阅读说明：本技术 一种东毕吸虫病检测用虫卵抗原的制备及胶体金标记的检测试纸条 (Preparation of worm egg antigen for detecting Tokyo fascicularis and colloidal gold-labeled detection test strip ) 是由 朱传刚 沈元曦 纪荣毅 周志平 冒丽 刘冀 于 2019-08-28 设计创作，主要内容包括：本发明涉及一种东毕吸虫病检测用虫卵抗原的制备及胶体金标记的检测试纸条,所述的东毕吸虫抗体检测胶体金免疫试剂条设有样品吸垫、金标垫、NC膜、吸收垫、PVC底板、检测线(T线)、控制线(C线),所述胶体金标记的检测试纸条的硝酸纤维素膜上包被有上述的东毕吸虫虫卵纯化抗原作为检测物质。本发明提供的这种检测试剂条检测效果准确可靠,操作简便易行,成本降低,更适用于东毕吸虫病低度流行区现场流行病学调查、疾病筛查以及免疫学诊断,适于大规模推广应用。(The invention relates to a preparation method of an egg antigen for detecting east schistosomiasis and a colloidal gold-labeled detection test strip, wherein the east schistosomiasis antibody detection colloidal gold immunoassay reagent strip is provided with a sample absorbing pad, a gold-labeled pad, an NC membrane, an absorbing pad, a PVC bottom plate, a detection line (T line) and a control line (C line), and the nitrocellulose membrane of the colloidal gold-labeled detection test strip is coated with the above purified egg antigen of east schistosomiasis as a detection substance. The detection reagent strip provided by the invention has the advantages of accurate and reliable detection effect, simple and easy operation and low cost, is more suitable for field epidemiological investigation, disease screening and immunological diagnosis in low-grade epidemiological areas of the east schistosomiasis, and is suitable for large-scale popularization and application.)

1. A colloidal gold immune test strip containing an antigen of an egg of a Diptera donnaeus.

2. The method of making a test strip of claim 1, comprising the steps of:

Preparing a soluble antigen of the egg for detection;

Spotting of the nitrocellulose membrane: diluting the soluble antigen of the east flatworm egg to 0.5mg/ml for marking off a detection line (T line), and diluting the mouse anti-His tag monoclonal antibody to 0.5mg/ml for marking off a quality control line (C line); attaching an NC film to a bottom plate, then marking with a colloidal gold sample application system, wherein the marking amount is 1 mu l/cm, placing the bottom plate in a 37 ℃ drying oven for drying for 2 hours after marking, sealing in a tin foil bag, and internally arranging a drying agent with the validity period of 15 months;

Preparing colloidal gold: sintering the colloidal gold according to a sodium citrate reduction method: all glassware for firing and storing colloidal gold should be clean; adding deionized water and chloroauric acid with the final concentration of 0.01% into the dried Erlenmeyer flask, and heating to boil; 1 percent of trisodium citrate with the volume 1.5 times that of the chloroauric acid is rapidly added, the color change is observed, the solution is changed into black and dark purple from light yellow, and finally the solution is gradually stabilized into stable red, and the color tends to be stable after about 3 minutes; boiling for 15 minutes, cooling to room temperature, and recovering the volume to the original volume by using deionized water;

The method comprises the steps of labeling colloidal gold and antigen, namely labeling the colloidal gold of recombinant streptococcal protein G, adjusting the pH of a prepared colloidal gold solution to be 5.0 by K ₂ CO ₃, dropwise adding the recombinant streptococcal protein G to enable the final concentration to be 10 mug/ml, shaking for 15 minutes at room temperature, standing for 15 minutes, adding confining liquid into the solution to enable the final concentration to be 1%, shaking for 15 minutes at room temperature, standing for 15 minutes, centrifuging at 11000r/min for 40 minutes, discarding supernatant, re-dissolving sediment with re-dissolving liquid to 1/5 of the volume of the original solution, wherein the color should be uniform wine red, preparing a gold-labeled pad, preparing the gold-labeled pad by a gold immersion method, uniformly dropwise adding the prepared colloidal gold probe onto the gold-labeled pad to enable the gold-labeled pad with 1.0cm ² to contain 100 mu l of the colloidal gold probe, placing the gold-labeled pad into a multifunctional freeze dryer, drying, sealing the gold-labeled pad into a tin bag after freeze-drying is finished, and the effective period is 15 months;

Preparing a reagent strip for detecting the Dongbi trematoda: the coating film, the gold label pad, the sample pad, the absorption pad, the protective film and the like are sequentially stuck on a PVC base plate, cut into strips with the width of 0.3cm on a strip cutting machine, filled into an aluminum foil bag together with a drying agent, sealed, pasted with labels and placed into an outer packing box together with a specification.

3. The method for preparing a test strip according to claim 2, wherein the step (1) is specifically as follows: putting the east schistosoma eggs into a grinder, adding normal saline according to the volume ratio of 1:1-5, grinding in the grinder, and performing microscopic examination to obtain intact eggs; repeatedly freezing and thawing at-40 deg.C to 10 deg.C for 3 times, performing ultrasonic treatment on ice for 2 seconds at intervals of 9 seconds for 0.5-2 hr, centrifuging at 12000r/min for 5 min after ultrasonic treatment, collecting soluble supernatant as crude extract of soluble antigen of ovum, and freezing at-20 deg.C; before production, the egg soluble antigen is centrifuged again at 12000r/min for 5 minutes, and the protein concentration is measured by a supernatant BCA method.

Technical Field

The invention relates to the technical field of protein purification and detection, in particular to the technical field of egg antigen purification and antibody detection for detecting Toyobo schistosomiasis, and specifically relates to a Toyobo schistosomiasis egg crude antigen purification method, a related purified antigen and a schistosome antibody detection colloidal gold test strip.

Background

East schistosomiasis is a schistosomiasis disease caused by east schistosomiasis species parasitizing in the portal and mesenteric veins of the host. Is distributed worldwide. This disease occurs in india, mongolia, irak, hassakestan in asia and russia, france, etc. in europe. In China, east fasciolosis is mainly distributed in vast areas such as Gansu, Xinjiang, Sichuan, Guizhou, Nemeng, Heilongjiang and the like.

The Toxoplasma donax is subdivided into Peng-Toxoplasma donax, Cheng-Toxoplasma donax, Turkey Stantendo Pixoplasma donax and Turkey Stantendo Pixoplasma nodulation. The development process of east birch requires two hosts: an end host and an intermediate host. After the east birch larva cercaria penetrates into the skin of the livestock such as the final host cattle, sheep and the like, the larva finally develops into a hugged adult through migration and spawns in the host blood vessel; the eggs enter the intestinal tract from the blood vessels, and are discharged out of the body along with the excrement, so that miracidium is hatched in the water body; the coenuruses enter the middle host otophycaceae to carry out asexual propagation, and a large amount of cercaria is generated; mature cercaria is released into water and can penetrate into the skin of the host when meeting the suitable host, thus completing the life cycle of east birch fluke.

Once the domestic animals are infected with the east schistosoma, the larvae of the east schistosoma move in vivo, so that the host tissues are often damaged mechanically; in the adult stage and the oviposition process, the feed not only contends for nutrient substances but also generates toxin, and influences the normal body function of the livestock. The lamb shows serious malnutrition, inappetence, listlessness, slow development, edema and weakness of lower limbs, discharges loose stool or bloody stool, gradually becomes thin and finally falls down; the ewes may also be infertile or aborted or dead. The livestock fasciolosis not only seriously damages the economic benefits of herders, but also cercaria can infect human beings, generate cercaria dermatitis and damage the health of the human beings, so the fasciolosis is an important parasitic disease of zoonosis.

The traditional diagnosis of east Dipteroides is etiological diagnosis, mainly based on autopsy and egg examination, including feces washing and precipitation method, egg larva hatching method and the like. However, pathogen detection takes a long time and has a low detection rate, which causes a certain detection missing rate and affects the control of the disease.

Disclosure of Invention

The invention mainly solves the technical problem of providing a east schistosome ovum and ovum antigen purification method, a related purified antigen and a schistosome antibody detection colloidal gold immune test strip, the east schistosome ovum antigen purification method has the advantages of ingenious design and simple and convenient operation, and the obtained purified antigen is used as the east schistosome antibody detection antigen, can reduce the serum consumption, increase the detection sensitivity, reduce the cross reaction rate, expand the application range, have accurate and reliable detection effect, simple and easy operation and low cost, is more suitable for site epidemiological investigation, disease screening and immunological diagnosis of east schistosome epidemic areas, and is suitable for large-scale popularization and application.

In order to achieve the above object, in a first aspect of the present invention, a method for collecting eggs of east schistosoma is provided, which is characterized in that after it is judged by a dung hatching method that a sheep is infected by east schistosoma, the sheep is dissected to obtain small intestines, and the eggs are collected.

Preferably, the worm egg collection efficiency is improved by using a NaOH erosion method.

Further, the invention provides an extraction method of soluble antigens of the east-birch insect eggs.

Preferably, the east schistosoma insect eggs are placed in a grinder, normal saline is added according to the volume ratio of 1:1-5, the grinding is carried out in the grinder, and no complete insect eggs are detected by microscopy; repeatedly freezing and thawing at-40 deg.C to 10 deg.C for 3 times, performing ultrasonic treatment on ice for 2 seconds at intervals of 9 seconds for 0.5-2 hr, centrifuging at 12000r/min for 5 min after ultrasonic treatment, collecting soluble supernatant as crude extract of soluble antigen of ovum, and freezing at-20 deg.C. Before production, the egg soluble antigen is centrifuged again at 12000r/min for 5 minutes, and the protein concentration is measured by a supernatant BCA method.

further, the invention provides a purified antigen of the east Dipteroides sinensis egg, which is characterized by being prepared by adopting the crude antigen purification method of the east Dipteroides sinensis egg.

Further, the invention provides an application of the purified antigen of the east schistosoma japonicum katsurada egg in preparing a detection product for detecting a schistosome antibody.

Further, the invention provides a colloidal gold immunoassay reagent strip for detecting the antibody of the east schistosoma japonica, which is characterized by comprising a colloidal gold immunochromatographic card for detecting the antibody of the east schistosoma japonica, wherein a nitrocellulose membrane of the colloidal gold immunochromatographic card for detecting the antibody of the east schistosoma japonica is coated with the purified antigen of the egg of the east schistosoma japonica as a detection substance.

Furthermore, the colloidal gold immunity reagent strip for detecting the antibody of the east flathead is provided with a sample absorbing pad, a gold label pad, an NC membrane, an absorbing pad, a PVC bottom plate, a detection line (T line) and a control line (C line). The sample suction pad, the gold label pad, the NC membrane and the absorption pad are sequentially adhered to the upper surface of the PVC base plate, one end of the sample suction pad is arranged on one end of the gold label pad, the other end of the gold label pad is arranged on one end of the NC membrane, one end of the absorption pad is arranged on the other end of the NC membrane, and the detection line and the quality control line are sequentially arranged on the NC membrane; soluble antigens of the east schistosoma ova are coated at the detection line, and streptococcus recombinant protein (rSPG) is coated at the quality control line.

Advantageous effects

By researching the feasibility of the east bibulous worm eggs as diagnostic antigens and obtaining the distribution rule of the east bibulous worm eggs through research, the invention adopts a chemical erosion method to obtain the worm eggs which are difficult to obtain by strictly controlling the concentration, the temperature and the action time of an erosion liquid and balancing the PH of the worm eggs to a neutral state through a buffer solution, and the purity of the worm eggs is high; the egg prepared by the method is further used for obtaining soluble antigens of the east schistosoma japonicum; removing useless components in the antigen by a denaturation method to finally obtain the antigen for detecting the ovine east schistosoma antibody; the antigen can realize the detection of the east schistosoma antibody in sheep bodies by test paper and other schemes.

Drawings

fig. 1 is a schematic structural diagram of a colloidal gold labeled test strip, wherein 1 is a sample to be tested, 2 is a sample absorption pad, 3 is a gold labeled pad, 4 is an NC film, 5 is an absorption pad, 6 is a PVC bottom plate, 7 is a detection line (T line), and 8 is a control line (C line).

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.