JP5582521B2 - Purification method of antigen for cystosis diagnosis - Google Patents

Description

  The present invention relates to a method for purifying an antigen for diagnosing parasitic diseases.

  Mangeous cysticesis is a disease caused by infection with larvae (cystic worms) of arachnid worms, and hatched eggs taken from orally ingested eggs or hatched from adults that have infested the intestines, resulting in multiple cysts throughout the body. Arise. In particular, cerebral cystic cysts caused by cysts infesting the central nervous system are fatal and are very important as emerging and re-emerging infections. Diagnosis of cysticesis is performed by combining image analysis such as X-ray and serodiagnosis. As an antigen for human serum diagnosis, a 10-26 kDa glycoprotein present in cyst fluid is used worldwide from the viewpoint of sensitivity and specificity.

  Currently, antigen glycoprotein purification methods for serodiagnosis include 1) affinity chromatography using lentil lectin (Non-patent Document 1), 2) preparative electrophoresis apparatus using protein charge. A method of separating by rotophore (Non-patent Document 2), 3) a method by affinity chromatography using polyclonal and monoclonal antibodies against glycoprotein (Non-patent Document 3) are known. The antigen obtained by method 1) can be used for immunoblotting because it contains non-specific components, but it cannot be used for ELISA. In contrast, antigens obtained by methods 2) and 3) can be used in both methods. However, in the method of 2), a special apparatus (for example, a BioRad Rotophore) that separates and purifies proteins by performing isoelectric focusing in a liquid while rotating a cylindrical chamber partitioned by a membrane is used. Since the method 3) requires a specific antibody against glycoprotein, there is a problem that the facilities capable of purifying the antigen are limited.

  On the other hand, research on cloning and cloning of genes encoding antigenic glycoproteins to identify and characterize antigenic proteins and development of serodiagnostic methods using recombinant proteins or their partial sequence peptides are still underway. It has not been put into practical use (patent document 1, non-patent document 4).

JP2005-265510

Tsang, V. C., J. A. Brand, and A. E. Boyer. 1989. An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J. Infect. Dis. 159: 50-59. Ito, A., A. Plancarte, L. Ma, Y. Kong, A. Flisser, SY Cho, YH Liu, S. Kamhawi, MW Lightowlers, and PM Schantz. 1998. Novel antigens for neurocysticercosis: simple method for preparation and evaluation for serodiagnosis. Am. J. Trop. Med. Hyg. 59: 291-294. Sato, MO, Y. Sako, M. Nakao, H. Yamasaki, K. Nakaya, and A. Ito. 2006. Evaluation of purified Taenia soliumglycoproteins and recombinant antigens in the serologic detection of human and swine cysticercosis. J Infect Dis. 194 : 1783-90. Sako, Y., M. Nakao, T. Ikejima, XZ Piao, K. Nakaya, and A. Ito. 2000, Molecular Characterization and Diagnositic Value of Taenia solium Low-Molecular-Weight Antigen Genes. J. Clin. Microbiol. 38 , 4439-4444

  An object of the present invention is to provide a simple method for purifying an antigen for serum diagnosis from cyst fluid without using affinity chromatography or a special isoelectric focusing device.

  The present inventors can purify antigens for serum diagnosis from cyst fluid by ion chromatography using a cation resin, and antigens purified by this method can be purified by conventional isoelectric focusing. It was found to show sensitivity and specificity equivalent to the resulting antigen. That is, the present invention is a method for purifying a 10-26 kDa glycoprotein from cyst fluid, wherein the cyst fluid is adsorbed to a cation exchanger under the conditions of pH 8-9, pH 8-9, and salt concentration 0-500 mM. A method comprising eluting at conditions is provided. Preferably, the cation exchanger is a sulfopropyl cation exchanger.

  In another aspect, the present invention relates to a 10-26 kDa sugar obtained by adsorbing cyst fluid on a cation exchanger under conditions of pH 8-9 and elution under conditions of pH 8-9 and salt concentration 0-500 mM. Provided is an antigen composition for serodiagnosis of cyst infection, comprising a protein.

  In yet another aspect, the present invention provides a method for detecting cystic infections. In this method, a body fluid obtained from a subject is contacted with a 10-26 kDa glycoprotein purified by the method of the present invention, and the presence of an antibody that binds to the glycoprotein antigen in the body fluid is measured. If it is detected, each step of determining that the subject is infected with a cyst is included.

  According to the method of the present invention, a serum diagnostic antigen can be easily purified from cyst fluid using a commercially available column. The method of the present invention is a simple and inexpensive method that does not require a special isoelectric focusing preparative electrophoresis apparatus and can be applied in endemic areas of cysticesis, and is extremely useful for serodiagnosis of cysticesis.

FIG. 1 shows the results of SDS-PAGE analysis of an antigenic glycoprotein purified from the cyst fluid of a manicidal cyst by the method of the present invention. FIG. 2 shows the detection of antibodies in sera from mandibular cystosis patients using the antigen glycoprotein purified by the method of the present invention. FIG. 3 shows the results of SDS-PAGE analysis of the antigen glycoprotein purified from the cyst fluid of Aspergillus cyst by the method of the present invention.

  As an antigen for serodiagnosis of cysticesis, a glycoprotein having a molecular weight of 10-26 kDa and an isoelectric point of 9.2 to 9.6 present in cyst fluid is known as an antigen with high sensitivity and specificity. . This is a mixture of multiple glycoproteins, and its detailed composition has not yet been clarified.

  In the method of the present invention, cyst fluid is used as a starting material. The cyst is a tapeworm larva having a sac-like structure filled with cyst fluid. The cyst fluid is prepared by removing the cyst from a naturally infected or experimentally infected pig and then physically destroying the worm body or using a syringe to collect the cyst fluid inside the worm body. It is desirable to store the collected cysts in a freezer until purification is started.

  Next, cyst fluid is adsorbed onto the cation exchange column. As the cation exchanger, any of various commercially available cation exchangers may be used. For example, a sulfopropyl cation exchanger, a carboxymethyl cation exchanger, or the like may be used. From the viewpoint of stability, a sulfopropyl cation exchanger is preferred. After equilibrating the cation exchange column with the starting buffer, the cyst fluid is loaded onto the column and adsorbed. Since the isoelectric point of the antigen to be purified is 9.2 to 9.6, the pH of the starting buffer is selected to be pH 9.0 or lower. Preferably, the pH of the starting buffer is set to pH 8.0 or higher, which can prevent the porcine antibody and the like contained in the parasitic material from binding to the cation exchanger. The cyst fluid is preferably preliminarily dialyzed or desalted and adjusted to a pH and ionic strength comparable to those of the ion exchange chromatography starting buffer. As the buffer, any buffer usually used for dissolving glycoprotein, such as Tris-HCl, HEPES, phosphate buffer, etc. can be used.

  After the antigen glycoprotein is adsorbed on the column, the cation exchange column is washed with a starting buffer to wash away unadsorbed components. For washing, a buffer different from the starting buffer may be used. Next, the glycoprotein is eluted using an elution buffer containing salt. As the elution buffer, a salt concentration gradient in which a salt of 0-1M NaCl is added to the start buffer can be used. Alternatively, stepwise concentration gradient elution or isocratic elution may be used. The target glycoprotein is eluted with 0-500 mM NaCl. The collected antigen glycoprotein can be stored at low temperature or frozen after concentration if necessary.

  Thus, the glycoprotein antigen purified from the cyst fluid by ion exchange chromatography using a cation exchange resin is a sugar protein purified using a conventional isoelectric focusing electrophoresis apparatus with respect to the reactivity with the antibody. Shows sensitivity and specificity equivalent to protein antigens. That is, according to the method of the present invention, it was possible to easily purify an antigen for serum diagnosis from cyst fluid using a commercially available column. The glycoprotein antigen obtained by the method of the present invention can be used in a serum diagnostic method by an immunoblot method or an ELISA method, similarly to the antigen obtained by a conventional method.

  The antigen obtained by the method of the present invention can be used for detection and diagnosis of cyst infections in humans or animals such as pigs and cows. That is, in another aspect of the present invention, a method for detecting cyst infection is provided. This method involves contacting a body fluid obtained from a subject with a glycoprotein purified as described above and measuring the presence of an antibody in the body fluid that binds to the glycoprotein antigen. If the presence of the antibody is detected, the subject is determined to be infected with cysts.

  Using the antigen glycoprotein obtained by the method of the present invention, the antibody titer in the blood of a subject can be measured using an antigen-antibody reaction well known in the art. Examples of antigen-antibody reactions include ELISA, immunochromatography, immunoblot, and dot blot. For example, when the antibody titer in human blood is measured using a typical ELISA method, it can be performed as follows. Antigen glycoprotein is bound to a conventional ELISA plate such as 96 well, and the plate is appropriately blocked to prevent non-specific adsorption. Next, serum prepared from the blood of the subject is appropriately diluted, added to each well of the plate, and incubated for a predetermined time. After washing the plate to remove unbound components, an antibody (eg, goat anti-human antibody) that is detectably labeled and capable of binding to a human antibody is added. Labeling can be performed by methods well known to those skilled in the art using enzymes such as peroxidase and alkaline phosphatase, fluorescent dyes, chemiluminescent substances, biotin, radiation compounds and the like. After the plate is incubated for a predetermined time, the label is detected. Detection can be performed by adding a suitable substrate and measuring the decrease of the substrate or the increase of the product, or by measuring the fluorescence, luminescence or radioactivity. In order to increase detection sensitivity or specificity, a secondary antibody (for example, anti-goat IgG) may be further used. In this manner, the amount of antibody against the antigen glycoprotein in the serum of the subject can be measured.

  When using the immunochromatography method, the antigen glycoprotein is first immobilized on a test line of a test paper made of a carrier such as nitrocellulose, and a labeled antibody is attached to another place on the test paper. When the sample is dropped on the test paper, the sample moves on the test paper, contacts the labeled antibody, and further moves to the test line. When the antibody is present in the sample, the complex of the antibody and the labeled secondary antibody binds to the antigen glycoprotein on the test line, and the label is detected. As the label, an enzyme that catalyzes a coloring reaction or colloidal gold particles can be used so that the color of the test line can be distinguished. The immunochromatography method is particularly useful for bedside testing and group testing because it is simple and does not require special equipment or technology.

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.
Example 1 Purification of an antigenic protein from cyst fluid The cyst fluid derived from an Ecuadorian isolate and a Chinese isolate was used as a starting material for purification. Both cyst fluids are cyst fluids obtained by removing the cysts from naturally infected pigs and then physically destroying the worm bodies. 2 ml of cyst fluid dialyzed against 20 mM HEPES buffer (pH 8.0) was added to a 1 ml bed volume cation exchange column (HiTrap SP XL, GE Healthcare Biosciences) equilibrated with the same buffer. Applied at a flow rate of / min. Then, after washing with the same buffer (more than 10 times the bed volume), the bound protein was eluted with a salt concentration gradient of HEPES buffer (pH 8.0) containing 0-1M NaCl. At the time of elution, 10 fractions of the eluted fraction were collected under the condition of 1 ml / fraction. The elution curve obtained by measuring OD280 was a single peak, and all the bound proteins were eluted below 500 mM NaCl. The elution curves were similar for both Ecuadorian and Chinese isolates of cyst fluid.

  All fractions eluted at 500 mM NaCl or less were mixed, and purified protein was analyzed by SDS-PAGE. The results are shown in Figure 1. A plurality of bands of about 10-20 kDa are observed.

Example 2 Evaluation of Purified Glycoprotein by ELISA In order to confirm the usefulness of the purified protein as a diagnostic antigen, an immunoblot method and an ELISA method were evaluated.
The antigen was electrophoresed at 50-60 μg / gel using a 4-20% polyacrylamide gradient gel (TEFCO: SDS-PAGE mini) and then electrically transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was treated with a blocking buffer (20 mM Tris-HCl, 1% casein, 150 mM NaCl, pH 7.4) for 1 hour at room temperature. Then, after reacting with test serum diluted 20-fold with blocking buffer at room temperature for 2 hours, a washing solution (surfactant Tween 20 was added to phosphate buffered saline at a concentration of 0.05%). Washed (3 washes for 5 minutes). After discarding the final washing solution, the peroxidase-labeled protein G solution (Zymed, 1000-fold diluted with blocking buffer) was allowed to react at room temperature for 1 hour, and then washed with the washing solution (4 washings for 5 minutes). . Thereafter, a chromogenic substrate (4-chloro-1-naphthol dissolved in 100 mM phosphate buffer (pH 7.4) at a concentration of 0.6 mg / ml and 0.003% hydrogen peroxide added) was reacted. After standing at room temperature (about 30 minutes), the reaction was stopped by washing with distilled water. The results are shown in FIG. Immunoblot analysis revealed that the purified protein was recognized in the sera of mandibular cysticesis patients. Of the patient sera, those that show a negative result are considered to have no antibody or the amount of antibody is below the detection limit. It is known that there are a certain percentage of patients in which antibodies are not detected even by conventional methods.

Next, ELISA was performed using negative control human serum, echinococcosis patient serum, and scabies patient serum. Antigen was adsorbed to a 96-well plastic plate (Nalge Nunc: MaxiSorpPlate) at 100 ng / hole. The antigen solution was discarded and washed with phosphate buffered saline. After adding 300 μL of blocking buffer (20 mM Tris-HCl, 1% casein, 150 mM NaCl, pH 7.4) to each well, it was sealed again and allowed to stand at 37 ° C. for 1 hour. The blocking buffer was discarded, and 100 μL of test serum diluted 200-fold with blocking buffer was added to each well. The plate was gently shaken to mix the serum. Then, it was made to react at 37 degreeC for 1 hour, and the test serum was thrown away. A washing solution (surfactant Tween 20 added to phosphate buffered saline at a concentration of 0.05%) was added to each well and washed. The above process was repeated 3 times. After discarding the washing solution, 100 μL of peroxidase-labeled protein G solution (Zymed, diluted 4000 times with blocking buffer) was added to each well. The plate was gently shaken to mix the peroxidase-labeled protein G solution. Then, it was made to react at 37 degreeC for 1 hour. Thereafter, the peroxidase-labeled protein G solution was discarded, and a washing solution was added to each hole for washing. The above was repeated 4 times. A chromogenic substrate (2,2'-azino-di- (ethyl-benzthiazoline sulfonic acid) was dissolved in 100 mM citrate buffer (pH 4.7) to a concentration of 0.4 mM in each hole, and 0.003% hydrogen peroxide was added. 100 μL was added. After standing at room temperature (about 30 minutes), 1% SDS solution was added to each hole (100 μL / hole) to stop the reaction.
Absorbance (OD value) was measured with an ELISA reader and judged based on the OD value. In the analysis, antigens purified by rotophore were used as standard antigens, and antigens derived from Ecuador isolates and Chinese isolates purified by this method were compared. The results are shown in Table 1. The results showed that the antigen purified by this method was not different in sensitivity and specificity from the standard antigen (p> 0.1, Pearson's chi-square test), indicating that this method is useful for the purification of antigens for serodiagnosis. It was. The kappa coefficients of Ecuador isolate and rotophor purified antigen, and Chinese isolate and rotophor purified antigen were 0.92 and 0.87, respectively, indicating a strong correlation.

Example 3 Purification of Antigen Glycoprotein from Aspergillus cyst cyst fluid As in Example 1, antigen glycoprotein was purified from Aspergillus cyst fluid and analyzed by SDS-PAGE. As in the case, multiple bands of about 10-20 kDa were observed (FIG. 3).

  According to the method of the present invention, an antigen glycoprotein can be purified easily and inexpensively in an endemic area of cysticesis, and is useful for serodiagnosis of cystosis.

Claims (4)

A method for purifying a 10-26 kDa glycoprotein from cyst fluid, wherein the cyst fluid is adsorbed on a cation exchanger at pH 8-9 and eluted at pH 8-9 at a salt concentration of 0-500 mM. Including methods. The method according to claim 1, wherein the cation exchanger is a sulfopropyl cation exchanger. For serodiagnosis of cyst infection, containing 10-26 kDa glycoprotein obtained by adsorbing cyst fluid on a cation exchanger under pH 8-9 conditions and eluting under pH 8-9, salt concentration 0-500 mM conditions Antigen composition. A method for detecting cyst infection in vitro, wherein a body fluid obtained from a subject is contacted with a glycoprotein, the presence of an antibody that binds to the glycoprotein antigen in the body fluid is measured, and the subject is infected with a cyst. And detecting the presence of an antibody indicating the presence of the antibody, wherein the glycoprotein adsorbs the cyst fluid to the cation exchanger under the condition of pH 8-9, and the condition of pH 8-9, salt concentration 0-500 mM. A glycoprotein of 10-26 kDa obtained by eluting with a method as described above.