ELISA TEST EQUIPMENT FOR THE DIAGNOSIS OF CLONORQUIASIS, PARAGONIMIASIS, CISTICERCOSIS AND SPARGANOSIS DESCRIPTION OF THE INVENTION The present invention relates to a serological diagnosis of parasitic infections residing in the tissue. More specifically, the invention relates to an Enzyme-Linked Immunosorbent Assay Kit (hereinafter, abbreviated "ELISA"), which allows simultaneous diagnosis of infections with Clonorchis sinensis, Paragonimus westermani, Cysticercus cellulosae and Sparganum. In Korea, human helminth parasites, such as Ascaris 1 imbricoides, Txichurus trichiura, Ancylostoma canmum and Trichostrongylus orientalis, which are usually detected by a fecal analysis, have been markedly diminished, and thus are now insignificant. Currently, such general fecal analysis is carried out only in a medical institution such as a hospital. In contrast, to date, infections with parasites of permanence of tissue occur continuously and thus become relatively more and more important. In addition, parasites of tissue permanence cause more serious damage than enteric parasites and thus, infections which require appropriate medical treatment. An accurate diagnosis is the first stage of proper medical management. Therefore, this is the key to obtaining an appropriate diagnostic tool. Particularly, infections with Clonorchis sinensis, Paragoni us westermaní, Cisticercus cellulosae and Sparganum are important endemic diseases in many developing countries. Many people have been infected with them now, but not treated efficiently. Clonorchiasis, of which more than 15 million patients suffer, has been generally diagnosed through a fecal analysis. However, it is very difficult to diagnose a mild case with a light burden of infection. In addition, many people have a tendency to avoid a fecal analysis. In addition, an intradermal test, which has been used for mass screening, has very low specificity to be used as a diagnostic tool. Therefore, the intradermal test tends to be repd with a serological test. On the other hand, paragonimiasis, cysticercosis and sparganosis, of which several tens of thousands of patients suffer each year, have been diagnosed only by a serological test. Such a serological test uses an ELISA comprising the steps of preparing each parasitic antigen and using the prepared antigen. Currently, in Korea, several laboratories have set their own standards for the test and carry out the test on their terms. However, such known evidence may not have been standardized. In addition, the method can detect only one kind of parasite at a time, and must be repeated several times to detect infections with several parasites. Therefore, this has been required to develop a new equipment capable of diagnosing parasites of greater residence in the tissue in a time by a simple and standardized method. Extensive studies have been conducted to solve the above problems in the diagnosis of parasitic infections residing in the tissue. As a result, immunochemical properties of an antigen antibody reaction are contemplated. Unpurified antigens of Clonorchis sinensis, Paragonimus westermani, Cysticercus cellulosae and Sparganum, and human serum are used for positive or negative reference antibodies, for this reason to simultaneously diagnose clonorchiasis, paragonimias is, cysticercosis and sparganosis. Accordingly, an object of the present invention is to provide a diagnostic equipment that allows standardized serological diagnosis of clonorchiasis, paragonimiasis, cysticercosis and sparganosis at the same time. The present invention relates to an ELISA equipment for the diagnosis of clonorchiasis, paragonimiasis, "cicercosis and sparganosis" comprising: (a) a strip or plate-multiple wells whose antigenes of Clonorchis sinensis, Paragonimus westermani, CJysticercus cellulosae and Sparganum are coated (b) a human serum, which does not react with the previous antigens, as a negative reference; and, (c) human serum, each of which contains antibodies especially reactive with the above antigens, as positive references. The present equipment may further comprise a dilution and wash solution, a conjugate solution, a substrate regulator, a substrate and a blocking solution. In a preferred embodiment, the dilution and washing solution contains phosphate buffered saline (PBS). The conjugated solution contains goat anti-human IgG serum labeled with horseradish peroxidase. The substrate is 0-phenylenediamine (OPD). In this case, the equipment also comprises hydrogen peroxide as an oxidizing agent. The blocking solution contains sulfuric acid. Preferably, the present kit contains Clonorchis sinensis and Paragonimus westermani antigens in an amount of 1.5-2 pg / well, and antigens of Cysticercus: elsae and Sparganum in an amount of 2-2.5 μg / well, respectively, as proteins. Also, preferably, -rr. It is of use, the serum reactive with Clonorchis sinensis and, 'Y5 cicercus cellulosae are 26 times diluted, and the serum reactive with Paragonimus westenuani and Sparganum are 101 times, respectively. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing coated antigens in each well of an antigen-coated plate. The present invention provides equipment for simultaneous detection of 4 classes of helminthic parasites that have been usually detected by a serological method, Clonorchis sinensis, Paragonimus westermani, Cysticercus cellulosae and Sparganum. More specifically, the invention provides an ELISA kit for the simultaneous diagnosis of infections with the above parasites using antigen antibody reactions between Clonorchis sinensis, Paragonimus westermani, Cysticercus ellulosae and Sparganum antigens, and human serum containing antibodies in the antigens. Therefore, the present equipment is characterized for containing only a microplate for diagnosis of infections with Clonorchis sinensis, Paragonimus westermani, Cysticercus cellulosae and Sparganum. That is, a 96 well microtiter plate, or a 12x8 well strip is coated and immobilized Clonorchis sinensis, Paragonimus westermani, Cysticercus cellulosae and Sparganum antigens. Then, a document from an examiner is added to it to diagnose the
parasitic infections. Since several kinds of reagents are required, they are provided at given quantities as a package along with the plate. This allows a standardized diagnosis at the same time. One embodiment of the present invention is an ELISA kit containing human serum as sample solutions, goat serum anti-human IgG labeled with horseradish peroxidase as a conjugated solution, 0-phenylenediamine as a substrate, and hydrogen peroxidase as an oxidizing agent . The team includes the following contents. It contains adequate amounts of antigens and undiluted serum, which will be diluted properly depending on the kinds of parasites before use. (i) A microplate in which 4 parasitic antigens are coated: 96-well plate or 12x8 well strips. Ii) A standard negative serum: one bottle (iii) Standard positive serum: one bottle for each of Clonorchls sinensis, Paragonimus westermani, Cysticercus cellulosae and Sparganum (a total of 4 bottles) (iv) A solution of dilution and washing ( concentrated x5): two vials (v) A conjugate solution (x350 diluted): one (vi) One substrate regulator: one vial | (vile) One substrate (0-phenylenediamine): 4.6 mg (viii) Hydrogen peroxide: a bottle (ix) A blocking solution: a bottle The above contents will be described specifically in the following: (i) An antigen-coated microplate: Table 1: Ingredients and contents in a 96-well microplate well or a 12x8 strip wells
- No antigen is coated in the wells of line A, B, C & D in the 1- row (ii) A standard negative serum: one bottle Table 2: Ingredients and contents in a standard negative serum bottle (15 μ?)
(iii) Standard positive serum: one bottle, respectively (a total of 4 bottles) (iii-1) Clonorchis sinensis positive standard serum Table 3: Ingredients and contents in a standard positive serum bottle of Clonorchis sinensis (6 μ?) Ingredients Contents
Inactivated human serum containing high concentration of 6 μ? no diluted antibody reagent specifically with Clonorchis sinensis antigen Proclin 300 0.05 v / v% (iii-2) Paragonimus westermani positive standard serum Table 4: Ingredients and contents in a positive standard serum vial of Paragonimus westermani (2 μ?) Ingredients Contents Human serum that has a high concentration of 2 μ? no diluted antibody reagent specifically with Paragonimus westermani antigen Proclin 300 0.05 v / v% (iii-3) Cysticercus cellulosae positive standard serum Table 5: Ingredients and contents in a positive standard serum bottle of Cysticercus cellulosae (6 μ?) Ingredients Contents
Inactivated human serum containing high concentration of 6 μ? no diluted antibody reagent specifically with Cysticercus cellulosae antigen Proclin 300 0.05 v / v% (iii-4) Sparganum positive standard serum Table 6: Ingredients and contents for a standard serum vial of Sparganum (2 μ?) Ingredients Contents
Inactivated human serum containing high concentration of 2 μ? no diluted antibody reagent specifically with Sparganum Proclin 300 antigen 0.05 v / v%
(iv) A concentrated diluted and washed solution (concentrated xlO): 2 bottles (x5 dilution before use) Table 7: Ingredients and contents in a bottle of a diluted and washed solution (25 ml) Ingredients Contents 0.1 M PBS 25 ml Tween 20 0.5% Proclin 300 0.05 v / v% (v) Diluted conjugate solution (x350): one bottle (xlOO dilution before use) Table 8: Ingredients and contents in a bottle of diluted conjugate solution
(vi) A regulated solution of substrate dilution: a bottle Table 9: Ingredients and contents in a bottle of substrate regulator (6 ml)
- A substrate regulator (6 ml): citrate-phosphate regulator (vii) One substrate: one vial Table 10: Ingredient and content in a substrate bottle, OPD Ingredient Content 0-Phenylenediarrhene Hydrochloride ... 4.6 mg - O-Hydrochloride phenylenediamine (OPD) (4.6 mg) was supplied as powder and diluted in 5 ml of PCB just before use. (viii) Acid peroxide: one bottle, containing 30 μ? of undiluted solution at 30¾, diluted in a substrate solution at a concentration of 0.1% just before use (xi) A blocking solution: a vial Table 11: Ingredient and contents in a vial of blocking solution (15 ml)
In the previous team, the following cases are defined as positive: Clonorchis sinensis; > 0.20; Paragonimus wester ani; 0.25, Cysticercus cellulosae > 0.25; and, Sparganum > 0.24. A process for manufacturing the present equipment is as follows: (i) Preparation of an antigen coated plate: The purified crude antigens of the 4 parasites are coated in a 96-well plate, and blocked and dried, and then cryopreserved. . The reference wells, from wells containing standard negative serum and 4 kinds of wells containing positive standard serum, are included in a plate. In the residual wells, 21 samples were tested. That is, no antigen is coated in the line wells?, B, C & D of the 1- row, as empty wells. The Clonorchis sinensis antigen is coated in wells of line A of the 2- to 12th row and wells of line E of the 1- to 12-row. The paragonimus westermani antigen is coated in the B-wells of the 2- to 12-row and wells of line F of the 1- to 12-row. The antigen of Cysticercus celllulosae is coated in the wells of line C from 2- to 12- rows and wells of line G from 1- to 12-rows. The Sparganum antigen is coated in the wells of line D of the 2 = to 12th rows and wells of line H of the 1- to 12-row. Figure 1 shows antigens coated in each well of the microplate. Other reagents are prepared according to a standard method. (ii) Preparation of standard serum - a standard negative serum which does not react with the 4 antigens - a serum reagent with Clonorchis sinensis antigen - a serum reagent with Paragonimus westermani antigen - a serum reagent with Cysticercus cellulosae antigen - a serum reagent with Sparganum antigen This invention will be better understood from the following examples. However, one skilled in the art will readily appreciate that the specific materials and the results described are simply illustrated, "and are not intended, nor should be intended to, limit the invention as described more fully in the claims, which follow below Example 1: Preparation of antigen-coated microplates (1) Preparation of antigens of Clbnorchis sinensis, Páragonimus westermani, Cysticercus cellulosae and Sparganum The metacercariae of Clonorchis sinensis and
Páragonimus westermani were isolated from naturally infected intermediate hosts (Clonorchis sinensis: Pseudorasbora parva, Páragonimus westermani: Cambaroides similis). Rabbits and dogs were orally infected with Clonorchis sinensis and Páragonimus westermani, respectively. The adult worms of Clonorchis sinensis and Páragonimus westermani were obtained from those animals in an amount of 2 g or more after 3 and 4 months, respectively. The larvae of Taenia solium were separated from pigs which had been naturally infected with ysticercus cellulosae in endemic areas and the cystic fluid was collected from the larval cyst. Sparganum larvae of approximately 2 g or more were collected from snakes which had been naturally infected. The adult worms obtained from Clonorchis sinensis and Paragonimus westermani, and spargana were washed with 10 ml of PBS (8 g / l of NaCl, 0.2 g / l KC1, 1.13 g / l of Na2HP04 and 0.2 g / l of KH2P04, pH 7.5 ) five times. The parasites were added to 15 ml of PBS and homogenized in a homogenizer for 1 to 2 minutes. The homogenized crude parasitic antigens were centrifuged at 4 ° C, 10,000 g for 60 minutes. The supernatant was taken to measure protein concentrations. This was then injected in a volume of 100 μ? per bottle and cryo-served at -70 ° C. (2) Coating antigens to microplate wells A microplate, Costar ™ product (96-well, flat bottom, polystyrene, Corning) assay plate was used. Each parasitic antigen was diluted with 50 mM of sodium carbonate buffer (1.5 g / l of Na2CO3, 2.93 g / l of NaHCO3, pH |. O) to adjust an amount of protein to 20-25 g / ml. Each antigen of 100 μ? of Clonorchis sinansis, • ragonimus westermani, Cysticercus cellulosae and sparganum were prayed at online wells A & amp;; E, B & F, C & G, and D & H (except lines Al, Bl, Cl and DI) in the microplate. Then, the microplate was allowed to stand at -10 ° C overnight for the antigens to coat the bottom of the plate. (3) Washing and blocking Residual antigens were removed from the wells. Each well was washed with 0.2 ml of PBST (0.01 M of phosphate buffer, 0.0027 M of potassium chloride, 0.137 m of sodium chloride, pH 7.4, 0.05 of Tween 20) three times. To each well was added 0.1 ml of an immobilization agent (3% skim milk / PBST, pH 7.6). Immobilization was performed at 37 ° C for 1 hour. Each well was washed with 0.2 ml of PBST three times. The plate was evacuated sufficiently to have no residual solution. This was dried and sealed, and then cooled. Example 2: Preparation of negative standard serum Blood of 10 ml was accumulated from a human without showing reaction with 4 antigens of Clonorchis s inensis, Paragoni us westermani, Cysticercus cellulosae and sparganum, and the serum was separated from the blood. The serum was inactivated by heating at 56 ° C for 30 minutes, and filtered with an aseptic filter. This was taken in a volume of 1000 μ? and proclin 300 of 0.5 μ? to obtain 1 ml of a standard negative serum. The serum was divided into 15 μ? in each bottle and was frozen until use. Example 3: Preparation of a positive standard serum (1) Positive reference serum of Clono-rchiasis Blood of 10 ml was accumulated from a human, who had been determined to have eggs of Clonorchis sinensis in a fecal analysis. The serum was separated from the blood. The serum was inactivated by heating at 56 ° C for 30 minutes, and filtered with an aseptic filter. This was taken in a volume of 1000 μ? and added to this one proclin 300 of 0.5 μ? to obtain 1 ml of a standard positive serum of clonorchiasis. The serum was divided into 5 μ? in each jar, and it was cooled. The serum was used after dilution (4 μ of serum in 100 μ of regulator). (2) Positive reference serum to Paragonimiasis Blood of 10 ml was accumulated from a human, who had been determined to have an infection with Paragonimus westermani or to react with a specific antigen. The serum was separated from the blood. The serum was inactivated by heating at 56 ° C for 30 minutes and filtered with an aseptic filter. This was taken in a volume of 1000 μ? and added to this one proclin 300 of 0.5 μ? to obtain 1 ml of a positive paragonimiasis reference serum. The serum was divided into 2 μ? in each jar, and it was cooled. The serum was used after dilution (1 μl of serum in 100 μl of regulator). (3) Positive reference serum of Cysticercosis Blood of 10 ml was accumulated from a human, who had been determined to have an infection with Cysticercus cellulosae or to react with an antigen from its cystic fluid. The serum was separated from the blood. The serum was inactivated by heating at 56 ° C for 30 minutes, and filtered with an aseptic filter. This was taken in a volume of 1000 μ? and added to this one proclin 300 of 0.5 μ? to obtain 1 ml of a positive reference serum for cysticercosis. The serum was divided into 5 μ? in each jar, and it was cooled. The serum was used after dilution (4 μ of serum in 100 μ of regulator). (4) Positive reference serum of Esparganosis Blood of 10 ml was accumulated from a human, who had been determined to have an infection with sparganum or to react with an unpurified antigen from sparganum. The serum was separated from the blood. The serum was inactivated by heating at 55 ° C for 30 minutes, and was filtered with an aseptic filter. This was taken in a volume of 1000 μ? and added to this one proclin 300 of 0.5 μ? to obtain 1 ml of positive reference serum of sparganosis. The serum was divided into 2 μ? in each jar, and it was cooled. The serum was used after dilution (1 μl of serum in 100 μl of regulator). Example 4: Determination of the presence of antibodies in Clonorchls sinensis, Paragonimus vrestermani, Cysticercus cellulosae and / or sparganum in the blood
ÍáÁ¿: ¡ás¡bÁ, í,? », JÜÜat.; ¾ii-« i «faaA» ¾ A plaque gave its own ID number. The serum to be tested was written on a record sheet and a recognition number was designated for registration. The diluted standard serum and serum samples were filled into the wells of the microplate coated with antigen prepared in Example 1. The wells Al, Bl, Cl and DI, as empty wells, were not loaded with any sample. The standard negative serum of 100 μ? was loaded in wells A2, B2, C2 and D2 and the positive reference serum of 100 μ? It was loaded in the following wells. The reference serum of clonorchiasis (diluted 1:26) was loaded into well A3; serum paragonimiasis (diluted 1: 101), in B3; serum of cysticercosis (diluted 1:26) in C3; serum of esparganosis (diluted 1: 101) in D3. From wells A4, B4, C4 and D4 to E12, F12, G12 and H12 were loaded with diluted serum 21 to be tested in a 4-well unit. At the base of the recognition number, to the wells when a reaction with Clonorchis sinensis or Cysticercus cellulosae occurs where the diluted serum is added 26 times (obtained by diluting 10 μ? Of serum with 250 μ? Of a regulator dilution) of 100 μ ? . To the wells where a reaction with paragonimus westermani or sparganum occurs, serum diluted 101 times (obtained by diluting 2.5 μ? Of the serum with 250 μ? Of a regulator dilution) of 100 μ? . Wells A & ?, 3 & F, C & G and D & H were to detect Clonorchis ir.ensis, Paragonimus westermani, Cysticercus cellulosae, and 9
sparganum, respectively. Therefore, an appropriately diluted serum should be added to each well according to the identification number. The microplate was covered with a lid and incubated at 37 ° C for 2 hours. Then, it was washed with diluted PBST (0.01 M of phosphate buffer, 0.0027 M of potassium chloride, 0.137 M of sodium chloride, pH 7.4, 0.05% Tween 20) of 200 μ? three times. The microplate was inversely struck on an absorbent paper to completely remove the washed regulator. A diluted solution of 100 μ? Containing the conguete (goat serum (specific Fc) of anti-human IgG with horseradish peroxidase diluted 100 times) a final protein concentration: 110-140 ng / ml in lxPBS, 0.05% Proclin 300 ) was added to the wells. The wells were covered with caps and a reaction was performed in a dark place at 3 ° C for 2 hours. The reaction solution was removed from the wells and the wells were washed with a washing regulator three times. A substrate solution of 50 μ? (prepared by adding O-phenylenediamine to the citrate buffer and adding acid peroxide to it just before use) to each well using a multichannel pipette. A reaction was performed in a dark place for 10 minutes. A blocking solution (4 N.H2S04) of 50 μ? was added to each well;;;::: by pipetting a multichannel pipette to stop the reaction.
The absorbency of each fiber was measured at 490 nm. In the following test the above procedure, a ca s or where the negative and positive standard serum had absorbances of 0.1 or less than 0.3 or more, respectively, was determined to be valid. Serum that has a designated standard value for each antigen or more was determined to be positive. The absorbances of the reference serum for parasitic infections are listed in the following Table 11. Table 12: Absorbances of reference serum for 4-parasitic infections.
Infections Clonorchiasis Paragonimiasis Cysticercosis Sparganosis - / + - + - + - + - + 1 0. 126 0 409 0.060 0.410 0 109 0.512 0. 102 0. 16 2 0 058 0 255 0.035 0.555 0.070 0.395 0.032 0.583 3 0.049 0.314 0 030 0.408 0.060 0.41 1 0.029 0 490 4 0. 103 0.345 0.069 0.413 0 128 0.520 0. 152 0.27.3 5 0.071 0.503 0.046 0.515 0 048 0.374 0.086 0.417 r > 0.054 0.261 0.033 0.375 0 040 0 391 0.039 0 419 7 0 066 0.258 0.033 0.305 0.070 0.303 0.033 0.383 8 0 051 0.282 0.036 0.365 0 161 0.299 0.086 0.395 9 0.037 0.404 0.042 0.329 0.068 0.335 0.029 0.527 10 0.042 0.351 0.049 0.3 13 0 062 0.419 0.028 0.428 or 0.050 0.444 0.035 0.3 14 0. 138 0.405 0.026 0.436 12 0.070 0.254 0.042 0.286 0.137 0.442 0.039 0.443 13 0.040 0.365 0.025 0.351 0.037 0.306 0.024 0.453 14 0 069 0.337 0.038 0.448 0.080 0.485 0.033 0.490 15 0 062 0.301 0 030 0.333 0.058 0.504 0.032 0.521 16 0.071 0.254 0.033 0.383 0.073 0.503 0.035 0..303 17 0.041 0.258 0.027 0.434 0.046 0. 15 0.020 1 0.466
1
INDUSTRIAL APPLICABILITY The equipment of the present invention allows a simple, fast and accurate diagnosis of infections with greater parasites that reside in the tissue, that is to say,
Clonorchis sinensi, Paragonimus westermani, Cysticercus cellulosae and sparganum at a time by a standardized method.