KR20020084850A - ELISA kit for diagnosis of clonorchiasis, paragonimiasis, cysticercosis and sparganosis - Google Patents

Abstract

PURPOSE: An enzyme-linked immunosorbent assay kit of Clonorchis sinensis., Paragonimus westermani, Cysticerus cellulosae and Sparganum infection syndrome is provided, thereby rapidly and correctly diagnosing the infection of main tissues with parasites. CONSTITUTION: The enzyme-linked immunosorbent assay kit of Clonorchis sinensis., Paragonimus westermani, Cysticerus cellulosae and Sparganum infection syndrome comprises a multi-well microplate or strip containing antigens of Clonorchis sinensis. Paragonimus westermani, Cysticerus cellulosae and Sparganum; a human serum which doesn't react with the antigens of Clonorchis sinensis. Paragonimus westermani, Cysticerus cellulosae and Sparganum as a negative standard serum; and a human serum which specifically and independently reacts with the antigens of Clonorchis sinensis., Paragonimus westermani, Cysticerus cellulosae and Sparganum as a positive standard serum.

Description

ELISA kit for diagnosis of clonorchiasis, paragonimiasis, cysticercosis and sparganosis

The present invention relates to a serological diagnosis of parasitic infectious diseases in tissues, and more particularly, enzyme immunoassay (ELISA: Enzyme-Linked ImmunoSorbent Assay) for diagnosing hepatic insects, pulmonary insects, rhizome cysts and Sparganum infections. ) Kit.

In the past, human parasites diagnosed mainly by screening, roundworms, worms, duodenal insects and oriental nematodes, have been reduced to almost no problem in Korea. At present, the general test is rarely performed except in hospital-level medical institutions. However, parasitic infections in tissues still persist, and tissue damage is greater than intestinal parasitic infections, and appropriate medical measures are urgently needed. Since the first step in these medical measures is an accurate diagnosis, it is important to first ensure proper diagnosis.

Among them, hepatic worms, pulmonary worms, rhizome worms and worm infections are important endemic diseases, but many patients are occurring, but effective management is not achieved. Currently, hepatomegaly with more than 1 million people in Korea has been diagnosed mainly by screening, but it is becoming increasingly difficult to diagnose less infected people with less infection, and both test subjects and testers tend to evade the screening itself. In addition, the intradermal response test is too low in specificity and thus has no diagnostic value. Diagnosis of hepatic pneumonia is in the process of gradually being replaced by serologic tests. Meanwhile, the diagnosis of pulmonary pneumoconiosis, rhinocytopenia and grievances, which still occur in thousands of patients annually, is entirely dependent on serological examination. The serological diagnosis of these infectious diseases is made by enzyme-immunoassay (ELISA) using each parasite antigen and using it. Currently, several laboratories in Korea have set their own standards. However, the existing methods are not standardized, and only one parasite infection can be detected at a time, and it is cumbersome to repeat several tests in order to confirm the infection of several parasites. . Therefore, it was urgent to develop a new diagnostic kit for diagnosing parasitic infections in major tissues at once.

Accordingly, the present inventors have conducted continuous research to solve the diagnostic problem of the existing parasitic infectious diseases in tissues. As a result, hepatomegaly, pneumococcus, larvae and worms, using the immunochemical properties of the antigen-antibody response, using human sera containing polyclonal antibodies against the provisions of hepatococcus, pneumococcal larvae and worms It was confirmed that the infection can be diagnosed at the same time, and came to complete the present invention.

Accordingly, it is an object of the present invention to provide a diagnostic kit capable of performing the serological diagnosis of hepatic insects, pulmonary nematodes, mole cysts and worm infections in a standardized method at one time.

1 is a schematic representation of an antigen attached to each well of an antigen-attached plate.

The present invention

Iii) Meltiwell plates or strips to which hepatitis, pneumococcal, motile cysts and grievous antigens are attached;

Ii) negative standard serum, human sera that does not react with hepatobacterium, pulmonary nematodes, mole cysts and grievances antigens; And,

Iii) human serum containing an antibody that specifically reacts with hepatitis, pulmonary worm, rhizome worm and worm antigen, respectively, as positive standard serum:

The present invention relates to an ELISA kit for hepatic insects, pulmonary insect repellents, mole cysts and worm infections.

The kit according to the present invention may further include a dilution and washing liquid, a conjugate liquid, a substrate liquid and a reaction stopping liquid. Preferably, the dilution and washing liquid contains phosphate-buffered saline (PBS). It contains anti-human IgG goat serum labeled with horseradish peroxidase, and the substrate solution contains O-phenylenediamine, wherein the kit further contains hydrogen peroxide. It includes, the reaction stop solution contains sulfuric acid.

In addition, the kit of the present invention preferably contains hepatococcus and pulmonary insufficiency antigens at a protein amount of 1.5 to 2 μg / well, and the mosquito cyst and worm antigens are preferably at a protein amount of 2 to 2.5 μg / well, respectively. It is preferable to add 26-fold diluted serum to the wells that react with the larvae antigen, and 101-fold diluted serum to the wells that react with the pulmonary vermin and worm antigens.

Hereinafter, the present invention will be described in detail.

The present invention relates to a kit for the simultaneous diagnosis of four parasites, namely hepatic, pulmonary, insect, and worm infections, diagnosed mainly by serological methods. Specifically, the infection of the parasites can be diagnosed at one time by using hepatitis, pulmonary insects, mole cysts and grievances antigens, human serum containing antibodies to each antigen, and antigen-antibody reactions between them. It relates to an ELISA kit.

Therefore, the kit of the present invention is characterized by using a single microplate for the diagnosis of hepatic insects, pulmonary insects, mole cysts and worm infections. That is, each of the parasite infections is diagnosed by attaching and fixing the antigens of hepatic insects, pulmonary insects, mole cysts and grievances to one 96-well microplate or 12 × 8 well strip, respectively. At this time, several types of reagents are required, and each of them is packed and provided in a required amount per plate, so that a standardized test can be performed at a time.

One example of the present invention is an ELISA kit using human serum as a sample solution, horseradish peroxidase conjugated anti-human IgG goat serum, and O-phenylenediamine as substrate and hydrogen peroxide as oxidant.

The kit contains the following contents and contains the appropriate amount of antigen and serum diluted to the appropriate concentration for each parasite:

Iv) Microplates with four parasite antigens attached: 96 well plates or 12 × 8 well strips

Ii) negative standard serum: 1 bottle

Iii) Positive Standard Serum: 1 bottle (positive 4 bottles) of positive standard serum of hepatococcus, pulmonary worm, rhizome cyst and worm

Iv) Dilution and washings (10-fold concentrate): 2 bottles

Iv) conjugate fluid (350-fold dilution): 1 bottle

기질) Substrate buffer: 1 bottle

Viii) Substrate (O-phenylenediamine): 13.6 mg

Iii) hydrogen peroxide: 1 bottle

Iii) reaction stopper: 1 bottle

The content is described below in more detail.

Iii) antigen-attached microplates:

No antigens are attached to rows A, B, C and D in column 1

Ii) negative standard serum: 1 bottle

Viii) Positive standard serum: 1 bottle each (4 bottles total)

Ⅲ-1) Hepato-insect positive standard serum

Ⅲ-2) Pulmonary Insufficiency-positive Standard Serum

Ⅲ-3) Molar cyst positive standard serum

Ⅳ-4) Opinion positive standard serum

V) Concentrated Dilution and Wash Solution (10-fold concentrate): 2 bottles (10-fold dilution when used)

Dilution conjugate fluid (350-fold dilution): 1 bottle (100-fold dilution when used)

Iv) Substrate solution: 1 bottle

Substrate buffer (15 mL): citric acid-phosphate buffer

Hydrochloric acid O-phenylenediamine (13.6 mg) was supplied as a powder and then dissolved in a buffer by the user immediately before use.

Vi) Hydrogen peroxide: 1 bottle, containing 100 μl of 30% stock solution. Dilute 0.1% in substrate solution immediately before use.

Iii) reaction stopper: 1 bottle

In the kit, a microplate reader is used to diagnose infection when the absorbance at absorbance of 490 nm is 0.25 for hepatococcus, 0.28 for pulmonary worms, 0.27 for mosquitoes, and 0.24 for worms.

Referring to the process of manufacturing the kit according to the invention in detail.

Iii) Preparation of the antigen-attach plate:

Purified clauses of four parasites are attached to a 96 well plate, blocked, dried and cryopreserved. One plate contains control wells, negative standard serum-containing wells and four positive standard serum-containing wells, based on the test of 18 specimens in the remaining wells. That is, the wells of rows A, B, C, and D of the first column do not attach the antigen as a control well for the test, and the hepatitis antigens of the rows A to B and the rows E to 1 to 12 of the columns are not attached. In the second row to the 12th row B row and the first row to the 12th row F row, the pneumococcal antigen is in the second row to the 12th column C and the first to the 12th column G row to the Eurasian cystic insects In the second row to the twelfth row D row and the first row to the twelfth row H row, the worm antigen is attached. 1 shows the antigen attached to each well of the microplate. Other reagents are prepared based on criteria.

Ii) Preparation of Standard Serum:

A 26-fold dilution of serum that does not react with the four antigens, as a negative standard serum

-26-fold dilution of serum reacting with hepatotoxic antigen

-101-fold dilution of serum reacting with pneumococcal antigen

-26-fold dilution of serum reacting with E. coli antigen

-101-fold dilution of serum that reacts with grievance antigen

Hereinafter, the present invention will be described in detail by way of examples, which are only intended to help the understanding of the present invention and are not intended to limit the scope of the present invention.

Example 1: Preparation of Antigen Attachment Microplates

1) Preparation of Hepatosporicidal, Lung Insecticidal, Dermal Cystic Insecticidal and Grievous Antigens

Hepatitis larvae and cyst larvae were isolated from naturally infected intermediate hosts (hepatitis: true fish, pulmonary lobster: crayfish). More than 2 g of adult hepatosperm over 3 months and adult lung respiratory tract over 4 months were obtained from these experimental animals. On the other hand, pigs naturally infected with E. coli were obtained, and the larvae were separated to collect the larvae. In addition, the larvae were isolated from the snakes naturally infected with the worms to secure more than 2 g.

Adults and grievances of hepatic and pulmonary insects obtained were transferred to 10 ml PBS (8 g / l NaCl, 0.2 g / l KCl, 1.13 g / l Na ₂ HPO ₄ and 0.2 g / l KH ₂ PO ₄ , pH 7.5), respectively. Washed twice. Each parasite was added to 15 ml PBS and homogenized in a homogenizer for 1-2 minutes. Each homogenized parasite member was placed in a centrifuge and centrifuged at 10,000 g for 60 minutes at 4 ° C. Each supernatant was taken, the protein concentration was measured, aliquots of 100 μl were stored frozen at -70 ° C.

2) Attachment of antigen to microplate wells

Microplates use Costar ™ products (96-well assay plates, bottoms, polystyrene, Corning) and each 50 mM sodium carbonate buffer (1.5 g / l Na ₂₎ so that the antigen of each parasite is 20-25 μg / ml protein. CO ₃ , 2.93 g / l NaHCO ₃ , pH 9.6).

Except for A1, B1, C1, and D1 in each microplate well, rows A and E are hepatococcal antigens, rows B and F are pneumococcal antigens, rows C and G are e.g. In rows and H, 100 μl of worm antigen was added, and the microplates were stored at 4 ° C. overnight to allow antigen proteins to adhere to the plate bottom.

3) washing and blocking

The antigen remaining in the plate wells was removed and each well was washed three times with 0.2 ml PBST (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4, 0.05% Tween 20), and then 0.1 ml in each well. A fixative (3% skim milk / PBST, pH 7.6) was added and fixed at 37 ° C. for 1 hour. Each well was washed three times with 0.2 ml PBST and the plate was shaken sufficiently to leave no solution, dried, sealed and cryopreserved.

Example 2: Preparation of Negative Standard Serum

Serum was isolated by collecting 10 ml of human blood that did not react with the four antigens of hepatic, pulmonary, fungal cyst, and gritty, and heat-treated at 56 ° C. for 30 minutes to make it inactive and filtered with a sterile filter. 40 μl of the mixture was mixed with 1,000 μl of the diluted buffer solution, 0.5 μl of ProClean 300 was added to prepare 1.04 ml of negative standard serum, and the resultant was frozen and stored.

Example 3: Preparation of Positive Standard Serum

1) Hepatosporicidal positive standard serum

Blood was collected from 10 ml of blood of humans identified as hepatosporosis in the test, serum was isolated, heat treated at 56 ° C. for 30 minutes, inactivated, and filtered through a sterile filter. 40 μl of the mixture was mixed with 1,000 μl of the diluted buffer solution, and 0.5 μl of ProClean 300 was added to prepare 1.04 ml of hepatosiosis positive standard serum.

2) Pneumoconiosis Positive Standard Serum

10 ml of blood of a person whose lung pneumococcal infection was confirmed or who responded to a specific antigen was collected and serum was isolated, and heat-treated at 56 ° C. for 30 minutes to make it inactive and filtered with a sterile filter. 10 μl of the solution was mixed with 1000 μl of the diluted buffer solution, and 0.5 μl of ProClean 300 was added to prepare 1.01 ml of pulmonary worm-positive standard serum.

3) Seropositive cystosis positive standard serum

Blood was collected from 10 ml of humans having been identified as E. coli or infected with cystic antigens, and serum was isolated. Then, heat treatment was performed at 56 ° C. for 30 minutes to make it inactive and filtered with a sterile filter. 40 μl of the mixture was mixed with 1000 μl of the diluted buffer solution, and 0.5 μl of ProClean 300 was added to prepare 1.04 ml of E. coli larvae positive standard serum.

4) Opinion positive standard serum

Serum was isolated by collecting 10 ml of blood from a person whose grievance was confirmed or reacted with a medicinal member. The serum was isolated, heat treated at 55 ° C. for 30 minutes, inactivated and filtered through a sterile filter. 10 μl of the mixture was mixed with 1000 μl of the diluted buffer solution, and 0.5 μl of ProClean 300 was added to prepare 1.01 ml of worm-positive standard serum.

Example 4: Determination of the presence of antibodies in the blood of hepatic worms, pulmonary worms, globules and worms

First, a plate identification number was given, and the serum to be tested was recorded on a recording sheet to give a recognition number and a test record was made. The antigen-attached microplates prepared in Example 1 were filled with standard serum and diluted samples. The A1, B1, C1 and D1 wells were control wells and did not fill the samples. Negative standard serum for A2, B2, C2, and D2 wells, hepatotoxic positive standard serum for A3, B3, C3, and D3 wells, and pneumonia positive standard serum for A4, B4, C4, and D4 wells, and A5, B5, C5, and D5 wells were filled with molar positive standard serum and A6, B6, C6 and D6 wells were filled with 100 μl of worm positive standard serum. On the other hand, the other wells were filled with dilutions of 18 sera to be tested in units of four wells from rows A7, B7, C7 and D7 to E12, F12, G12 and H12. For wells that will react with hepatitis and E. coli antigens based on their identification numbers, dilute serum 26-fold (10 μl of serum in 250 μl of diluent buffer) and add 100 μl to each well. For the wells to be reacted, the serum was diluted 101-fold (2.5 μl of serum in 250 μl of dilution buffer) and 100 μl of which was added to each well. Lines A and E are hepatitis, rows B and F are pulmonary insects, rows C and G are urocytic larvae, and lines D and H are gritty test wells. Was added.

The microplate was covered with a plate cover and incubated for 2 hours at 37 ° C., and then washed three times with diluted 200 μl PAST (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4, 0.05% Tween 20). Turn the microplate upside down and tap gently to remove all plate wash buffer on the absorbent paper, then use a multichannel pipette to conjugate diluents (horseradish peroxidase conjugated anti-human IgG (F _c specific) chlorine included in the kit). 100-fold dilution of serum, final concentration of protein: 1 × PBS, 110-140 ng / ml in 0.05% ProClean 300) was added to 100 μl of all wells, covered with plate cover, and then at 37 ° C. for 2 hours in the dark. Reacted for a while. The reaction solution of the microplate wells was removed and the wells washed three times with wash buffer.

50 μl of substrate solution (prepared by adding O-phenylenediamine to citric acid buffer and hydrogen peroxide solution) was added to each well using a multichannel pipette and allowed to react in the dark for 10 minutes. 50 μl of reaction stopper solution (4 NH ₂ SO ₄ ) was added to each well by a multichannel pipette to stop the reaction, and the absorbance of each well was measured at 490 nm.

When tested according to the method described above, it was effective only when the absorbance was measured as negative standard serum 0.1 or less, positive standard serum 0.3 or more, it was determined that the serum showing the absorbance above the predetermined value for each antigen was positive. The absorbance of the reference serum for four parasitic infections is shown in Table 11.

Using the kit of the present invention, it is possible to diagnose the parasitic worms of major tissues, ie, hepatic insects, pulmonary insects, mole cysts and worms, in a simple, quick and accurate manner.

Claims (7)

Meltiwell microplates or strips to which hepatitis, pneumococcal, motile cyst, and grievous antigens are attached; As a negative standard serum, human serum that does not react with hepatobacterium, pulmonary nematodes, mole cysts and worm antigens; And, Human serum containing positive antibodies, each of which specifically reacts with hepatitis, pulmonary worms, rhinocysts and grievances antigens: Enzyme Immunodiagnostics (ELISA) kit of hepatic worms, pulmonary worms, horseshoe worms and worm infections comprising a. The kit of claim 1, further comprising dilution and washing liquid, conjugate liquid, substrate liquid, and reaction stopping liquid. The kit of claim 2, wherein the dilution and wash solution contains phosphate-buffered saline (PBS). The conjugate solution of claim 2, wherein the conjugate solution contains an anti-human IgG goat serum labeled with horseradish peroxidase, and the substrate solution contains O-phenylenediamine, and further hydrogen peroxide solution. Kit comprising a. The kit according to claim 2, wherein the reaction stopper solution contains sulfuric acid. The kit according to claim 1, wherein the kit contains hepatobactericidal and pulmonary repellent antigens at a protein amount of 1.5 to 2 [mu] g / well, and the glomerulent worm and the worm antigen at a protein amount of 2 to 2.5 [mu] g / well, respectively. The kit of claim 1, wherein serum is diluted 26-fold to the wells that react with hepatococcus and rhinocytomegaly antigens and 101-fold diluted to the wells that reacts with pulmonary pneumococci and worm antigens.