CN102393461A - Colloidal gold immunochromatography kit for detecting circulating antigen of schistosomiasis and preparation method thereof - Google Patents
Colloidal gold immunochromatography kit for detecting circulating antigen of schistosomiasis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold immunochromatography kit for detecting a circulating antigen of schistosomiasis, which comprises a box body, wherein a base plate is arranged in the box body; a sample pad is arranged at one end of the upper side of the base plate, and a tail-end water absorption pad is arranged at the other end; a detection layer is arranged at the middle part of the base plate; a gold mark pad is arranged between the sample pad and the detection layer; the detection layer is wrapped with a detection line and a quality control line; and a sample adding hole and an observation hole are formed on the front surface of the box body. The invention also provides a preparation method of the colloidal gold immunochromatography kit for detecting the circulating antigen of schistosomiasis. The kit disclosed by the invention is convenient and quick to operate, has high sensitivity, strong specificity and good repeatability, provides an intuitive result, does not need special equipment, avoids interference from environmental conditions and has high practical values.
Description
Technical field
The present invention relates to bioengineering field, relate in particular to a kind of kit, particularly a kind of colloidal gold immunochromatographimethod kit and preparation method of the detection snail fever CAg based on IgG2b, two kinds of monoclonal antibodies of IgM.
Background technology
1, the importance of immunodiagnosis in the Japanese schistosomiasis control
Snail fever is that a kind of being widely current suffered from parasitic disease altogether in the people beast of the torrid zone and subtropical zone, and about 200,000,000 populations of 76 countries and regions, the whole world are infected, and the infected threat of 600,000,000 populations is arranged.China is the popular district of Japanese schistosomiasis, has once had a strong impact on people's ordinary production and life in the early days of foundation.Through the control in more than 50 years, obtained remarkable achievement, to total several 843007 people of snail fever patient in the whole nation in 2003, (1161.2 ten thousand people) reduced 92.74% more in the early days of foundation.5 provinces (district, city) such as existing Guangdong, Shanghai, Fujian, Guangxi, Zhejiang have blocked the propagation of snail fever; 5 province lake regions such as only surplus Hunan, Hubei, Jiangxi, Anhui, Jiangsu and river, mountain area, Yunnan are not controlled.We also are faced with the huge challenge of further preventing and controlling in joyful.Along with going deep into of preventing and controlling, the population infection degree constantly descends, and low popular district enlarges, and the etiological diagnosis of snail fever is used more and more difficult at the scene on a large scale, not only susceptibility poor, waste time and energy, and be difficult for by the masses and staff's acceptance.On the other hand; The main means of control snail fever are the praziquantel chemotherapy now; But on a large scale, single medicine chemotherapy repeatedly, possibly produce drug resistance, according to abroad; Therefore the Schistosoma mansoni strain of tool praziquantel resistance occurred, the development susceptibility and the higher diagnostic means of specificity that are used for the low popular district of snail fever classified as the prevention and cure of snail fever of the third-largest world and planned preferential research project.
2, the effect of CAg in the Japanese schistosomiasis immunodiagnosis
Through the development of decades, the snail fever immune diagnostic technique has had higher susceptibility, specificity and feasibility.Detecting the blood fluke circulating antibody is the detection low, simple to operate of a kind of cost; Use the widest approach when also being present on-the-spot epidemiology investigation; But circulating antibody still keeps higher level in significant period of time after treatment; The previous infection that is not easily distinguishable infects with existing disease, confirms target chemotherapy crowd in the time of can't using at the scene; And be difficult to reflect curative effect of medication, should not be as the evaluation index of efficacy assessment.Another kind of approach is to detect CAg; CAg (circulating antigen; CAg) be metabolic product and the secretion that is present in the blood fluke polypide in the body fluid such as blood samples of patients, saliva, urine; Detect the CAg in patient's serum or the urine, can assess worm load number and activity and infect, can be used as the foundation of diagnosis.And; After treatment; CAg turns out cloudy comparatively fast, detects the foundation that CAg can be used as efficacy assessment, and the whole bag of tricks that detects based on CAg emerges in an endless stream; But all perplex on the problem low in susceptibility, that specificity is not strong, CAg detection method and the technology of therefore seeking susceptibility height, high specificity are the new directions of snail fever immunodiagnosis.
3. the application of monoclonal antibody technique in the snail fever immune diagnostic technique
Since Kohler and Milstein (1975) invention Monoclonal Antibody technology, the monoclonal antibody technology extensively applies to medical science and biological every field, for generation, development, diagnosis and the treatment of disease provides important means.Monoclonal antibody is produced by a cell, its composition (type, subclass, type and combination P position etc.) be homology; Only be directed against an antigenic determinant of complicated antigen, and identical compatibility is arranged, antibody titer is also very high; Detect the specificity that CAg has height, antigen and monoclonal antibody are produced on a large scale, and method is simple and practical; And have efficacy assessment value preferably, can distinguish existing disease and infect and previous infection, can be used for the immunodiagnosis and the epidemiology field investigation of snail fever.
Summary of the invention
The object of the present invention is to provide the colloidal gold immunochromatographimethod kit and the preparation method that detect the snail fever CAg, the colloidal gold immunochromatographimethod kit of described this detection snail fever CAg and preparation method will solve the technical matters that method susceptibility is low, specificity is not strong that detects circulating antigen of schistosome in the prior art.
The invention provides a kind of colloidal gold immunochromatographimethod kit that detects the snail fever CAg; Comprise a box body, be provided with a backing plate in the described box body, an end of the upside of described backing plate is provided with a sample pad; An other end of the upside of described backing plate is provided with a terminal adsorptive pads; The middle part of described backing plate is provided with a detection layers, and described detection layers closely is connected with described terminal adsorptive pads, between described sample pad and described detection layers, is provided with a gold mark pad; The anti-schistosomiasis CAg monoclonal antibody that contains colloid gold label on the described gold mark pad; Described sample pad, gold mark pad closely are connected with described detection layers, on described detection layers, are coated with detection line and nature controlling line, and a front of described box body is provided with a well and a viewport; Described well is positioned at the top of sample pad, and described viewport is positioned at the top of described detection line and nature controlling line.
Further, an end of described detection layers is arranged on the downside of described gold mark pad, and an end of described gold mark pad is arranged on the downside of described sample pad.
Further, an other end of described detection layers is arranged on the downside of described terminal adsorptive pads.
Further, described detection layers is a nitrocellulose filter.
Further, described detection line is after to be that 1～3% formaldehyde fixed is liquid-solid decide the monoclonal antibody IgG2b of anti-schistosome soluble antigen that concentration is 0.8～1.5mg/ml with mass percent concentration, to be sprayed on the detection layers stage casing equably and to process.
Further, the monoclonal antibody IgG2b of described anti-schistosome soluble antigen is the mouse hybridoma cell generation of CGMCC NO:4978 by preserving number.
Further; Described nature controlling line is after to be that 1～3% formaldehyde fixed is liquid-solid decide sheep anti-mouse igg or IgM that concentration is 2～2.5mg/ml with mass percent concentration; Be sprayed on the detection layers equably and process, described nature controlling line is near described terminal adsorptive pads, distance detecting line 0.5～1cm.
Further, described gold mark pad comprises a tunica fibrosa, and spray is provided with the anti-schistosome soluble antigen monoclonal antibody IgM of the good collaurum of mark on the described tunica fibrosa, and spouting liquid is 0.8～2.0 μ l/cm.
Further, described tunica fibrosa is a glass fibre membrane.
Further, described anti-schistosome soluble antigen monoclonal antibody IgM is the mouse hybridoma cell generation of CGMCC NO:3642 by preserving number.
The present invention also provides above-mentioned a kind of preparation method who detects the colloidal gold immunochromatographimethod kit of snail fever CAg; Comprise a step that adopts hybridoma technology to prepare the monoclonal antibody of anti-schistosome soluble antigen; The specific monoclonal antibody of described anti-schistosome soluble egg antigen is used to prepare the monoclonal antibody and detection monoclonal antibody of colloid gold label, comprises the step of a preparation collaurum, comprises the step of the monoclonal antibody IgM for preparing a colloid gold label anti-schistosome soluble antigen; The step that comprises a preparation gold mark pad; When the collaurum for preparing with after monoclonal antibody to be marked successfully combines, be sprayed on equably on the detection layers with the monoclonal antibody of the moving appearance of two dimension spray the good collaurum of mark, comprise the step of detection line and nature controlling line in the preparation detection layers; The monoclonal antibody IgG2b of anti-schistosome soluble antigen is sprayed on the detection layers stage casing equably; Obtain detection line,, be sprayed on the detection layers equably sheep anti-mouse igg or IgM; Distance detecting line 0.5～1cm place obtains nature controlling line; Comprise the step of a preparation box body and backing plate, detection layers is sticked on the backing plate, backing plate is arranged in the described box body.
Further, described detection layers is a nitrocellulose filter.
Further, in a step that adopts the monoclonal antibody that hybridoma technology prepares the anti-schistosome soluble antigen, comprise the steps,
1) step of an animal immune, in the step of described animal immune, the bilharzial soluble antigen of preparation through bilharzial soluble antigen immune animal, is obtained immune spleen cell then earlier;
2) step of a Fusion of Cells is got myeloma cell SP2/0 and immune spleen cell and is merged under the PEG4000 effect in 1: 5～1: 10 ratio;
3) step of filtering hybridoma and cloning obtains the mouse hybridoma cell strain that preserving number is CGMCC NO:4978 and 3642;
4) above-mentioned monoclonal cell strain is injected the step of the monoclonal antibody IgG2b of the monoclonal antibody IgM that produces a kind of anti-schistosome soluble antigen in the animal body and a kind of anti-schistosome soluble antigen respectively;
5) step of the monoclonal antibody of the above-mentioned two kinds of anti-schistosome soluble antigens of purifying respectively.
Further, in the step of a described preparation box body and backing plate, prepare a backing plate earlier; The detection layers that will contain detection line and nature controlling line earlier sticks on the backing plate, pastes gold mark pad earlier at an end of detection layers then, contains the anti-schistosomiasis CAg monoclonal antibody of colloid gold label on the gold mark pad; One side of filling up at the gold mark away from detection layers is again pasted sample pad; Described sample pad, gold mark pad closely are connected with described detection layers, paste terminal adsorptive pads at the other end of detection layers, and detection layers closely links to each other with terminal adsorptive pads; Backing plate is arranged in the box body; Well and viewport are set on the front of box body, and described well is positioned at the top of sample pad, and described viewport is positioned at the top of described detection line and nature controlling line.
Further, described detection line is after to be that 1～3% formaldehyde fixed is liquid-solid decide the monoclonal antibody IgG2b of anti-schistosome soluble antigen that concentration is 0.8～1.5mg/ml with mass percent concentration, to be sprayed on the detection layers stage casing equably and to process.
Further, the monoclonal antibody IgG2b of described anti-schistosome soluble antigen is the mouse hybridoma cell generation of CGMCC NO:4978 by preserving number.
Further; Described nature controlling line is after to be that 1～3% formaldehyde fixed is liquid-solid decide sheep anti-mouse igg or IgM that concentration is 2～2.5mg/ml with mass percent concentration; Be sprayed on the detection layers equably and process, described nature controlling line is near described terminal adsorptive pads, distance detecting line 0.5～1cm.
Further, described gold mark pad comprises a tunica fibrosa, and spray is provided with the anti-schistosome soluble antigen monoclonal antibody IgM of the good collaurum of mark on the described tunica fibrosa, and spouting liquid is 0.8～2.0 μ l/cm.
Further, described anti-schistosome soluble antigen monoclonal antibody IgM is the mouse hybridoma cell generation of CGMCC NO:3642 by preserving number.
Principle of work of the present invention is: when liquid samples such as test serum sample drip on sample pad; Liquid promptly constantly spreads to the nature controlling line place; When liquid arrives gold mark pad place; Anti-schistosome soluble antigen monoclonal antibody gold label is dissolved; Simultaneously with sample in the CAg reaction and form compound, liquid continues to move forward to the detection line place, the compound of colloid gold label again with film on the anti-schistosome soluble antigen monoclonal antibody of the detection line lines that combine to form gold mark monoclonal antibody-CAg-monoclonal antibody sandwich complex and present redness; The antibody of unnecessary colloid gold label continues to move forward to the nature controlling line place and combines and appear red lines with sheep anti-mouse igg or IgM, and the liquid of last remnants is absorbed by terminal adsorptive pads.Positive sample all has band to occur at detection line and nature controlling line, and feminine gender then has only band of nature controlling line, if no band explains that then test strips lost efficacy.
The present invention also provides the monoclonal cell strain of a kind of anti-schistosome soluble antigen (SEA); Its called after mouse hybridoma cell (Japanese schistosomiasis cell strain of monoclonal antibody A1E3) of classifying; This cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); The address at China Committee for Culture Collection of Microorganisms common micro-organisms center is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City (institute of microbiology of the Chinese Academy of Sciences); Preservation date be on March 3rd, 2010 preserving number be CGMCC NO:3642, this cell line is to merge the hybridoma cell strain that obtains by murine myeloma cell SP2/0 and immune spleen cell.
The present invention also provides the monoclonal cell strain of a kind of anti-schistosome soluble antigen (SEA); Its called after mouse hybridoma cell (cell line D9G8) of classifying; This cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); The address at China Committee for Culture Collection of Microorganisms common micro-organisms center is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City (institute of microbiology of the Chinese Academy of Sciences); Preservation date is on June 21st, 2011, preserving number CGMCC NO:4978.This cell line is to merge the hybridoma cell strain that obtains by murine myeloma cell SP2/0 and immune spleen cell.
The present invention compared with prior art has following advantage: kit of the present invention is easy and simple to handle, quick; Detect acute and chronic snail fever patients serum; Recall rate is high; Be fit to clinical diagnosis, efficacy assessment and epidemiology survey that departments such as hospital laboratory, disease prevention and control center, She Kang center are used for snail fever, have very high practical value.The double antibody sandwich method that adopts kit of the present invention to detect circulating antigen of schistosome is a single stage method, and susceptibility height, high specificity, good reproducibility are fit to the detection and the efficacy assessment of sample in batches respectively.
Description of drawings
Fig. 1 is a kind of surface structure synoptic diagram that detects the colloidal gold immunochromatographimethod kit of snail fever CAg of the present invention, and wherein: 1 is box body; 2 is well; 3 is viewport.
Fig. 2 is a kind of inner structure synoptic diagram that detects the colloidal gold immunochromatographimethod kit of snail fever CAg of the present invention, and wherein: 4 is sample pad; 5 is gold mark pad; 6 is the nitrocellulose membrane detection layers; 7 is detection line; 8 is nature controlling line; 9 is terminal adsorptive pads, and 10 is the PVC backing plate.
Fig. 3 is a kind of positive findings that detects the colloidal gold immunochromatographimethod kit of snail fever CAg of the present invention, and sample liquid is a serum of acute schistosomicide patient.
Fig. 4 is a kind of negative findings that detects the colloidal gold immunochromatographimethod kit of snail fever CAg of the present invention, and sample liquid is a normal human serum.
Embodiment
Embodiment 1 anti-schistosome SEA MONOCLONAL ANTIBODIES SPECIFIC FOR method is following:
(1) animal immune
1. antigen preparation: every 7 new zealand rabbits that infect 1500 schistosoma japonicum cercariaes (from Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C) were dissected separated and collected worm's ovum and freeze-drying from liver by conventional method after 42 days.Get dried ovum and weigh, the ratio in 1% is dissolved in 0.9% the physiological saline, after the grinding, puts 4 ℃ of cold soakings, during jolting every day 2 times, each 2 minutes.Ice-bath ultrasonic is pulverized after 4 days, and each 3 minutes, intermittently 3 minutes, totally 3 times.Went up cold soaking for another example 2 days, 10000rpm4 ℃ centrifugal 30 minutes, get supernatant, collect packing, with Braford method mensuration antigen protein concentration ,-20 ℃ of preservations are subsequent use.
2. the selection of immune animal: the female BALB/c mouse (from Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C) of selecting 6～8w for use.Initial immunity blood fluke soluble antigen (SEA) is mixed with Freund's complete adjuvant and emulsification complete, by 25～100 a μ g/ dosage intraperitoneal injection of mice.Booster immunization is carried out with SEA and the incomplete Freund mixing and emulsifyings of same dose in 4 week backs, and is later whenever at a distance from 2 weeks, again with the independent booster immunization of SEA 2 times; Detect immunizing potency with the ELISA method; Reach 1/10000 when above, carry out last tail vein booster immunization, get spleen after three days and merge.
(2) Fusion of Cells: get myeloma cell SP2/0 and immune spleen cell and under the PEG4000 effect, merge in 1: 5～1: 10 ratio; Merge good back hybrid cell through containing hypoxanthine (hypoxanthine; H), aminopterin-induced syndrome (aminopterin; A) and thymidine (thymidine selects growth in nutrient culture media T) (HAT), treat that it grows to hole floorage 1/10 sucking-off supernatant confession antibody test when above.
(3) filtering hybridoma and cloning: hybridoma is promptly screening with SEA about 2 weeks after the fusion, and the hybridoma cell strain of picking out to SEA carries out cloning, up to obtaining the stable monoclonal cell strain to SEA of secretion.The monoclonal cell strain of a kind of anti-schistosome soluble antigen (SEA); Its called after mouse hybridoma cell (Japanese schistosomiasis cell strain of monoclonal antibody A1E3 and cell line D9G8) of classifying; This cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); The address at China Committee for Culture Collection of Microorganisms common micro-organisms center is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City (institute of microbiology of the Chinese Academy of Sciences); Preservation date is on March 3rd, 2010 and on June 7th, 2011, and preserving number is 3642 and 4978.
(4) above-mentioned mouse hybridoma cell (CGMCC 3642 and 4978) is injected the BALB/c mouse intraperitoneal respectively; The a large amount of antibody of 1～2 week back mouse peritoneal output; The careful ascites that extracts; The centrifugal monoclonal antibody ascites supernatant that obtains being directed against in a large number SEA; Anti schistosoma soluble egg antigen (SEA) monoclonal antibody (Anti-SEA-IgG2b) is the mouse hybridoma cell strain generation of CGMCC NO:4978 by preserving number, and anti schistosoma soluble egg antigen (SEA) monoclonal antibody (Anti-SEA-IgM) is the mouse hybridoma cell strain generation of CGMCC NO:3642 by preserving number.
(5) the Purification of Monoclonal Antibodies method of described blood fluke soluble egg antigen (SEA) is following:
1. the preparation of saturated ammonium sulfate solution: 500g ammonium sulfate adds in the 500ml distilled water, is heated to dissolving fully, ambient temperature overnight, and the crystallization of separating out is let alone to stay in the bottle.Face with before getting required amount, transfer pH to 7.8 with 2mol/L NaOH.
2. saltout: the ascites that absorption 10ml handles well moves in the small beaker, under agitation, drips saturated ammonium sulfate solution 5.0ml; Continue slowly to stir 30min; The centrifugal 15min of 10000r/min; Abandoning supernatant, sediment suspends with 1/3 saturation degree ammonium sulfate, and beating action 30min is centrifugal with method; Repeat back 1～2 time; Sediment is dissolved in 1.5ml PBS (0.01mol/L pH7.2) or the Tris-HCl damping fluid.
3. desalination: column chromatography commonly used or dialysis.Column chromatography is that the sample of saltouing is crossed the SephadexG-50 chromatographic column, with PBS or Tri s-HCl damping fluid as equilibrium liquid and eluent, flow velocity 1ml/min.First protein peak is the antibody-solutions of desalination.Dialysis is in 2%NaHCO with bag filter
3, boil 10min in the 1mmol/L EDTA solution, clean the bag filter surfaces externally and internally with distilled water, boil bag filter 10min with distilled water again, be chilled to room temperature and can use (also can be in 0.2mol/L EDTA solution, 4 ℃ of preservations are subsequent use).The sample of will saltouing is packed in the bag filter, to the PBS of 50～100 times of volumes or Tris-HCl damping fluid dialysis (4 ℃) 12～24 hours, changes dislysate therebetween 5 times; With Nai Shi reagent (mercuric iodixde 11.5g; Potassium iodide 8g, adding distil water 50ml is after waiting to dissolve; Add 20%NaOH50ml again) detect, till extracellular fluid dialysis does not have yellow formation.
4. the mensuration of protein content: (Pr) (mg/ml)=(1.45 * 0D
280-0.74 * 0D
260) * extension rate; Or (Pr)=0D
280* extension rate/3
5. packing is frozen subsequent use.
As depicted in figs. 1 and 2, the invention provides a kind of colloidal gold immunochromatographimethod kit that detects the snail fever CAg, comprise a box body 1; Be provided with a backing plate 10 in the described box body 1; The upside of backing plate 10 is provided with a sample pad 4, and the other end of backing plate 10 upsides is provided with a terminal adsorptive pads 9, and the middle part of backing plate 10 is provided with a detection layers 6; Between sample pad 4 and detection layers 6, be provided with a gold mark pad 5; One end of detection layers 6 is arranged on the downside of gold mark pad 5, and an end of gold mark pad 5 is arranged on the downside of sample pad 4, is coated with detection line 7 and nature controlling line 8 on the detection layers 6; The front of box body is provided with a well 2 and a viewport 3; Well 2 is arranged on the top of sample pad 4, and viewport 3 is arranged on the top of detection line 7 and nature controlling line 8, and an other end of detection layers 6 is arranged on the downside of terminal adsorptive pads 9.
Further, described detection layers 6 is a nitrocellulose filter.
Further, described backing plate 10 materials are PVC.
Further, described detection line 7 is after to be that 1～3% formaldehyde fixed is liquid-solid decide the monoclonal antibody IgG2b of anti-schistosome soluble antigen that concentration is 0.8～1.5mg/ml with mass percent concentration, to be sprayed on detection layers 6 stage casings equably and to process.
Further; Described nature controlling line 8 is after to be that 1～3% formaldehyde fixed is liquid-solid decide sheep anti-mouse igg or IgM that concentration is 2～2.5mg/ml with mass percent concentration; Be sprayed on equably on the detection layers 6 and process; Described nature controlling line 8 is near described terminal adsorptive pads 9, and distance detecting line 7 is 0.5～1cm.
Further, described gold mark pad 5 comprises a tunica fibrosa, and spray is provided with the anti-schistosome soluble antigen monoclonal antibody IgM of the good collaurum of mark on the described tunica fibrosa, and spouting liquid is 0.8～2.0 μ l/cm.
Further, described tunica fibrosa is a glass fibre membrane.
Further, the monoclonal antibody IgG2b of described anti-schistosome soluble antigen is the mouse hybridoma cell generation of CGMCC NO:4978 by preserving number.
Further, described anti-schistosome soluble antigen monoclonal antibody IgM is the mouse hybridoma cell generation of CGMCC NO:3642 by preserving number.
Further, described sample pad 4 is formed by the thieving paper cutting with terminal adsorptive pads 9.
Embodiment 3
The present invention also provides a kind of method for preparing the colloidal gold immunochromatographimethod kit of above-mentioned detection snail fever CAg; This method comprises a step that adopts hybridoma technology to prepare the monoclonal antibody of anti-schistosome soluble antigen; The specific monoclonal antibody of described anti-schistosome soluble egg antigen is used to prepare the monoclonal antibody (IgM) of colloid gold label and detects monoclonal antibody (IgG2b); The step that comprises a preparation collaurum; The step that comprises the monoclonal antibody for preparing a colloid gold label anti-schistosome soluble antigen comprises the step of a preparation gold mark pad 5, when the collaurum for preparing with after monoclonal antibody (IgM) to be marked successfully combines; Use the moving appearance of two dimension spray to be sprayed on the monoclonal antibody of the good collaurum of mark on the glass fibre membrane equably; Comprise the step of detection line 7 and nature controlling line 8 in the preparation nitrocellulose filter detection layers 6, will detect monoclonal antibody (IgG2b) and be sprayed on the nitrocellulose membrane stage casing equably, obtain detection line 7; With sheep anti-mouse igg or IgM (sigma company or other companies buy), be sprayed on equably on the nitrocellulose membrane, distance detecting line 70.5～1cm place obtains the step that nature controlling line comprises a preparation box body and backing plate, and backing plate is arranged in the described box body.
Further, in a step that adopts the monoclonal antibody that hybridoma technology prepares the anti-schistosome soluble antigen, comprise the steps,
1) step of an animal immune, in the step of described animal immune, the soluble antigen of preparation Schistosoma japonicum through the soluble antigen immune animal of Schistosoma japonicum, is obtained immune spleen cell then earlier;
2) step of a Fusion of Cells is got myeloma cell SP2/0 and immune spleen cell and is merged under the PEG4000 effect in 1: 5～1: 10 ratio;
3) step of filtering hybridoma and cloning obtains the mouse hybridoma cell strain that preserving number is CGMCC NO:4978 and 3642;
4) monoclonal antibody of the anti-schistosome soluble antigen that a kind of colloid gold label of generation is used in the animal body and the step of the monoclonal antibody of the anti-schistosome soluble antigen that detects usefulness are injected in above-mentioned monoclonal cell strain respectively;
5) step of the monoclonal antibody of the above-mentioned two kinds of anti-schistosome soluble antigens of purifying respectively.
Further, in the step of a described preparation box body and backing plate 10, prepare a backing plate 10 earlier; The detection layers 6 that will contain detection line 7 and nature controlling line 8 earlier sticks on the backing plate 10, pastes gold mark pad 5 earlier at an end of detection layers 6 then, contains the anti-schistosomiasis CAg monoclonal antibody of colloid gold label on the gold mark pad 5; One side of filling up at the gold mark away from detection layers 6 is again pasted sample pad 4; Described sample pad 4, gold mark pad 5 closely are connected with described detection layers 6, paste terminal adsorptive pads 9 at the other end of detection layers 6, and detection layers 6 closely links to each other with terminal adsorptive pads 9; Backing plate 10 is arranged in the box body; Well and viewport are set on the front of box body, and described well is located at the top of sample pad 4, and described viewport is positioned at the top of detection line 7 and nature controlling line 8.
Principle of work of the present invention is: when liquid samples such as test serum sample dripped in 4 last times of sample pad; Liquid promptly constantly spreads to nature controlling line 8 places; When liquid arrives gold mark pad 5 places; Anti-schistosome soluble antigen monoclonal antibody gold label is dissolved; Simultaneously with sample in the CAg reaction and form compound, liquid continues to move forward to detection line 7 places, the compound of golden mark again with film on the anti-schistosome soluble antigen monoclonal antibody of detection line 7 lines that combine to form gold mark monoclonal antibody-CAg-monoclonal antibody sandwich complex and present redness; Unnecessary golden labeling antibody continues to move forward to nature controlling line 8 places and combines and appear red lines with sheep anti-mouse igg or IgM, and last remaining liquid is by terminal adsorptive pads 9 absorptions.Positive sample all has band to occur at detection line 7 and nature controlling line 8, and feminine gender then has only 8 one bands of nature controlling line, if no band explains that then test strips lost efficacy.(like Fig. 3, shown in Figure 4)
Embodiment 4
Kit specificity, sensitivity detect:
1. specific detection: draw 20 μ l serum with pipettor, be added drop-wise on the sample pad, put room temperature about 10 minutes, treat that color reaction appears in nature controlling line 8, gets final product observations.Every kind of combination all detects identical serum, detects 20 parts of normal human serums altogether, 20 parts of acute disease human serums, 40 parts of chronic patient serum, 5 parts of Paragonismus westermani fluke disease human serums, 5 parts of toxoplasmosis human serums, 5 parts of clonorchis sinensis patients serums.5 parts of cysticercosis human serums.Result such as table 1, it all is 100% that this detection kit detects positive rate to the acute patient of Schistosoma japonicum and chronic patient, also is 100% to normal person's specificity.But a little cross reaction is arranged with cysticercosis human serum and paragonimiasis westermani human serum.
Table 1 colloidal gold immuno-chromatography test paper strip detects blood fluke SEA antigen in the different blood serum samples
2. sensitivity detects: with blood fluke SEA antigen diluent during to 1ng/mL; Still visible colloidal gold immuno-chromatography test paper strip two red line all occur at detection line 7 (T) and nature controlling line 8 (C), and other parasite antigen samples only a purplish red colo(u)r streak occurs at nature controlling line 8 (C).Show that colloidal gold immuno-chromatography test paper strip has very high sensitivity, detectability can reach ng level (seeing table 2).
The sensitivity determination result of table 2 colloidal gold immuno-chromatography test paper strip
Claims (18)
1. colloidal gold immunochromatographimethod kit that detects the snail fever CAg; Comprise a box body, it is characterized in that: be provided with a backing plate in the described box body, an end of the upside of described backing plate is provided with a sample pad; An other end of the upside of described backing plate is provided with a terminal adsorptive pads; The middle part of described backing plate is provided with a detection layers, and described detection layers closely is connected with described terminal adsorptive pads, between described sample pad and described detection layers, is provided with a gold mark pad; The anti-schistosomiasis CAg monoclonal antibody that contains colloid gold label on the described gold mark pad; Described sample pad, gold mark pad closely are connected with described detection layers, on described detection layers, are coated with detection line and nature controlling line, and the front of described box body is provided with a well and a viewport; Described well is positioned at the sample pad top, and described viewport is positioned at the top of described detection line and nature controlling line.
2. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 1; It is characterized in that: an end of described detection layers is arranged on the downside of described gold mark pad, and an end of described gold mark pad is arranged on the downside of described sample pad.
3. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 2 is characterized in that: the other end of described detection layers is arranged on the downside of described terminal adsorptive pads.
4. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 1 is characterized in that: described detection layers is a nitrocellulose filter.
5. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 1; It is characterized in that: described detection line is sprayed on the detection layers stage casing equably and processes after to be that to use mass percent concentration be that 1～3% formaldehyde fixed is liquid-solid decide the monoclonal antibody IgG2b of anti-schistosome soluble antigen that concentration is 0.8～1.5mg/ml.
6. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 5 is characterized in that: the monoclonal antibody IgG2b of described anti-schistosome soluble antigen is the mouse hybridoma cell generation of CGMCC NO:4978 by preserving number.
7. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 1; It is characterized in that: described nature controlling line is after to be that 1～3% formaldehyde fixed is liquid-solid decide sheep anti-mouse igg or IgM that concentration is 2～2.5mg/ml with mass percent concentration; Be sprayed on the detection layers equably and process; Described nature controlling line is near described terminal adsorptive pads, distance detecting line 0.5～1cm.
8. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 1; It is characterized in that: described gold mark pad comprises a tunica fibrosa; Spray is provided with the anti-schistosome soluble antigen monoclonal antibody IgM of the good collaurum of mark on the described tunica fibrosa, and spouting liquid is 0.8～2.0 μ l/cm.
9. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 8 is characterized in that: described tunica fibrosa is a glass fibre membrane.
10. the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 8 is characterized in that: described anti-schistosome soluble antigen monoclonal antibody IgM is the mouse hybridoma cell generation of CGMCC NO:3642 by preserving number.
11. the preparation method of the colloidal gold immunochromatographimethod kit of the described detection snail fever of claim 1 CAg; It is characterized in that: comprise a step that adopts hybridoma technology to prepare the monoclonal antibody of anti-schistosome soluble antigen, the specific monoclonal antibody of described anti-schistosome soluble egg antigen is used to prepare the monoclonal antibody and detection monoclonal antibody of colloid gold label, comprises the step of a preparation collaurum; The step that comprises the monoclonal antibody IgM for preparing a colloid gold label anti-schistosome soluble antigen; The step that comprises a preparation gold mark pad, when the collaurum for preparing with after monoclonal antibody to be marked successfully combines, move appearance with the two dimension spray and be sprayed on the monoclonal antibody of the good collaurum of mark on the glass fibre membrane equably; The step that comprises detection line and nature controlling line in the preparation detection layers; The monoclonal antibody IgG2b of anti-schistosome soluble antigen is sprayed on the detection layers stage casing equably, obtains detection line, with sheep anti-mouse igg or IgM; Be sprayed on the detection layers equably; Distance detecting line 0.5～1cm place obtains nature controlling line, comprises the step of a preparation box body and backing plate, and backing plate is arranged in the described box body; Detection layers is sticked on the backing plate, backing plate is arranged in the described box body.
12. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 11; It is characterized in that: prepare in the step of monoclonal antibody of anti-schistosome soluble antigen in described employing hybridoma technology; Comprise the steps
1) step of an animal immune, in the step of described animal immune, the bilharzial soluble antigen of preparation through bilharzial soluble antigen immune animal, is obtained immune spleen cell then earlier;
2) step of a Fusion of Cells is got myeloma cell SP2/0 and immune spleen cell and is merged under the PEG4000 effect in 1: 5～1: 10 ratio;
3) step of filtering hybridoma and cloning obtains the mouse hybridoma cell strain that preserving number is CGMCC NO:4978 and 3642;
4) step of above-mentioned monoclonal cell strain being injected the monoclonal antibody IgG2b of the monoclonal antibody IgM that produces the anti-schistosome soluble antigen in the animal body and anti-schistosome soluble antigen respectively;
5) step of the monoclonal antibody of the above-mentioned two kinds of anti-schistosome soluble antigens of purifying respectively.
13. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 11; It is characterized in that: in the step of a described preparation box body and backing plate, prepare a backing plate earlier, the detection layers that will contain detection line and nature controlling line earlier sticks on the backing plate; Paste gold mark pad earlier at an end of detection layers then; The anti-schistosomiasis CAg monoclonal antibody that contains colloid gold label on the gold mark pad, a side of filling up at the gold mark away from detection layers is again pasted sample pad, and described sample pad, gold mark pad closely are connected with described detection layers; Paste terminal adsorptive pads at the other end of detection layers; Detection layers closely links to each other with terminal adsorptive pads, and backing plate is arranged in the box body, and well and viewport are set on the front of box body; Described well is positioned at the top of sample pad, and described viewport is positioned at the top of detection line and nature controlling line.
14. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 11; It is characterized in that: described detection line is after to be that 1～3% formaldehyde fixed is liquid-solid decide the monoclonal antibody IgG2b of anti-schistosome soluble antigen that concentration is 0.8～1.5mg/ml with mass percent concentration, to be sprayed on the detection layers stage casing equably and to process.
15. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 11 is characterized in that: the monoclonal antibody IgG2b of described anti-schistosome soluble antigen is the mouse hybridoma cell generation of CGMCC NO:4978 by preserving number.
16. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 11; It is characterized in that: described nature controlling line is after to be that 1～3% formaldehyde fixed is liquid-solid decide sheep anti-mouse igg or IgM that concentration is 2～2.5mg/ml with mass percent concentration; Be sprayed on the detection layers equably and process; Described nature controlling line is near described terminal adsorptive pads, distance detecting line 0.5～1cm.
17. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 11; It is characterized in that: described gold mark pad comprises a tunica fibrosa; Spray is provided with the anti-schistosome soluble antigen monoclonal antibody IgM of the good collaurum of mark on the described tunica fibrosa, and spouting liquid is 0.8～2.0 μ l/cm.
18. the preparation method of the colloidal gold immunochromatographimethod kit of detection snail fever CAg as claimed in claim 17 is characterized in that: described anti-schistosome soluble antigen monoclonal antibody IgM is the mouse hybridoma cell generation of CGMCC NO:3642 by preserving number.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011102945971A CN102393461A (en) | 2011-09-29 | 2011-09-29 | Colloidal gold immunochromatography kit for detecting circulating antigen of schistosomiasis and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011102945971A CN102393461A (en) | 2011-09-29 | 2011-09-29 | Colloidal gold immunochromatography kit for detecting circulating antigen of schistosomiasis and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103454418A (en) * | 2013-06-24 | 2013-12-18 | 广西大学 | Immunochromatography rapid detection test strip for fasciola gigantica and preparation method thereof |
| CN107356762A (en) * | 2016-05-10 | 2017-11-17 | 吉林大学 | Detect toxplasmosis in pigs circulating antigen immunity colloidal gold strip and preparation method |
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| CN102043053A (en) * | 2009-10-22 | 2011-05-04 | 华中科技大学 | Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103454418A (en) * | 2013-06-24 | 2013-12-18 | 广西大学 | Immunochromatography rapid detection test strip for fasciola gigantica and preparation method thereof |
| CN103454418B (en) * | 2013-06-24 | 2016-10-05 | 广西大学 | A kind of Fasciola gigantica immune chromatography rapid detecting test paper strip and preparation method thereof |
| CN107356762A (en) * | 2016-05-10 | 2017-11-17 | 吉林大学 | Detect toxplasmosis in pigs circulating antigen immunity colloidal gold strip and preparation method |
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Application publication date: 20120328 |