阅读说明：本技术 曼氏血吸虫虫卵的分离纯化方法及虫卵可溶性抗原及其胶体金免疫层析卡的制备 (The preparation of the isolation and purification method and worm's ovum soluble antigen and its colloidal gold immunochromatographimethod card of Schistosoma mansoni worm's ovum ) 是由 朱传刚 沈元曦 纪荣毅 林矫矫 洪炀 于 2019-08-26 设计创作，主要内容包括：本发明涉及曼氏血吸虫虫卵的分离纯化方法及虫卵可溶性抗原及其胶体金免疫层析卡的制备。所述血吸虫抗体检测胶体金免疫层析卡包括样品垫、PVC底板、胶体金垫、测试线、控制线、硝酸纤维素膜(NC膜)、吸水垫。应用本发明胶体金免疫层析卡检测,操作简单、方便、快速、简捷,不需特殊仪器设备,不需专业培训,结果清晰易辨,操作简单,易于推广,适合基层,适合于突发事件的大批量现场检测,适合流行病学调查。(The present invention relates to the preparations of the isolation and purification method of Schistosoma mansoni worm's ovum and worm's ovum soluble antigen and its colloidal gold immunochromatographimethod card.The schistosome antibody detection colloidal gold immunochromatographimethod card includes sample pad, PVC bottom plate, colloidal gold pad, p-wire, control line, nitrocellulose filter (NC film), water absorption pad.It is detected using colloidal gold immunochromatographimethod card of the present invention, it is simple, convenient, quick, simple and direct, it is not required to special instruments and equipment, is not required to professional training, as a result it clearly easily distinguishes, it is easy to operate, it is easy to spread, it is suitble to base, is suitable for the mass field detection of emergency event, is suitble to epidemiological survey.)

1. a kind of method that suitable Schistosoma mansoni worm's ovum isolates and purifies, the method are as follows:

(1) it is suitable for host that S. mansoni cercariae, which infects, and host includes but is not limited to mouse；

(2) the host animal organ in 6 to 10 week of infection, the preparation for worm's ovum are taken；

(3) separate and purify the worm's ovum of Schistosoma mansoni.

2. the method that suitable Schistosoma mansoni worm's ovum isolates and purifies as described in claim 1, specifically:

60d dissects mouse after infection, takes the viscera tissues such as stomach, liver, spleen, the intestines of each mouse, is carefully separated off and is adhered to respectively The external tissue such as blood vessel, the mesenterium of tissue；Intestines are subdivided into small intestine, cecocolon, three sections of rectum according to function, Small Intestine is again Upper and lower two sections are equably subdivided into, intestinal contents are emptied；

It cuts open and kills, take the biomaterials such as liver, the fritter of 1g or so, the biologies such as every mouse liver are cut into after the connective tissues such as rejecting blood vessel Material adds the most suitable 10ml of 5-15ml() physiological saline (pH value should be 7.0~7.5), homogenate is made using pulper under room temperature, It is homogenized revolving speed 500-2000r/min, (most suitable 2000r/min) Homogenization time 1 minute, interval 3 minutes repeats aforesaid operations 1-5 Secondary (being most suitable for 3 times), obtains the biomaterials homogenates such as the liver containing worm's ovum；

The biomaterials such as above-mentioned liver homogenate is molten in the 10%NaOH that 45-60 DEG C of preheating is added in 1:1-5 ratio (most suitable 1:1) Liquid (be most suitable for 50 DEG C, similarly hereinafter) mixes, and it is 5-15 minute digested (being most suitable for 8 minutes) to be placed in 45-60 DEG C of water bath chader, shakes Swing revolving speed 50-200r/min(and be most suitable for 100r/min), after digestion, the homogenate of digestion is inserted into rapidly 2~8 DEG C of ice Cooling down in water, 3000r/min is centrifuged 1-5 minutes, and supernatant is abandoned in 2~8 DEG C of operations, after adding PBS (0.01M pH6.8) that precipitating is resuspended, It repeats aforesaid operations centrifuge washing 1-3 times (being most suitable for 3 times), after adding PBS (0.01M pH7.2) that precipitating is resuspended, repeats above-mentioned behaviour Make centrifuge washing 1-3 times；After precipitating finally is resuspended with physiological saline (0.9%NaC), repeat aforesaid operations centrifuge washing 1-3 times, Collect worm's ovum；

Above-mentioned tissue is put into centrifuge tube after weighing respectively, adds suitable distilled water, is homogenized after constant volume with high-speed homogenization machine, with thin Born of the same parents' tally counts worm's ovum number, and every sample counts three times.

3. a kind of preparation method of Schistosoma mansoni worm's ovum soluble antigen, the method are as follows:

(1) physiological saline, dismembyator inner powder is added by the worm's ovum precipitating for being centrifuged acquisition after washing plus according to the volume ratio of 1:1-5 Broken, microscopy is without complete worm's ovum；- 40 DEG C to 10 DEG C multigelation 3 times, it is ultrasonic on ice, be spaced 9 seconds within ultrasound 1 second, it is 0.5-1 hours total, 12000r/min is centrifuged 5 minutes after ultrasound, takes soluble upper, as worm's ovum soluble antigen crude extract, -20 DEG C freeze；

Before production, by worm's ovum soluble antigen, 12000r/min is centrifuged 5 minutes again, and supernatant is that detection worm's ovum solubility is anti- It is former；

(2) to the inspection of worm's ovum soluble antigen, wherein supernatant character should be limpid translucent liquid；Concentration Testing: pass through Folin- phenol reagent process measurement antigen concentration should be not less than 1mg/ml.

4. a kind of preparation method of Schistosoma mansoni worm's ovum soluble antigen as claimed in claim 3, the method it is optimal Reaction condition are as follows:

Physiological saline is added by the worm's ovum precipitating for being centrifuged acquisition after washing plus according to the volume ratio of 1:3, is crushed in dismembyator, microscopy Without complete worm's ovum；- 40 DEG C to 10 DEG C multigelation 3 times, ultrasonic on ice, ultrasound intensity 500w, ultrasonic 1 second intervals 9 seconds, total 0.5- 1 hour, 12000r/min was centrifuged 5 minutes after ultrasound, took soluble upper, as worm's ovum soluble antigen crude extract, -20 DEG C of jellies It deposits；

Before production, by worm's ovum soluble antigen, 12000r/min is centrifuged 5 minutes again, and supernatant is that detection worm's ovum solubility is anti- It is former.

5. a kind of Schistosoma mansoni worm's ovum purifying antigen, which is characterized in that graceful using one kind according to claim 3 or 4 The preparation method of family name's japonice ovum soluble antigen is prepared.

6. japonice ovum purifying antigen according to claim 5 is in the testing product of preparation detection schistosome antibody Using.

7. a kind of schistosome antibody detects colloidal gold immunoassay kit, which is characterized in that detect colloidal gold including schistosome antibody Immunity chromatography card is coated on the nitrocellulose filter of schistosome antibody detection colloidal gold immunochromatographimethod card and is wanted according to right Japonice ovum purifying antigen described in asking 5 is as detection substance.

8. schistosome antibody as claimed in claim 7 detects colloidal gold immunochromatographimethod card, the schistosome antibody detects colloid Golden immunity chromatography card includes sample pad, PVC bottom plate, colloidal gold pad, p-wire, control line, nitrocellulose filter (NC film), water suction Pad.

9. the preparation method of schistosome antibody detection colloidal gold immunochromatographimethod card as claimed in claim 8, the method are as follows:

(1) firing of colloidal gold

All glasswares that is used to fire, save colloidal gold are placed in sour cylinder and are impregnated for 24 hours, it is standby to rinse drying after taking-up well With；100mL deionized water and 1mL1% gold chloride are taken, is heated to boiling；1.5mL1% trisodium citrate is added dropwise, until color Stablize into red；Hereafter continue to boil 15min, be cooled to room temperature；

(2) preparation of colloidal gold probe

The colloidal gold solution prepared 0.2%K2CO3 is adjusted into pH to 5.0, recombinant protein rSPG is added dropwise, shakes 15min stands 15min；10mL10%PEG20000 stabilizing solution is added, room temperature shakes 15min, stands 15min；Solution carries out 2000rpm is centrifuged 20min, takes supernatant to carry out 10000rpm centrifugation 30min again, takes precipitating, precipitating is resuspended with 20mLTBS；By this For colloidal gold probe uniform fold in gold-labelled pad, -20 DEG C of freezings 2h, -80 DEG C of freezing 2h are placed on vacuum in multi-functional freeze dryer Freeze-drying；

(3) test strips assemble

With 0.5mg/ml concentration, the speed of 4 μ l/cm is drawn for graceful formula japonice ovum soluble antigen and streptococcus protein G (SPG) Line is fixed on nitrocellulose filter (CN95), as detection line (T) and nature controlling line (C)；With glass fibre element film, PVC bottom plate, Blotting paper and gold-labelled pad are assembled into schistosome antibody test strip together.

Technical field

The invention belongs to parasitology diagnostic fields, isolation and purification method and worm more particularly, to Schistosoma mansoni worm's ovum The preparation of ovum soluble antigen and its colloidal gold immunochromatographimethod card.

Background technique

Blood fluke is also known as schistosome or bilharzia, is under the jurisdiction of Trematoda, Digenea, Schistosomatidae, Schistosoma.It is parasitic The blood fluke of human body mainly has 6 kinds, i.e. Schistosoma mansoni (Schistosoma mansoni), Schistosoma japonicum (S.japonicum), Schistosoma haematobium (S.haematobium) interleaves blood fluke (S. intercalatum), and river bank public affairs blood is inhaled Worm (S.mekongi) and Malaysia blood fluke (S.malayensis), wherein with Schistosoma mansoni, Schistosoma japonicum and Egyptian blood Schistosomiasis endemic caused by fluke is widest in area, and harm is maximum.Wherein Schistosoma mansoni is widely current in the African (Nile Delta, including Egypt, Kenya, Tanzania etc.), South America (states such as Brazil, Caribbean), Asia (Arabic half Island).

Though it is Chinese at present without disease prevalence, in recent years, closed with being established with Africa and pushing forward novel strategic partner comprehensively System, the exchange of personnel is increasingly frequent, and Introduced cases Schistosoma mansoni patient happens occasionally, and southern china some areas have found Schistosoma mansoni intermediate host's Biomphalaria straminea, there are the potential risks that Manson's schistosomiasis is propagated.But China is for this at present Class Africa Introduced cases disease and its still planless monitoring of related host and detection architecture, in relation to Existed in Labor Services Dispatching mechanism and entry and exit Administrative department payes attention to the diseases prevention inadequate communication of inward and outward personnel, and non-personnel is gone to lack consciousness of self-protection, without corresponding inspection after coming back home Epidemic disease monitoring system, therefore can not early detection.And primary care and Disease Control Agency lack phase during daily diagnosis and treatment and prevention and treatment The training for closing knowledge, without corresponding fast diagnosis reagent and method, therefore does not know about disease incidence feature yet, cannot identify and make a definite diagnosis disease Substance often results in mistaken diagnosis, misdiagnosis.Meanwhile finding that double navel spiral shell distribution areas are wide during prevention and treatment, it is not easy to eliminate control；Mouse, The reservoir hosts such as monkey and baboon are more, and concurrent chemotherapy difficulty is big；Reinfection rate is high after the Population And Cattle Chemotherapy of Endemic Area；Praziquantel chemotherapy is to again There is no protective effects for subinfection, and being used for a long time has certain toxic side effect, and is also easy to produce drug resistance, need to study more effective Control measure.

Summary of the invention

It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of suitable Mans blood to inhale The method that worm worm's ovum isolates and purifies, and based on this, establish the gold-marking immunity test strips diagnostic method of Schistosoma mansoni；It should Method can be used for the diagnosis of Manson's schistosomiasis.

The purpose of the present invention can be achieved through the following technical solutions:

In the first aspect of the present invention, the present invention provides a kind of method that suitable Schistosoma mansoni worm's ovum isolates and purifies, the side Method are as follows:

(1) it is suitable for host that S. mansoni cercariae, which infects, and host includes but is not limited to BALB/c mouse；

(2) the host animal liver in 6 to 10 week of infection, the preparation for worm's ovum are taken

(3) separate and purify the worm's ovum of Schistosoma mansoni.

Further, the present invention provides a kind of preparation method of Schistosoma mansoni worm's ovum purifying antigen；

Further, the present invention provides a kind of Schistosoma mansoni worm's ovum purifying antigen and produces in the detection of preparation detection schistosome antibody Application in product；

Further, the present invention provides a kind of schistosome antibody detection colloidal gold immunoassay kit, including schistosome antibody detection Colloidal gold immunochromatographimethod card is coated with blood suction on the nitrocellulose filter of schistosome antibody detection colloidal gold immunochromatographimethod card Worm worm's ovum purifying antigen is as detection substance；

Further, schistosome antibody of the invention detects colloidal gold immunochromatographimethod card, wherein the schistosome antibody detects glue Japonice ovum purifying antigen conduct according to claim 5 is coated on the nitrocellulose filter of body gold immunity chromatography card Detect substance.

Further, schistosome antibody of the invention detects colloidal gold immunochromatographimethod card, and the schistosome antibody detects glue Body gold immunity chromatography card includes sample pad, PVC bottom plate, colloidal gold pad, p-wire, control line, nitrocellulose filter (NC film), inhales Water cushion.

Detailed description of the invention

Fig. 1 different tissues of mice deposits worm's ovum percentage (infection 60d)

Fig. 2 colloidal gold strip ideograph, wherein 1. sample pads；2.PVC bottom plate；3. colloidal gold pad；4. p-wire；5. control Line；6. nitrocellulose filter (NC film)；7. water absorption pad；

The graceful formula blood fluke test strips sensitivity technique of Fig. 3,1-12: graceful formula blood fluke positive serum gradient dilution, respectively 1:5, 1:10、1:20、1:40、1:100、1:200、1:400、1:800、1:1000、1:2000、1:5000、1:10000。

Embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified It is commercially available from routine biochemistry reagent shop.