MXPA99011805A - Methods for assaying antibody and device for assaying antibody - Google Patents

Abstract

Methods for accurately, conveniently and specifically assaying an antibody against a source of infection contained in a body fluid. More particularly speaking, a methodfor immunologically assaying an antibody by effecting an antigen-antibody reaction between the target antibody in a specimen and an antigen in the presence of a component originating in i(Escherichia coli);and a method for assaying an antibody by using as an antibody-detecting reagent one having a binding specificity to the Fc region of the antibody IgG.

Description

METHOD FOR ANALYZING ANTIBODIES AND DEVICE FOR ANTIBODY ANALYSIS TECHNICAL FIELD The present invention relates to a method for detecting or quantifying antibodies in samples and, more particularly, to a method by which antibodies against sources of infection such as bacteria and viruses can be detected or analyzed with great precision, when they occur in clinical samples of body fluids, particularly urine samples, in an expeditious manner and with good specificity. The present invention, in another aspect, relates to a device for detecting or quantifying an antibody present in a sample, and more particularly, to a device with which the antibody can be detected or analyzed with great precision against a source of infection that occurs in clinical samples of body fluids, particularly urine samples, in an expeditious manner and with good specificity. The invention further relates to a kit or kit of reagents for antibody analysis, which is useful for the above-mentioned antibody analysis method, and for the method of analysis using said antibody analysis device.

THE BACKGROUND TECHNIQUE The detection of antibodies, specific for various sources of infection (pathogens), such as bacteria and viruses, which can occur in body fluids, is an indirect means useful for diagnosing an infection. Therefore, until now, immunological analysis techniques and immunological analysis devices have been used to detect an antibody, using a pathogen or a component of the pathogen as the antigen of the analysis, in a broad field of diagnosis. Said method of immunoassay which uses a pathogen or a component thereof as the analysis antigen, is advantageous, since the necessary analysis system can be easily established, but it is not totally satisfactory in terms of sensitivity or specificity, which leaves need for improvements. As an immunoanalysis device for use in said immunological assays, there can be mentioned a strip of porous material, on which a binding analysis (antigen-antibody reaction) is carried. An analysis device of this type takes advantage of the capillary property of a porous substrate, that is, a body fluid applied to one end of a porous strip migrates to the other end. In this way, when a test sample (liquid) containing a substance to be analyzed is applied to one end of the strip carrying various reagents arranged successively in strategic positions, the sample migrates by capillary action along the length of the sample. pulls it and finds those reagents in these positions, in succession, to undergo reactions. The existence of the substance to be analyzed can be confirmed and its amount determined by detecting a detectable marker signal included in the ligand-receptor coupling system. The immunoassay technique using the above principle is often referred to as "immunocapillary analysis" or "immunochromatographic analysis," and has been described in WO No. 87/02774, EP No. 0306772, and in other publications. As regards the modifications of the technique, mention may be made of the inventions described in Japanese Unexamined Patent Publication No. 63865/1989, Japanese Unexamined Patent Publication No. 299464/1989 and Japanese Unexamined Patent Publication No. 167497/1994. The aforementioned device is advantageous since a specific instrument for the determination is not necessary, and the analysis can be easily completed, and within a short time, but there is a need for improvements in sensitivity and specificity. In addition, because the device performs only one test, a negative or positive control sample can not be determined concurrently; with the consequent disadvantage that it is impossible to judge whether the result is a reliable data, generated by the determination itself.

In general terms, urine and saliva, among the fluids of the body, are favored as samples for clinical analysis, because their collection does not require an invasive procedure and is easy and safe, compared to blood. However, it is usual for the concentrations of antibodies present in said samples to be extremely low, for example, of the order of one thousandth to one ten thousandth of the concentrations with which they appear in the blood. Additionally, urine samples collected from subjects who have taken large amounts of water are extremely thin, with the result that a large variation in antibody titer is inevitable between samples. In such cases, with the conventional analysis device described above, the test will be negative when the sample is too thin to detect an antibody, so that the problem arises that the case of a "true negative" can not be differentiated of the case of "negative (false negative)", caused by the low concentration that appears in the sample. Additionally, when testing lean samples for antibodies, a highly sensitive analysis system is needed; but in that case, there is a problem that the by-products formed by non-specific reactions, due to contaminants present in the samples, are susceptible to being detected simultaneously to give false positive results.

Therefore, an antibody analysis system that guarantees high enough detection sensitivity is necessary, even when body fluids such as urine and saliva are used, that is, a reliable analysis system that contributes to reduced probabilities of false negative tests. and false positives, thanks to its high specificity. It is the main objective of the present invention to provide a technology for antibody analysis (antibody analysis method and device for antibody analysis) that is capable of detecting antibodies against sources of infection that occur in test samples, such as fluids. of the body with high sensitivity and high specificity. It is the second objective of the present invention to provide an antibody analysis method that allows high precision determinations, by suppressing the "false positive" reactions arising from contaminants present in the samples, even when the samples are those of urine or other body fluid that are substantially poor in the antibody being sought. It is the third objective of the present invention to provide an antibody analysis method as an improvement in immunocapillary analysis or immunochromatographic analysis, by which the existence and quantity of the antibody being sought can be accurately determined as an object of detection in a sample, with a clear demarcation between a "false negative reaction" that is due to the nature of the sample, and a "true negative" reaction.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing a solid phase support, in the form of a strip of a constituent element of the device for analysis of antibodies of the invention. In Figure 1, the code 1 represents a first region; 2, a tracer region; 3, a second region; 4, a third region; 5, a test zone and 6, a control zone. Figure 2 is a diagram illustrating the principle of analysis of the antibody that is sought, in a sample, with the device for antibody analysis of the invention. The respective codes used have the same meanings as in Figure 1. Figure 3 is a schematic diagram showing a solid phase support strip (A), and a housing (B) accommodating the solid phase support included in the device for analysis of antibodies of the invention. In Figure 3, codes 1 to 6 have the same meanings as in Figure 1; and the code 7 represents a top section of the housing; 8, a lower section thereof; 9, a sample admission port; and 10, a detection window. Figure 4 is a diagrammatic representation of the determination results of the anti-/ - / antibody. pylori in the urine, of example 1 (5) (i).

In figure 4, the light circles represent data about the urine samples of subjects with H. pylori infection, who gave a positive 13C-UBT test; and the dark circles represent data on urine samples from subjects who gave a negative 13C-UBT test. Figure 5 is a diagrammatic representation of the anti-H antibody data. pylori in the urine, as determined in example 1 (5) (ii). In figure 5, the ordinate represents the absorbance (O. D. 450 nm) and the abscissa represents the groups positive for H. pylori and negative for H. pylori, established according to the 13C-UBT test. Figure 6 is a diagrammatic representation of the data about the anti-H antibody. pylori in urine, as determined in example 1 (6). In figure 6 the light circles represent data on urine samples of subjects who tested positive on 13C-UBT; and dark circles represent data on urine samples from subjects who tested negative for 13C-UBT. Figure 7 is a diagrammatic representation of data about anti-HBc antibody in urine, as generated in example 2 (2). In Figure 7, the dark circles represent data on urine samples from subjects who tested positive for anti-HBc antibodies in the blood, and clear circles represent data on urine samples from subjects who were negative in the analysis of anti-HBc antibodies in the blood. Figure 8 is a diagram showing the gel permeation chromatograms of urine samples giving false positive reactions in the determination of anti-HIV antibody in urine and the antibody reactivity of each fraction (example 3 (1)). In Figure 8, the ordinate represents the absorbance (O.D.) and the abscissa represents the chromatographic fraction of gel permeation (fraction number). The solid line represents the absorbance of the protein at 280 nm; the line of black dots represents the data generated with the human anti- (IgG + IgM) antibody; the line of clear triangles represents the data generated with anti-human IgG antibody (specific for Fe); and the line of dark triangles represents data generated with the anti-human IgG antibody (specific for Fab). Figure 9 is a diagrammatic representation of data about the anti-H antibody. pylori in the urine, as determined in example 5. In figure 9, the ordinate represents the absorbance (OD 450-650 nm) and the abscissa represents the group positive for H. pylori (+: n = 56) and the negative group to H. pylori (-: n = 44), as classified by the 13C-UBT analysis. Figure 10 is a diagrammatic representation of the anti-rubella antibody data in the urine, as generated in example 6. In figure 10 the ordinate represents the absorbance (OD 450-650 nm) and the abscissa represents the group positive to the anti-rubella antibody (+: n = 76) and negative to the anti-rubella antibody (-: n = 23), according to was classified according to the level of serum measured with a commercial team. Figure 11 is a diagram showing the assay site and the control site in the second region of the antibody assay device of the present invention (example 7 (3)).

Figure 12 is a histogram showing the analysis data on anti-H antibody. pylori in the urine, in whole blood and in plasma, as generated by the device for antibody analysis of the invention, in comparison with the corresponding data, generated with control devices (A-E). In figure 12, "specificity" represents the percentage of negative analyzes (negative rate), with respect to the total number of analyzes, when it was determined that the samples of subjects verified by the 13C-UBT analysis were negative, for each line of the analysis; with each analysis device; and "sensitivity" represents the percentage of positive analyzes (positive rate) with respect to the total number of analyzes, when it was determined that the samples of subjects verified by the 13C-UBT analysis were positive, for each line of analysis, with each analysis device. The control devices A-H mean the following devices: A) Helitest (manufactured by Cortees Diagnostics), B) H. pylori-Check-1 (manufactured by Bio-Medical Products) C) First Check H. pylori- (manufactured by Worldwide Medical Corp). D) Biocard Helicobacter pylori IgG (manufactured by Anti Biotech Oy) E) Insta Test H. pylori (manufactured by Cortez Diagnostics, Inc.) F) One Step H. pylori Test (manufactured by Teco Diagnostics G) H. pylori SPOT (manufactured by International Immuno-Diagnostics) H) Quick Stripe H. pylori (manufactured by Diatech Diagnostics, Inc.).

DESCRIPTION OF THE INVENTION The inventors of the present invention carried out much research to establish an analysis system that would allow the determination, with great precision, of the desired antibodies, even when the samples were poor in antibodies, for example, urine samples; and found that there exists in the assay system an antibody component that binds non-specifically to the antigen in an antigen-antibody reaction (hereinafter referred to as the non-specifically binding antibody component) that results in nonspecific reactions, thus causing a false positive result and, consequently, decreasing the accuracy of the detection. Based on the above-mentioned discovery, the inventors continued to investigate and found that such non-specific reactions can be suppressed by carrying out the antigen-antibody reaction between the antibody sought on which the analysis is to be made, and the antigen specific for the Particular antibody, in the presence of a component of Escherichia coli (E. col?), which can reduce the false positive rate, to obtain a significant improvement in the accuracy of detection. The inventors further discovered that the non-specifically binding antibody component comprises fragments of IgG and / or its denaturing products, which retain the antigenicity of the light chain (L) or the F (ab) region of the IgG and that this component of the antibody cross-reacts with the ordinary reagents of antibody assays (eg, secondary antibodies) used in the serum antibody analysis systems, thereby giving false-positive analyzes. Based on the above findings, the inventors of the present invention further confirmed that non-specific reactions in an antibody analysis system can be inhibited by using a reagent having specific affinity for the Fe region of the antibody IgG sought in the assay , as a reagent of the antibody analysis, with which the false positive rate can be reduced to improve the detection accuracy, to an important degree. Meanwhile, the inventors devoted themselves to improving the instrument for the analysis of the antibody (the immunocapillary analysis device and the immunochromatographic analysis device) and found that the "true negative" reactions could be accurately detected excluding the "false negative" reactions. by establishing a "control site" to detect an arbitrary antibody in the samples, in addition to the site (site of analysis) to detect the antibody sought in the reaction zone (evaluation zone) of the strip, as part of the analysis device . Thus, in such an analysis system, when the sample is an improper sample that can not be analyzed for reasons such as too low antibody concentration (ie, the total amount of the antibody is too small), the "control site" "gives a negative signal, which indicates that the sample is not analysable. On the other hand, when the sample has an appropriate concentration of antibody, the "control site" gives a positive signal, which indicates that the sample is appropriate for the intended analysis of the antibody sought. Then, according to the results in this "site of analysis", one can know with certainty the presence or absence of the antibody sought in the sample; that is, if the sample is "positive" or "true negative". In this context, both Unexamined Japanese Patent Publication No. 299464/1989 and Japanese Unexamined Patent Publication No. 167497/1994 describe improvements in artifacts for antibody analysis (the immunocapillary analysis device and the immunochromatographic analysis device. ); describe devices that include a control site in addition to an analysis site. Nevertheless, the control site in those devices is intended to determine whether a label disposed in a region upstream of the strip, has passed or not through the analysis site, due to capillary action and, therefore, is quite different from the site of control in accordance with the invention. The inventors hereby additionally confirmed that, when the coupling reaction between the antibody being sought and the corresponding antigen, by means of the above improved device for antibody analysis, is carried out in the presence of the component of £. coli, the non-specific reaction in this antigen-antibody reaction system is inhibited; and that when a reagent having specific affinity for the Fe region of the IgG is used, as a reagent of the antibody analysis, the non-specific reaction with the antigen-antibody complex is inhibited, which leads to a significant decrease in the incidence of the false positive result of the analysis. The present invention has been developed on the basis of the various discoveries noted above. In a first aspect thereof, the present invention provides a high precision method for analyzing an antibody with reduced incidence of false positive reaction. (1-1) .- As a mode of this, the above-mentioned antibody analysis method, for detecting a desired antibody in a sample, using an antigen-antibody reaction is characterized in that the reaction is carried out between the antibody and the antibody. antigen analysis in the presence of an E. coli component. This method for analyzing an antibody includes the following specific methods: (a) the antibody analysis method, in which the E. coli component is at least one member selected from the group consisting of the soluble fraction and the fraction of lipopolysaccharide of Escherichia coli. (b) The method of antibody analysis, wherein the E. coli component is used in a proportion of about 0.1-100 μg, preferably about 0.5-50 μg per microgram of the test antigen. (1-2) .- As another mode, the method of antibody analysis comprises detecting a desired antibody in a sample by the sandwich technique, characterized in that a reagent comprising a secondary antibody having specific affinity for the Fe region is used. of the IgG of the antibody sought, as a reagent of the antibody analysis. This method for analyzing an antibody includes the following specific methods: (a) the antibody analysis method, wherein the secondary antibody is an anti-IgG antibody specific for Fe; (b) the method of antibody analysis comprising an antigen-antibody reaction step, wherein the antibody sought from the sample is coupled to an immobilized antigen, specific for the antibody that is immobilized on a support; and a reaction step in which the target antibody, captured by the immobilized antigen, is reacted with a secondary antibody having specific affinity for the Fe region of the antibody IgG; (c) The above antibody analysis method, in which the antigen-antibody reaction is carried out in the presence of an E. coli component.

In a second aspect, the present invention relates to a device for the analysis of antibodies. This device includes the following modalities: (2-1) .- A device for antibody analysis comprising a solid phase support having at least: (a) a first region to which a sample is applied, and (b) ) a second region in which the antibody present in the test sample is reacted, when arranged in such a sequence, that the sample is transported from the first region to the second region, by capillary action; and a marker means for detecting the reaction result in the second region; said (b) second region has: (i) an analysis site in which a ligand has been immobilized to capture the antibody sought; and (i) a control site in which a ligand has been immobilized to capture an arbitrary antibody present in the sample. (2-2) .- The device for antibody analysis in which the ligand immobilized at the site of analysis is an antigen for the antibody sought, which occurs in the sample. (2-3) .- The device for antibody analysis in which the ligand immobilized at the control site is a human anti-immunoglubulin antibody, capable of capturing an arbitrary antibody present in the sample. (2-4) .- The device for antibody analysis comprising a labeled ligand that is going to bind both the desired antibody and the arbitrary antibody, as said marker means. (2-5) .- The device for antibody analysis, in which the labeling means is a labeled ligand that is going to bind both the desired antibody and the arbitrary antibody, as detachably supported upstream of the second support region of the antibody. solid phase, in such a way that, when contacted with a sample, it reacts with the intended antibody and with the arbitrary antibody to form a searched antibody / labeled ligand complex and an arbitrary antibody / labeled ligand complex, respectively, which are then transported by capillary action to the second region, where they are fixed at the analysis site and the control site, respectively. (2-6) .- The device for antibody analysis in which the labeled ligand is supported in a region (the tracer region) intermediate between the first region and the second region of the solid phase support. (2-7) .- The device for antibody analysis, wherein the labeled ligand to be bound to both the antibody sought and the arbitrary antibody, is a labeled anti-human immunoglobulin antibody. (2-8) .- The device for antibody analysis, wherein the anti-human immunoglobulin antibody is an anti-IgG antibody having specific affinity for the Fe region of immunoglobulin G. (2-9) .- The device for antibody analysis, wherein the solid phase support is additionally provided with an absorption region current below the first and second regions, so that the sample transported from the first region to the second region, is transported further, by capillary action, to the absorption region. (2-10) .- The device for antibody analysis in which the coupling reaction of the desired antibody is carried out in the test site, in the second region, in the presence of an E. coli component. In a third aspect, the present invention relates to a method for solid phase analysis of a desired antibody, in a sample. This method includes the following modalities: (3-1) .- A method for solid phase analysis of a desired antibody, which comprises applying the sample to the first region of the device for antibody analysis, and detecting the development of a color in the analysis site in the second region, under the condition of the control site in the second region, which develops a color. ((3-2) .- The method for solid phase analysis of a desired antibody, wherein the coupling reaction of the antibody sought at the site of analysis in the second region of the device for antibody analysis is carried out in presence of an E. coli component In a fourth aspect, the present invention relates to a kit or kit of reagents for antibody analysis, for use in association with the device for antibody analysis. of antibodies can include the following modalities: (4-1) .- A kit or kit of reagents for antibody analysis, characterized in that it comprises an E. coli component. (4-2) .- The kit of reagents for analysis of The antibody additionally comprises an antigen or an antibody analysis reagent, which is optionally immobilized. (4-3) .- The reagent kit for antibody analysis is characterized in that it contains an antibody. anti-IgG specific for Fe, as an antibody analysis reagent. (4-4) .- The reagent kit for antibody analysis contains the device for antibody analysis according to the present invention. (1) .- THE ANTIBODY ANALYSIS METHOD First, the method of antibody analysis is now described in detail, as the first aspect of the present invention. The method of antibody analysis of the present invention represents an improvement in the immunoassay method of antibodies, and is characterized in that the incidence of false positive results in the reaction can be decreased by inhibiting the non-specific reaction. (1-1) .- As a modality of the aforementioned method of antibody analysis, a method can be mentioned in which the antigen-antibody reaction between the searched antibody present in a sample and an antigen specific for that antibody, is carried out in the presence of an E. coli component. In accordance with this method, the non-specific reaction in the antigen-antibody reaction is significantly inhibited, with the result that the incidence of a false positive in the analysis can be decreased. The E. coli component is not particularly restricted, as long as it is a component of Escherichia coli; thus, it includes, but is not limited to, the protein component, the carbohydrate component or the lipid component thereof, or a mixture of such components. As a preferred example, there may be mentioned a soluble fraction or the lipopoiisaccharide (LPS) fraction of E. coli. There is no particular limitation as to the method for preparing said E. coli component, but a variety of methods can be selectively used. A common procedure may involve developing an arbitrary strain of E. coli in a medium suitable for proliferation; harvest the developed cells, and physically disintegrate the cells by means of an apparatus for sonic treatment, or by solubilizing them with a surfactant or the like, to provide a soluble fraction (extract). The LPS mentioned above can be prepared by an extraction process, using an organic solvent, for example, phenol, chloroform or ether, or a mixture of two or three different organic solvents. It can also be prepared artificially, using a genetic engineering technique. Additionally, commercial products can be used expeditiously (for example, the E. coli liposaccharide, which can be obtained from Difco or from Sigma). The preferred sample to which the invention can be applied is a sample of body fluid. The fluid of the body is not restricted, as long as it is a body fluid derived from a human or other animal, in which the antigen sought is supposedly contained. Thus, the term "body fluid" covers a wide variety of biological fluids that are used as samples in routine laboratory tests. More particularly, the body fluid includes: blood, including serum and plasma; urine, cerebrospinal fluid, amniotic fluid, saliva, sweat, and others. In particular, the present invention solves the problem of poor detection accuracy, associated with non-invasive samples, which are favored as samples for the detection of antibodies, such as urine, saliva and sweat, particularly urine and, for Consequently, these biological materials can be mentioned as the preferred examples of body fluid. The "target antibody", the object of the determination, is not restricted in particular, as long as it is an antibody whose detection is desired; including, in that way, antibodies against different sources of infection, which are foreign bodies to the host.

The sources of infection are not particularly restricted, but include many different pathogens that infect man and other animals and give rise to antibodies in the hosts. More particularly, the aforesaid pathogens include a variety of viruses, such as HIV (human immunodeficiency virus), hepatitis viruses types A, B, C and others; rubella virus, influenza virus, mumps virus, cytomegalovirus, simplex virus, varicella-zoster virus, adenovirus, entorovirus, etc .; bacteria, such as Helicobacter pylori (hereinafter abbreviated H. pylori), Clamydia spp., Mycobacterium tuberculosis, spirochetes, gonococci, Treponema pállidum, Mycoplasma spp., etc. (excluding Escherichia coli), and protozoa, such as Toxoplasma gondii, Entamoeba histolytica, Rickettsia tsutsugamushi, and others. Preferred are viruses, such as HIV, hepatitis virus, rubella virus, influenza virus, mumps virus, herpes virus, etc .; and the bacteria, represented by Helicobacter pylori, etc .; bacteria being particularly preferred, such as H. pylori. The antigen for use in the antibody analysis method of the present invention is not restricted in particular, as long as it is an antigen capable of undergoing the antigen-antibody reaction with the desired antibody to be detected. Thus, for example, any of the antigens used in the conventional antibody analysis system in serum can be used successfully. These antigens can not only be the pathogens themselves, such as the viruses and bacteria mentioned, but, in addition, they can also be the antigens that have the antigen-determining groups, intrinsic to the respective pathogens. Thus, for example, the inactivated pathogens, available by heat treatment or by irradiation of the pathogens, the antigens prepared by extracting the pathogens with a surfactant or the like, and the antigens artificially prepared by chemical synthesis or by recombinant DNA technology. Incidentally, whether a candidate antigen can be used satisfactorily or not in the method of analysis of the present invention can be easily determined, typically by analyzing its reactivity with the desired antibody, in the conventional manner. In the method of analysis of the invention, the immobilized antigen can optionally be used in an arbitrary solid phase, in advance. The solid phase mentioned just above may be any of the various solid phases that are routinely used in this field of the art, including, but not limited to, sticks, pellets, plates (including microtiter plates) and the test tubes, made of various materials, for example: glass, cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethylcellulose, ion exchange resins, dextran, plastic film, plastic tubes, nylon, pearls of glass, silk, polyamine-methylvinyl ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, and others.

The method for immobilization is not particularly restricted either; but it can be any of between physical union and chemical union. For example, chemical binding methods, such as covalent binding methods, for example, the diazo method, the peptide method (the acid amide derivative method, the carboxyl chloride resin method, the of the carbodiimide resin, the maleic anhydride derivative method, the isocyanate derivative method, the bromocyano activated polysaccharide method, the cellulose carbonate derivative method, the condensation reagent method, etc.), the alkylation method, the method of coupling with the entanglement agent (the method for coupling to a support using glutaraldehyde, hexamethylene isocyanate or the like, as an entanglement agent); the coupling method by the reaction of Ugi, etc .; the ionic bonding methods, which use ion exchange resins and similar supports; and physical adsorption methods, which use glass beads or other porous glass supports. The amount of the antigen to be used in the analysis system is not restricted in particular, but can be freely selected in accordance with the amount of the antigen that is being routinely used for the particular analysis system. For example, when the sandwich method is used, the antigen in excess is generally used with respect to the target antibody. Taking as an example the case in which the reaction is carried out in a 100 μl reaction system, the antigen can be used in a proportion generally of about 0.1 to 100 μg / ml, preferably about 1-10 μg / ml. ml. The conditions of the antigen-antibody reaction between the antigen and the antibody sought are not particularly restricted, but may be the same as in routine use for conventional immunoassays, except that the reaction must be carried out in the presence of a component of E. coli. A typical procedure may comprise incubating, or allowing said antigen, said antibody, and said E. coli component to remain together, at a temperature generally not exceeding 45 ° C, preferably from about 4 to 40 ° C, more preferably, about 25 to 40 ° C, for about 0.5-40 hours, preferably, about 1 to 20 hours. The solvent for use in the reaction, and its pH, are not particularly restricted, as long as they do not interfere with the reaction. Thus, conventional regulators showing a regulatory action on the pH scale of about 5 to 9 can be used, such as: citrate regulator, phosphate regulator, tris regulator, acetate regulator, etc., in general, in a routine manner . The proportion of the E. coli component in this reaction system is not particularly restricted, but can be, for example, in general, from about 0.1 to 100 μg, preferably about 0.5 to 50 μg, per microgram of the antigen present in the reaction system. The method for practicing the antibody analysis method of the present invention is not restricted in the particular, except for the basic requirement that it must comprise a reaction step of the antigen and the antibody; wherein the antibody sought is reacted with the corresponding antigen, which may be an immobilized antigen, in the presence of said component of E. coli. However, in general, the method further comprises a step of detecting the target antibody captured by said antigen (antigen-antibody complex), i.e., a step of reacting the antigen-antibody complex with the assay reagent. antibody. The method for detecting and quantifying the antigen-antibody complex obtained by said antigen-antibody reaction, and the conditions thereof are not particularly restricted, but may be those that are routinely used for immunoassays in general. It is preferred to practice the present invention by means of the sandwich method. In the solid-phase sandwich method, for example, the antibody sought in a sample can be analyzed, typically, by the following procedure: Firstly, an E. coli component and a sample that is supposed to contain the desired antigen are added. (a body fluid, such as urine), to a solid-phase antigen, which is an immobilized antigen, capable of being subjected to a specific antigen-antibody reaction, with the desired antibody, in order to carry out in that way a Antigen-antibody reaction. After unbound substances are removed by washing, which were not coupled to the solid phase antigen, for example, an antibody analysis reagent is added to react with the desired antibody, coupled to the solid phase antigen (antigen-antibody complex) and an antigen complex is detected -anti-body or is analyzed quantitatively by means of detection that corresponds to the analytical reagent in particular. The selection and modification of various means for such analyzes are well known to those skilled in the art, and any of those techniques can be used in the practice of the present invention (For example, Rinsho Kensa-hou Teiyo (Guidelines for Analysis Clinical), Kanehara Publishing Co., 1995) The reagent for antibody analysis, which is to be used herein, is not particularly restricted, but includes a variety of reagents that are routinely used in the art. For example, secondary antibodies, such as the human anti-immunoglobulin antibody, capable of binding to the target antibody (immunoglobulin) can be mentioned. The anti-human immunoglobulin antibody mentioned above includes antisera, purified products thereof (polyclonal antibodies) and monoclonal antibodies, obtainable from arbitrary animals, immunized using an immunoglobulin, of the class corresponding to the immunoglobulin sought. , as an immunogen. Additionally, as an antibody assay reagent, an anti-IgG antibody having a specific affinity for the Fe region of the target antibody (IgG) can also be used. As such, an anti-IgG antibody, an anti-IgG antibody specific for Fe, which is not reactive with the IgG light chain or the F (ab) region of IgG or protein A, protein G may also be used. or similar, that is specifically reactive for the IgG Fe region. These can be used with particular advantage when the antibody sought is an IgG. These antibody analysis reagents can be prepared in a conventional manner, or they can be purchased from commercial sources. For detection, the antibody analysis reagent can be directly modified with a conventional labeling agent, or it can be modified indirectly by some additional detection means. The labeling agent is not particularly restricted, but any of the agents known up to now, or which are expected to come into use in the future, may be employed. To mention some specific examples, radioisotopes such as 125l, 3H, 14C, etc. can be used.; enzymes such as alkaline phosphatase (ALP), peroxidase (eg, HRP), etc .; fluorescent substances, such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (RITC), etc .; 1 N- (2,2,6,6-tetramethyl-1-oxy-1,4-piperidyl) -5 N- (aspartate) -2,4-dinitrobenzene (TOPA), etc. The immunoassay methods, which utilize the marker agents mentioned hereinabove, are referred to, respectively, as radioimmunoassay, enzyme immunoassay, fluoroimmunoassay, and centrifugation immunoassay, respectively. The immunochromatoanalysis method can also be used, which uses an antibody analysis reagent, prepared by latex marker particles, stained with colloidal gold. Labeling with these marker agents, modifications by indirect labeling, and their detection, can be carried out by methods known per se (Tatsuo Iwasaki and co-authors: Monoclonal Antibody, Kodansha Scientific, 1984, and Eiji Ishikawa and co-authors: Enzyme Immunoassay , 2nd edition, Igaku Shoin, 1982, among others). In the method of analysis of the invention it is essential that an E. coli component be included in the reaction system comprising the desired antibody to be analyzed, and the corresponding antigen, and to the extent that this requirement is met. , the remainder of the basic procedure is not particularly restricted, but may be the same as that used in conventional immunoassays, or that is routinely employed in the art. Accordingly, the conditions of the reaction between said antigen-antibody complex and the antibody analysis reagent are also not particularly restricted, but may be the same as those used in immunoassays in general. The most common procedure comprises incubating or allowing the analysis system to remain together, under the same condition as described hereinabove for the antigen-antipobody reaction; that is, in general, at a temperature not exceeding 45 ° C, preferably at a temperature of about 4 to 40 ° C, more preferably, at about 25-40 ° C; and at a pH level of about 5 to 9, for about 0.5 to 40 hours, preferably about 1 to 20 hours. The presence or absence of the desired antibody in a sample, or its content, is evaluated by measuring the activity of the label or label, which depends on the class of labeling agent used in the labeling or labeling of the antibody analysis reagent (or the label). indirect), in a routine manner, or in terms of the antibody titer, calculated from the measured value. (1-2) .- As an alternative mode of the antibody analysis method of the present invention, there may be mentioned an immunological method for the analysis of the desired antibody, in a sample, which comprises using a reagent having a specific affinity for the Fe region of the IgG of the antibody sought, as an antibody assay reagent. According to this method, the reaction between the antibody component that does not bind specifically, and the antibody analysis reagent, can be suppressed significantly, so that the frequency of false positives in the assays can be decreased. In this way, this method of antibody analysis can be considered as an improvement in the sandwich method, for the immunoassay of antibodies. This antibody assay reagent of the present invention is reactive for the antibody sought (the IgG) and, therefore, can be used for the detection of the antibody; and is characterized in that it is not reactive for the light chain of the IgG of the antibody sought, nor for the F (ab) region of the IgG sought; that is, it has specific affinity for the Fe region of the IgG sought. More particularly, said antibody analysis reagent, for example, can be an anti-IgG antibody specific for Fe, which can be prepared using the Fe region of the IgG of the desired antibody, as an immunogen; and includes antisera, their purified products (polyclonal antibodies) and monoclonal antibodies, which can be obtained from arbitrary animals, immunized with said immunogen. This reagent is not limited to said antibody preparations, but may be protein A, protein G or the like, which are especially reactive for the Fe region of the antibody IgG. These antibody assay reagents can be prepared in the routine manner, or can be purchased from commercial sources (eg, Sigma, Cappel or Jackson Immuno Research Laboratories, Inc.).

For detection, the antibody assay reagent can be modified directly with a conventional labeling agent or can be modified indirectly by means of an additional detection means. The marker agent class, the method to mark and the method of detecting the label or marker can be the same as those mentioned under (1-1). The method of antibody analysis of the present invention comprises the use of the antibody analysis reagent mentioned above, as an essential aspect thereof, in the detection of the antibody sought, ie, the antigen-antibody complex; and as far as this requirement is concerned, the rest of the basic procedure can be released in the same way as is used in conventional immunoassays by the sandwich technique. Basically, the antibody analysis method of the present invention is practiced by reacting an antigen that is capable of reacting with the desired antibody present in the sample, and detecting the desired antibody bound to the antigen (antigen-antibody complex) with said antigen. antibody analysis reagent. The test sample and the antigen may be, respectively, equal to those mentioned under (1-1), and as for the antibody sought, the same antibodies that were mentioned may also be used (1-1), provided that when the antibody is an IgG of antibody against the source of infection. In the method of the present invention, the source of infection may include Escherichia coli. When necessary, the antigen or antibody assay reagent of the present invention can be used by being immobilized on an arbitrary solid phase. The solid phase for use and the immobilization method, for example, may be the same as those mentioned under (1-1). The antigen-antibody reaction between the antigen and the antibody sought is not restricted in particular, but can be carried out under the conditions of routine use for conventional immunoassays. A typical method comprises incubating, or allowing a reaction system comprising the desired antigen and antibody to remain together, generally at a temperature not exceeding 45 ° C, preferably about 4 to 40 ° C, more preferably, about 25 to 40 ° C, for about 0.5 to 40 hours, preferably about 1 to 20 hours. Although not imperative, an E. coli component can be made to be present in the antigen-antibody reaction, as mentioned under (1-1). The resulting antigen-antibody complex is then tested and subjected to a step in which it is reacted with said specific reagent for antibody analysis. This reaction can be carried out under the same conditions that are generally used in, or in substantially the same manner as in, conventional immunoassays (sandwich analysis). For example, the solvent is not restricted in particular, as long as it does not interfere with the reaction; including, but not limited to, regulators for pH from about 5 to 9, such as the citrate buffer, the phosphate buffer, the tris buffer and the acetate buffer, to mention only a few examples. The time of the reaction and the temperature of the reaction are also not restricted in the particular, but, for example, may be the same as those mentioned above for the antigen-antibody reaction. The presence or absence of the desired antibody in a sample is evaluated, or its content is evaluated by measuring the activity of the label or label, which depends on the kind of labeling agent used in the labeling of the reagent of the antibody analysis (or the label or label). indirect) in the routine manner, or in terms of the antibody titer, calculated from the measured value, just as mentioned under (1-1). (2) .- THE DEVICE FOR ANTIBODY ANALYSIS The device for antibody analysis according to the second aspect of the present invention will now be described in detail.

The device for antibody analysis of the invention is an improved method by which the presence and / or quantity of the desired antibody, which is to be detected in a sample, can be determined with good precision, by means of an analysis procedure. in solid phase. More particularly, the device for antibody analysis of this invention is a device for analysis comprising a solid phase support having at least (a) a first region, for contacting a sample; and (b) a second region for reacting the antibody present in the sample, when arranged in such a sequence, that the sample is transported by capillary action from the first region to the second region; and a label or label means for detecting the result of the reaction in said second region; characterized in that said (b) second region has: (i) an analysis site, wherein a ligand is immobilized for the desired antibody to be analyzed; and (ii) a control site, wherein a ligand is immobilized to capture an arbitrary antibody present in the sample. The outstanding aspect of the antibody analysis device of the invention is that a control site is provided, independent of the analysis site, in the second region; and said control site is of such a nature that, when an appropriate sample is applied and analyzed in an appropriate manner, it forms an indication that represents a positive analysis in the presence of a label or label, regardless of whether or not it is present in the desired antibody sample, while, when an inappropriate sample is applied or a sample is inappropriately analyzed, it forms an indication representing a negative analysis in the presence of a label or label, regardless of whether the antibody sought is present or not in the sample. Thus, the control site in the second region of this device is a site that gives an indication of whether the result (particularly the negative result) at the analysis site is a valid result of analysis, regardless of the presence or absence of the antibody sought in the sample. With the device for antibody analysis of the invention, thanks to the above construction, it is possible to determine, both qualitatively and quantitatively, the antibody of the sample with high precision (high reliability) with a clear distinction between false negative and true negative results. Additionally, the antibody analysis device of this invention can be designed in such a way that the reaction of the desired antibody is carried out at the site of analysis, in the presence of an E. coli component, or it is designed in such a way that a reagent having specific affinity for the Fe region of the IgG of the target antibody, as an antibody assay reagent, is used to detect the result of the reaction at the site of analysis. With the device for antibody analysis of the present invention, thanks to the construction described above, non-specific reactions are inhibited, so that the incidence of a false positive analysis is significantly decreased, thereby making it possible to determine both qualitative as quantitatively, the antibody sought in the sample, with good precision and high sensitivity. As a sample to be analyzed and as a sought antibody, for this device for antibody analysis of the invention, it is possible to use, for example, those mentioned under (1). The present invention first provides a solid phase support comprising at least a first region and a second region. The first region is an area in which the applied sample comes into contact with the device; and the second region is an area in which the antibody (the total antibody that may contain the desired antibody) present in the sample is subjected to the reaction and to the coupling in the ligand-receptor mode, or in the antigen- antibody; and the result of the reaction is displayed in the presence of a label or marker (reaction zone and evaluation zone). These regions are arranged on a solid phase support, in such a way that the applied sample, which enters the first region, is transported by capillary action from said first region to the second region. It is preferred that these regions are arranged in such a way that all, or at least a portion, of the sample entering the first region, is displaced by capillary action, through a substantially flat layer of the solid phase support, to the second region. Optionally, the solid phase support may have a third current region below the second region, such as a region that absorbs the sample (liquid) that migrates, by capillary action, from the first region to the second region, and further downstream. The preferred solid phase support is formed in the form of a strip, and said first and second regions are disposed in one and the same plane of the strip, in such a way that the applied sample is displaced, by capillary action, from a first band (first region) to a second band (second region) and, optionally, further to a third band (third region). While the preferred form of the solid phase support is a strip, as mentioned above, any other shape or geometry may be employed, provided it is possible to implement the expected functions of the solid phase support in the present invention. The solid phase support is capable of absorbing the sample (liquid) and, when moistened with the sample, allowing at least the antibody present in the sample to be displaced, by capillary action, from the first region to the second region of the sample. solid phase support and, optionally, further, to the third region. In addition, the solid phase support is preferably one that is capable of supporting and immobilizing a ligand that reacts with the antibody (including the desired antibody) contained in the sample to capture the latter. The solid phase support itself includes a variety of porous materials, for example, polyethylene, fiberglass, cellulose, rayon, nylon, interlaced dextran, various types of chromatographic paper, nitrocellulose and filter paper.The first region, the second region and the third region, for example, of the solid phase support, can be constituted, respectively, by the same members or by different members, selected from among the above-mentioned materials; depending on the selection of the materials of the papers and the functions that the respective regions have. The first region of the solid phase support, preferably, is constituted by a porous material adapted to absorb the sample applied on its surface and leave it to be displaced, by capillary action, to the second region. The porous material suitable for the first region is not particularly restricted, but, in general, polyethylene (e.g., POREX, Porex Technologies, Fairbum, GA, USA), glass fiber, rayon, nylon and cellulosic materials can be used, including paper. The preferred material is a porous polyethylene or a cellulosic material, such as filter paper. The second region of the solid phase support is preferably constituted by a porous material, which is capable of allowing the sample (the liquid) to be transferred by capillary action from the first region to the second region, and which bears a ligand for the antibody (in which the desired antibody is included) that is found in the sample, in a condition not dislodged by capillary action. The porous material that has such properties includes: filter paper, chromatogram paper, fiberglass, interlaced dextran, nylon, nitrocellulose, etc. The preferred material is nitrocellulose, because the ligand can be easily immobilized on it. The second region is provided with an analysis site, in which a ligand, adapted to specifically recognize the target antibody present in the sample and capture it, has been immobilized; and a control site, in which a ligand adapted to recognize an arbitrary antibody present in the sample and capture it has been immobilized. The control site is disposed away from the analysis site, with a given interval between them, preferably downstream in the direction of capillary flow, and is preferably arranged in such a way that both sites come into contact with the front of the sample liquid , under identical conditions. The ligand for the analysis site is not restricted in particular, provided that it is specifically coupled to the desired antibody to be analyzed, but preferably it is an antigen that is specifically recognized and bound by the antibody sought (the antigen reaction). -antibody). As the antigen that has just been mentioned here above, those antigens which are of routine use in a conventional serum antibody assay system can be used liberally. These antigens can be the pathogens themselves, such as viruses and bacteria, but they can also be substances that contain the antigenic determinant groups, intrinsic to the respective pathogens. Thus, for example, pathogens inactivated by heat or by irradiation can be mentioned; the antigens obtained by extracting the pathogens with a surfactant or the like, and the antigens that are artificially prepared by means of chemical synthesis or by recombinant DNA technology. The ligand for the control site is not particularly restricted, as long as it is coupled with an arbitrary antibody in the sample; but it is preferred that it be an antibody (anti-immunoglobulin antibody) that specifically recognizes an antibody present in the urine, and binds to it. These ligands are immobilized on the porous material at the site of analysis and at the control site, respectively, so that they are not dislodged from the respective sites by the capillary flow of the liquid sample. Thus, each ligand is bound to the corresponding site, on the porous support, so that it is not made to diffuse when the second region is wetted by the sample containing the desired antibody, but remain stationary at the site, without being transported to the third region of the solid phase support. The immobilization of the ligands on the porous support can be achieved by techniques well known to those skilled in the art, that is, by physical binding or by chemical bonding. Thus, for example, chemical bonding methods, such as covalent binding methods, for example, the diazo method, the peptide method (the acid amide derivative method, the chloride resin method) may be mentioned. of carboxyl, the carbodiimide resin method, the maleic anhydride derivative method, the isocyanate derivative method, the bromocyano activated polysaccharide method, the cellulose carbonate derivative method, the condensing reagent method, etc.) , the alkylation method, the coupling method with interlacing agent (the method for coupling to a support, using glutaraldehyde, hexamethylene isocyanate or the like, as an entanglement agent); the coupling method by the reaction of Ugi, etc .; ionic bonding methods, and physical adsorption methods. When nitrocellulose is used as the porous support for the second region, said ligand can be immobilized by non-covalent binding. The amount of the ligand (antigen) for use in the analysis site of the second region is preferably in excess, so that essentially all of the desired antibody, presumably present in the sample, binds to the site of analysis. The amount of the ligand (anti-immunoglobulin antibody) for use at the control site of the second region is also, preferably, in excess, so that essentially all of the arbitrary antibody (which may also contain the desired antibody) present In the sample, you join the control site. The coupling reaction between the antibody sought and the ligand (in particular an antigen) present at the site of analysis is preferably carried out in the presence of an E. coli component. The E. coli component can be supplied to the reaction system by incorporating it into a dilution of the test sample, and applying the mixture to the first region, or it can be detachably immobilized at the solid phase support analysis site, upstream of the analysis site (for example, said first region, or the tracer region that will be described later). The component of E. coli is not restricted in particular, as long as it is derived from Escherichia coli as mentioned previously. In such a way, it can be the protein fraction, the carbohydrate fraction or the lipid fraction of the cells, or a mixture of those fractions. The soluble fraction obtained by means of extraction of the cells, or the lipopolysaccharide fraction (LPS) can be mentioned as a preferred example thereof. When the sample is applied to the first region, the sample migrates, by capillary action, to the second region, in which the desired antibody, which is present in the sample, is bound and fixed to the analysis site, within the second region; and then the remaining arbitrary antibody (which includes the residue of the sought antibody that had not been bound to the site of analysis) is bound and fixed to the control site, located downstream from the site of analysis. The labeling or labeling means of the present invention is used to detect whether the antibody sought and the arbitrary antibody, present in the sample, have been coupled and fixed to the test site and to the control site, respectively.

The label means may consist of a ligand for the antibody and a detectable label, coupled to the ligand. The ligand for the antibody is not particularly restricted as long as it is a molecule that recognizes the antibody present in the sample and binds to it; but, in the present invention, it is preferred that it be one that binds not only to the searched antibody to be analyzed, but also binds to the arbitrary antibody present in the sample. As an example of said ligand, the same ligand as mentioned for the control site can be mentioned, particularly the same anti-immunoglobulin antibody that was adopted as a ligand at the control site. The anti-immunoglobulin antibody mentioned above includes antisera available from arbitrary animals immunized with a relevant immunoglobulin, for example, a human immunoglobulin, as an immunogen; its purification products (polyclonal antibodies) and monoclonal antibodies. The anti-immunoglobuin antibody, used as a ligand in the control site, can optionally be an antibody directed to all kinds of antibodies, so that it can capture and detect all the antibodies present in the sample, or it can be an antibody directed to a desired class of anibodies, such as immunoglobulin G (IgG). It is preferable that the ligand is an anti-immunoglobulin antibody directed to antibodies of the same class as the class to which the desired antibody belongs and this arrangement is preferred, in that antibodies of the same class as the antibody sought are detected from that way at the control site, and the optimal indication is obtained there to judge whether the analysis has been carried out properly, not only when a urine sample is used, but also when other body fluids, such as sera, are used as samples. . When the antibody sought is IgG, it is more preferable that the ligand used as the labeling medium be a ligand characterized in that it has specific affinity for the Fe region of the IgG antibody and, as preferred examples of said ligand, a specific anti-IgG antibody can be mentioned. for Fe, which is not reactive for the IgG antibody light chain neither for its F (ab) region, or protein A, G protein or the like, which have a specific reactivity for the Fe region of the IgG antibody.

Said anti-immunoglobulin antibodies, or ligands, can be prepared routinely, or can be purchased from commercial sources. The detectable label or tag component is not restricted in the particular, provided that it is a detectable tag that is known to be useful for specific binding assays, in particular for immunoassays, or that may be used in the future ( Tatsuo Iwasaki and coauthors: Monoclonal Antibody, Kodansha Scientific, 1984; Eiji Ishikawa and co-authors: Enzyme Immunoassay, 2a. edition, Igaku Shoin, 1982, etc.). The preferred label is one that undergoes color changes at the analysis site or at the control site of the second region. Although not restricted, a label that undergoes a color change that can be visually recognized without the aid of any instrument is particularly preferred. For example, various chromogens can be mentioned, such as fluorescent substances and absorbent dyes. Still more preferred is a label in the form of a powder containing a visually detectable label. Suitable particulate label includes polymer particles (e.g., latex or polystyrene beads), sacs, liposomes, metal gels (e.g., colloidal silver, colloidal gold, etc.) and polystyrene dye particles. Among them, metal gels, such as colloidal silver and colloidal gold, are preferred. While said label may be coupled to a ligand, either chemically or physically, in the conventional manner to provide a ligand with label or marker, commercial products can also be used. The label or label means that can be used in the present invention can be any label that, when applied to the second region (including the analysis site and the control site) of the solid phase support, indicates the result of the reaction with the antibody, which occurs in the sample that has been placed in the second region and, insofar as this function can be achieved, there is no particular limitation on how it is present. For example, when the assay device of the present invention is provided in the form of a flow through the device, the labeling means can be included in the kit of reagents for antibody analysis, independently of the solid phase support. It is preferable that the labeling means be immobilized detachably on a solid phase support; and it is more preferred that it be detachably immobilized upstream of the second region of the solid phase support. The mode that is even more preferred is such that the marker means is supported in a region (hereinafter referred to as the tracer region) located between the first and second regions of the solid phase support. In this mode of use, the sample applied to the first region is transferred, by means of capillary action, to the tracer region, where it comes in contact with the labeled or labeled ligand, to form the desired antibody / labeled ligand complex, and the arbitrary antibody / labeled ligand complex. After passing through the tracer region, the sample containing these complexes migrates, by capillary action, further down to the second region. The ligand (antigen) disposed at the site of analysis, within the second region, is specific for the antibody sought; and the ligand (anti-immunoglobulin antibody) arranged at the control site is specific for the arbitrary antibody. Accordingly, the searched antibody / labeled ligand complex, which has migrated by capillary action, is captured first at the site of analysis; and the remaining arbitrary antibody / labeled ligand complex (which includes the searched antibody / labeled ligand complex) present in the sample, is then captured at the control site.

The tracer region of the solid phase support is not particularly restricted, as long as it is a support capable of transporting the test sample containing the desired antibody and the arbitrary antibody, from the first region to the second region, and of supporting said antibody. Ligand marked in such a way that the latter can be released by capillary flow. In general, it is a porous member, made of polyethylene, fiberglass, rayon, nylon or a cellulose material, including paper. A material that hardly allows non-specific adsorption is preferred, for example, glass fiber optionally treated with polyvinyl alcohol (PVA). Among the preferred embodiments of the invention is a mode in which the tracer region and the analysis site, in the second region, are arranged with a given interval between them, on a solid phase support. When said region of interval is provided between the tracer region and the site of analysis, the antibody present in the sample, which comes into contact with the labeled ligand that is in the tracer region, is mixed with the labeled ligand therein. , before the sample reaches the analysis site of the second region; with which the coupling reaction between the antibody and the labeled ligand is more positively assured. Thus, this region provides an incubation environment (time and space) for the coupling of the desired antibody and the arbitrary antibody with the labeled ligands, before they come into contact with the desired antibody and the arbitrary antibody found in the sample. , with the analysis site and with the control site, respectively.

Additionally, the solid phase support of the present invention may have a third region, located downstream of the second region. The sample continues to migrate from the first region to the optional tracer region, to the second region and, subsequently, to the third region, through capillary action. Thus, the third region functions as a region that receives the liquid that arrives from the second region by capillary action. In general, the third region only needs to discharge the function of receiving the unbound liquid component in the second region; but it may be additionally provided with a site that reports that the analysis has been completed as the capillary flow of the sample (ie, the liquid front) advances to a predetermined end-point zone on the solid phase support. For the purpose stated above, a visible indicator zone containing a water-soluble dye, such as erythrosin B, saffranin O, phenol red or the like, current below the second region can be provided on a solid phase support. In this case, when the liquid front of the sample crosses the second region towards the third region, it flows through the indicator zone and the fintee disposed in that area is carried downstream by the capillary flow of the sample, liquid, informing of that way the sample has already passed through the first region, the tracer region and the second region (analysis and control sites) and, consequently, that the analysis has just been completed. The material for the third region is not particularly restricted, as long as it is capable of absorbing the sample (liquid), including, for example, porous films or porous sheets of polyethylene, rayon, nylon and cellulosic materials, including paper. The preferred material is a cellulosic material, such as paper. The present invention will now be described in detail, reference being made to the appended drawings, which show the antibody analysis device and its constituent members. However, it should be understood that the illustrated device is merely one embodiment of the invention and in no way defining the present invention. Figure 1 is a diagram showing a solid phase support in the form of a strip (60 x 5 mm) as a member of the device of the present invention. In figure 1 the code 1 represents a first region (16 x 4 mm, 0.92 mm thick) in which a sample of analysis is applied and it is put in contact with the strip; code 2 represents a tracer region (5 x 5 mm, 0.79 mm thick), in which a labeled ligand is immobilized (eg, human IgG antibody labeled with colloidal gold); code 3 represents a second region (18 mm x 5 mm, 0.1 mm thick) in which the reaction with the antibody present in the sample takes place and the result is indicated; and the code 4 represents a third region (22 x 5 mm, 1.46 mm thick) in which the sample that has migrated from the first region and the second region is absorbed. The thickness of the first region is generally between 0.2 and 2 mm, and preferably between 0.8 and 1.2 mm; and the thickness of the tracer region is generally between 0.2 and 1.5 mm, and preferably between 0.5 and 1 mm. The thickness of the second region is generally between 0.03 and 0.2 mm and preferably between 0.08 and 0.12 mm, and the thickness of the third region is generally between 0.5 and 3 mm, and preferably between 1 and 2 mm. However, these scales are not critical. Arranged within said second region (3) is an analysis site (line of analysis, approximately 1 x 5 mm) (5), where a ligand having specific affinity for the desired antibody is supported in that position; and a control site (control line, approximately 1 x 5 mm) (6), where a ligand having affinity for the arbitrary antibody (human anti-immunoglobulin antibody) is supported in that position. The analysis site (5) is arranged at a certain dnce (about 6 mm) from the tracer region; and the control site (6) is arranged at a certain dnce (approximately 6 mm) from the analysis site (5). The site of analysis has the function of reacting specifically with the desired antibody present in the sample, and indicating in the presence of a label, whether or not the antibody sought ex in the sample; and the control site has the function of indicating in the presence of a label, whether the applied sample is appropriate or not.

The materials (supports) for the first region, the tracer region, the second region and the third region constituting the solid phase support strip, may simply have been connected in series, in the longitudinal direction of the strip, along which the sample migrates, and there is no restriction about the connection mode. However, it is preferred that the connection between the longitudinally frontal end of the first region and the trailing end of the tracer region; the connection between the front end of the tracer region and the trailing end of the second region; and the connection between the front end of the second region and the rear end of the third region, are, respectively, in superposed relation. It is more preferred that, as illustrated in FIG. 1, the longitudinally front end portion of the first region is superimposed on the trailing end portion of the tracer region; and that the longitudinally front end portion of the tracer region is superimposed on the rear end portion of the second region. The rear end portion of the third region may be superimposed on the front end portion of the second region. In said connection mode, the sample applied to the first region is allowed to migrate unimpeded in the longitudinal direction of the strip. The width of the strip, in its longitudinal direction, is not particularly restricted; but it can be, for example, 0.5 to 10 mm, preferably 1 to 2 mm, more preferably, 0.8 to 1.2 mm. It should be understood that the upper / lower relations of the respective regions, in the superimposed strip structure, are not limited to those mentioned above, but can not be reversed. For convenience of use, the above solid phase support is preferably packaged in the form of a unit of analysis. The analysis principle for the analysis device of the present invention will now be explained with reference to Figure 2. (3) .- THE SOLID PHASE ANALYSIS METHOD The third aspect of the invention is directed to a method of solid phase analysis, which uses the device described above; which is specifically a solid phase method for analyzing the desired anficibody in a sample, comprising bringing the sample into contact with the first region of the device for antibody analysis, and detecting the development of a color at the site of analysis of the second region, under conditions in which the control site of the second region is indicating a color. In this analysis, a sample suspected of containing the desired antibody is first applied to the first region (1) of the solid phase support. For application of the sample, a body fluid, such as urine, as obtained, or after diluting it with an appropriate diluent may be used. There is no particular restriction on the diluent, but it includes various regulators having regulatory action within the approximate pH range of 5 to 9, preferably, about 6.5 to 8.5 (eg, citrate buffer, phosphate buffer, tris buffer). , acetate regulator, borate regulator, etc.); surfactants, etc. Since non-specific reactions can be suppressed in the antigen-antibody reaction, to reduce the incidence of false positives in the analysis, by carrying out the antigen-antibody reaction in the presence of an E. coli component, it is preferred to use a diluent containing said component of E. coli. The amount of the E. coli component (LPS) to be incorporated in the diluent is not particularly restricted, but is preferably such that it is available from about 0.1 to 10 μg, preferably about 0.5 to 5 μg. of said component, per microgram of the ligand (antigen) present in the reaction system, that is, at the analysis site. The amount of the E. coli component (LPS) that will be incorporated into the sample, for example, can generally not be less than 5 μg / ml, preferably from 5 to 100 μg / ml, more preferably, from 10 to 50 μg / ml. Although the use of a substance of more than 100 μg / ml is not prohibitive, the effect of the present invention can be obtained at an addition level up to 100 μg / ml. When the sample is applied to the first region (1), this first region is wetted. The applied sample then flows through the first region (1) to the tracer region (2), by means of capillary action, and comes into contact and reacts with the labeled ligand (human anti-IgG antibody, labeled with colloidal gold ), detachably supported in the tracer region. When a suitable sample is used and the desired antibody is contained in the sample, both the antibody sought and the arbitrary antibody contained in the sample are coupled with the labeled ligand, which is in the tracer region (2), to form a complex of searched antibody / labeled ligand and an arbitrary antibody / labeled ligand complex, respectively. After their passage through the tracer region (2), the respective complexes, or the labeled ligand that does not form said complexes, are transported together with the current sample below the tracer region (2). In a preferred embodiment, the labeled ligand which is not yet complexed, nevertheless receives sufficient time (space) to form the complexes as it moves with the sample containing the antibody, from the tracer region (2) to the site of analysis ( 5) of the second region (3), by means of capillary action. When the sample arrives at the site of analysis (5) in the second region, the targeted antibody / labeled ligand complex is coupled to the ligand supported at the site of analysis (5) and immobilized in situ. The sample migrates further downstream by capillary action, until it reaches the control site (6), where the arbitrary antibody / labeled ligand complex is coupled to the ligand (anti-human immunoglobulin antibody) which is in that site and immobilized there. Then, when detecting the immobilized complexes in the analysis site (5) and in the control site (6) of the second region, according to the labeling component of the labeled ligand, the result of the analysis can be indicated as a positive analysis . In contrast, when a sample that does not contain the desired antibody is applied, said searched antibody / labeled ligand complex that is to be immobilized at the site of analysis (5), is not formed so that the label or label is not detected. in this analysis site (5) (negative analysis). In connection with this, the indication of negative analysis at the site of analysis (5) includes both a negative (false negative) analysis due to a low concentration of the sample (ie, a small total amount of antibody present in the sample) as a negative analysis (true negative) due to the absence of the antibody sought in the sample. These negative analyzes can not be differentiated from one another according to the result of the analysis site (5) only. However, in the case of a false negative analysis, said arbitrary antibody / labeled ligand complex to be immobilized at the control site (6) is not formed, so that the control site gives a negative indication; whereas in the case of a true negative analysis, said arbitrary antibody / labeled ligand complex is formed, so that the control site gives a positive indication. Therefore, according to whether a negative or positive indication is given at the control site, it is possible to say whether the negative result at the analysis site (5) is a false negative analysis or a positive negative analysis. Additionally, when a labeled ligand that has been deactivated is used or otherwise is not carried out an appropriate analysis in an appropriate system, the control site (6) gives a negative indication, so that a finding can be prevented. false negative analysis, due to such causes. The sample liquid containing all the unbound antibodies, the labeled ligands, etc., continues to migrate further downstream from the second region (3) to the third region (4). An indicator zone can optionally be provided and, in that case, the liquid front carries with it the dye of the indicator zone up to the endpoint zone; with what indicates the passage of the liquid and the dye through the third region and that the analysis has been completed. Figure 3 shows an example of a device for antibody analysis of the present invention, for use in horizontal position. The solid phase support (A) comprising the first region (1), the tracer region (2), the second region (3) (which includes the analysis site (5) and the control site (6)) and the third region (4) is accommodated in a housing (B), made of a suitable material. The housing material is preferably a moldable plastic material, such as polystyrene, although other materials, such as glass, metal and paper can also be used. The housing consists of an upper section (7) having several openings and a lower section (8) and the solid phase support is disposed in the lower section of the housing, and is covered with the upper section which is at its upper end . The openings (9) and (10) in the upper section of the housing are arranged in alignment with the series of regions of the solid phase support and in the positions corresponding to the first region (1) and the second region (3), respectively, of the solid phase support. The sample can be applied to the first region (1) of the support from the opening (9) (sample feed port). The opening (9) is preferably provided with a peripheral projecting wall, around it, so that the wall can help to drip the liquid sample on the first region of the support. The method for contacting the sample with the first region of the solid phase support is not restricted in a particular way, but the sample is preferably dripped from the sample feed port, perpendicular to the plane of the solid phase support. The opening (10) is arranged in a position that allows visual access to the analysis and control sites in the second region of the solid phase support; so that the binding of the labeled ligand / antibody complex sought at the test site can be visually confirmed, and the formation of the labeled ligand complex / arbitrary antibody at the control site can be visually confirmed (detection port, harbor of evaluation). The opening (10) does not necessarily have to be a single port that allows visual access to both the analysis site and the control site, but can consist of two independent ports, respectively, one for the analysis site and another for the site of control. With the antibody analysis device of the present invention, the presence or absence of the desired antibody in a sample can be determined, as well as the amount of the antibody, with precision, allowing the analysis system to remain at rest for a few minutes. , up to thirty minutes, preferably from 5 to 20 minutes, after the application of the sample; in general at a temperature not exceeding 45 ° C, preferably from 4 to 40 ° C, more preferably, from about 15 to 30 ° C. (4) .- THE REAGENT EQUIPMENT FOR ANTIBODY ANALYSIS The antibody analysis method described under (1), or the solid-phase analysis method using the device for antibody analysis described under (2), can be carried out more expeditiously when a kit or kit is used. of reagents for antibody analysis, which contains a complete set of various reagents and the equipment necessary for the determination of the antibody. Thus, the present invention provides a kit or kit of reagents for antibody analysis, to carry out the practice of said method of antibody analysis and said method of solid phase analysis more easily.

The kit or kit of reagents for antibody analysis according to the present invention is intended to be used for the purpose of detecting and quantifying an antibody present in a sample, by means of an antigen-antibody reaction, as a preferred mode; and includes a device that contains said E. coli component as a component of the device. This kit of reagents additionally contains an optionally immobilized antigen, adapted to undergo antigen-antibody reaction with a desired antibody to be analyzed, an antibody assay reagent, and others. Additionally, for convenience of analysis, this kit of reagents may additionally include a suitable reaction medium, a diluent, a washing buffer, a reaction stopper and / or a reagent for analyzing the activity of the label or label. In addition, as another embodiment, the kit of reagents for antibody analysis of the present invention may include a reagent having specific affinity for the Fe region of the desired antibody IgG, preferably an anti-IgG antibody specific for Fe. As yet another alternative embodiment, the reagent kit for antibody analysis of the present invention may contain said device for antibody analysis. The reagent kit may additionally contain the E. coli component, or the substance having specific affinity for the Fe region of the IgG of the target antibody, or both, as reagents. Additionally, the equipment may also contain accessories such as a pipette and an ampoule (tube) to be used to dilute the sample, in addition to said reaction medium, a diluent, a washing regulator, a reaction stop and a reagent to test the activity of the marker, among other reagents.

THE IMPROVEMENT WAY OF PUTTING THE INVENTION INTO PRACTICE The following examples are intended to illustrate the present invention in greater detail, and in no way should they be considered as a definition of the technical scope of the present invention. It should be understood that those skilled in the art can make many changes and modifications in the art, easily, based on the foregoing description of the invention, without departing from the technical scope of the invention.

EXAMPLE 1 ANALYSIS OF H. PYLORI (1) .- PREPARATION OF THE ANTIGEN OF H. PYLORI H. pylori (a clinical isolate) was cultured on Brucella agar medium (Becton) for 48 hours (10% CO2, 5% O2, 37 ° C), and the developed cells were harvested, in cold PBS. The cells were washed centrifugally with cold PBS, a total of five times; and cold PBS was added in order to bring the concentration of the cells to 100 mg / ml. An equivalent of 0.2% Triton X-100 / PBS was added under stirring. The mixture was stirred for 5 minutes, and then centrifuged and the supernatant recovered as a solution of H. pylori antigen, and stored at -80 ° C. (2) .- PREPARATION OF AN ANTIGEN PLATE OF H. PYLORI The above H. pylori antigen solution (2.5 μg protein / milliliter) was added to a plate of 96 concavities, 100 μl / concavity; and incubated at 4 ° C overnight. After the concavities were washed once with PBS, a blocking solution was added (Dulbecco-PBS [D-PBS], 1% BSA, 5% sorbitol, 0.05% NaN3 [pH 7.4], at 300 μl / concavity , and the plate was incubated at 4 ° C overnight After the blocking solution was discarded, the plate was dried at 25 ° C overnight, sealed, together with a desiccator, in an aluminum bag and stored at 4 ° C until used. (3) .- PREPARATION OF A COMPONENT OF E. COLI Escherichia coli (pvc18 / JM109, Takara Shuzo) was grown in liquid LB medium containing ampicillin (Luria-Bertani medium, Nippon Seiyaku) at 37 ° C for 18 hours. The culture was centrifuged to harvest the cells, which were washed with two portions of PBS. Cold PBS was added to the washed cells to make 100 mg / ml, and the mixture was broken and extracted with a sonic treatment apparatus (10 seconds x 3). The supernatant was recovered for use as the E. coli component and stored at -80 ° C (hereinafter referred to as the E. coli extract). (4) .- DETERMINATION OF THE ANTI-H ANTIBODY. PYLORI IN THE URINE Using urine as a sample, the anti-H antibody was determined. pylori present in the sample. To each concavity of the H. pylori antigen plate, prepared according to (2), was added 25 μl of a first regulatory solution (200 mM Tris-HCl regulator, 0.14 M NaCl, 2% casein, 0.5% of BSA, 0.05% of Tween 20, 0.1% of NaN3 [pH 7.3]), which contained 20 μg of protein / ml of the E. coli extract and 100 μl of the urine sample. The mixture was stirred for 10 seconds and allowed to stand at 37 ° C for one hour. The concavities were washed with six portions of PBST (0.05% Tween 20 and 0.05% NaN3 in PBS) and 100 μl of an 11,000 fold dilution of a human anti-IgG antibody, labeled with an enzyme (HRP) ( anti-lgG (Fe) human goat Affini Puré, conjugated with peroxidase, Jackson Immuno Research), in a second regulator (50 mM regulator Tris-HCl, 0.14 M NaCl, 0.5% BSA, 5% goat serum , 0.05% Tween 20, 0.1% XL-II [pH 7.3]). The plate was allowed to stand at 37 ° C for one hour and then washed with six portions of PBST. Then 100 μl of a color developer solution (50 mM citrate-Na 2 HP04) 50% TMB solution, 0.0075% H202) was added, and it was reacted at room temperature for 20 minutes, at the end of which 100 μl of a reaction stopper (50% TMB stop solution, 50% 1N H2SO4) was added, and the absorbance was measured. (5) .- RESULTS (i) .- In five examples of positive cases of H. pylori, and the same number of negative cases for H. pylori, which were diagnosed by analysis with 13C-UBT [J. Gastroenterol., 33, pages 6-13 (1998)], which is considered to be the most accurate of all the diagnostic methods currently available for H. pylori infection, urine was sampled and the antibody was analyzed anti-H pylori by the procedure described in (4). As a control experiment, the same procedure was applied to the same urine samples, except that the addition of the E. coli extract was omitted and, based on the results, the effect of adding an extract of E. coli was evaluated. .coli, in accordance with this invention. The data are presented in figure 4.

In Figure 4 the ordinate represents the absorbance (O. D., 450, 650 nm) and the abscissa represents the addition (+) or the omission (-) of the E. coli extract. Additionally, clear circles represent the results in patient / - / - p / or / '- positive urine samples, by 13C-UBT analysis, and dark circles represent the results in urine samples from H. p patients. / ori-negatives, by means of analysis with 13C-UBT. It will be evident from Figure 4 that carrying out the analyzes in the presence of an E. coli extract according to this invention, one could clearly discriminate the positive and negative cases of H. pylori, coinciding with the results of the analysis with 3C-UBT, which confirms the high accuracy of the method of the present invention. (ii) - Using urine samples from ninety-nine healthy volunteers who have no history of an eradication treatment against H. pylori and 20 patients with stomach diseases (7 cases of gastric ulcer and 13 cases of gastritis), the anti-H antibody. urinary pylori in the presence of E. coli extract according to the procedure described under (4). The results are presented in the figure . In Figure 5, the ordinate represents the absorbance (O. D. 450 nm) and the abscissa represents the groups according to the analysis with 13C-UBT (positive and negative cases). The results indicate that, in the analysis system containing an extract of E. coli, all the urine samples from the negative patients gave definitively negative results, without a false positive result. (iii) .- The amounts of anti-H antibody were determined. pylori in the sera of the same subjects that participated in the experiment (ii), with commercial ELISA kits, and the results were compared with the results of the urinalysis, by the method of the present invention, in the same subjects, with respect to sensitivity, specificity and precision. Simultaneously, serum and urine samples were collected from each subject and prepared for analysis. The commercial ELISA kits were the following: the HM-CAPMR team, Enteric Products (HP-CAP); the Helico GMR team, Shieid Diagnostic (Helico G); and the HEL-p-TestMR team, Amrad Biotech (HEL-p). Each analysis was carried out in accordance with the protocol included with each team. The results are shown in table 1.

TABLE 1 Referring to Table 1, "sensitivity" means the positive rate generated with each kit in H-py / or / v'-positive subjects (positive to the infection according to the analysis with 13C-UBT: n = 70); "specificity" means the negative rate generated with each team in H. pylori-negative subjects (negative to the infection according to the analysis with 13C-UBT, n = 49) and "precision" means the percentage of accurate results with each team , in the total population (70 + 49 = 119 patients). It is clear that, despite the use of urine samples containing only traces of the antibody, the determination of the anti-H antibody. pylori by the method of the present invention gave greater sensitivity to detection and greater specificity, as well as a significantly higher precision, compared with conventional blood antibody analysis equipment. (6) ANALYSIS OF THE ANTI-H ANTIBODY. PYLORI IN THE URINE Using the first regulator containing E. coli LPS (Difco) (concentration of LPS: 5 μg / concavity) instead of the extract of E. coli, the procedure of (4) was repeated otherwise, to determine the presence of antibody anti-H pylori in urine samples, and the results were evaluated in the same way as under (5) (i). The results are presented in Figure 6. It is clear from the diagram that similar results can be obtained by using E. coli LPS in place of the aforementioned E. coli extract.

EXAMPLE 2 HEPATITIS B ANTI-VIRUS ANTIBODY ANALYSIS (HBC) (1) .- ANALYSIS OF THE ANTIBODY ANTI-VIRUS OF HEPATITIS B (HBC) An antigen plate was prepared using the HBc antigen (Chemicon International), in accordance with the procedure of example 1 (2) and the analysis of anti-heptatitis antibody B (core (HBc) in urine samples, according to example 1 (4)) was carried out. this way, 25 μl of the first regulator, which contained the E. coli extract in various concentrations, and 100 μl of sample urine, were added to each concavity of the HBc antigen plate, and after shaking for 10 seconds, allowed the plates to settle at 37 ° C for one hour.After the plate was washed with six portions of PBST, 100 μl of an 11,000 fold dilution of enzyme-labeled human anti-IgG antibody (HRP) was added. , in second regulator, and the plate was allowed to settle at 37 ° C for one hour, and then washed (six times with PBST), then 100 μl of a color developer solution was added, and the reaction was carried out. at room temperature for 20 minutes, after which 100 was added μl of a reaction stopping solution, and absorbance was measured. (2) .- THE RESULTS * In five cases of positive anti-HBc blood antibody and five cases of anti-HBc antibody in blood-negative, which were classified by analysis using the commercial anti-HBc antibody analysis equipment (Dinabott), the anti-HBc antibody was determined in urine, by the procedure described under (1). The concentrations of the E. coli extract were established in the reaction mixtures, at 0, 0.78, 1.56, 3.13, 6.25 and 1.25 μg / ml, and the effect of the addition of the extract was evaluated. The results are presented in Figure 7. In Figure 7, the ordinate represents the absorbance (O. D. 450 to 650 nm), and the abscissa represents the addition level of the E. coli extract.

Additionally, the dark circles represent the data about the urinary antibody in patients with anti-HBc antibody in positive blood, and the light circles represent data about the urinary antibody in patients with anti-HBc antibody in the blood, negative. It is evident from the diagram that, even when urine samples are used, the difference in the level of detection of the anti-HBc antibody between the group of patients with anti-HBc antibody in positive blood, and the group of those with anti-HBc antibody. HBc in negative blood, became more prominent in relation to the level of addition of E. coli extract.

EXAMPLE 3 Using urine samples that gave false-positive analyzes in the determination of the anti-HIV antibody in urine, as a known method of analysis [CalypteMR HIV-1 Uriñe ElA: Arch. Pathol. Lab. Med., 119, 139-141 (1995); Clinical Infectious Diseases, 19, 1100-1104 (1994)], an exploratory experiment was carried out to identify the component supposedly responsible for a non-specific reaction in the same analysis system. (1) .- Each of the previous urine samples was adjusted to pH 7.4 with a 1 M phosphate buffer (pH 7.7) and filtered through filters with 5.0, 0.8 and 0.2 μm cutoff. A 20 ml portion of the filtrate was concentrated, by ultrafiltration (a 10 kDa cutoff membrane) to 2 ml. The concentrated urine was subjected to gel permeation chromatography (Sephacryl S-300, Pharmacia) and each fraction was tested for its reactivity with the HIV antigen. Reactivity to the HIV antigen was confirmed by causing each fraction to react with a plate with an immobilized HIV antigen, prepared by immobilizing the HIV antigen (gp160) and detecting the conjugate (non-specific binding component) with human anti (IgG + lgM) antibody. goat, labeled with ALP, goat anti-IgG antibody (specific for Fe), labeled with ALP, or goat anti-IgG antibody (specific for Fab), labeled with HRP (all available from Jackson ImmunoResearch Labs) . The results are presented in Figure 8. In Figure 8 the ordinate represents the absorbance (O. D.) and the abscissa represents the gel permeation chromatographic fractions (fraction numbers). The solid line represents the absorbance of the protein at 280 nm and the line of dark circles represents the result of the detection with human anti- (lgG + lgM) antibody, the line of clear triangles represents the result of the detection with anti- lgG (specific for Fe) human, and the line of dark triangles represents the result of the detection of the anti-human IgG antibody (specific for Fab). It is evident from Figure 8 that, in the detection with the human anti-IgG antibody (specific for Fab), the mode of reaction (the reactivity with the non-specific binding component) is the same as in the detection with the anti-IgG antibody. (IgG + IgM) human, while no reactivity is found with the human anti-IgG antibody (specific for Fe). This discovery suggests that the non-specific binding component is a fragment or denaturing product of human IgG, which retains the reactivity with the human anti-IgG antibody (specific for Fab), without the Fe region. Therefore, it was clear that, when a human anti-IgG antibody (specific for Fe) is used is not reactive with said non-specific binding component as an assay reagent, the non-specific reaction in the antibody detection system can be inhibited and, therefore, the incidence of a false positive analysis due to said non-specific reaction, with the result that a very specific and very precise method of analysis of the antibody can be provided.

EXAMPLE 4 ANTI-HIV ANTIBODY ANALYSIS IN URINE To each concavity of a plate of HIV antigen (CalipteMR HIV_IJJrine ElA, Calypte Biomedical Corp), 25 μl of a first regulator (the sample regulator of CalypteMR HIV-I Uriñe ElA) and 200 μl of sample urine was added.; and after stirring for 10 seconds, the plate was allowed to settle at 37 ° C for one hour. Afterwards, this plate was washed six times (washing buffer: D-PBS, 0.05% Tween 20), 100 μl of a 11,000-fold dilution of human anti-IgG antibody (specific for Fe) was added. goat, labeled with HRP (pure goat anti-human IgG, Affini, conjugated with peroxidase, specific for the Fe fragment, Jackson ImmunoResearch Labs.), in the second regulator (50 mM Tris-HCl regulator, 0.14 M NaCl, 0.5% BSA, 5% goat serum, 0.05% Tween 20, 0.1% XL-II (pH 7.3)) and the plate was allowed to settle at 37 ° C for one hour and was washed six times the same way as before. Then 100 μl of a color developer (50% TMB solution, 50 mM citrate-Na2HPO4, 0.0075% H2O2) was added, and it was reacted at room temperature for 10 minutes, and at the end of that time 100 was added. μl of a reaction stop solution (50% TMB stopper, 50% 1N H2SO4), and absorbance (OD 450 nm) was measured. As a control experiment, the sample was analyzed with the known analysis method [CalypteMR HIV-1 Uriñe. ElA: Arch. Pathol. Lab. Med., 119, 139-141 (1994); Clinical Infectious Diseases, 19, 1100-1104 (1994)] (control method), using goat anti-human immunoglobulin antibody, labeled with ALP, as the second antibody. Additionally, the negative control and the positive control of the above analysis equipment were measured by means of the above analysis method of the present invention. Since the absorbency values found in this way were comparable with those found with the previous equipment, the cut-off point for the method of the invention was set at the value found by adding 0.180 to the average absorbance of the previous negative control, in accordance with the method to calculate the cut-off value of said equipment. The results of the analyzes in 100 samples (of urine) of subjects with seropositive anti-HIV antibody (two cases) and subjects with seronegative anti-HIV antibody (98 cases) are presented in table 2 below.

TABLE 2 Oe these 4 subjects, two are subjects with sero-positive anti-HIV antibody.

It can be seen from table 2 that, although both the sensitivity of the method of the present invention and that of the control method were 100% (2/2), the specificity was 71.4% (70/98) for the control method, against 98% (96/98) for the method of the invention. The above results indicate that, compared to the control method, the antibody analysis method of the invention is remarkably low in the incidence of a false positive analysis and very high specificity.

EXAMPLE 5 ANTI-HERB ANTIBODY ANALYSIS. PYLORI IN THE URINE (1) .- PREPARATION OF AN ANTIGEN PLATE OF H. PYLORI To a 100 mg / ml suspension of Helicobacter pylori (a clinical isolate) in cold Dubelcco-PBS, as routinely prepared (J. Clin. Microbiol., 29: 2587-2589 (1991)), a volume was added. equal to 0.2% solution of Triton X, under constant agitation with a stirrer, and the mixture was further stirred for 5 minutes and centrifuged (3,000 rpm, 20 minutes). The supernatant was transferred to a new tube for use as an extract (1 to 1.5 mg / ml as protein). This extract was diluted with D-PBS (2.5 μg / ml) and the dilution was distributed in a plate of 96 concavities, 100 μl per concavity, and incubated at 25 ° C overnight. After each concavity was washed, 300 μl of a blocking solution (D-PBS, 0.5% casein, 5% sorbitol, 0.05% NaN3 (pH 7.4)) was added, followed by incubation at 25 ° C overnight. The blocking solution was then discarded and the plate dried at 25 ° C overnight, sealed together with a dissector in an aluminum bag and stored at 4 ° C until used. (2) .- ANALYSIS Using the immobilized antigen (plaque) prepared as stated above, the anti-H antibody was analyzed. pylori in urine samples, as in example 4. Thus, 25 μl of a first regulator (200 mM Tris-HCl regulator, 0.14 M NaCl, 2% casein, 0.4% BSA, was added to each concavity). 0.05% of Tween 20, 0.1% of NaN3, 20 μg / ml of E. coli extract (pH 7.3)) and 100 ml of sample urine, and after shaking for 10 seconds the plate was allowed to settle to 37 ° C for one hour, and then washed six times. As in example 4, 100 μl of said dilution of goat-labeled human amphiphile IgG (specific for Fe) was added to the second regulator, and the plate was allowed to settle at 37 ° C for one hour , to detect the antibody. The results obtained are represented in figure 9 and table 3.

TABLE 3 In Figure 9 the ordinate represents the absorbance (OD 450-650 nm) and the abscissa represents the positive group of infection with H. pylori (+; n = 56) and the negative group of infection with H. pylori (-; n = 44), according to the analysis with 13C-UBT (J. Gastroenterol., 33: pages 6-13 (1998)). In Table 3, "sensitivity" denotes the percentage of cases detected as positive among positive cases according to the analysis with 13C-UBT; the "specificity" denotes the percentage of cases detected as negative among the negative cases according to the analysis with 13C-UBT; and "precision" denotes the percentage of cases detected as positive and negative, respectively, between positive and negative cases according to the 13C-UBT analysis; and the respective figures corresponding to the cut-off point of M + 3SD or M + 4SD means. As a control experiment, the same urine samples were analyzed with an anti-H antibody analysis kit. serum pylori [HM-CAP; EPI / Kyowa Medies (K.K.)], and the results are also tabulated (control method).

The above results indicate that the method of the invention is superior to the control method in sensitivity and precision, particularly.

EXAMPLE 6 ANTI-ANTIBODY ANTI-VIRUS ANTI-VIRUS OF RUBEOLA IN URINE (1) .- PREPARATION OF A RUBEOLA ANTIGEN PLATE Using a commercial antigen for rubella [obtainable from BIO-DESIGN], at a concentration of 1 μg / ml and a blocking agent composed of D-PBS, 1% BSA, 5% sorbitol and 0.05% NaN3 (pH 7.4) , the procedure of example 5 (1) was repeated, to provide an antigen plate for rubella. (2) .- THE ANALYSIS Using the aforementioned antigen plate, the rubella antibody was analyzed in urine samples, in the same manner as in Example 5 (2). The results are presented in figure 10 and that in table 4.

TABLE 4 In figure 10 the ordinate represents the absorbance (OD 450-650 nm) and the abscissa represents the group positive for the anti-rubella antibody in the serum (n = 76) and the group negative for the anti-rubella antibody in the serum (n = 23), according to the results of the determination with a case or commercial equipment (Rubella IgG (ll) -EIA, SEIKEN, obtainable from Denka Seiken). The data given in table 4 indicate a complete coincidence between the result obtained in the urine by the method of the present invention and the result obtained in the serum by the control method. In accordance with the above data, the degree of coincidence between the method of the invention and the control method is up to 100% (99/99), thus indicating that the present invention allows detection of the antibody with high sensitivity and great specificity, even in the urine; It is safe and convenient and, therefore, it is very useful in laboratory tests.

EXAMPLE 7 CONSTRUCTION OF A DEVICE FOR ANTIBODY ANALYSIS (1) .- PREPARATION OF AN ANTIGEN AGAINST H. PYLORI An antigen solution against H. pylori was prepared by the same procedure as that used in Example 1 (1), and stored at minus 80 ° C. (2) .- PREPARATION OF A DRY GLASS FIBER CONTAINING THE MARKED HUMAN ANTI-IGG ANTIBODY To a fiberglass sheet (5.0 mm x 260 mm x 0.8 mm thick, Whatman) was added 1 ml of a solution of human anti-IgG antibody (specific for Fe) labeled with colloidal gold, 40 nm (from diameter), and the sheet was dried overnight. This sheet was stored together with a desiccant at room temperature, until it was used. (3) .- PREPARATION OF A MEMBRANE 3 mg / ml of the antigen solution against H. pylori, prepared under (1) above, and 0.3 mg / ml of a solution of human anti-IgG antibody, were applied on a 26.5 mm nitrocellulose membrane. 260 mm x 0.1 mm thick; Advance MicroDevice), spraying 1.5 μl / cm in lines, at a predetermined spacing, as illustrated in Figure 11, and dried at 37 ° C for 120 minutes. After drying, the membrane was immersed and washed in a Borax regulator (pH 8.2) containing skim milk for 30 minutes. The washed membrane was dried at 37 ° C for one hour, and stored in the presence of a desiccant, at room temperature. (4) .- ASSEMBLY (A SOLID PHASE SUPPORT) As illustrated in Figure 3 (A), the anterior membrane (3), a pad (4) of absorbent filter paper (22 x 260 mm x 1.5 mm thick, Whatman), the sheet of paper were glued together. glass fiber containing the labeled antibody (2) and a sample pad (1) (15 x 260 mm x 1.0 mm thickness, filter paper, Whatman), with an adhesive, and cut to 5 mm wide . The solid phase support thus prepared was placed in position on a lower section (8) of a plastic housing, and an upper section (7), provided with a port (9) for the admission of plastic, was placed on the solid phase support. sample and a detection window (10), in series, and fixed securely on the lower section (8).

EXAMPLE 8 ANTI-H ANTIBODY ANALYSIS. PYLORl IN URINE Using the device constructed in Example 7, anti-H antibody analysis was carried out. pylori in three classes of urine samples, namely: urine samples from subjects with H. pylori infection, urine samples from subjects without infection with H. pylori, and extremely poor urine samples, from subjects with H. pylori infection. First, 500 μl of urine sample was added to 500 μl of a sample diluent [200 mM Tris-HCl buffer, 0.14 M NaCl, 2% casein, 0.5% BSA, 0.05% Tween 20, 0.1% NaN3 (pH 7.3), E. coli LPS (Difco), 50 μl / ml], and then mixed. Six drops (approximately 150 μl) of the resulting dilution was then added from the sample admission port (9) of the device constructed in example 7, for adsorption on the support, which was then allowed to stand for twenty minutes. As a result, when a qualified urine sample was used, a pink-red band appeared in the control region of the detection window (10). On the other hand, when an extremely poor unqualified urine sample was used, neither the region of analysis nor the control region of the detection window (10) showed a color development, indicating that the sample was not evaluable . When the sample was a qualified urine sample, from a subject without H. pylori infection, a pink-red band appeared only in the control region of the detection window (10), showing a negative analysis (true negative). ) for H. pylori infection, whereas, in the presence of infection with H. pylori, a pink-red band appeared both in the test region and in the control region of the detection window (10), which shows a positive analysis for H. pylori.

EXAMPLE 9 ANTI-H ANTIBODY ANALYSIS. PYLORI IN URINE (1) .- PREPARATION OF A COMPONENT OF E. COLI Escherichia coli (pvc18 / JM109; Takara Shuzo) was grown in liquid LB medium containing ampicillin (Luria-Bertani medium; Nihon Seiyaku) at 37 ° C for 18 hours, and the developed cells were harvested, by means of centrifugation, and he washed them with two portions of PBS. Then cold PBS was added to a final cell concentration of 100 mg / ml, and the cells were broken and extracted, using a sonic treatment apparatus (10 seconds x 3 times). The resulting supernatant was used as an E. coli extract protein. (2) .- Using the E. coli extract protein prepared in (1) above, instead of the E. coli LPS added to the sample diluent of example 8, the procedure of example 8 was repeated, determine the anti-H antibodies. pylori in urine samples. As 3 result the same results were obtained as were described in example 8.

EXAMPLE 10 Using whole blood, plasma and urine from twenty-one subjects positive for H. pylori infection and the same number of subjects negative for H. pylori infection (a total of 42 cases) according to the 13C-UBT analysis [J . Gastroenterol., 33: 6-13 (1998)], anti-H antibodies were analyzed. pylori by the procedure that was described in Example 8. As a control experiment, samples from the same subjects were respectively analyzed, using the commercial antibody analysis kits for H. pylori, directed to whole blood or plasma, and evaluated the effectiveness of the device of the invention, based on the data. The results are presented in Figure 12. In Figure 12, the control equipment A to H were the following: A) Helitest (manufactured by Cortees Diagnostics) B) H. py / or / -Check 1 (manufactured by Bio- Medical Products) C) First Check H. pylori (manufactured by Worldwide Medical Corp.) D) Biocard Helicobacter pylori IgG (manufactured by Anti Biotech Oy) E) Insta Text H. pylori (manufactured by Cortez Diagnostics, Inc.) F) One Step H. pylori Test (manufactured by Teco Diagnostics) G) H. pylori SPOT (manufactured by International Immuno-Diagnostics) H) Quick Stripe H. pylori (manufactured by Diatech Diagnostics, Inc.). In Figure 12, the "specificity" denotes the percentage of negative analyzes (negative rate) that was found when analyzing negative samples to the 13C-UBT analysis with the corresponding equipment, and "sensitivity" denotes the percentage of positive analyzes (positive rate). ) that was found when analyzing samples positive to 13C-UBT, with the corresponding equipment. It is evident from the data shown in Figure 12 that the device for antibody analysis and the solid phase analysis method of the present invention provide excellent analysis systems, with high specificity of detection and high accuracy, even when Apply to urine samples, not to mention blood samples (whole blood, plasma). It is also clear from the above results that, although the sample is a urine sample that is safe and convenient, the present invention allows the detection of antibodies, with high sensitivity and great specificity, which is why it is very useful in examinations. from laboratory.

EXAMPLE 11 EFFECT OF AN E. COLI COMPONENT ON ANTIBODY ANALYSIS IN URINE (1) .- The effect of a component of E. coli on the analysis of antibodies in the urine was evaluated using the analysis device built in example 8. In this way, using the urine of positive and negative subjects to the infection by H. pylori, selected by analysis with 3C-UBT, anti-H antibodies were analyzed. pylori in urine samples, in a system that uses a sample diluent that does not contain E. coli LPS (diluent 1) and in systems that use the same diluent, supplemented with E. coli LPS at the various concentrations that appear in Table 5. Linear color development in the region of analysis and in the control region was evaluated from the line intensity measured with a densitometer (manufactured by ATTO). The results are shown in table 5.

TABLE 5 It was found that, when E. coli LPS was added to the sample at 11.1 μg / ml, and at higher levels, the non-specific reaction observed with diluent 1 disappeared, so that a false-positive analysis (detection error) could be prevented. ).

INDUSTRIAL APPLICATION The present invention provides an antibody analysis technology by which desired antibodies, specific for infection sources, can be detected with high sensitivity and high specificity, even when urine samples that are comparatively poor in antibodies are used as samples for analysis. . According to the method of antibody analysis of the invention, the "false positive" reactions due to contaminants present in the samples can be significantly inhibited, so that very accurate and reliable analysis results can be obtained. Additionally, the present invention provides improvements in immunocapillary or immunochromatographic analyzes, by means of which the existence of the desired antibodies and their quantity in the samples can be accurately detected, with a clear distinction between "false negative" and "true negative".

Claims (32)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for analyzing an antibody comprising detecting a desired antibody in a sample, using an antigen-antibody reaction, characterized in that said antigen-antibody reaction is carried out between the antibody and a test antigen, in the presence of a component of E. coli.

2. The antibody analysis method according to claim 1, further characterized in that the E. coli component is at least one member selected from the group consisting of the soluble fraction and the lipopolysaccharide fraction of E. coli.

3. The method of antibody analysis according to claim 1, further characterized in that the component of E. coli is used in an approximate ratio of 0.1 to 100 μg per microgram of the antigen being analyzed.

4. A method for analyzing an antibody comprising detecting a desired antibody in a sample, by means of the sandwich technique, characterized in that a substance having specific affinity for the Fe region of IgG is used as a reagent for the analysis of antibody. of the antibody that is sought.

5. - The antibody analysis method according to claim 4, further characterized in that the antibody analysis reagent is an anti-IgG antibody specific for Fe.

6. The method of antibody analysis according to claim 4, characterized also because it comprises an antigen-antibody reaction step, wherein the antibody sought in the sample is coupled to an immobilized form of an antigen for said antibody, which is immobilized on a support, and a reaction step in which the The target antibody, captured by the immobilized antigen, is reacted with an antibody analysis reagent having specific affinity for the Fe region of the antibody IgG.

7. The method of antibody analysis according to claim 6, further characterized in that the antigen-antibody reaction is carried out in the presence of an E. coli component.

8. The method of antibody analysis according to claim 1 or claim 4, further characterized in that the sample is a urine sample.

9. The method of antibody analysis according to claim 1 or claim 4, further characterized in that the antibody sought is the antibody against a source of infection.

10. The method of antibody analysis according to claim 1 or claim 4, further characterized in that the source of infection is a member selected from the group consisting of human immunodeficiency virus, hepatitis viruses, rubella virus , influenza virus, mumps virus, herpes virus, cytomegalovirus, Clamydia spp., gonococci, Helicobacter pylori and Toxoplasma gondii.

11. The method of antibody analysis according to claim 1 or claim 4, further characterized in that the antigen is a member selected from the group consisting of bacteria, viruses, protozoa and components of bacteria, viruses or protozoa that contain at least the antigenic determinants of said bacteria, viruses or protozoa.

12. The method of antibody analysis according to claim 1 or 4, further characterized in that the antigen is the microorganism Helicobacter pylori or a component thereof, which contains at least the antigenic determinant group of that microorganism.

13. A device for analyzing an antibody, characterized in that it comprises a solid phase support having at least: (a) a first region to which a sample is applied; and (b) a second region in which the antibody of the test sample is reacted, which are arranged in such a sequence, that the sample is transported from the first region to the second region by capillary action; and half markers to detect the result of the reaction in the second region; said (b) second region has (i) a test site, in which a ligand has been immobilized to capture the desired antibody to be detected, and (i) a control site in which an antibody has been immobilized. ligand to capture an arbitrary antibody present in the sample.

14. The device for antibody analysis according to claim 13, further characterized in that the ligand immobilized at the site of analysis is an antigen for the desired antibody that occurs in the sample.

15. The device for antibody analysis according to claim 13, further characterized in that the ligand immobilized at the control site is a human anti-immunoglobulin antibody, capable of capturing an arbitrary antibody present in the sample.

16. The device for antibody analysis according to claim 13, further characterized in that it comprises a labeled ligand to be linked both to the antibody sought and the arbitrary antibody, as a marker medium.

17. The device for antibody analysis according to claim 13, further characterized in that the labeling means is a labeled ligand that is going to bind both the desired antibody and the arbitrary antibody, detachably supported upstream of the second support region. of solid phase, in a manner such that, when contacted with a sample, it reacts with the antibody sought and with the arbitrary antibody to form a complex of searched antibody / labeled ligand and an arbitrary antibody / labeled ligand complex, respectively, which they are then transported by capillary action to the second region, where they are fixed at the analysis site and at the control site, respectively.

18. The device for antibody analysis according to claim 17, further characterized in that the labeled ligand is supported in a region (tracer region) intermediate between the first region and the second region of the solid phase support.

19. The device for antibody analysis according to claim 17, further characterized in that the labeled ligand to be bound to both the desired antibody and the arbitrary antibody, is a labeled, anti-human immunoglobulin antibody.

20. The device for antibody analysis according to claim 19, further characterized in that the anti-human immunoglobulin antibody is an anti-IgG antibody that has specific affinity for the Fe region of immunoglobulin G.

21.- The device for antibody analysis according to claim 13, further characterized in that the solid phase support is additionally provided with a current absorption region below the first and second regions, so that the sample transported from the first region to the second region is transported further, by capillary action, to the absorption region.

22. The device for antibody analysis according to claim 13, further characterized in that the coupling reaction of the antibody sought at the site of analysis, in the second region, takes place in the presence of an E. coli component.

23. The device for antibody analysis according to claim 13, further characterized in that the sample is a urine sample.

24. The use of the device for antibody analysis claimed in any of claims 13 to 22, to analyze a target antibody, directed to a source of infection, which occurs in a sample.

25. The use according to claim 24, further characterized in that the sample is a urine sample.

26.- A method for solid phase analysis of a desired antibody in a sample, characterized in that it comprises applying the sample to the first region of the device for antibody analysis claimed in claim 13, and detect the development of a color in the analysis site, in the second region, under the condition that the control site in the second region develops a color.

27. A method for solid phase analysis of a desired antibody in a sample, characterized in that it comprises applying the sample to the first region of the device for antibody analysis claimed in claim 20, and detecting the development of a color in the blood of analysis of the second region, under the condition that the control site of the second region develops a color.

28. - The method for solid phase analysis of a desired antibody, according to claims 26 or 27, further characterized in that the coupling reaction of the antibody sought at the site of analysis in the second region of the device for antibody analysis, takes place in the presence of an E. coli component.

29. The method for solid phase analysis of a desired antibody, according to claim 26, further characterized in that the sample is a urine sample.

30. A kit or kit of reagents for antibody analysis, for use in the analysis of an antibody in a sample, using an antigen-antibody reaction, characterized in that the kit or kit of reagents for antibody analysis contains a component of E. coli. 31.- A kit or kit of reagents for antibody analysis, characterized in that it contains a substance that has a specific affinity for the Fe region of the IgG of the desired antibody, as a reagent for the antibody analysis. 32. A kit or kit of reagents for antibody analysis, characterized in that it contains the device for antibody analysis claimed in claim 13. SUMMARY OF THE INVENTION The invention relates to a technology by means of which antibodies directed to sources of infection, present in body fluids, can be analyzed with high precision, very quickly and with great specificity; more in particular, the invention provides an immunoassay method for antibodies, wherein the antigene-antibody reaction between a target antibody in a sample and an assay antigen is carried out in the presence of an E. coli component; and an antibody assay method comprising using a reagent having specific affinity for the Fe region of an antibody IgG, as a reagent for antibody analysis; additionally, the invention provides a device for antibody analysis, comprising a solid phase support having at least (a) a first region to which a sample is applied; and (b) a second region in which the antibody present in the mixture is reacted, which are arranged in such an arrangement, that the sample is carried by capillarity from the first region to the second region; and a marker means for detecting the reaction result in the second region; and characterized in that the second region (b) is provided with (i) an analysis site in which a ligand is immobilized to capture the desired antibody to be analyzed; and (ii) a control site in which a ligand is immobilized to capture an arbitrary antibody that occurs in the sample.