MXPA96002274A - Procedure for the preservation of antigens without using enzymatic inhibitors and their application in methods inmunologi - Google Patents

Abstract

The present invention relates to the method with the preservation of a complex antigenic system of Entamoeba histolytica molecules, without using enzymatic inhibitors and using these preserved Entamoeba histolytica molecules as a reagent in a diagnostic assay system and as a starting material for the Protein isolation from Entamoeba histolyti

Description

PROCEDURE FOR THE PRESERVATION OF UNUSED ANTIGENS ENZYMATIC INHIBITORS AND THEIR APPLICATION IN METHODS IMMUNOLOGICAL.

DESCRIPTION PE THE INVENTION BACKGROUND OF THE INVENTION Amoebiasis is a parasitic disease caused by the protozoan Entamoeba histolytica. It mainly affects the inhabitants of developing countries. Under suitable conditions, which are not well understood, the trophozoites differentiate into an infective or cyst form. The cysts are present in the stool and by this route can infect a new host by oral ingestion of contaminated food and water or by transmission from person to person. The majority of people infected with histolytic Entamoeba are asymptomatic, but in 10% of people with amoebiasis, the protozoan produces disease when it invades the intestinal mucosa, producing amoebic colitis or more serious damage when the protozoan is outside the body. intestine and there is a spread of the protozoan to the liver, causing amoebic liver abscess. In cases where perforation of the liver or intestine occurs, it can cause pleural damage, pericarditis, peritonitis and even death. In Mexico, amoebiasis ranks sixth among the most frequent causes of death. In Mexico, as in Venezuela, it has been reported that of the cases of children hospitalized with diarrhea, 2 to 15% had infections associated with histolytic Entamoeba. To carry out correct epidemiological studies, it is necessary to develop diagnostic methods that are sensitive and specific. The coproparasitoscopic diagnosis of Entamoeba histolytica is especially difficult because in order to prevent false interpretations highly trained workers are required, besides it is impossible to differentiate in this way the pathogenic from the non-pathogenic strains. Serological diagnosis is not effective because existing tests are not sensitive enough, especially when used in areas of high endemicity. To develop useful diagnostic tests, it is necessary to know the amoebic molecules actively involved in cases of invasive amoebiasis and use these molecules to design effective diagnostic tests. Once the role of these molecules is known, studies can be carried out to determine their role in the mechanisms of immune protection generated against amoebiasis and its possible use in the instrumentation of vaccines. A major impediment to achieving this goal is the high enzymatic activity of the proteases present in the amoebic extracts (see McLaughlin et al., Canadian Journal of Microbiolos? 23: 420-425 (1977) and Pérez-Monfort, et al. , Molecular and Biochemical Parasitology, 26: 87-89 (1987)). Proteases degrade proteins in amoebic extracts producing degradation products and making it impossible, or at least very difficult, to standardize the methods of analysis of the antigenicity of these proteins. Enzyme inhibitors are used worldwide to prevent this enzymatic activity, however, the inhibitors used are not totally effective and the protein degradation is carried out even in the presence of the inhibitors. In general, amoebic extracts containing enzyme inhibitors can only be worked for relatively short periods of time and do not give the opportunity to store the same samples for later testing. The study reported by Araüeyo-García et al .. Arch. Invest. Med. (Méx.) 21: 3-9 (Supl. 1) (1990) Different methods of extraction of antigenic proteins of £ were evaluated. histolytica using. The highest yield of cell lysates of E. histolytica was obtained by homogenizing trophozoites in the presence of 10 M p-hydroxymercuribenzoate (pHMB) and by lysis with Triton X-100 and a mixture of protease inhibitors. It was found that the frozen extracts were stable for a period of two or three months. Nevertheless, those extracts were not subject to repeated freezing and thawing (personal communication). The authors found that different methods for preparing amoebic extracts resulted in differences in the yield of protein concentration, as well as in the antigenic composition, as observed by electrophoretic patterns. These authors point out the need to select a standardized procedure to prepare amoebic extracts for use in serological tests. The present invention was based on the method to delipidate amoebic extracts described by Said-Fernández et al., Zeitschrift fur Parasitenkunde. 56: 219-225 (1978). These authors showed that the electrophoretic analysis of the trophozoite proteins of four different Entamoeba strains showed a similarity between the two strains studied of E. histolytica. The authors propose that the electrophoretic analysis of amoebic proteins could be used as a taxonomic criterion, after confirming their use by studying a larger number of Entamoeba strains. The present inventor has determined that amoebic antigens subjected to extraction with organic solvents, traditionally used to remove lipids from biological material, but also subjected to heating (article by the applicant) exhibit very low levels of proteolytic enzymatic activity, which does not cause amoebic proteins no extensive degradation. The amoebic extracts prepared with this method remain stable for at least two years without any enzymatic degradation. Therefore, the antigens prepared with this method are stable for longer periods of time than the extracts containing protease inhibitors and are ideal for use in diagnostic tests, such as ELISA, IHA, agglutination, Western-blot, dot-blot. The possibility of longer storage provides clear advantages for commercial diagnostic products that use amoebic antigens prepared according to this method. The applicant has demonstrated that amoebic extracts produced according to this method can be used as stable antigens in diagnostic tests, such as ELISA, IHA, dot-blot, Western-blot without the need to use protease inhibitors to prevent the degradation of amoebic extracts traditionally used as standard or standard treatment by experts in the area.

Therefore, the present invention relates to diagnostic tests and methods for detecting the presence of antibodies directed to E. histolytica in biological samples. In addition, the applicant has demonstrated that amoebic extracts produced according to this method can also be used as starting material to produce and characterize specific E. histolytica proteins associated with different clinical manifestations of amoebiasis; for example, amoebic liver abscess (AHA) and intestinal amoebiasis (AI). These specific proteins can be used as stable antigens in diagnostic tests, such as ELISA, IHA, agglutination, dot-blot, Western-blot to detect the presence of antibodies to E. histolytica in biological samples from patients or experimental animals and subsequently used in the production of a vaccine against E. histolytica.

BRIEF DESCRIPTION OF THE INVENTION The method of the invention relates to the preservation of the antigens of microorganisms, such as, for example, E. histolytica. without using enzymatic inhibitors, although by no means restricted to E-histolytica. The enzymatic activity of the proteases contained in E-histolytica makes the study of antigenically important amoebic molecules difficult. Therefore, one of the objects of the present invention is to use E-histolytica as a model for other parasites. Another objective of the present invention is to reduce the proteolytic action of the protozoan enzymes without adding enzymatic inhibitors. A further object of the present invention is to provide amoebic extracts of better quality containing antigens preserved from E. histolytica to study amoebic proteins. In the present invention, the amoebic extracts obtained using the method of the present invention are compared with conventional methods in which enzymatic inhibitors are used. The present method is based on obtaining E-histolytica molecules preserved antigenically from an amoebic extract insoluble in polar solvents or their mixtures and heating the extract in a boiling water bath for 5 to 20 minutes to obtain a greater reduction of the enzymatic activity than the one obtained with enzymatic inhibitors. The method of this invention preserves the antigenicity of amoebic molecules, since the antibodies present in the serum of individuals with amoebic liver abscess recognize them and react with them. The procedure can be of great help in the analysis and identification of amoebic antigen molecules and in the study of the immune response directed against E. histolytica but also has many uses: for example, but without limitation, to obtain amoebic molecules for prepare a vaccine or to prepare equipment or diagnostic kits for detection and support in the diagnosis of amoebiasis, invasive or non-invasive, allowing an adequate treatment of these diseases. The present application relates to a composition, diagnosis, equipment and method of use thereof, to detect the presence of antibodies and anti-histolytica. The preparation of the preserved antigenic extract of E-histolytica identified as IC-MC, the insoluble fraction of trophozoites of E-histolytica obtained from the extraction with a mixture of chloroform and methanol subsequently heated, is used as the antigen in the diagnostic composition contained in the team. The English version of the abbreviation for antigen preparation is C: MIH, derived from chloroform: methanol, insoluble, heat. "Heat" in Spanish, is warming, hence the original designation IC: MC. Alternatively, the antigen used in the diagnostic kit may be a < 8-12 kDa obtained from preparative gels by SDS-PAGE starting from preparations IC: MC or from the culture media where E-histolytica was grown. The present application relates to a method for purifying the molecular weight protein between < 8 + 12 kDa by SDS-PAGE of the preparative gels.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents a graph in which the relationship between the protein content of the crude extracts and their proteolytic activity on azocasein is analyzed and compared with the IC: MC fraction in the presence and absence of 2% SDS and 5% 2 - ME. The figure shows the activity of the crude extract -IC.MC and IC: MC + 2% SDS and 5% 2-ME (2-mercaptoethanol) * - *. The activity of 0.5 mg. of crude extract protein is normalized to unity (Unit = 0.320 O.D.). Figure 2 shows I. Electrophoretic patterns in SDS-PAGE II. Electroinblot blot (Western-blot) immunoblot patterns using serum from patients with amoebic liver abscesses (AHA). A. Fraction IC: MC. B.

Crude amoebic extract C. Crude amoebic extract with the iodoacetamide inhibitor. Figure 3 shows the patterns of trophozoite extracts of E-histolytica HK-9 I. Electrophoretic patterns in polyacrylamide-SDS gel stained with silver nitrate. II. Western-blot patterns using serum from a patient with amoebic liver abscess. A. Raw amoebic fraction. B. IC extract: MC. Figure 4 shows the Western-blot patterns obtained with the anti-ameal antibodies present in the serum of individuals with amoebiasis. Amoebic liver abscess (AHA) 3, 4, 6, 11, 12 and 15; amoebic dysentery (DA) 8 and 9; Intestinal amoebiasis (A. I.) 1, 2, 10, 13 and 14; sera for negative control. 5 and 7. Figure 5 shows protein bands and particularly the < 8-12 kDa separated by SDS-PAGE and Western-blot (immunoblot) using serum from patients with AHA. A and B; known molecular weight markers; C - unused culture medium; D and F culture medium where E. histolytica was cultured; E- unused culture medium, with bovine serum and proteins; G - bovine serum and H - IC: MC. Figure 6 shows on lines A and B a Western blot protein band of < 8-12 kDa electroeluted from a preparative SDS-PAGE. Figure 7 shows an isoelectric focusing test with a pH range of 3.5 to 10.0 performed on polyacrylamide gels of the electroeluted fraction. A to G - known markers of pl and H - protein bands obtained by electroelution from preparative SDS-PAGE. Figure 8 shows the antigenic stability of the IC: MC fraction after being incubated with buffer solutions at different pHs. A - pH 6.8; B - pH 10.0; C - pH 9.0; D - pH 5.0; E-pH 3.0; F - markers of known molecular weight. Figure 9 shows a Western-blot of IC: MC ammonium sulfate precipitates and culture media where E. histolytica A - molecular weight indicators were cultured; B - supernatant of the culture medium; C - precipitate from the culture medium; D - culture medium after cultivation E-histolytica (control) and E-IC: MC (control). Figure 10 shows a Western blot of the < 8-12 kDa precipitated with different concentrations of ammonium sulfate. A - (80%) precipitate; B - (80%) supernatant; C - (70%) precipitated; D - (70%) supernatant; E - (60%) precipitate and F - (60%) supernatant. Figure 11 shows a Western-blot of SDS-PAGE with the supernatants obtained from precipitation with rivanol, from culture media where E-histolytica was grown and which contained different concentrations of proteins. A - 14% proteins; B - 7% proteins; C-proteins at 3.5%; Known molecular weight markers. Figure 12 shows a Western-blot of culture medium where it was cultured from E-histolytica after being passed through ion exchangers. C - SP - SEPHADEX and D - DEAE - SEPHADEX.

DETAILED DESCRIPTION OF THE INVENTION In the present invention the trophozoites of Entamoeba histolytica were axenically cultured. The trophozoites were immediately used or lyophilized and stored until use. The protein content was determined according to the method of Lowry et al. (Journal of Bioloaical Chemestry, 193: 265-276 (1951)). The extract containing the insoluble fraction in polar solvents or their mixtures was obtained (chloroform, methanol, ether, ethanol). The fraction insoluble in polar solvents can be obtained by treating the trophozoites with a polar solvent such as chloroform, methanol, ethanol, ether or mixtures of these. Preferably, the trophozoites are resuspended in ranges comprised between 5 to 100 volumes of chloroform: methanol in volume to volume ratios comprised between 1: 1 and 20: 1 and even 1:20. The mixture is stirred and centrifuged for at least 2 minutes at 400 X to 4 C. The supernatant is discarded and the operation may be repeated several times. When the IC: M fraction is obtained, it is dried under vacuum and refrigerated or frozen until use. When needed, the IC: M fraction is resuspended in a buffer solution with a pH in a pH range of 4 to 9, and heated in a boiling water bath for several minutes for a maximum of 20 minutes. The heated fraction IC: M is called fraction IC: MC, and is ready for use and can be stored at -70 ° C, -20 ° C or 4 ° C for up to three years without enzymatic degradation of proteins. If the IC: MC fraction is kept lyophilized it can be stored at -70 ° C, -20 ° C or 4 ° C for up to three years without protein degradation. If the IC: MC fraction is kept frozen in solution at -70 ° C or -20 ° C it can be stored for up to three years without protein degradation being observed. In addition, the preparation can be frozen and thawed repeatedly without enzymatic degradation of the proteins. As stated in the objectives, the reduction in proteolytic activity in the IC: MC fraction was demonstrated in the absence of enzyme inhibitors and the method of the present invention was compared with conventional methods using an enzyme inhibitor (iodoacetamide) to reduce the proteolytic activity of crude amoebic extracts. Crude extracts were useful as a positive control of enzymatic activity. To obtain crude extracts, the trophozoites were resuspended and diluted at a concentration of 5 mg / ml of protein or more; the mixture was stirred and subjected to ultrasonic vibration and then centrifuged at 400 X g for 5 minutes, the pellet was discarded and the supernatant was used in the experiments. Two methods were used to determine the proteolytic activity in the crude extracts and in the IC: MC fraction. In the first method, the azocasein substrate was used and hide powder azure was used in the second method. In the first method, the experiments were based on reports by McLaughlin et al., Canadian Journal of Microbiology. 23: 420-425 (1977) and Avila et al., Journal of Protozoology. 32: 163-166 (1985). The crude extract was resuspended 0.05M Tris-HCl, pH 7.6 at a range of 0.5% Triton X-100 was incubated for 1 hour at 4 ° C. The samples were centrifuged at 400 X g for 2 minutes and the supernatants were recovered for analysis. The IC: MC fraction was treated in the same manner as the crude extract. To some samples 2% SDS and 10% 2 -ME were added. These samples were used in electrophoretic techniques in polyacrylamide gels in the presence of sodium dodecylsulfate (SDS-PAGE). Avila et al. (1985) reported that SDS and 2 -ME stimulate the proteolytic activity of amoebic enzymes. With each sample of the different extracts, serial dilutions of 0.5 to 1.5 mg of total proteins were made with 150 ul of 0.05 M Tris-HCL pH 7.5 and 100 ul of 1% azocasein were added and incubated for 90 minutes at 37 ° C. 250 μl of 10% trichloroacetic acid was added to each test and centrifuged at 7000 × g for 6 minutes. To each 200 uls of supernatant 1.8 ml of .5N NaOH was added and the absorbance of each sample was read at 420 nm on a PMQ II Zeiss PMQ III spectrophotometer (Zeiss, Germany). Trypsin was used as a positive control (300 ug for each reaction mixture).

Figure 1 shows that the proteolytic activity of the crude extract on azocasein corresponds to a linear relationship with respect to the total protein content. In contrast, in the IC: MC fraction with the protein content equivalent to that of the crude extracts, no enzymatic activity was observed, even in the presence of SDS and 2 -ME. The second method was carried out according to Rinderknecht et al., Clinica Chimica Acta .. 21: 197-203 (1968). The activity corresponding to 6 million trophozoites was tested. Crude amoebic extracts, IC: M and IC: MC were used. The samples were resuspended in 1.5 ml 0.05M Tris-HCL pH 7.5 containing 0.5% Triton X-100 and incubated for one hour at 4 C and centrifuged at 11,000 X g for 15 minutes. One ml of the supernatant was added to 1 ml Tris-HCL containing 5 mg hide powder azure and 100 ul of 0.2 M cysteine. The samples were incubated at 37 C for 1 hour and at the end the reaction was stopped by placing the tubes in a bath of ice water. They were centrifuged in cold for 5 minutes at 450 X g. The absorbance of the supernatants was determined at the wavelength of 600 nm. The first treatment was also performed with samples that were incubated in the presence of 2% SDS and 2 -ME 5% or 4 mM iodoacetamide. The positive control was taken as the activity obtained with 0.23 mg of papain, which was activated with 2 -ME 0.7 mM in Tris-HCL 0.05M at pH 8.0 at 4 C for 10 minutes and then at 37 ° C for 15 minutes in shaking frequent. Reaction mixtures without extract were used as a negative control. Using the insoluble substrate "hide powder azure" (Table 1), the activity of the crude extract was inhibited approximately 70% with 4 mM iodoacetamide. On the other hand, fraction IC: M showed a reduction in proteolytic activity of 87% with respect to the activity of the crude extract. The IC: MC fraction showed a slight increase in proteolytic activity in the presence of 2% SDS and 5% of 2-ME (0.322 units). As can be seen in Table 1, the increase was almost completely eliminated when the fraction IC: M was previously heated for 5 minutes (fraction IC: MC) and the proteolytic activity was not increased neither in the presence of SDS and 2 -ME, TABLE 1 (a) Reduction based on the activity of the crude extract. (b) 1.00 = 0.750 + 0.09 UDO (optical density units) As shown in Table 1, fraction IC: MC prepared according to the method developed in the present invention contains antigens preserved from E-histolytica. fraction IC: MC does not have any of the problems of proteolytic degradation of amoebic extracts made by the methods traditionally used by experts in the area, as shown in Table 1. As explained above, fraction IC: MC is stable when frozen in a state of lyophilization or in solution and can be used as an antigen in diagnostic tests for up to three years, without proteolytic degradation, which is an advantage over what is commercially available, such as the amebiasis microassay made by Diamedix Co. , Miami Florida. As a result of the stability of the IC: MC fraction, the present inventor has determined that the IC: MC fraction is an excellent amoebic antigen to be used in immunoassays, such as ELISA and IHA, agglutination, Western blot. Antibodies against all antigens, even those recognized by the sera of negative control patients can be detected by the ELISA using IC: MC as antigen. However, serum from patients with AHA and AD have higher titers of anti-E histolytica antibodies than negative control sera. The diagnosis is made after knowing the antibody titers, as well as the clinical symptoms. The ELISA using the IC: MC fraction is just another instrument to help the doctor diagnose the patient after taking into account exams as well as clinical symptoms. The IC: MC fraction can be used in an ELISA as follows: The IC: MC fraction is obtained as stated above, and subsequently sonicated in a sonicator, centrifuge at 1100 X g for 3 - 5 minutes and recover the supernatant. As for example, the following was done: the concentration of the supernatant containing the E. histolytica proteins was adjusted between 25-250 ug / ml, preferably between 50 ug 0 100 ug / ml in 0.1M acetate buffer, pH 5.0 or the buffer suitable for the type of plate used in the ELISA. Then the wells of the polystyrene microtiter plates were not covered with 100 ul of the adjusted supernatant and the samples that were supposed to contain antibodies directed against E-histolytica were allowed to dry overnight at 4 ° C in the covered wells. After incubation, the plates were washed three times with a washing solution (0.1M PBS, pH 7.4 containing 1: 1000 Tween 20). The plates were then incubated with 1% BSA in the wash solution for 2 hours at 37 ° C to block the plates and avoid non-specific binding. Other blocking reagents such as gelatin or skim milk can be used. The BSA solution was discarded and the plates were washed 3 times with washing solution. The plates were washed and incubated with a second antibody conjugated to an enzyme, for example, with 100 ul / ml of immunoglobulin conjugated to horseradish peroxidase, for 2 hours at 37 ° C. The conjugate was discarded and the plates were washed three times with washing solution. The plates were incubated in the dark with 100 ul of substrate and chromogen (for horseradish peroxidase H202 and orthophenylenediamine O.P.D. are used) for 30 minutes. The reaction that generates color was stopped with 40 ul of H2SO4 and the absorbance was read at an Optical Density of 492 nm. Any known combination of enzyme, substrate and chromogen conjugate known to the personnel trained in the method can be used and the color reaction can be stopped with other known reagents. Table 2 shows the results of the examination of 34 sera from patients, some of them suspected of having antibodies directed against amoebas due to amoebic liver abscess. The ELISA method was used and the results were expressed in EU / ml (of ELISA units / ml).

TABLE 2 EU / ml was calculated as follows: Calibrator = Absorbance of the calibrator 100 X Absorbance of the sample = EU / ml of the sample examined The calibrator is a known positive serum. Less than 50 EU / ml is a negative result for invasive amoebiasis. The ELISA test described above was used to identify the isotypes of the antibodies against E. histolytica present in 36 sera from patients with AHA and in 10 sera used as a negative control in healthy people. Patients with AHA had the positive IHA test for anti-E histolytica antibodies. clinical symptoms of AHA and amoebic liver abscess confirmed by ultrasound.

The negative control subjects had the negative IHA test for E-histolytica. the negative coproparasitoscopic examination and they did not have clinical symptoms. The results are shown in Table 3.

Table 3 The figures supporting these results show that the ELISA units of anti-E. histolytica antibodies from patients with AHA are greater than the ELISA units obtained with anti-E histolytica antibodies from negative controls. The data show that it is very important to measure IgG and IgA isotypes in ELISAs, to detect amoebiasis to obtain the best results in examinations and that IgM isotype measurements should be avoided in endemic zones of amoebas. IC: MC extract can also be used in an IHA (Indirect Hemagglutination Assay) to cover red blood cells using diethylmethylcarbodiimide as the coupling agent or glutaraldehyde. Red blood cells covered with IC: MC were incubated with samples that were presumed to have antibodies against E. histolytica. in polystyrene plates for microtitre. The positive samples resulted in agglutination of the red blood cells that could be detected visually. The IC: MC fraction is also useful for application in studies of anti-E-histolytica antibodies induced in animal models, such as mice. The DOT-BLOT method was used to detect E. histolytica trophozoites in the serum of mice immunized with lyophilized E. histolytica trophozoites. The mice were immunized intraperitoneally (IP) or orally (VO). The sheet of the nitrocellulose membrane was covered with points from the IC: MC fraction and incubated with the mice serum in PBS 0.1M, pH 7.4. The reaction sites were developed with an operoxidase conjugate directed against the mouse antibodies, H202 and 3,3 diaminobenzidine. The results of DOT-BLOT showed that mice immunized intraperitoneally developed a greater immune response of anti-E antibodies. histolytica than mice immunized orally. In addition, the ELISA described above was used to identify the isotypes of the anti-E-histolytica antibodies and to determine their levels in the sera of mice immunized with lyophilized E-histolytica trophozoites, intraperitoneally (IP) or via oral (VO) The results showed no difference in the ELISA Units between the immunization routes for IgM and IgA and whether they showed differences between the immunization routes for polyvalent IgG and Ig. The study of amoebic molecules is difficult, because they are rapidly degraded by the enzymatic activity of the proteases contained in • histolytica. In the present invention it has been verified that many molecules of the parasite are preserved. As a result of the electrophoretic analysis of the crude extracts, of the crude extracts with iodoacetamide and the fraction IC: MC, it was demonstrated that the fraction IC: MC preserves a great variety of molecules of low, medium and high molecular weights, which are dyed with silver nitrate. The crude extract and the crude extract with iodoacetamide contain an enzymatic degradation product. The different extracts were analyzed electrophoretically in polyacrylamide gels in a gradient of 8-18% T 2. 10., in the presence of SDS, with the modified Maizel technique (Maizel, JR., J.V. In: Methods in Virology Vol. V, 198-246, Maramorosch, K. &; Kaprowski, H (editors), New York: Academic press (1971). Briefly: 5% packing gels were used. After a pre-shift of 20 minutes at 50V, IC: MC was applied in a buffer solution Tris-HCl 0.049M pH of 6.8 containing 10% glycerol, 2% SDS, 0.012% bromophenol blue and 5% 2 -ME . In each shift, well-known molecular weight markers were also run. After shifting, part of the gel subjected to electrophoresis was separated to be stained with silver nitrate, with the modified Merril technique (Merril, C.R. et al., Science 211, 1434-1538 (1981)). This portion of the gels was fixed with a 50% methanol: 12% acetic acid solution for 20 minutes; the gels were washed to remove the excess SDS with a solution containing 10% ethanol: 5% acetic acid, followed by three washes of 5 minutes each, with distilled water. Silver nitrate (0.02M) was added for 30 minutes. The bands were developed with 0.28M sodium carbonate with 0.05% formalin. The reaction was stopped with 1% acetic acid. In illustration 2, lanes IB and IC show the electrophoretic patterns corresponding to the crude extract and the crude extract + iodoacetamide and lane IA corresponds to the fraction IC: MC. The IC: MC fraction presents the best resolution with a great variety of bands of different molecular weights. In the lane of the crude extract (IB), few bands are observed; but at the front of the shift an important stain is observed that contains peptides of low molecular weight coming from the enzymatic degradation of molecules of greater molecular weight. The lane of the crude extract with iodoacetamide (IC) has fewer bands than the crude extract alone, but even so, it has fewer bands than the fraction IC: MC. The crude extract with the enzyme inhibitor iodoacetamide should be used rapidly, while, as mentioned above, the IC: MC fraction can be preserved for at least three years. The method of the present invention is directed to immunoassays using the antigens preserved from E-histolytica contained in the IC: MC fraction which does not require the use of enzymatic inhibitors and the purification of the amoebic proteins obtained from the IC: MC fraction. One of the objectives of the present invention is the use of Entamoeba histolytica as a model to design a methodology for preserving parasite antigens and another objective is to compare the preserved antigens of the present invention with the antigens obtained by conventional methods using enzyme inhibitors. Therefore, a test was performed to verify that antibodies present in the serum of patients with amoebic liver abscess (AHA) recognize and react with the molecules present in the extracts that are electrophoretically separated. Figure 2, II shows that amoebic molecules, which were electrophoretically isolated according to their molecular weight, were electrotransferred to a nitrocellulose sheet in an electrotransfer chamber (R) for 90 minutes at 1.0 final amp, according to Towbin et al. to the. (PNAS 76: 4350-4354 (1979)). After transfer, the nitrocellulose membrane was blocked with 30% fish gelatin (R) in PBS 0.15M pH 7.4. The serum to be analyzed was diluted in PBS with 0.3 of gelatin and Tween 20 (R) 1: 1000 and incubated with the nitrocellulose strip overnight at 4 C. The next day it was revealed with a polyvalent conjugate directed against immunoglobulins. human, bound to peroxidase, in the presence of H202 substrate and 3,3 diaminobenzidine tetrahydrochloride as chromogen (R). Other conjugates and other enzymes with their respective substrates and chromogens can also be used. Furthermore, it was demonstrated that the antigenic structure of the amoebic molecules was preserved in the IC: MC fraction obtained with the method described in this invention, since antibodies from the serum of patients with amoebic liver abscess (AHA) give antigen-antibody reaction bands with IC: MC preparations electrotransferred to nitrocellulose strips (Figure 2 -II). More bands corresponding to antigen-antibody reactions were observed in the IC: MC fraction (Figure 2-IIA), than in the crude extracts (Figure 2-IIB) in which only few recognition bands were observed, since most of the of the antigens had been degraded by the action of amoebic enzymes. In addition, more bands were observed in the IC: MC fraction than in the crude extract with iodoacetamide (Figure 2-IIA, 2-IIC). These results show that even in the presence of the inhibitor, many molecules are degraded, while with the method of the invention in the IC: MC fraction they are preserved. Identification through immunoblotting of Entamoeba histolytica antigens, recognized only by sera from patients with invasive amoebiasis Sera were obtained by venipuncture, from 32 adults of both sexes who attended the "Dr. José Eleuterio González" University Hospital of Monterrey, Nuevo León, Mexico. Aliquots were taken and frozen at -20 until use. For this study the sera were classified into the following groups: Group 1. (32 cases). This group included the sera of patients with amoebic liver abscess (AHA) with a clinical picture compatible with AHA; with ultrasound also consistent with AAH and with positive IHA test. Group 2. (21 cases). This group included the sera of individuals with luminal intestinal amoebiasis (LA) with positive coproparasitoscopic examination and no clinical symptoms. To select this group, 3 serial coproparasitoscopic exams were administered to 554 university students. Group 3. (15 cases). Negative control The sera were selected from individuals who followed the following criteria: absence of symptoms of amoebiasis, coproparasitoscopic examination in negative series and negative IHA examination and without any evidence of having symptomatic amoebiasis.

COPROPARASITOSCOPIC EXAMINATION TO SELECT THE INDIVIDUALS OF GROUPS 2 AND 3 ANALYZED POPULATION: 554 students from the Autonomous University of Nuevo Leon, whose ages ranged between 16 and 19 years old and belonging to low-income classes, were studied.

FECAL SAMPLES: 3 fecal samples were taken in series from each individual and 5% formalin were preserved at 5% at 4 C until microscopic analysis.

METHOD USED: The method of concentration and flotation of Faust was used to obtain cysts, eggs and parasite larvae. (Faust E.C. et al., Clinical Parasitology of Craia and Faust, 2nd Ed., Mexico: UTEHA, 1056 (1961)).

ANTIGENS OF Entamoeba histolytica: The trophozoites of Entamoeba histolytica strain HK-9 were cultured in PEHPS medium. (Said-Fernandez, S. et al., Trans R Soc. Trop Med Hys 83:29 (1988)). The trophozoites were donated by Dr. Salvador Said-Fernández of the Biomedical Research Center of Northeast IMSS, Mexico.

AMIBIAN EXTRACTS: The fraction IC: MC was obtained according to the process of the present invention. Fraction IC: MC was lyophilized and dissolved in 0.049M Tris HCl buffer solution pH 6.8, containing 10% glycerol, 2% SDS, 0.015% Bromophenol blue. The sample was subjected to a water bath for 5 minutes. Aliquots were then frozen at -70 C. Before use, 5% of 2-mercaptoethanol was added.

POLYACRILAMIDE GELS (SDS-PAGE) AND IMMUNOELECTROTRANSFERENCE (WESTERN-BLOT): These experiments were performed as described above.

DATA ANALYSIS: Each paper strip was examined individually and the migration distances were measured, to calculate the Rf- value of each antigenic band. Molecular weights were calculated by interpolation on the curves obtained with commercially available molecular weight markers (Rf = distance of protein migration / distance of dye migration).

PROOF OF INDIRECT HEMAGLUTINATION (IHA): It was done according to the instructions of the commercial team (Behring). In the IHA, red blood cells coated with the solubilized antigen of IC: MC can also be used, as previously explained.

RESULTS To select the serum of the individuals that would form groups 2 and 3 (intestinal amoebiasis without symptoms and negative control), serial coproparasitoscopic examinations of 554 students were made and the results are shown in Tables 2 and 3. 74 individuals presented Entamoeba histolytica . what the 13. 35% of the population analyzed. To form group 2, sera from individuals were selected only with Entamoeba histolytica and cases in which the protozoon was found with other pathogenic or commensal species were discarded from this study. (Flores-Castañeda M.S. et al., Border Epidemiological! Bulletin O.P.S. (W.H.O. No. 5: 1-5 (1991)).

TABLE 4 RESULTS OF COPROPARASITOSCÓPICOS EXAMINATIONS REALIZED IN 554 INDIVIDUALS RESULTS NUMBER NEGATIVE 382 (69%) POSITIVE IN PATHOGENIC SPECIES * 109 (19.7%) POSITIVE IN COMENSALES 63 (11.3%) * Including Entamoeba histolytica TABLE 5 PROPORTION OF INDIVIDUALS WITH COPROPARASITOSCÓPICO POSITIVO A Entamoeba histol? Tica SPECIES NUMBER OF INDIVIDUALS Entamoeba histmettica 36 Entamoeba hístQlytÍca + 5 Any pathogenic species 53 (a) (a) association with G_. lamblia or Hymenolepis nana. (b) association with E-coli. Dwarf. I. butschlii Electrophoresis was done in gels of polyacrylamide (SDS-PAGE), from crude extracts of Entamoeba crude histolytica and fraction IC: MC. The results are shown in Figure 3. A better resolution was obtained with the fraction IC: MC, both in the gels stained with nitrate of silver (IB) as in the Western-blot (IB, IIB). In the Figure 3 (IB) can be observed that the pattern The obtained electrophoretic is very complex and has bands of molecules with molecular weights between < 8 kDa and 200 kDa.

Also, it was verified that not all amoebic molecules are recognized by the serum of patients with AHA used in this Western-blot (IIC compared with IB), which seems to indicate that not all amoebic molecules are immunogenic. The proteases of Entamoeba histolytica are very active; The spot appearing in front of the raw extract is probably a product of random enzymatic degradation, which was not observed in the IC: MC fraction. To determine the relative molecular weights of the immunodominant bands, the fraction IC: MC was analyzed by SDS-PAGE and Western-blot and the antigen-antibody reaction bands obtained with serum of Groups 1, 2 and 3, were correlated with the Protein bands on SDS-PAGE gels. From these data, the reference antigenic patterns for each group of sera were identified. A more intense reactivity was always obtained with AHA sera compared to that obtained with AI sera (Figure 4). The sera of AI and the sera of the negative controls give reaction bands, but weak. This finding was expected because Mexico is an endemic area of amoebiasis. The results allowed the identification of bands that are recognized by all sera and have relative molecular weights of 45, 43, 42, 39, 38, 32, 31 and 12 kDa. The following bands are recognized only by the AHA and AI sera and have the relative molecular weights of: 150, 29, 21, 20, 17, 16, 14 and 13 kDa. One of the most relevant aspects of this invention is the identification of an antigenic pattern recognized exclusively and selectively by the sera of patients with amoebic liver abscess (AHA). The exclusive Western-blot pattern of AAH includes bands with relative molecular weights of 23, 25, 37, 11, 10, 9 and 8 kDas, which are recognized only by the serum of patients with amoebic liver abscess. No sera from AI patients recognized these bands (Figure 4: strips 1, 10, 13). Assuming that only invasive amoebiasis (AHA) induces protection against subsequent amoebic reinfections, it can be assumed that the antigens of Western blot patterns exclusive of AAH can play an important role in immune protection against amoebas. The Western-blot pattern characteristic of AHA identified is useful for differentiating serum from patients with invasive amoebiasis (AHA or DA) from those with non-invasive intestinal amoebiasis (AI). The Western-blot patterns identified could help to increase our knowledge about the antigens involved in the induction of the immune response against Entamoeba histQlyticct.

PURIFICATION AFTER MOLECULAR WEIGHT RELATIVE TO THE PROTEIN 8 kPa One of the proteins that was found to be recognized exclusively by the serum of patients with invasive amoebiasis, using the Western-blot technique described above, is the relative molecular weight protein of 8 kDa identified in Figures 3 - 11; however, this protein band appears to have a molecular weight located between < 8-kDa and 12-kDa with the different procedures, gels and chromatography columns made by the present inventor. Electrophoresis was performed on SDS-PAGE, fraction IC: MC and culture medium where the amoebas grew. Subsequently, Western blotting was performed, using sera from patients with AHA, as previously described. These analyzes gave results as shown in Figure 5. The < 8-12 kDa was identified in lanes D, F and H. D and F show the antigen-antibody reaction bands obtained with the culture medium. H those obtained with IC: MC. This figure shows that the low molecular weight band is found both in IC: MC and in the culture medium where the amoebas grew. This is confirmed by the results of lane E, where the virgin culture medium is shown and the absence of the low molecular weight protein is observed. The protein band of < of 8-12 kDas was obtained from a preparative gel subjected to electrophoresis with SDS, using fraction IC: MC or the culture medium where E-histolytica grew as starting material. From this preparative gel, the < 8-12 kDa and a partially purified fraction was obtained. To confirm that this fraction contained the protein < 8-12 kDa the partially purified fraction was run electrophoretically again on an analytical gel and a Western Blot was made using serum from patients with AHA (See Figure 6) which showed that the purified band subjected to migration had a molecular weight of < 8-12 kDa. Isoelectric focusing was performed with a pH range of 3-10 on a 5% polyacrylamide gel of the electroeluted fraction. In Figure 7, lanes A-G represent the known pl-markers and lane H represents the electroeluted fraction after dyeing with Coomassie Blue. Two bands indicated by arrows can be observed in lane H. The first band has a pH between 6.5 and 7.5 and the second band has a pl between pH 5.5 and 6.5 IC phase isoelectric focusing was also performed: MC (data not shown). The IC: MC fraction was incubated in water, and several buffer solutions, including acetate buffer, MES, phosphate buffer, NaCl, phosphate salts, Tris-HCl, HEPES, carbonates and bicarbonates at pHs of 10.0, 9.0, 8.0, 7.5, 7.0, 6.8, 6.5, 5.5, 5.0, 4.5, 3.6 and 3.0 for several hours. After the gel electrophoresis and immunoblot or Western-Blot were performed and positive antigen-antibody reactions were observed (see Figure 8). This proved the preservation of the antigenicity of the low molecular weight antigen in different buffer solutions at different pH, water and different precipitating salts values. It was found that the antigenicity was preserved since the antigen could be recognized by the antibodies present in the serum of patients with AHA. The protein < 8-12 kDa is shown with an arrow in lanes A-E. As shown in Figure 9, the culture medium in which E-histolytica grew. as well as IC: MC can be precipitated with ammonium sulfate at different concentrations. An 8 kDa band can be seen in lane B (where E-histolytica was grown) and lane E (IC: MC). The protein < 8-12 kDa can be precipitated with ammonium sulfate at 30, 50, 60, 70 and 80% saturation and probably with sodium sulfate. Figure 10 shows a Western-Blot of the < 8-12kDa precipitated at 60, 70 and 80% saturation and is present in both the precipitate and the supernatant of each of these treatments. Figure II shows a Western-Blot of an SDS-Page electrophoresis of the supernatants obtained after precipitating with rivanol (6,9-diamino-3-lactate of etoxiacridine) culture media where the amoebas grew, containing different concentrations of proteins . With this method, a large amount of albumin is precipitated, which is found in the culture medium and in the IC: MC. Some of the < protein is precipitated 8-12 kDa, but most remain in solution (see Figure II). Therefore, this method could be used as a previous step to precipitate undesirable proteins by conserving the proteins < 8-12 kDa in solution. The proteins of < 8-12 kDa can be bound to cationic or anionic exchange resins, such as Sp-Sephadex (Figure 12-C) or DEAE-Sephadex (See Figure 12-D) (See Figure 12, where the lower arrow is indicated). The methods described above can be used to isolate proteins < 8-12 kDa, as well as other proteins of interest that can be separated and purified from the medium where E. histolytica or IC: MC grew. Those proteins and in particular the < 8-12 kDa can be used as an antigen in diagnostic equipment for invasive amoebiasis, to investigate the liver damage caused by amoebas and as a basis for a vaccine against Entamoeba histolytica. The proteins < 8-12 kDa can be isolated from the culture medium where E. histolytica grew. precipitating it with rivanol at 3.5-14%. Taking the supernatant and dialyzing it against distilled water or a suitable buffer, concentrate the proteins. Then electrophoresis of the proteins in a preparative SDS-PAGE and electroeluted to obtain a protein band between 8-12kDa. The IC: MC fraction can be run electrophoretically directly in a preparative SDS-PAGE and be electroeluted to obtain a protein band between 8-12 kDa. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (43)

1. A diagnostic composition for the detection of antibodies directed against Entamoeba histolytica in a sample comprising: antigens preserved from Entamoeba histolytica that do not require the presence of enzymatic inhibitors to prevent proteolytic degradation, where said antigens are produced by: anoxic culture of E trophozoites. -histolytica: extracting an insoluble fraction of the trophozoites with polar solvents; drying the insoluble fraction to eliminate residues of the solvents; suspending the dry fraction in a buffer solution; heating the damped suspension for sufficient time and at a temperature sufficient to obtain the said antigens preserved from E-histolytica: and a suitable medium, wherein said antigens preserved from E. histolytica are present in sufficient amounts and are capable of binding to said antibodies directed against E-histolytica in said sample.

2. The diagnostic composition of claim 1, wherein the polar solvent is chloroform, methanol, ether, ethanol or mixtures thereof.

3. The diagnostic composition of claim 2, wherein the polar solvent is a mixture of chloroform and methanol.

4. The diagnostic composition of claim 1, wherein the heating of the quenched suspension is done in a water bath for approximately 2 to 20 minutes.

5. A method for detecting antibodies directed against E. histolytica in a sample comprising: incubating said diagnostic composition of claim 1 with a sample suspected of containing antibodies directed against Entamoeba histolytica. for a sufficient period of time and under conditions sufficient to allow antigen-antibody binding and to detect said binding or antibody-antigen binding.

6. The method of claim 5 also comprises: binding the preserved E. histolytica antigen of the diagnostic composition to a solid support, prior to said incubation step and detecting the antigen-antibody binding by adding a labeled antibody to the antigen-bound antibody and measuring labeling on said labeled antibody.

7. The method of claim 5, further comprising: attaching the preserved Entamoeba histolytica antigen of the diagnostic composition to a solid support prior to the incubation step; and detect antigen-antibody binding visually observing the agglutination of said antigen bound to a solid support.

8. An Entaba histolytica protein having a molecular weight of < from 8 to 12 kDa on an SDS-PAGE subjected to electrophoresis.

9. A method for purifying the protein of claim 8.

10. A diagnostic kit for detecting the presence of antibodies directed against Entamoeba histolytica in a sample including in separate containers: the diagnostic composition of claim 1; and labeled antibodies to detect the binding of said antigens preserved from Entamoeba histolytica in said diagnostic composition against said antibodies against Entamoeba hjstolytica.

11. The diagnostic equipment of claim 10 wherein said antigens preserved from Entamoeba histolytica are added to a solid support.

12. A diagnostic composition for the detection of antibodies against Entamoeba histolytica in a sample including: said gn to oeba histolytica protein of claim 8; and an appropriate medium, wherein said Entamoeba histolytica protein is present in sufficient amounts and is capable of binding said anti-Entamoeba histolytica antibodies to said sample.

13. A method for detecting anti-Entamoeba histolytica antibodies in a sample including: incubating said diagnostic composition of claim 12, with a sample suspected of containing anti-Entamoeba histolytica antibodies. for a sufficient period of time and under conditions sufficient to allow antigen-antibody binding; and detecting said antigen-antibody binding.

14. The method of claim 13, further comprising: attaching said Entamoeba histolytica protein of said diagnostic composition, to said solid support, prior to said incubation step; and detecting said antigen-antibody binding by the addition of labeled antibodies to said antibody bound to said antigen and measuring said labeling on said labeled antibody.

15. The method of claim 13, further comprising: attaching said Entamoeba histolytica protein of said diagnostic composition to a solid support prior to said incubation step; and detecting said antigen-antibody binding by visual observation of agglutination said antigen bound to a solid support.

16. A diagnostic kit to detect the presence of antibodies directed against Entamoeba histolytica. in a sample including in separate containers: said diagnostic composition of claim 8; and labeled antibodies for the detection of the binding of said Entamoeba histolytica protein. in said diagnostic composition, said antibodies directed against Entamoeba histolytica.

17. A method of purifying the protein of claim 8, comprising: adding IC: MC or culture medium where E-histolytica trophozoites were axenically cultured. in a preparative gel and running said gel under sufficient and appropriate conditions, to obtain the separation of said protein: by electroeluting said protein band.

18. A method of purification of the proteins of claim 8, characterized in that they perform isoelectric focusing techniques of the proteins.

19. A method of purifying the proteins of claim 8, characterized in that it comprises: the use of protein precipitating reagents.

20. A method of purifying the proteins of claim 8, characterized in that it includes: the use of ion exchangers.

21. A method of purifying the proteins of claim 8, characterized in that it includes: use of gel permeation chromatographs.

22. A method of purification of the antigen proteins of Entamoeba histolytica. identified in the Western blot pattern indicative of amoebiasis and recognized by the antibodies present in the sera of patients with amoebiasis, both of invasive amoebiasis and intestinal amoebiasis and whose antigens have the relative molecular weights in kDa of: 150, 29, 21, 20, 17, 16, 14 or 13.

23. A method of purification of the antigen proteins of Entamoeba histolytica. identified in the Western blot pattern indicative of invasive amoebiasis, and selectively recognized by the antibodies present in the sera of patients with invasive amoebiasis, said antigens have the relative molecular weights in kDa of: 8, 9, 10, 11, 23, 25 or 37.

24. A diagnostic kit to detect the presence of anti-E-histolytica antibodies. in a sample comprising in separate containers: the Entamoeba histolytica proteins of claim 22; and labeled antibodies to detect the binding of said proteins to said anti-E histolytica antibodies.

25. A diagnostic kit to detect the presence of anti-E histolytica antibodies. in a sample comprising in separate containers: said Entamoeba histolytica proteins of claim 23; and labeled antibodies to detect the binding of said proteins to said anti-E histolytica antibodies.

26. A diagnostic kit, wherein said proteins of claim 22 are fixed to a solid support.

27. A diagnostic kit for the detection of antibodies directed against Entamoeba histolytica in a sample including: said Entamoeba histolytica proteins of claim 26; and an appropriate medium, wherein said Entamoeba histolytica proteins are present in sufficient amounts and are capable of binding said anti-Entamoeba histolytica antibodies in said sample.

28. A diagnostic kit, wherein said proteins of claim 23 are fixed to a solid support.

29. A diagnostic kit for the detection of antibodies directed against Entamoeba histolytica in a sample including: said Entamoeba histolytica proteins of claim 28; and an appropriate medium, wherein said Entamoeba histolytica proteins are present in sufficient amounts and are capable of binding said anti-Entamoeba histolytica antibodies in said sample.

30. A vaccine against E. histolytica characterized because it comprises the fraction IC: MC.

31. A vaccine against E-histolytica characterized in that it comprises a mixture of the immunogens of relative molecular weights in kDa of: 8, 9, 10, 11, 23, 25 or 37.

32. A vaccine against E. histolytica characterized in that it comprises a mixture of immunogens of relative molecular weights of: 150, 29, 21, 20, 17, 16, 14 or 13 kDa.

33. A vaccine against E. histolytica characterized in that it comprises a mixture of the immunogens of relative molecular weights of 8 kDa (from 5 kDa to 12 kDa).

34. Antibodies capable of reacting with fraction IC: MC of claim 1.

35. Antibodies capable of reacting with the proteins that form the specific Western blot pattern of invasive amoebiasis, of claim 23.

36. Antibodies capable of reacting with the proteins that form the Western blot pattern characteristic of invasive amoebiasis, of claim 32.

37. Antibodies capable of reacting with the proteins forming the protein band of relative molecular weight of 8 kDa (5 to 12 kDa) included in the Western blot pattern characteristic of invasive amoebiasis, of claims 8 and 23.

38. A diagnostic composition of claim 1, comprising the following steps: obtaining fraction IC: MC; sonication of fraction IC: MC; mixtures of the resulting product with a diluent.

39. Antigens preserved from degradation, characterized by resisting repeated freezing-thawing and being stable for periods of more than one year.

40. A preserved antigen of the claim 39, characterized by a protein extract.

41. A preserved antigen of claim 39, characterized in that the protein extract comes from a biological sample having proteolytic activity.

42. A preserved antigen of claim 39, characterized in that the protein extract comes from a parasite.

43. A preserved antigen of claim 39, characterized in that the protein extract is stable in a pH range of 3-10. EXTRACT OF THE INVENTION The method of the invention relates to the preservation of a complex antigenic system of Entamoeba histolytica molecules. without using enzymatic inhibitors and using these preserved Entamoeba histolytica molecules as a reagent in a diagnostic assay system and as a starting material for the isolation of Entamoeba histolytica proteins.