ELISA detects CAg variation in the infection trichina mouse different time serum.
Fig. 2 is that the IgY-McAb sandwich ELISA detects infection trichina mouse treatment back different time change of serum C Ag variation.
Embodiment
IgY-McAb sandwich ELISA of the present invention detects the method for trichina CAg, it be with anti-trichina muscle larvae ES antigen chicken yolk antibody IgY as capture antibody, with anti-trichina ES antigen McAb as detecting antibody; Said trichina muscle larvae ES antigen chicken yolk antibody IgY and anti-trichina ES antigen McAb are prepared from through following method respectively:
(1) the anti-trichina muscle larvae ES antigen chicken yolk antibody IgY of preparation:
(1.1) preparation of trichina muscle larvae ES antigen
The ratio of the trichina muscle larvae of improveing the collection of Bei Shi method in 5 000 larva/mL added in 1640 nutrient culture media, add penicillin (l00U/mL) and streptomysin (100U/mL), 37 ℃ of 5%CO during cultivation
2Cultivate 18h in the incubator, 4 ℃ of supercentrifuges are centrifugal, and with 4 ℃ of supernatants dialysis, 3 d, fast vacuum concentration systems concentrate drying promptly get ES antigen, packing after the mensuration concentration, and it is subsequent use to put-80 ℃ of preservations.Whole process must the Strict aseptic operation.
(1.2) preparation of anti-trichina muscle larvae ES antigen chicken yolk antibody IgY
Get the fully emulsified after vein immunity Luo Man laying hen under the wing of 100 μ g trichina muscle larvae ES antigens and isopyknic Freund's complete adjuvant, 2 weeks added the immunity of incomplete Freund vein with same dosage at interval, and immunity is 4 times altogether.Begin to collect egg behind the first immunisation 7d, get yolk and mix fully stirring 15min of back with distilled water with 1:9,4 ℃ are spent the night; Get supernatant, 4 ℃ of 10 centrifugal 25min of 000g; 50%, 33% saturated ammonium sulphate is 2 times, abandons supernatant, and an amount of PBS washes deposition; Put in the bag filter with sterilized distilled water dialysis 3d, 6h changes water 1 time, collects supernatant; After fast vacuum concentration systems drying concentrated, the Bradford method was surveyed its concentration, and-80 ℃ of packing postposition are subsequent use.
(2) the anti-trichina ES antigen McAb of preparation:
(2.1) trichina muscle larvae ES antigen immune BALB/c mouse
Select female BALB/c mouse in 6 ~ 8 ages in week; Get trichina muscle larvae ES antigen 50 μ g and equivalent Freund's complete adjuvant fully emulsified back groin and the subcutaneous multi-point injection of nape portion; After this 3 weeks of every interval with 25 μ g ES antigens and the abundant mixing and emulsifying of incomplete Freund equal-volume after with same way as injection, immunity is 3 times altogether; Last is after 2 weeks of immunity, and 3d does not directly have adjuvant ES antigen 50 μ g booster immunizations to the immune mouse intrasplenic injection before merging.
(2.2) foundation of hybridoma cell strain
(2.2a) preparation of immune spleen cell: wash out above-mentioned immune mouse spleen cell with the incomplete nutrient culture media of RPMI-1640 (Roswell Park Memorial Institute-1640); Splenocyte is transferred in the 50mL centrifuge tube; The centrifugal 5min of 1 000g; With suitably dilution after the nutrient culture media washing 2 times, count subsequent use.
(2.2b) recovery of SP2/0 cell and preparation: merge the frozen SP2/0 myeloma cell strain of preceding 2 weeks recovery, add 8-AG (20 μ g/mL), 5% CO
237 ℃ of Continuous Cultivation are more than 3 generations, with screening and maintenance hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency in the incubator.Merge preceding 24 ~ 48h, select the cultivation of partly going down to posterity of the SP2/0 cell that is in the logarithm division stage of well-grown, complete form.
(2.2c) Fusion of Cells: the SP2/0 in the growth period of taking the logarithm and immune spleen cell; The centrifugal 5min of difference 1 000g; After abandoning supernatant, with counting behind the incomplete nutrient culture media suspendible of the RPMI-1640 cell, the mixed that myeloma cell and splenocyte are pressed 1:10 ~ 1:5 together; With nutrient culture media washing 1 time, the centrifugal 10min of 1 000g abandons supernatant to the greatest extent; Centrifuge tube is placed 37 ℃ of water-baths, dropwise slowly splash into 37 ℃ of warm in advance fusion agent 50% polyglycol (Polyethylene Glycol, PEG of 1mL; Molecular weight 4 000 adds in the 30s), make itself and cell mixing; Stop the PEG effect; The centrifugal 5min of cell suspension 1 000g with reaction terminating; Abandon and gently be suspended from the HAT that contains 20% hyclone behind the supernatant (Hypoxanthine-Aminopterin-Thymidine HAT) in the nutrient solution, adds and has been covered with in 96 orifice plates of feeder cells merging the back cell suspension; 100 μ L/ holes, 5%CO
237 ℃ of cultivations of incubator.
(2.3) limiting dilution assay carries out hybridoma cloning
The hybridoma colony (strong positive hole) that antibody-secreting is positive blows afloat gently, and mixing carries out viable count, calculates cell density; Get the cell suspension of mixing, accurately carry out serial dilution, with cell dilution to 5,10,50 cell/mL with complete medium; The cell suspension that dilution is good adds respectively in 96 well culture plates of having completed feeder cells, and every hole 100 μ L make every hole contain 0.5,1 and 5 cell respectively; Put 5% CO
2The upgrowth situation of observation of cell is noted in 37 ℃ of cultivations in the cell culture incubator, treats that cell colony partly changes liquid when growing at the bottom of the hole 1/10 ~ 1/5 area; When the naked eyes visible cell is cloned, draw suitable nutrient solution supernatant and carry out antibody test; The hybridoma of first cloning need in complete medium, add HT (Hypoxanthine-Thymidine, HT); In the time of each cloning remaining cell is carried out enlarged culture and in time frozen, detect positive rates until all cloning cell holes and reach 100%; Generally, can confirm to have obtained the hybridoma cell strain of secrete monoclonal antibody through 2 ~ 3 time cloning operations.
(2.4) induce the ascites legal system in the body and be equipped with ascites
Get 8 age in week BALB/c mouse, every mouse carries out pre-service through lumbar injection sterilized liquid paraffin 0.5mL, after 1 week again lumbar injection 0.5mL be in the hybridoma suspension (1 ~ 2 * 10 of exponential phase
6Individual/mL), mouse web portion obviously expands behind the 10d, and aseptic condition is collected ascites down.
(2.5) monoclonal antibody in sad-ammonium sulfate method purifying ascites
With the ascites 4 ℃ of 10 centrifugal 20min of 000g in high speed low temperature centrifugal machine that collects, collect supernatant after discarding surperficial grease, with 4 times of volume acetate buffer solutions dilutions, the slow caprylic acid that dropwise adds 2.5 times of volumes, 4 ℃ leave standstill the centrifugal 30min of 10 000g behind the 1h; Get supernatant, add 10 * PBS of 1/10 volume, dropwise add 45% saturated ammonium sulphate after 4 ℃ of precoolings, 4 ℃ leave standstill 1h, the centrifugal 20min of 10 000g; Abandon supernatant, deposition is suspended among a small amount of PBS, change bag filter after the dilution over to, 4 ℃ of dialysis 3d in the PBS of 100 times of volumes, every 6h changes liquid 1 time; Reclaim the dialysis product, 4 ℃ of 10 centrifugal 30min of 000g removes the McAb that insoluble sediment promptly gets prepared anti-trichina muscle larvae ES antigen, measures its concentration and packing with Coomassie brilliant blue G-250 method, and-80 ℃ of preservations are subsequent use.
(3) the IgY-McAb sandwich ELISA detects the trichina CAg:
(3.1) encapsulate IgY: the chessboard titrimetry is confirmed the righttest concentration and the serum optimum dilution degree of encapsulating of IgY; Encapsulate damping fluid (PH9.6) with carbonate and will survey the IgY of concentration and be diluted to the righttest concentration that encapsulates, 100 μ L/ hole coated elisa plates, 4 ℃ are spent the night.
(3.2) washing: the liquid in the ELISA Plate hole of vertically turning, clap and do, fill it up with cleansing solution (PBST) in the hole, leave standstill 4min, 3 times repeatedly, at last ELISA Plate is upside down on the thieving paper, clap and do.
(3.3) sealing: with the sealing of 5% skimmed milk power, 200 μ L/ holes, 37 ℃ of 1h.
(3.4) washing: the same.
(3.5) add seized serum: with dilution seized serum is diluted with optimal proportion, get 100 μ L dilute serums and add ELISA Plate, every plate is all established the positive, feminine gender and blank, 37 ℃ of 1h.
(3.6) washing: the same.
(3.7) adding one resists: monoclonal antibody is diluted 100 μ L/ holes, 37 ℃ of 1h with dilution with optimum dilution degree.
(3.8) washing: the same.
(3.9) add ELIAS secondary antibody: the HRP-goat anti-mouse igg that uses dilution is two anti-, and every hole adds enzyme mark goat anti-mouse IgG 100 μ L, 37 ℃ of 1h.
(3.10) washing: the same.
(3.11) add substrate: add freshly prepared substrate solution 100 μ L, 37 ℃ of 20min in each reacting hole.
(3.12) add stop buffer: add stop buffer 50 μ L in each reacting hole, survey the OD value immediately.
(3.13) observations: (λ=490nm) surveys light absorption value (A490 value) with enzyme-linked immunosorbent assay instrument after the visual inspection.
(3.14) result judges: visual inspection is faint yellow negative, and salmon pink is positive.Survey the OD value with enzyme-linked immunosorbent assay instrument, positive with the average OD value of testing sample OD value/negative control >=2.1.
Beneficial effect of the present invention is mainly reflected in:
1, the IgY of anti-trichina muscle larvae ES antigen involved in the present invention and McAb be by trichina muscle larvae ES antigen-immunized animal preparation, and be with strong points, has the specificity of height when trichina CAg catches in the serum.
2, ELISA method involved in the present invention is a mature technology, is easy to grasp, and is widely used at biomedical sector, can promote on a large scale.
3, IgY-McAb sandwich ELISA method involved in the present invention not only has the susceptibility and the specificity of height when trichina CAg detects in to serum, and stability is good with repeatability; Early stage, fast detecting be can realize, and discriminating, efficacy assessment and the seroepidemiological survey of the past and existing disease infection can be used for trichinzation.
The sandwich ELISA method susceptibility that trichina CAg detects in to serum:
Get the trichina muscle larvae ES antigen of concentration known,,, note being provided with simultaneously negative control and blank according to the method for IgY-McAb sandwich ELISA described in the instructions of the present invention procedure by certain gradient doubling dilution successively.Mensuration result shows, when trichina muscle larvae ES antigen diluent during to 1ng/mL, its S/N value is still greater than 2.1, and the result is still positive, explains that the present invention can be low to moderate 1ng/mL to the minimum detectable activity of ES antigen, has higher susceptibility, and is as shown in table 1.
Table 1 IgY-McAb sandwich ELISA sensitivity Detection (S/N value)
The IgY-McAb sandwich ELISA method detects the dynamic change of CAg in the trichinzation mice serum:
Detect CAg situation of change in the trichinzation mice serum with the IgY-monoclonal antibody sandwich ELISA of setting up, according to the IgY-McAb sandwich ELISA method procedure described in the instructions of the present invention.Testing result finds that the 3d after infection of the CAg in the infecting mouse serum examines first, and positive rate is 6% (1/15); Along with the prolongation of infection time, CAg also increases thereupon in the infecting mouse serum, infects back 24d peak (positive rate 100%); Lower gradually then; Infect back 32d and the 2nd peak value occur, reduce rapidly subsequently, like table 2 and shown in Figure 1.Explain that the present invention can be used for the variation of the CAg level in the dynamic monitoring infection animal serum in the scientific research; In implementation process, promptly detect the CAg in the serum in infecting back 3d; This time, prompting the present invention can realize the early diagnosis to trichinzation far beyond at present existing method in advance.
Table 2 IgY-McAb sandwich ELISA detects and infects CAg variation in the trichina mouse different time serum
The IgY-McAb sandwich ELISA method is used for the efficacy assessment of albendazole to the treatment of trichinzation mouse:
With albendazole 7 all mouse after infecting trichina are treated, treatment back 2d begins tail vein blood and detects CAg situation of change in the serum, and the result finds; CAg raises rapidly in the mice serum of treatment back, and 8d reaches peak value after treatment, lowers gradually then; 42d after treatment; All detect in the treatment group mice serum, do not make the CAg that then still can detect in the control group mice that heals with medicine in the serum less than CAg, the difference between two groups have statistical significance (
P<0.05), prompting the present invention can realize to the efficacy assessment after the trichinzation treatment, like table 3 and shown in Figure 2 through the CAg that detects in the serum.
Table 3 IgY-McAb sandwich ELISA detects and infects CAg variation in the different time serum of trichina mouse treatment back
Claims (1)
1. an IgY-McAb sandwich ELISA detects the method for trichina CAg, it is characterized in that: it be with anti-trichina muscle larvae ES antigen chicken yolk antibody IgY as capture antibody, with anti-trichina ES antigen McAb as detecting antibody; Said trichina muscle larvae ES antigen chicken yolk antibody IgY and anti-trichina ES antigen McAb are prepared from through following method respectively:
(A) the anti-trichina muscle larvae ES antigen chicken yolk antibody IgY of preparation:
The trichina muscle larvae is added axenic cultivation in the nutrient culture media, and supernatant purifying dialysis back concentrate drying promptly gets ES antigen, and packing is for use behind the mensuration protein concentration; With above-mentioned trichina muscle larvae ES antigen immune Luo Man laying hen, the egg yolk that produces collect IgY with the saturated ammonium sulphate method, measure concentration behind the purifying, promptly get and resist trichina muscle larvae ES antigen chicken yolk antibody IgY, subsequent use after the packing;
(B) the anti-trichina ES antigen McAb of preparation:
Set up the hybridoma cell strain of the anti-trichina muscle larvae ES antigen of secretion, use limiting dilution assay to carry out cloning, use sad-ammonium sulfate method purifying McAb, packing is subsequent use after the mensuration concentration.
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| 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 |
| CN109946448A (en) * | 2017-12-20 | 2019-06-28 | 欧蒙医学诊断技术有限公司 | The antigen of hair shape nematode species detects |
| CN114163525A (en) * | 2020-09-11 | 2022-03-11 | 吉林大学 | Yolk antibody for resisting trichina excretion and secretion antigen, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109946448A (en) * | 2017-12-20 | 2019-06-28 | 欧蒙医学诊断技术有限公司 | The antigen of hair shape nematode species detects |
| CN109946448B (en) * | 2017-12-20 | 2023-10-03 | 欧蒙医学实验诊断股份公司 | Antigen detection of trichostrongylus species |
| CN114163525A (en) * | 2020-09-11 | 2022-03-11 | 吉林大学 | Yolk antibody for resisting trichina excretion and secretion antigen, preparation method and application thereof |
| CN114163525B (en) * | 2020-09-11 | 2023-11-10 | 吉林大学 | Egg yolk antibody for resisting excreted and secreted antigen of trichina, and preparation method and application thereof |
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Application publication date: 20120125 |