METHOD FOR DETECTING INFECTION-PHASE-SPECIFIC ANTIBODIES

Abstract: The present invention relates to a method for the detection of antibodies and/or antibody isotypes specific for at least two different phases of an infection. The method of the invention is characterized by the simultaneous incubation of a sample from a patient with at least two preferably antigenic compounds. These compounds comprise an epitope that is recognized by an antibody/antibody belonging to a particular isotype wherein said antibody is specifically occurring in one of said phases of said infection. The method of the invention also requires that at least two epitopes are present in the incubation reaction that are specific for different phases of the infection and thus bind antibodies/antibody isotypes specific for said at least two different phases of said infection. The invention also relates to a kit useful for carrying out the method of the invention.

Description: METHOD FOR DETECTING INFECTION-PHASE-SPECIFIC ANTIBODIES The present invention relates to a method for the detection of antibodies and/or antibody isotypes specific for at least two different phases of an infection. The method of the invention is characterized by the simultaneous incubation of a sample from a patient with at least two preferably antigenic compounds. These compounds comprise an epitope that is recognized by an antibody/antibody belonging to a particular isotype wherein said antibody is specifically occurring in one of said phases of said infection. The method of the invention also requires that at least two epitopes are present in the incubation reaction that are specific for different phases of the infection and thus bind antibodies/antibody isotypes specific for said at least two different phases of said infection. The invention also relates to a kit useful for carrying out the method of the invention. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention. The early diagnosis of infections with pathogens is desirable for a variety of reasons. For example, results published in recent years allow the conclusion that detection of HIV infection at an early stage significantly increases the prognosis of the patient. In addition, with the increasing information available on the etiology of a large number of diseases, a detailed knowledge on the stage of the disease would be highly advantageous. Thus, in the case of infections of pregnant women by pathogenic viruses, information on the phase of the infection may be essential for the welfare of the fetus. Various studies have been carried out to differentiate between or identify distinct phases of pathogenic infections. For example, Vornhagen et al., J. Clin. Microbiol. 34 (1996), 1020-1023 have identified immunoglobulin A-specific reactivities of recombinant human cytomegalovirus antigens indicative of the acute phase of infection. The same group has identified a major target antigen for the IgM antibody response during acute infection, Vornhagen et al., J. Clin. Microbiol. 33 (1995), 1927- 1930). EP-B1 0 328 588 describes a method of determining the amount of toxoplasmosis-associated IgM in a sample wherein a high IgM content is indicative of an acute infection of Toxoplasma. US 4,877,726 discloses a method for distinguishing acute and chronic infection with Toxoplasma on the basis of the properties of a specific antibody. Angrano et al., J. Clin. Microbiol. 19 (1984), 1905- 1910 published a principle of simultaneously detecting total antibody and IgM specific for the hepatitis B core antigen. Japanese Examined Patent Application 2523332 describes a method for differentiating between IgM and IgG antibodies. The different isotype can be measured on the basis of different activities in agglutination reactions to these isotypes. Finally, US 5,670,310 discloses a method for distinguishing between acute and chronic HCV infection by employing a collection of distinct peptides and measuring the binding strength of said peptides with antibodies in a sample. All these prior-art methods are either useful for the detection of only one specific phase of an infection or they rely on rather time-consuming and/or costly analyses for differentiating between said different phases, including ELISAs, RIAs, Western blots etc. They are thus suboptimal for a day-to-day routine analysis of a large number of samples. On the other hand, in WO92/21024, a method has been established that allows for the simultaneous assay of multiple analytes in a single fluid sample. Said method may be employed for the simultaneous assay for human IgG, human IgA and human IgM in analytes. However, WO92/21024 provides no teaching or suggestion that the disclosed method could be applied to distinguish between various phases of infections with pathogens. Thus, the technical problem underlying the present invention was to provide an improved method for the convenient analysis of phases of an infection by a pathogen. The solution to said technical problem is achieved by providing the embodiments characterized in the claims. Accordingly, the present invention relates to a method for the detection of antibodies/antibody isotypes specific for at least two different phases of an infection comprising (a) incubating a sample from a patient simultaneously with at least two types of compounds, each type of compound comprising a different epitope under conditions that allow binding of said antibodies to said epitopes, wherein each epitope is recognized by antibodies/antibodies belonging to a particular isotype specific for one of said phases of said infection, wherein further at least two epitopes are present in the incubation reaction recognized by antibodies/antibodies belonging to a particular isotype specific for at least two different phases of said infection, wherein further each type of compound/all types of compounds comprising epitopes recognized by antibodies/antibody isotypes indicative of the same phase of infection is/are affixed to the same type of support and wherein each type of support has properties that allow the physical distinction of said type of compounds from other types of support by physical means; (b) physically distinguishing said compounds; and (c) assessing whether antibodies/antibodies belonging to a particular isotype are bound to said types of compounds. In accordance with the present invention, it has surprisingly been found that various phases of an infection can be identified in a convenient manner by incubating a sample from a patient known or suspected to suffer from infection by a pathogen with at least two compounds comprising different epitopes, wherein each epitope reacts with antibodies that are specifically found in a certain phase of the infection. This, in turn, will usually mean that the epitope is exposed to the immune system of this phase of the infection. Generally, the present invention as outlined above envisages two different modes of determining the status or phase of an infection: either, two (or more) antibodies, e.g. of the μ isotype (i.e. IgM antibodies) recognize two (or more) different epitopes wherein these two (or more) epitopes are characteristic of different phases of the infection; alternatively, two (or more) antibodies represent different isotypes and recognize different epitopes wherein said epitopes are again characteristic of different phases of the infection. In other words, the antibodies specific for different phases of an infection alternatively belong to different isotypes. Thus, it is known that an early phase of an infection is often associated with the occurrence of antibodies of the IgM and IgA classes, whereas in later phases of an infection, IgG may be more prominent. In accordance with the invention, binding by antibody is preferably specific, i.e. the antibodies do not cross-react with other epitopes. It is essential that antibodies reacting with one epitope used in the method of the invention do not cross-react with another epitope used in said method wherein said other epitope is indicative of a different phase of the infection. The above recited two different modes are reflected by the wording "antibodies/antibody isotypes" or "antibodies/antibodies belonging to a particular isotype" etc. The term "isotype" is well established in the art; see, e.g., Paul, "Fundamental Immunology", 2 nd ed. 1989, Raven Press, New York. It is important to note in accordance with the invention that fragments or derivatives of antibodies (Fab, F(ab) 2 , scFv etc.) may also be employed as long as the physical distinction of said compounds (in step (b)) is not compromised. In certain cases, there may be a simultaneous detection of different types of antibodies, wherein each type of antibody would be considered indicative of a certain phase of infection. Nevertheless, the person skilled in the art will usually be in a position to identify the phase of infection, for example by evaluating the ratio of the different antibodies. The reason for this type of finding may be, for example, that a certain type of antibody/antibody isotype is raised during one phase of infection, such as an early phase of infection, but may persist in the body during later phases of infection. In such a case, the actual phase of infection may be identified by way of determining the ratio of the amount of an antibody/antibody isotype indicative of a later phase of infection in relation to the amount of persisting antibodies. Alternatively, the concomitant presence of two types of antibodies/antibody isotypes or the absence of one type of antibody/antibody isotype may be evaluated. The method of the invention is also suitable for identifying the phase of an infection by detecting more than two of said antibodies/antibody isotypes. By applying the above principles and his common general knowledge, the person skilled in the art will interpret the data to determine the actual stage of disease of a patient. He is also capable of establishing test conditions that allow a detectable antibody-epitope- binding; see for example Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, New York. The allocation of epitopes/antibodies of a specific isotype directed thereto to contain phases of an infection with a pathogen can be effected by the skilled artisan without undue burden on the basis of his common knowledge, optionally in combination with the teachings of the present invention. For example, previous studies (Vornhagen et al.) reported that recombinant fragments mainly derived from the teguments (pp150) detected IgM antibodies in sera without acute infection. These observations were confirmed in accordance with the present invention with the COPALIS technology by evaluating several pp150 fragments, the pp65 and p28: whilst the pp150 fragments were positive for nearly all the subjects anti-CMV positive for both IgG and IgM, the anti pp65 response was poorly or not detectable in most patients and the anti p28 was found all in a limited fraction of sample (around 40-50%) CMV IgG positive. These data suggested that antigen composition for the specific "datation" purpose had to be selected as a function of IgM sensitivity at the early stage of the infection on one hand and the lack of reactivity after a certain period (typically after 90 days). Furthermore, for the specific intended use of the assay, by capitalizing the intrinsic capabilities of the COPALIS technology the unique opportunity was provided to improve the reliability of the datation combining the results coming from two analytical outputs, selecting one marker positive in a shorter window period (typically 60 days), the other one or a prolonged one (up to 90 days). It was found that the 2 antigens p53 and CM2 fulfilled these basic requirements and accordingly these antigens were selected for challenging the presented method against sequential serum samples that had been previously characterized with other methods. Finally, based on previous studies on the kinetic of the immunological response of various antigens where it was shown that anti glycoproteins were later compared phosphoproteins, the viral particle was selected as a source of late antigens because rich on glycoproteins. As is apparent from the above, the method of the invention further relies on the principle that the compounds comprising the various epitopes are affixed to different types of support that are distinguishable by their physical properties. The term "type of support" may also, but not necessarily, mean that one type of support is disinguishable from another type of support by its chemical constitution. The compound comprising the epitope may be affixed to said support by a variety of means known in the art. These means include chemical coupling, coating, attachment by van-der-Waals forces etc. It is important, however, that the epitope confirmation is not destroyed and that the epitope is accessible to binding by antibody. Comprised by the method of the invention are also embodiments wherein different epitopes recognized by antibodies/antibody isotypes indicative of the same phase of infection are coupled to the same type of support. With these embodiments, a more accurate determination of the phase of infection may be achieved. Also, a stage of progress within a certain phase of infection/disease may be determined if the antibodies/epitopes are indicative of certain time points/time ranges within a certain phase of an infection. Conveniently, the method of the invention can be combined with established prior art analytical techniques, as demonstrated, for example, in the appended examples to confirm or further analyze the results. In a preferred embodiment of the method of the present invention said antibodies recognizing said different epitopes are IgM antibodies. This embodiment is favored in cases where different types of IgM (i.e. IgM antibodies with different binding specificities) may be employed for distinguishing two different phases of infection. As has been explained in the above, the same type of antibody may first occur in a certain (first) phase of an infection and persist during the second phase of infection. IgM antibodies recognizing different epitopes may first occur in the same (first) phase, but not persist in the later phase. Thus, occurrence of the first and the second antibody in this scenario would be indicative of the early phase, whereas detection of only one (e.g. the first) antibody would be indicative of the later phase of the infection. The same principle applies to other isotypes, such as IgG and IgA, as well. In another preferred embodiment of the method of the present invention said antibodies belonging to a particular isotype are IgM, IgG or IgA antibodies. In this embodiment, usually IgM or IgA detection would be indicative of an earlier phase of infection, whereas IgG antibodies would be indicative of a later phase of infection. In a further preferred embodiment of the method of the present invention, said at least two different phases of an infection include the acute phase, the post-acute phase, the chronic phase, the remission phase of an infection or a phase past infection. The above indicated phases of an infection are well accepted in the medical field, but may, with respect to their distinction from earlier or later phases, in various diseases not be clear-cut for every type of infection. In such cases, the respective phase is to be interpreted in accordance with what the person skilled in the art understands by the respective term. The term "phase past infection" is intended to mean a phase where infectious pathogen is no longer detectable in the organism and pathogenic effects are no longer detectable. In an additional preferred embodiment of the method of the present invention said infection is an infection with a virus, a bacterium or a protozoon. In a particularly preferred embodiment of the method of the present invention said virus is human Cytomegalovirus, Hepatitis C virus, Hepatitis A virus, Hepatitis B virus, Epstein-Barr virus, HIV or Herpes simplex virus. In another particularly preferred embodiment of the method of the present invention said bacterium is Borrelia burgdorferi, Treponema pallidum or Helicobacter pylori. In a different particularly preferred embodiment of the method of the present invention said protozoon is Toxoplasma gondii or Trypanosoma cruzi. In one further preferred embodiment of the method of the present invention said sample from a patient is blood, serum or is derived therefrom. It is well-known in the art that blood and serum may be treated prior to analysis and/or that certain fractions thereof may be used for analysis. The term "is derived therefrom" is intended to include these treated body fluids or fractions thereof. In accordance with the invention, a number of different compounds comprising the epitopes of interest may be employed. For example, the compound may be a carrier of a natural or a non-natural origin. Preferably, the invention in another preferred embodiment relates to a method wherein said compound is an antigen. Preferably said antigen is a natural or a recombinant antigen. In accordance with the present invention, it is further preferred that said antigen is treated with detergents, like SDS, Triton ® -X (Triton ® X-100) or other detergents known to the person skilled in the art prior to step (a). Further detergents known to the skilled artisan comprise Tween ® 20, Tween ® 40, Tween ® 80, CHAPS and CTAB. In a most preferred embodiment of the method of the present invention said antigen is a (poly)peptide or DNA. The term "(poly)peptide", in accordance with the present invention, refers to either a peptide or a polypeptide. The term "(poly)peptide" also includes the term "protein". The (poly)peptide, in accordance with this invention, may comprise naturally occurring peptides or proteins, as well as synthetic or recombinantly produced peptides/proteins. The (poly)peptide may encompass amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such (poly)peptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention. In accordance with this invention, a (poly)peptide may comprise different (poly)peptide species. A (poly)peptide species is defined by its chemical composition and modifications of said peptide(s)/polypeptide(s) by, inter alia, glycosylations, acetylations, phosphorylations, lipidations or by amino acid exchanges. The term (poly)peptide species is therefore defined as the smallest unit of protein classification, defined by its chemical structure. Furthermore, the term (poly)peptide comprises, in accordance with this invention, antigenic fragments of said (poly)peptide(s) and/or fusion proteins. Additionally, the term refers also to a mixture of (poly)peptides, like the mixture in lysates. The term "DNA", in accordance with this invention refers to polynucleotides and/or nucleic acid molecules and refers to coding as well as to non-coding sequences. In accordance with the present invention, the term DNA comprises also any feasible derivative of a nucleic acid and peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkage (Nielson, Science 254 (1991 ), 1497-1500). In another most preferred embodiment of the method of the present invention said antigen or (poly)peptide is CM2 fusion protein (HCMV) (Vornhagen et al., J. of Virological Methods 60: 73-80 (1996) and Vornhagen et al., DE 4 435 789 C1 (1995)), p52 (HCMV) (Vornhagen et al., J. of Clinical Microbiology 34: 1020-1023 (1996)) or a viral particle from HCMV, gp36, gp33, gp27, p30 or p24 (hepatitis B virus) (Meisel et al., Intervirology 37: 330-339 (1994), Cambiaso; EP-B1 0 328 588 (1994)), a Toxoplasma gondii lysate, optionally proteinase K-treated (Milich, PNAS 82 (1985), 8168-8172; Milich, Science 228 (1985), 1195-1199) or deglycosylated, or 41 kDa protein (Coleman and Benach, J. of Infectious Diseases 155: 756-765 (1987)) or 31/34 kDa protein (Craft et al., J. Clin. Invest. 78: 934-939 (1986)) from Borrelia burgdorferi. A number of supports known in the art are suitable for serving the purposes of the present invention. Such supports may comprise, inter alia, plates, stripes, wells, microchips or containers. Suitable materials for such supports or materials for further coating of said supports include, but are not limited to, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, like nitrocellulose, polyacrylamide, agaroses, magnetide and metals, like colloidal gold. In a preferred embodiment of the method of the present invention said support is a bead. Said beads preferably have a range of diameter between 1.5 and 2 μm such as 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0 μm. Compounds comprising an epitope recognized by an antibody specific for an early phase of an infection may thus, for example, be coupled to beads having a diameter of 1.5 μm whereas compounds comprising an epitope recognized by an antibody specific for a later phase of an infection may be coupled to beads having a diameter of 1.9 μm. In one particularly preferred embodiment of the method of the present invention said bead is a latex bead, a colloid metal particle, such as a gold particle, or any combination thereof. In another preferred embodiment of the method of the present invention said property that allows the physical distinction of said types of compounds is mass, size, refractive index, a magnetic property, electric property or a combination thereof. In another preferred embodiment of the method of the present invention the physical distinction of said compounds is effected by measuring a change in said property caused by the agglutination of said compounds. The agglutination of said compounds is effected by their crosslinking by antibody binding of the epitopes. Depending on the concentration of specific antibody and/or antibody isotype present in the sample (and depending on the concentration of epitope), a smaller or larger degree of agglutination will occur. Thus, it is well known in the art hat naturally occurring IgM is usually in a pentameric structure whereas IgA or IgG are dimeric. IgM will thus normally give rise to larger agglutination structures. In a particularly preferred embodiment of the method of the present invention said agglutination is an agglutination of two or three compounds. In a further particularly preferred embodiment of the method of the present invention said measurement is the measurement of light scattering, magnetic field variation or electric field variation. With respect to the embodiment where measurement is effected by light scattering, the technology developed in WO92/21024 and WO 94/15193, which are herewith incorporated by reference, is preferably employed. The methodology as disclosed in WO 92/21024 relies on the use of a high resolution optical sheath flow cell, a single detector for measurement of pulse signals from unidirectional low angle forward light scatter from said differently-sized, differently-coated beads and their aggregated multimers, and a flow particle analyzer apparatus. Said method involves the mixing of samples with said coated monomeric particles and an incubation period of said samples with the coated monomeric particles, to allow agglutination reactions to occur. Said agglutination is measured in said flow particle analyzer, whereas monomeric, dimeric or n-meric particles pass in a sheath- type flow all through a finely focussed optical beam produced by a semiconductor laser. As each type of particle passes through the beam, a unique light scatter signal is produced, which is detected by a photodiode. These pulses are classified by amplitude into a histogram for electronic data analysis. Whereas this method provides qualitative and semiquantitative data, the improved technology developed in W094/15193, based on similar principles but employing binding molecule-coated polymeric microspheres and binding molecule-coated colloid metal particles, allows the simultaneous qualitative and quantitative determination of different analytes. This is achieved through the use of two different photodiodes which detect forward or sideward scattered light signals. In another embodiment, the method of the present invention relates to a method wherein in step (a) the sample from a patient is further incubated with an anti-human immunoglobulin antibody. In a particularly preferred embodiment said anti-human immunoglobulin antibody is an anti-lgM antibody. Said anti-human immunoglobulin antibody may be added prior, during or after contacting the sample from a patient with said at least two types of compounds comprising a different epitope. The addition of said anti-human immunoglobulin antibody may improve the sensitivity for the detection of antibodies/antibody isotypes, as documented in the appended examples. Said anti-human immunoglobulin antibody may be a monoclonal or polyclonal antibody. Said anti-human immunoglobulin antibody comprises also synthetic antibodies, as well as fragments of antibodies, such as F(ab') 2 or scFv fragments (e.g. scFv fragments expressed as "phagobodies" (Felici et al., Biotechnol. Annu. Rev. 1 (1995), 149-183). Such anti-human immunoglobulin antibodies or fragments thereof can be obtained by using methods which are described, e.g. in Harlow and Lane, (1988), -Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory. In an additional preferred embodiment of the method of the invention, fivalian/coupling/coating of said compound to said support is stabilized by surfactant treatment. An example of such a surfactant treatment is provided by appended Example 4. The invention also relates to a kit comprising different types of supports of distinguishable physical properties wherein to each type of support a different compound comprising an epitope is covered, wherein further each epitope is recognized by an antibody/antibody belonging to a particular isotype and each antibody/antibody belonging to a particular isotype is specific for a phase of a disease and wherein said different compounds and epitopes, respectively, are recognized by antibodies/antibodies belonging to a particular isotype specific for at least two different phases of said infection. The kit of the invention is particularly useful in carrying out the method of the invention that has been described in detail herein above. Preferably, said supports are beads. In a more preferred embodiment of the kit of the present invention said beads are latex beads, colloid metal particles, preferrably gold particles, or combinations thereof. In another preferred embodiment of the kit of the present invention said compound is CM2 fusion protein (HCMV), p52 (HCMV) or a viral particle from HCMV, gp36, gp33, gp27, gp30 or gp24 (hepatitis B virus), a Toxoplasma gondii lysate, optionally proteinase K-treated or deglycosylated, or 41 kDa protein or 31/34 kDa protein from Borrelia burgdorferi. In yet another preferred embodiment of the kit of this invention, said kit further comprises an anti-human immunoglobulin antibody. In a particularly preferred embodiment, said anti-human immunoglobulin is an anti-lgM antibody. It is also preferred in accordance with the present invention that the same type of support is coated with different epitopes or different compounds wherein said different epitopes or compounds are recognized by antibodies/antibody isotypes indicative of the same phase of infection. The kit of the invention may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the containers may comprise, inter alia, the anti-human immunoglobulin antibody in lyophilized form or in solution. In addition, the carrier means may also contain a plurality of containers each of which comprises, inter alia, different, predetermined amounts of the compound and/or beads useful in the method of the invention. These latter containers can then be used to prepare a standard curve into which can be interpolated the results obtained from the sample containing the unknown amount or unknown type of the detectable antibodies and/or antibody isotypes. The figures show: Figure 1 Seroconversion profiles evaluated with the Copalis I system, employing CM2 and p52 protein. Used beads are coated with CM2 protein or p52 protein. The Y axis depicts a signal in CTR, whereas on the X axis the days of the infection are blotted. The cut-off lines are printed for the two different beads (see legend). Figure 2 Seroconversion profiles evaluated with the Copalis I system, employing CM2 and VP. Used beads are coated with CM2 protein or viral particle (VP). The Y-axis depicts the signal in CTR, whereas on the X axis the days of infection are blotted. The cut-off lines are printed for the two beads (see legend). Figure 3 Evaluation of patient sera, positive for anti-Toxoplasma gondii IgG and negative for anti-Toxoplasma gondii IgM. Top, left: Distribution frequency of sera which are negative for anti- Toxoplasma gondii-lgM and -IgG, as evaluated with the Copalis I system. The used beads are coated with the Toxoplasma gondii lysate which was treated with proteinase K. Top, right: Comparative results obtained with sera which are negative for anti Toxoplasma gondii-lgM and -IgG, employing the Copalis I system and a reference test (Toxo Vidas IgG, # 30209 (TXG), Toxo Vidas IgM, # 30202 (TXM)). Bottom, left: Distribution frequency in sera which are negative for anti- Toxoplasma gondii-lgM and positive for anti-Toxoplasma gondii-lgG, as evaluated with the Copalis I system. Again, the used beads have been coated with Toxoplasma gondii lysate previously treated with proteinase K. Bottom right: Comparative results obtained with sera which were negative for anti-Toxoplasma gondii-lgM and positive for anti- Toxoplasma gondii-lgG, employing the Copalis I system and the reference test (Toxo Vidas IgG, # 30209 (TXG), Toxo Vidas IgM, # 30202 (TXM)). Figure 4 Top: Distribution of sera which are positive for anti-Toxoplasma gondii IgM as evaluated with the Copalis I system (left) and the reference system (right) (Toxo Vidas IgM, # 30202 (TXM)). Bottom: Comparative results obtained with sera positive for anti- Toxoplasma gondii IgM as evaluated with Copalis I system and a reference system (Toxo Vidas IgM, # 30202 (TXM)). Figure 5 Seroconversion profiles evaluated with the Copalis I system employing CM2, p52 and VP. Used beads are coated with CM2 protein, p52 protein or viral particle (VP). Left: the test has been carried out with the addition of the anti-lgM antibody. Right: the test has been carried out without the addition of the anti-lgM antibody. The Examples illustrate the invention Example 1 Differentiation of acute from post-acute phase of HCMV infection In order to differentiate the acute from the post-acute phase of an HCMV infection and to discriminate in a simultaneous immunoassay between IgM and IgG antibodies, differently sized latex microparticles were coated with various HCMV specific viral antigens. Latex beads of a diameter size of 1.8 μm were coated with CM2 fusion protein, latex beads of a diameter of 1.7 μm were coated with the p52 protein and latex beads with a diameter of 1.6 μm were coated with viral particle (VP). The coated latex beads were used for simultaneous measurement of IgM or IgG in a sheath flow optical Flow Particle Analyzer (Hansen (WO92/21204)). For the coating procedure, a coating buffer (CB), comprising 1.59 g/l Na 2 C0 3 , 2.93 g/l NaHC0 at pH 9.6 and an overcoating buffer (OB), comprising 7.51 g/l glycine, 10 g/l BSA fraction V protease free, 50 g/l sucrose and 2 g/l NaN 3 were used. In order to coat CM2 (from Biotest AG, Germany; Vornhagen et al., J. of Virological Methods 60:73-80 (1996), and DE 4 435 789 C1 ) to the 1.8 μm latex beads, a 0.5% solution of latex beads in coating buffer was prepared. After 3 washes of the beads in coating buffer comprising centrifugation steps for 20 minutes at 4,000 rpm, the beads were resuspended in CB to a final concentration of 1%. The recombinant CM2 protein was prepared in coating buffer at a final concentration of 20 μg/ml. Drop by drop, the recombinant protein solution was combined with the 1.8 μm latex beads. Hydrophobic coating of the latex beads with CM2 protein was carried out during a 1 hour incubation at room temperature on a drum roller. Coated latex beads were washed in OB by centrifugation for 20 minutes at 4,000 rpm. The coated 1.8 μm latex beads were resuspended in OB to a concentration of 1%. Post-coating in OB was carried out for 1 hour at room temperature on a drum roller. After centrifugation for 20 minutes at 4,000 rpm, the beads were resuspended in OB to a final concentration of 0.5% (w/v) and stored at 4°C until further use. In order to coat the 1.7 μm latex beads with p52, a 1 % solution of these beads was prepared in coating buffer. After 3 washing steps in CB, the 1.7 μm latex beads were resuspended in CB to a final concentration of 2%. In order to obtain p52, the p52 gene was isolated by PCR using the following primers: 5' GACTGGATCCGATCGCAAGACGCGCCTC 3' forward primer (SEQ ID NO: 1 ) 5' GACTAAGCTTCGCGCTCTAGCCGCACTT 3' reverse primer (SEQ ID NO: 2) The PCR product was inserted into pET30 (Kan R) (Novagen; USA) with a His tag at the N-terminus. The resulting plasmid was used to transform E. coli BL21 cells and the heterologous protein was obtained after 3 hours of induction. The cellular paste was processed as follows: the p52 pellet obtained from a 125 mL culture was resuspended in 5 mL of 50 mM Tris pH 8, adding 50 μl benzonase (Benzonuclease from Merch, Germany) and 50 μl MgCI 2 0.1 M. After the incubation at 37°C for 3 hours, the sample was spun at 10°C for 30 min 11 ,000 rpm in a JA20 rotor. The resulting pellet was resuspended in 3 mL of 50 mM Tris, 8 M urea, 10 mM beta- OH pH 9.4 (buffer A), dissolved and sonicated 5 times for 30 sec. The obtained lysate was shaken for 1 hour at RT, then heated at 70°C for 10 min and spun at 4°C for 30 min 11 ,000 rpm. The 8 M urea supernatant was gel-filtrated with the buffer A, and the pooled protein was dialyzed overnight at RT against 1.5 I of buffer 50 mM Tris, 6 M urea, 500 mM NaCI pH 8.8. The protein has been purified through chelating chromatography (# 17040901 PHARMACIA, Sweden), exploiting the His tail property. A 100 μg/ml p52 protein solution in coating buffer was prepared, SDS to a final concentration of 0.1% was added to the recombinant protein solution and incubated at room temperature for 30 minutes. After this incubation the 1.7 μm latex beads were combined, drop by drop, with the recombinant p52 protein solution. Hydrophobic coating of the latex beads with p52 was carried out by incubation for 2.5 hours at room temperature on a drum roller. The beads were washed twice in OB and post-coating was carried out for 1 hour at room temperature in OB. After a further centrifugation step at 4,000 rpm for 20 minutes, the p52-coated 1.7 μm latex beads were resuspended in OB to a final concentration of 1 % (w/v) and stored at 4°C until further use. Coating of 1.6 μm latex beads with viral particle (VP) was carried out by washing 1.6 μm latex beads three times in coating buffer. After the last washing step a 2% solution of 1.6 μm latex beads in OB was prepared. To 1 μg of viral particle (VP from AB USA) 80 μl 1 % SDS solution was added and incubated for 30 minutes at room temperature. The viral particle solution was prepared at a concentration of 160 μg/ml in coating buffer. Same volumes of viral particle solution and 1.6 μm latex beads solution were combined drop by drop and incubated for 1 hour at room temperature on a drum roller. After 2 washing steps in OB, the latex beads were resuspended in OB and post-coating was carried out for 1 hour at room temperature on a drum roller. After further centrifugation steps at 4,000 rpm for 20 minutes, the beads were resuspended in OB to a final concentration of 1 % (w/v) and stored at 4°C until further use. For assay procedures during a Copalis I run, 20 μl of viral particle-coated 1.6 μm latex beads, 5 μl of p52-coated 1.7 μm latex beads, 15 μl of CM2-coated 1.8 μm latex beads and 3 μl of irp (internal reference partial of 1.1 μm) were mixed and spun for 20 minutes at 4,000 rpm. After the centrifugation step, 23 μl of the supernatant were discarded and the beads were resuspended in 20 μl OB-buffer per reaction vessel. The reaction vessels were dried for 75 min by heating at 37°C under control of relative humidity (10%), and a stirring bar was added to each cup. Cups were stored at 4°C until further use. The dried latex-bead mixture was resuspended in 180 μl reaction buffer (comprising 0. 5 M KBr, 0.1 % BSA, 0.15% PEG-8000, 0.002% zwitterionic detergent, 1 mM EDTA and and 0.1 M Glycine at pH 9.0) and 20 μl of test sample was added. After an incubation for 10 minutes at RT, the sample was measured under Copalis I standard, procedures (Copalis™ System, Procedure Manual 1997, Sienna Biotech). The measurement in Copalis I is based on a monitoring of light scattered from single particles or particle aggregates the instrument belongs to the class of Flow Particle Analyzes (FPA) (Hansen (WO92/21024)). Latex beads and n-meric microparticles can be discriminated in size by the scattered photon energy. If coating on the latex beads caused an agglutination of the microparticles in the presence of IgG or IgM in the sample, the latex beads changed their size distribution. The number of monomeric latex beads was depleted in a sample, while the number of n-meric particles was increased. The comparison of the number of monomeric latex beads when exposed to a non-reactive sample and the same monomer number when exposed to a reactive sample is compared. To define the presence of a specific reactive component and the extent of their reaction, the Copalis Test Result (CTR) can be mathematically defined as 100 times the ratio between the number of monomeric microparticles counted in the presence of a non-reactive sample (negative control) and the same entity in the presence of a reacting sample (CTR = 100 x (negative control )/(sample under assay)). The CTR monitors the tested sample reactivity with respect to a non-reactive reference specimen/sample. CTR indicates the coated microparticle tendency to aggregate in the presence of the tested sample, i.e. the number of reacted monomers. During test-runs with negative sera, analytical or clinical cut-offs were defined, whereas the analytical cut-off was defined as the Signal (CTR) that induced 99.5% of an internal reference of normal negative control population and the clinical cut-off as the Signal (CTR) that allowed 80% sensitivity on the prediction of a recent infection as evaluated on positive reference population. In order to evaluate whether a sample was negative or positive, a S/CO ratio was defined. S was defined as the Signal-CTR and CO as the cut-off CTR. Negative samples showed a S/CO ratio <1.00, whereas positive samples showed a S/CO ratio >1.00. Test results with CM2-, p52-, viral particle-coated latex beads and different sera CM2 coated latex beads were supposed to react with IgM during acute and postacute phase of an HCMV infection, p52 coated latex beads were supposed to react with IgM of the acute phase of an HCMV infection and VP coated latex beads were supposed to have a high sensitivity for IgG after an HCMV infection. Accordingly, the specificity and sensitivity of the different coated latex bead was tested. These relevant data are summarized in Tables 1 to 5. Specificity and sensitivity of the HCMV-antigen coated latex beads was tested, using anti-CMV IgG and anti-CMV IgM negative sera. Table 1 : IgM NEGATIVE / IgG NEGATIVE SERA Table 2: IgM NEGATIVE / IgG POSITIVE SERA Table 3: IgM - LOW POSITIVE SERA Table 4: IgM MEDIUM POSITIVE SERA Table 5: IgM - HIGH POSITIVE SERA Not tested for VP Not tested for VP As shown in Table 1 , all 25 tested sera were negative and did not cause agglutination of neither CM2-coated, p52-coated nor VP-coated latex beads. No false positive reactions occurred. Table 2 shows the results for 25 Test-sera which were anti-CMV IgM-negative and anti-CMV IgG-positive. Only one serum (# 23208) showed reactivity with CM2- and p52-coated beads (probably due to anti-latex antibodies in the serum), whereas all tested samples showed reactivity with VP-coated latex beads, which documented the high sensitivity of VP-coated beads for anti-HCMV IgG. Tables 3, 4 and 5 document the high sensitivity of CM2- and p52-coated latex beads for anti-CMV IgM antibodies. For the documented results, different anti-CMV IgM positive sera of low (Table 3), medium (Table 4) or high (Table 5) titers have been used. In order to evaluate anti-HCMV seroconversion in patients, different blood samples of 9 individual patients (all pregnant women) have been analyzed. The day of HCMV infection had been evaluated using a standard test (Revello M.G: et al., J. Virol. Methods 35 (1991 ), 315-329) from Pavia Hospital and kits from DiaSorin: ETI- CYTOK-G (# 2860) and ETI-CYTOK-M reverse (# 3238) employed according to the manufacturers instructions) and by clinical information obtained by each patient. Table 6: Seroconversions Table 6 shows for each patient the test-results which were obtained at different days post-infection, using the CM2-, p52- and VP-coated latex beads in Copalis I (Copalis™ System, Procedure Manual 1997, Sienna Biotech). Data obtained with CM2-coated beads demonstrate that this fusion protein was able to detect all anti-HCMV IgM antibodies up to the end of the post-acute phase (6 to 7 months post infection). Results obained with p52-coated beads show (see e.g. seroconversion # 20675) that p52 was very specific for the acute phase of the HCMV infection: the signal generated by the p52-coated latex beads increased during the month of infection and decreased during the second month post infection. This decrease of p52-signal can be correlated to the end of the acute phase of HCMV infection. Results obtained with VP-coated latex beads demonstrated the appearance of anti- HCMV IgG antibodies in the tested sera during the progression of HCMV infection. The seroconversion data obtained from these 9 patient samples are summarized in the graphics of Figures 1 a, 1 b, 2a and 2b. These data demonstrate that the CM2- coated latex beads detected all anti-HCMV IgM antibodies up to the end of the post- acute phase of the infection, whereas for anti-HCMV IgM immunoreactivity with p52 is prominent during early phases/acute phases of an HCMV infection. As demonstrated in this example, multianalyte immunoassays for immunoglobulins of different subtypes can be carried out simultaneously, using specific antigen coated microparticles of different sizes, each of the microparticles being reactive towards a different antibody class in a biological fluid. These data show a high specificity (98%) and sensitivity (94%) of the latex beads coated with CM2 and p52 for anti-HCMV IgM antibody isotype and a high specificity (98%) and sensitivity (98%) of VP-coated latex beads for anti-HCMV IgG antibody isotype. In conclusion, it is possible to determine the stage of an HCMV infection (acute or post-acute phase) combining the data which can be obtained with CM2- and p52-coated beads: an acute phase of infection is identified by high p52 and high CM2 values, whereas the post-acute phase is defined by low p52 and high CM2 readings. Example 2 Hepatitis B, differentiation of acute from remission infection phase In order to provide antigens which could be used as marker systems for the discrimination between acute and remission phase of a Hepatitis B (HBV) infection and could be used for the detection of anti-HBV IgM and anti-HBV IgG in patient sera, different indirect Western blots have been carried out. The recombinant preS2-HBsAg protein (Sammata and Youn, Vaccine 7: 69-76 (1989); ay subtype), the so-called middle-protein, was expressed in human kidney cells 293. (ATCC, Maryland, USA) using a BK vector (Gallina et al., J. of General Virology 73:139-148 (1992)). PreS2-HBsAg is a Hepatitis B surface protein (Heermann, J. Virol. 52: 396-402 (1984)), using 3 different in-frame start codons (Gallina et al., J. of General Virology 73:139-148 (1992)). Initiation at the 3 rd AUG codon generates the major protein (HBsAg, p24 and gp27 in its glycosylated form); initiation at the 2 nd AUG codon generates the preS2-HBsAg protein (middle-protein p30, and its mono- and diglycosylated forms, gp33 and gp36 respectively) and initiation at the 1 st AUG codon adds a further 108 to 119 aa extension (preS1 region) to the N-term of the middle protein generating the so-called large-protein (p39). In order to purify preS2-HBsAg, the cell culture supernatant was neutralized with a solution containing 0.15 M NaCI, 0.01 M NaH 2 P0 4 , 1 mM MgCI 2 at pH 7.8. Endonuclease (445 U/μL, SIGMA) and 0.05 mM PMSF, 1 μg/mL Aprotinin, 1 μg/mL Leupeptin (anti-protease mixture) were added. A first precipitation was carried out by slowly adding PEG (polyethylene glycol) to a final concentration of 6%. After an incubation for 30 min at 37°C on a shaker, the solution was discarded and a second precipitation with PEG (final concentration of 14%) was carried out. After incubation at 37°C for 30 min on a shaker, the solution was centrifuged at 10,000 rpm for 25 min at room temperature. The resulting pellet was resuspended in 1/34 initial volume in PBS/0.05% NaN 3 at pH 7.4 and supplemented with the anti-protease mixture. 100 mL of the sample was dialyzed for 1 hour at room temperature with SpectraPor MWCO 50000 against 0.05 M citrate buffer at pH 2.4. After a centrifugation at 10,000 rpm for 15 min, the dialyzed samples were neutralized with 0.03 M NaHC0 3 . The pellet was again dialyzed with SpectraPor MWCO 50000 against PBS, 0.05% NaN 3 at pH 7.4 and anti-protease mixture. The solution was filtered (porosity of filter: 0.2 μM) and dispensed in aliquots until further use. Two 140 μl aliquots of purified preS2-HBsAg were analysed by SDS-PAGE and four bands could be detected: gp36, gp33, gp27 and p24 (Meisel et al., Intervirology 37: 330-339 (1994)). The gp36 polypeptide was the deglycosylated form of the preS2- HBsAg protein (Asn-123 within the preS2 region and Asn-320 within the S region), gp33 was the monoglycosylated form of the preS2 region (Asn-123), gp27 was the glycosylated form of the S region (Asn-320), p24 represented the non-glycosylated form of the S region (Meisel et al., Intervirology 37: 330-339 (1994)). One aliquot of the recombinant preS2-HBsAg protein was deglycosylated: Na 2 HP0 buffer pH 7.7 and a solution containing 2% SDS and 1 M 2-mercaptoethanol were added to the protein sample. After heating of the sample (140 μl) at 100°C for 5 minutes and immediately cooling with ice for 5 minutes, 12 μl Nonidet P-40 (99%) and 5 μl PNGaseF enzyme (BioRad, 2.5 Ul/ml) were added at 37°C for 2.5 hours. The obtained p30 protein was the non-glycosylated form of preS2-HBsAg protein. The two aliquots (untreated and deglycosylated) were divided into four parts (samples 1 , 2, 3, 4), separated on SDS-PAGE (polyacrylamide gel 15%) and transferred onto a PVDF membrane (IMMOBILON P, Millipore) in order to perform Western blot analysis. Transferred samples 1 and 2 were incubated with a 1 :50 dilution of PHM 907-09 serum, a seroconversion marker for HBV infection (provided by Boston Biomedica Inc.) in dry low-fat milk in PBS pH 7.4. Samples 3 and 4 were incubated with a serum representative of the remission phase (from AALTO, Ireland) diluted 1 :50 in dry skimmed milk in PBS pH 7.4. After incubation at 37°C for 1 hour, the membrane was thoroughly washed with PBS pH 7.4. Samples 1 and 3 were further incubated with a 1 :200 dilution of anti-human IgM monoclonal antibodies, labeled with HRP (horse-radish peroxidase) (from CSL, Australia). Samples 2 and 4 were incubated with a 1 :350 dilution of anti-human IgG goat antibodies (Atlantic Antibodies, Maine, USA), labeled with HRP. After an incubation period of 1 hour at 37 °C on a shaker, the membranes were washed and a revelation procedure was carried out, using 9-ethyl-amino-carbazol (AEC, Sigma). Table 7 Case 1. Acute phase. Tracer: anti-human IgM monoclonal antibodies (Mab HSP- P06, Silenum, ADM) Legend: ++ very high reaction, + high reaction, +/- weak reaction or very weak reaction, - null reaction Table 8 Case 2. Acute phase. Tracer: anti-human IgG goat antibodies (Atlantic Antibodies, Maine, USA) Legend: ++ very high reaction, + high reaction, +/- weak reaction or very weak reaction, - null reaction As shown in Tables 7 and 8, during acute phase of an HBV infection, two glycosylated forms of preS2, gp36 and gp33, were highly reactive with human IgG/lgM antibodies, whilst there was no reactivity towards the S region, the gp27 and p24 polypeptides. In sample 2 a very high reactivity with gp36 and p30 could be detected with anti-human IgG antibodies. No reactivity against gp27 and p24 could be detected with anti-human IgG antibodies. Table 9 Case 3. Remission phase. Tracer: anti-human IgM monoclonal antibodies (Mab HSP-P06, Silenum, ADM) Legend: ++ very high reaction, + high reaction, +/- weak reaction or very weak reaction, - null reaction Table 10 Case 4. Remission phase. Tracer: anti-human IgG goat antibodies (Atlantic Antibodies, Maine, USA) Legend: ++ very high reaction, + high reaction, +/- weak reaction or very weak reaction, - null reaction During remission phase of an HBV infection (see Tables 9 and 10), no reactivity of any of the tested HBV-specific antigens could be detected with anti-human IgM antibodies. In contrast, very high reactivity towards gp27 and p24, but no reactivity against gp27, p30 and p24 could be shown with anti-human IgG conjugates. In conclusion, PHM907-09 serum (DNA HBV positive, HBsAg positive and HBeAg positive), being diagnostic for an acute HBV infection phase, reacted very strongly with the gp36 and gp33 antigens, showing immunoreactivity of the patient towards the glycosylated forms of preS2 region. In contrast, the serum representative of the remission phase (anti-HBs positive, anti-HBc positive, anti-HBe positive) reacted mainly with gp27 and p24, showing high immunoreactivity towards the major protein (S region). Therefore, in analogy to example 1 , gp36, gp33 and p30 can be used as specific antigens for the detection of an acute phase of an HBV infection, whereas gp27 and p24 can be employed as antigens for the remission phase of an HBV infection. Example 3 Toxoplasma gondii isotype antibody differentiation and preparation of isotype specific antigen-beads In order to prepare latex beads which are coated with Toxoplasma gondii (Tg), specific antigens for the detection of anti-Tg IgMs, 1.8 μm latex beads (1 % w/v) were washed 3 times in washing buffer (0.1 M glycine, 0.17 M NaCI, 9.15 NaN 3 at pH 9.2, diluted in 1 :15 deionized H 2 0). After a last centrifugation step at 13 krpm for 4 minutes, 620 μl Toxoplasma gondii lysate (T. gondii were obtained from Gaslini Hospital Genua, Italy and the lysate was prepared according to conventional procedures) in NP40 (concentration 5.11 mg/ml, lot 076, Pharmacia, USA) was added to the latex bead pellet, to a final antigen concentration of 1.5 mg/ml. After the addition of 200 μl Glycine Buffered Saline (GBS: 0.1 M glycine, 0.17 M NaCI, 9.15 mM NaN 3 at pH 9.2) and 180 μl deionized H 2 0, the beads were incubated with the Toxoplasma gondii lysate for 30 minutes at room temperature. After centrifugation at 13 krpm for 4 minutes, the beads were washed twice in GBS (diluted 1 :15 with deionized H 2 0) and once in GBS containing 10g/l BSA (bovine serum albumine). Pelleted beads were resuspended in 1 ml GBS-BSA buffer and sonicated for 15 sec at 150 W. Beads were pelleted by centrifugation at 13 krpm for 4 minutes and washed three times in TRIS-buffer (0.1 M TRIS, 20 mM CaCI 2 at pH 7.5). After the last wash, beads were resuspended in 1 ml TRIS-buffer containing 200 μm/ml proteinase K. After a 2- hour incubation at 37°C on an orbital shaker, the proteinase K reaction was blocked by addition of 10 μl 0.1 M PMSF (Sigma, USA) and further incubation for 30 minutes at room temperature. After centrifugation at 13 krpm for 4 minutes, the coated beads were washed 3 times in 1 ml GBS-BSA buffer, and beads were dried according to example 1 and stored at 4°C until further use. Before their use in assays, beads were resuspended in reaction buffer (as described in example 1 ) and sonicated at 150 W for 15 sec. For the assay procedure during a Copalis I run (Copalis™ System, Procedure Manual 1997, Sienna Biotech), the resuspended Tg/proteinase K-coated latex beads 180 μl were combined with 20 μl test sample. After an incubation for 10 minutes at room temperature, the sample was measured under Copalis I standard procedures, as briefly outlined and described in Example 1. In order to analyze the specificity of the Tg/proteinase K-coated beads for anti-Tg IgM determination in sera, the Tg/proteinase K beads were tested using anti-Tg IgM and anti-Tg IgG negative control sera (doubly negative control sera). Furthermore, the beads were tested on sera, which were anti-Tg IgM negative and anti-Tg IgG positive. The results of the tests are shown in Figure 3. Of 93 doubly negative control sera, only 3 samples showed values above the cut-off CTR (123). In 24 samples which were anti-Tg IgG positive but anti-Tg IgM negative, no false positive signal could be detected. No cross reactivity of the proteinase K treated beads with anti-Tg IgG could be measured. All measured CTRs were below the cut-off CTR of 123. As illustrated in Figure 4,. when anti-Tg IgM positive sera were used in the Copalis analysis of Tg/proteinase K-coated beads, only 1 out of 96 samples was false positive, illustrating the high sensitivity of proteinase K treated Tg-coated beads for the detection of anti-Tg IgM antibodies. The results from Figures 3 and 4 are summarized in Tables 11 and 12. TABLE 11A IgG neg - IgM neg sera TABLE 11B IgG neg - IgM neg sera TABLE 12 IgM pos SERA A 42 +/- 170 A 43 +/- 256 A 44 ++ 320 A 45 ++ 373 A 46 + 405 A 48 +/- 212 A 50 + 425 A 51 + 244 A 52 +/- 266 A 53 + 233 A 54 + 375 A 55 + 460 A 56 + 447 A 57 + 446 A 58 +/- 278 A 59 ++ 498 A 60 ++ 576 A 61 +/- 349 A 62 +/- 419 A 63 +/- 275 A 64 + 337 A 65 + 140 A 66 +/- 201 A 67 +/- 210 A 68 + 237 A 69 + 450 A 77 + 445 B 2 ++++ 465 B 3 +++ 332 B 4 +++ 127 B 5 ++++ 359 B 7 +/- 195 B 13 +/- 184 B 14 +/- 197 B 25 ++ 158 B 31 ++ 500 B 32 ++++ 552 B 33 +++ 567 B 34 + 136 B 35 + 146 B 37 ++ 291 B 38 + 303 B 39 +++ 276 B 41 + 126 B 43 ++++ 341 B 44 +/- 218 B 46 ++ 374 B 47 + 343 B 48 +++ 487 B 49 +++ 330 B 50 + 127 B 65 +++ 580 B 70 ++ 616 B 71 ++ 167 B 78 ++ 165 B 80 ++ 172 B 84 ++ 105 In order to provide specific latex beads which were coated with Tg-antigens specific for anti-Tg IgG reaction, the following experiments were carried out. Since it was confirmed that the anti-Tg IgM reactivity is localized on glycosydic moieties of specific proteins from the pathogen (see the previous section), it was reasoned that the cleavage of these glycosydic moieties should lead to modified antigens which are specific for anti-Tg IgGs detection. The cleavage or disruption of the glycosydic part of the main Toxoplasma g. proteins has been done by two methods on different extracts of the protozoa: 1. enzymatic cleavage by an enzymatic deglycosylation kit (# 170.6500, BioRad, USA) 2. chemical disruption: oxidation by Nal0 4 . The enzymatic deglycosylation kit enzymatically cleaves N-linked and sialic acid substituted Gal (b1-3) GalNAc (a1 ) O-linked oligosaccharides from glycoproteins. The enzymatic deglycosylation was less harsh than chemical methods and provided deglycosylated glycoproteins that were suitable for further protein and carbohydrate analysis. Furthermore, the role of the glycosylation on protein bioactivity and antibody binding could be determined. Deglycosylation-reaction was carried out according to manufacturers' recommendation. Efficiency of the deglycosylation reaction was confirmed by running samples (before and after the deglycosylation) on SDS-PAGE gels and Western blots. With these techniques, a reduction of the molecular weights of the main antigens was observed, which resulted in a disruption of anti-TG IgM reactivity, while the anti-Tg IgG reactivity was completely maintained. The deglycolsylated protein mixture was coated onto polystyrene particles of appropriate size, ranging from 1.6 through 1.9 μm. A surfactant was added in order to stabilize the coating and a post-coating buffer (Overcoating buffer as described in example 1 ) was employed to reduce non-specific reactions. The coated beads were treated with the deglycosylating mixture at 37°C for 2.5 hours and stored at 4°C until further use. After drying of the beads, the beads were resuspended in reaction buffer and mixed with test samples (as described in example 1 ). After an incubation for 10 min at room temperature, the sample was measured under Copalis I standard procedures (Copalis™ System, Procedure Manual 1997, Sienna Biotech). The findings from these experiments confirmed the results obtained by the Western blot analysis: anti-Tg IgM reactivity was reduced, whereas anti-Tg IgG reactivity was maintained. The chemical disruption was performed by treating the main Toxoplasma g. protein with solutions of Nal0 4 at different concentrations (ranging from 0.01 to 40 mM). Reactions were carried out at room temperature for 30 minutes and at different pHs. The reactions were blocked by ethylenglycol treatment acid treatment. Residual aldehydic groups were blocked by the reduction with NaCNBH 3 in the presence of glycine or ethanolamine 0.1 M at basic pH. Efficiency of the reactions was confirmed by running samples (before and after the deglycosylation) on SDS-PAGE gels and Western blots. With these techniques, it was observed that the anti-Tg IgM reactivity was disrupted, whereas the anti-Tg IgG reactivity was completely maintained. Toxoplasma gondii lysate-coated polystyrene beads were also treated with the periodate. When agglutination assays were performed with the Nal0 treated beads, the results as described for the Western blot assays could be confirmed. Example 4 Differentiation of Borrelia burgdorferi infection phases At the onset of an infection with Borrelia burgdorferi, a rise of IgM antibody-titers against a flagellum protein of 41 kDa is observed (Coleman and Benach, J. of Infectious Diseases 155, 756-765 (1987)). Later during infection, IgG reactivity against the same protein is developed. The anti-41 kDa antigen reactivity is maintained either for short (weeks through months) periods, or for long (even years) and is supposed to be due to the continuous presence of the spirochete. The reactivity against 41 kDa is seen as a marker for a freshly contracted infection, but cannot be used as a marker for late infection since patient specific reactions are described which vary from patient to patient. Furthermore, later during infection reactivities against a broader spectrum of Borrelia antigens, the molecular weights of which can range from 14 to about 97 kDa, is documented. Mainly an immunoreactivity against two different outer membrane proteins of 31 kDa and 34 kDa respectively, is observed (Craft et al., J. Clin. Invest. 78, 934-939 (1986)). These two proteins can be used as markers for a latent infection. The reactivity against the 31/34 kDa couple is mainly sustained by IgG, but can also be due to the presence of IgM. A differentiation between a new and old Borrelia burgdorferi infection is based on the finding that no reactivity against the 31/34 kDa proteins can be detected during early phases of infection. The three Borrelia burgdorferi proteins are easily purified by separation of the outer membrane fraction (31 kDa and 34 kDa antigens) of Borrelia burgdorferi from the periplasmatic flageliae (41 kDa protein), and further electrophoretic separation or electroelution. In order to obtain outer envelope (OE) components of Borrelia burgdorferi, Borrelia burgdorferi cultures were centrifuged (7000 g for 20 min at 20°C). The resulting pellet was washed and resuspended in saline. SDS (0.03%) was added at RT for 15 min and the suspension was centrifuged (25000 g for 90 min at 4°C). The supernatant containing the OE fraction was filtered and dialyzed to remove, SDS. The pellet fraction resulting from the SDS treatment was resuspended in saline and blended in a Waring blender. The suspension was centrifuged (26000 g for 6 min at 4°C) and the resulting supernatant was collected. The procedure was repeated several times on the remaining pellets and the collected supematants were pooled and concentrated by TFUF (tangential-flow ultrafiltration, Millipore Minitom). This resulted in a periplasmic flageliae fraction (PF). A preparative SDS-PAGE gel was loaded either with OE or PF fraction and run overnight at 30 mA. The protein position was identified by Coomassie blue staining and subsequent isopropanol/acetic acid destaining. The desired proteins were identified by comparison of the resulting gel with an analytical SDS-PAGE. The gel portion containing the proteins was then electroeluted for 12 hours at 10 V in a BioRad Transblot. In order to remove SDS, a dialysis step followed. The purified proteins were coated onto polystyrene microparticles of appropriate size, ranging from 1.6 through 1.9 μm, under either acidic, basic or neutral conditions, using either sodium carbonate (pH 9.6), glycine (pH 4.0) or HEPES (pH 7.0) as buffer systems. A surfactant (SDS or Tween 20 at concentrations ranging from 0.01 to 0.05%) was added in order to stabilize the coating. In the same way, a lysate of a Borrelia burgdorferi culture was coated onto the polystyrene particles. The coated latex beads are then blocked by a neutral BSA-sucrose solution (1 % BSA, 5% sucrose), BSA concentration around 1% and the sucrose. The results of the agglutination immunoassay gives the following reactivity patterns: anti 41 kDa anti 31/34 kDa anti lysate reactivity interpretation reactivity reactivity neg neg neg not exposed patient pos neg neg or slightly pos new infection pos pos pos old infection, living spirochetes neg pos pos or slighlty pos old infection Example 5: Evaluation of a Cytomegalovirus (CMV, HCMV) Multiplex Reagent Kit on Copalis I The CMV Multiplex Reagent Kit is an homogeneous test for the simultaneous determination of different isotypes of anti-CMV antibodies developed by DiaSorin s.r.l. on the instrument Copalis I (see Example 1 ). The CMV Multiplex assay is a test able to differentiate between acute infection stages and immune status to Cytomegalovirus, using coupled particle light scattering. Sized micro-particles, coated with one of three antigens (p52, CM2, and viral particle (VP) see Example 1 ) are dried in the reaction cup. "Acute" IgM antibodies are detected by the CM2 and the p52 antigens, however, an antibody response to the CM2 antigen but not to the p52 antigen can only be detected during the convalescent stage of an acute infection. Furthermore, antibodies to the VP appear during acute infection and persist. Single reactivity to VP is, in contrast, characteristic of a prior infection and therefore indicative of an immune status. Antibody detection (as carried out by the Copalis I Immunoassay System) is here documented qualitatively for the IgM analytes and semi-quantitatively for the IgG analytes. Samples were obtained from different patient categories employing the following protocol: Expected negative subjects: 100 expected negative samples Past CMV infection: 150 expected IgG positive samples CMV infection follow-up: 20 with paired consecutive samples All the specimens were classified first on the basis of the laboratory routine test and by analysis of the available clinical information. Comparative assays which were used to test the samples comprised the DiaSorin ELISA kits: ETI-CYTOK-G and ETI-CYTOK-M reverse (see Example 1 : available from DiaSorin #2860 and #3238 and the test developed by Revello M.G. et al., J._ Virol. Methods 35 (1991 ), 315-329). These tests were employed according to the manufacturers instructions. The samples were tested in parallel employing the Copalis CMV multiplex kit and the above-mentioned comparative kits, according to the manufacturers instructions. Obtained results were expressed as "Copalis index" (Cl): This index monitors the tested sample reactivity with respect to the cut-off signal. The latter, in turn, is calculated on a basis of the monthly instrument calibration. The Copalis Index is the parameter of choice for clinical interpretation of results. The Cl is a positive number/figure defined by the following characteristics: The cut-off threshold (CO) corresponds to: Cl=1.00 The high-positive threshold (HP, were defined) corresponds to: Cl=3.00 In this study, a sample was considered positive when a Cl of 1 or higher was obtained; a "grey zone" of ±5%, ±15% and ±20% across the threshold was set for VP, CM2 and p52, respectively. In order to optimize the clinical information from the instrument, said grey zone ("equivocal") is set between negative and positive results. The equivocal zone setting is marker dependent: for example, in this CMV assay, a grey zone of ±5%, ±15%, ±20% on the Copalis Index is set for the considered markers (VP, CM2 and p52, respectively). In order to explain discrepancies between the CMV Multiplex Reagent kit and the comparative kits, the discordant and doubtful samples were tested by an additional Western Blot assays. This routine test (Western test) was the same as the above described comparative assay, employing the kits ETI-CYTOK-G and ETI-CYTOK-M reverse. The following results were obtained: 367 serum samples, selected from the required patient categories, were tested. According to the analysis of the routine test results (see above), the tested samples were classified as: 100 expected negative samples 198 expected IgG positive samples 21 subjects with at least two paired consecutive samples (69 samples) The classification of all samples was based on the reactivity for anti-CMV antibodies in the laboratory routine tests (see above). If further ambiguous results were obtained, additional tests were carried out in order to classify the sera samples. These tests comprised, inter alia, standard Western analysis on fluorotrans-membranes. Briefly, in this standard Western blot, viral (CMV) lysates were run on SDS-PAGE and transferred onto fluorotrans-membranes. Said membranes were incubated with the ambiguous sera and, correspondingly with defined negative and positive control sera in order to classify the ambiguous test sera. Development of the Western blot was carried out using standard HRP and chloroNaphtol H 2 0 2 developments. After clear classification of the test samples, all samples were stored frozen before Copalis testing. 1. Expected negative samples 100 serum samples were classified as negative for anti-CMV IgG and IgM on the basis of the above described routine tests and were tested on Copalis. The results are reported in the following table: expected value COPALIS Five samples gave a deviating result: they showed a positive Copalis result on CM2, but the routine tests classified them as negative. Detailed initial and repeat results are reported in the following table (the Cl is given for Copalis results and the signal to cut-off ratio (S/CO) for ELISA). Table 13: Deviating samples of expected negative sera These 5 discordant samples were classified as negative by the DiaSorin ELISA tests: three of them (#21784111, #21784VI and #22972VIII) gave only positive results by Copalis test-CM2, and the other two (#21784V and #22972VII) were classified as "equivocal", i.e. in the "grey zone" as defined herein above. The three truly negative samples were considered as false positive results by the Copalis test; the specificity of this marker (CM2) was 97%, whereas the other two markers (p52 and VP) showed a specificity of 100%. 2. Expected IgG positive samples 195 serum samples were selected (on the basis of the routine tests) as reactive for anti-CMV IgG (positive) and non-reactive for anti-CMV IgM (negative). These 195 samples were tested on Copalis. The results are reported in the following table. ETI IgG COPALIS Twenty-three samples showed deviating results: twenty a positive Copalis result for both VP and CM2, one a positive Copalis result for both VP and p52 and two a positive Copalis result for VP, CM2 and p52, however, the comparative tests classified them as positive by anti-CMV IgG only. Detailed initial and repeated results (confirmation test) are reported in the following table (the Cl is given for Copalis results and the signal to cut-off ratio (S/CO) for ELISA). Table 14: After the additional Western tests, the discrepancies were solved as follows: - sample #24582 was considered positive by Copalis for p52 and VP. Since the Western Blot for CMV-lgM was positive, this sample might be derived from to a patient at the onset of infection, not already detectable by the ELISA-lgM test. - out of the 19 samples that were positive in the Copalis test for CM2 and VP: ■ seven showed a positive result by Western Blot for CMV-lgM. They may be explained as "low" IgM-positive samples and therefore defined as a post- acute or recent infection. They correctly resulted "positive" in the Copalis , test for CM2; ■ three samples (#21122, #24655, #25594) were classified as equivocal and excluded from the analysis; ■ additional tests or information were not available for the remaining nine samples and can be considered as false positive result for CM2 by Copalis. Furthermore, these might be samples from patients with a post-acute infection, when the ELISA test is not able to detect IgM any more. - sample #24750 was positive in the Copalis test for VP, p52 and CM2. Since the Western Blot for CMV IgM resulted also positive, it was correctly detected classified in the Copalis test for the IgM markers and could be defined as an "acute infection". - samples #26597 resulted positive in the Copalis test for VP, p52 and CM2. Since the p52 specificity was correct, this sample might be a low IgM positive sample and defined as an acute infection. Then it correctly resulted as positive by Copalis. 4. CMV/HCMV infection follow-up Twenty-one patients (69 samples) with at least two consecutive blood withdrawals were selected and tested on Copalis. The patients were pregnant women with a primary CMV infection. The date of infection was estimated on the basis of the clinical and serological information collected by the investigator. Detailed results are reported in the following table (the Cl is given for Copalis results and the signal to cut-off ratio (S/CO) for ELISA tests). Table 15: ■ In twelve patients (identified as 'same pattern') the Copalis test detected the anti-CMV antibodies with the same pattern as in the comparative tests; ■ In seven patients (identified as 'same pattern CM2') the Copalis test result was positive for the CM2 marker and negative for the p52 marker identified as 'later CM2'. This means that the seroconversion window of CM2 is wider than the seroconversion window of p52 and correlates with the classification observed in the standard assay. ■ In another patient defined as later CM2 the sensitivity for CM2 was slightly higher in the Copalis test than in the comparative assays. ■ In one patient (identified as earlier positive), the Copalis test detected anti-lgM antibodies two blood withdrawals earlier than the comparison assay. The analysis of these serial samples showed that p52-reactivity typically diminished to negative levels by 60-90 days, whereas the CM2-reactivity could persist for more than 150 days. On this basis, these two different markers for IgM allow the differentiation between the acute and convalescent stages of a CMV infection. Negative reactivity to the CM2 and p52 antigens and additional reactivity to the viral particle (VP) is consistent with a previous infection and indicates the patient's previous exposure to the CMV virus. 5. Clinical evaluation summary In order to analyse the diagnostic sensitivity and specificity of every single marker, the results obtained after resolution of the discrepant cases can be summarised as follows. expected results COPALIS On the basis of these results, the diagnostic specificity of Copalis CMV multiplex test is for the three markers: . VP: 100.0% (all 102 expected negative were negative for VP) . CM2: 95.8% (273 observed negative out of 285) . p52: 100.0% (all 285 expected negative samples were negative for the p52) The diagnostic sensitivity of Copalis CMV Multiplex test can be calculated as follows: (a) apart for the VP marker (where 257 out of the 259 expected positive samples were observed positive in the CMV Multiple test): 99.2%; (b) for the p52 and CM2 markers together (where 75 out of the 76 expected positive samples were observed positive in the CMV Multiplex test): 98.7%. With regard to the p52 marker, the clinical significance of this marker is different from a typical IgM marker and the additional advantage of its presence is the ability for differentiation of the early phase of an acute CMV infection: the majority (90.9%; i.e. 40 out of 44 samples) of samples (having an infection in the range of 60-90 days) showed positive results. Considering that the three results are simultaneously available, the test efficacy of Copalis CMV Multiplex can be considered as 96.4% (348 samples out of the 361 tested were correctly classified by the test). Accordingly, the analysis of the obtained Copalis CMV Multiplex result(s) can be interpreted as follows: Therefore, the assay is able to simultaneously distinguish between the acute and convalescent stages of CMV infection. Example 6: Analysis of sera from transplantation patients with CMV Multiplex Copalis and CMV Multiplex Copalis IgM plus. In order to verify the efficacy of the CMV Multiplex Copalis test for detecting a CMV infection in transplantation patients, the serological profile of 3 transplantation patients was followed. Additionally, in order to demonstrate that the agglutinating factors of the p52 and CM2 antigens are immunoglobulin of the class M (IgM) a monoclonal murine anti-lgM antibody (60 μg/ml; Mab HSP-P 06, Silenum, ADM) was added to the dried beads (see example 1 ). The monomer reduction events in Copalis with and without the addition of the anti-lgM was compared. The addition of anti-lgM antibody did not significantly influence the agglutination on the VP particle, confirming that the major factor(s) mediating the agglutination with VP are immunoglobulins of class G (IgG). In order to test the sera from transplantation patients, two different versions of the CMV multiplex assay were used: • The first version consists of cups prepared according to the procedure described in Example 1 , i.e. beads were coated with the three different antigen p52, CM2 and viral particle. • For the second version of the assay the same coating procedures were used but before the drying step of the beads, the above described monoclonal anti-human IgM antibody was added. As shown in Fig 5, patient 1 (# VT-872562) VP (viral particle) was positive for all the withdrawals of blood in both cases, showing a higher COPALIS index (Cl) without the anti-lgM antibody; p52 was positive in 2 out of 3 bleedings in both cases (+/- anti- lgM), CM2 became positive in the 2 nd withdrawal with the anti-lgM antibody and positive only at the 3 rd without the anti-lgM antibody. In the second patient (#VT871802), CM2 was positive only in 3 out of 6 withdrawals without the anti-lgM antibody, whilst it was positive in all the withdrawals with the anti-lgM antibody. p52 coated bead was in the grey zone without the anti-lgM antibody and was above the cut-off with the anti-lgM antibody in the first withdrawal only; interestingly the VP was positive in all the bleedings without the anti-lgM antibody and became positive only in 3 out of 6 with the addition of the anti-lgM antibody. This means that the addition of the anti-lgM antibody mediates a better sensitivity for IgM detection, but gives also a better specificity. Taken together these results confirmed that IgM are the main agglutinating factors of the p52 and CM2 particles and IgG of the VP particle. Furthermore, the CMV Multiplex COPALIS assay is able to detect the appearance of CMV infection not only with immunocompetent subjects but also with immunocompromised subjects such as transplanted patients. Table 16: Sera from transplantation patients Tested without anti-lgM Table 17: Sera from transplantation patients Tested with anti-lgM VT-871802 3,62 + 0,15 2,03 + 30/05/97 VT-865172 11 ,11 + 2,40 + 40,32 + 08/11/96 VT-865172 5,15 + 0,55 7,78 + 10/01/97 VT-865172 27,53 + 0,55 9,25 . + 28/02/97 VT-865172 13,33 , + 0,18 1 ,68 : + 08/04/97

Claims: Claims 1. A method for the detection of antibodies/antibody isotypes specific for at least two different phases of an infection comprising (a) incubating a sample from a patient simultaneously with at least two types of compounds, each type of compound comprising a different epitope under conditions that allow binding of said antibodies to said epitope, wherein each epitope is recognized by antibodies/antibodies belonging to a particular isotype specific for one of said phases of said infection, wherein further at least two epitopes are present in the incubation reaction recognized by antibodies/antibodies belonging to a particular isotype specific for at least two different phases of said infection, wherein further each type of compound/all types of compounds comprising epitopes recognized by antibodies/antibody isotypes indicative of the same phase of infection is/are affixed to the same type of support and wherein each type of support has properties that allow the physical distinction of said type of compounds from other types of support by physical means; (b) physically distinguishing said compounds; and (c) assessing whether antibodies/antibodies belonging to a particular isotype are bound to said types of compounds. 2. The method of claim 1 , wherein said antibodies are IgM antibodies. 3. The method of claim 1 , wherein said antibodies belonging to a particular isotype are IgM, IgG or IgA antibodies. 4. The method of any one of claims 1 to 3, wherein said at least two different phases of an infection include the acute phase, the post-acute phase, the chronic phase, the remission phase of an infection or a phase past infection. 5. The method of any one of claims 1 to 4, wherein said infection is an infection with a virus, a bacterium or a protozoon. 6. The method of claim 5, wherein said virus is human Cytomegalovirus, Hepatitis C virus, Hepatitis A virus, Hepatitis B virus, Epstein-Barr virus, HIV or Herpes simplex virus. 7. The method of claim 5, wherein said bacterium is Borrelia burgdorferi, Treponema pallidum or Helicobacter pylori. 8. The method of claim 5, wherein said protozoon is Toxoplasma gondii or Trypanosoma cruzi. 9. The method of any one of claims 1 to 8, wherein said sample from a patient is blood, serum or is derived therefrom. 10. The method of any one of claims 1 to 9, wherein said compound is an antigen. 11. The method of any one of ciaims 1 to 10, wherein said compound or antigen is treated with (a) detergent(s) prior to step (a). 12. The method of claim 10 or 11 , wherein said antigen is a (poly)peptide or DNA. 13. The method of claim 12, wherein said (poly)peptide is CM2 fusion protein (HCMV), p52 (HCMV) or a viral particle from HCMV, gp36, gp33, gp27, gp30 or gp24 (hepatitis B virus), a Toxoplasma gondii lysate, optionally proteinase K-treated or deglycosylated, or 41 kDa protein or 31/34 kDa protein from Borrelia burgdorferi. 14. The method of any one of claims 1 to 13, wherein said support is a bead. 15. The method of claim 14, wherein said bead is a latex bead, a colloid metal particle, preferrably a gold particle, or any combinations thereof. 16. The method of any one of claims 1 to 15, wherein said property that allows the physical distinction of said types of compounds is mass, size, refractive index, a magnetic property, electric property or a combinations thereof. 17. The method of any one of claims 1 to 16, wherein the physical distinction of said compound is effected by measuring a change in said property caused by the agglutination of said compounds. 18. The method of claim 17, wherein said agglutination is an agglutination of two or three compounds. 19. The method of claim 17 or 18, wherein said measurement is the measurement of light scattering, magnetic field variation or electric field variation. 20. The method of any one of claims 1 to 19, wherein in step (a) the sample from a patient is further incubated with (an) anti-human immunoglobulin(s) antibody. 21. The method of claim 20, wherein said anti-human immunoglobulin is an anti- lgM antibody. 22. The method of any one of claims 1 to 21 wherein fixation of said compounds to said supports is stabilized by surfactant treatment. 23. Kit comprising different types of supports of distinguishable physical properties wherein to each type of support a different compound comprising an epitope is covered, wherein further each epitope is recognized by an antibody/antibody belonging to a particular isotype and each antibody/antibody belonging to a particular isotype is specific for a phase of a disease and wherein said different compounds and epitopes, respectively, are recognized by antibodies/antibodies belonging to a particular isotype specific for at least two different phases of said infection. 24. The kit of claim 23, wherein said support is a bead. 25. The kit of claim 24, wherein said beads are latex beads, colloid metal particles, such as gold particles, or any combinations thereof. 26. The kit of any one of claims 23 to 25, wherein said compound is CM2 fusion protein (HCMV), p52 (HCMV) or a viral particle from HCMV, gp36, gp33, gp27, gp30 or gp24 (hepatitis B virus), a Toxoplasma gondii lysate, optionally proteinase K-treated or deglycosylated, or 41 kDa protein or 31/34 kDa protein from Borrelia burgdorferi. 27. The kit of any one of claims 23 to 26 further comprising an anti-human immunoglobulin antibody. 28. The kit of claim 27, wherein said anti-human immunoglobulin is an anti-lgM antibody. 29. The kit of any one of claims 23 to 28 wherein the same type of support is coated with different epitopes or different compounds wherein said different epitopes or compounds are recognized by antibodies/antibody isotypes indicative of the same phase of infection.

Inventors: BARBERO LUCA MARIA, BARBIERI UGO, BONELLI FABRIZIO, CORNO FEDERICO, SOLETI ANTONIO, BARBERO, LUCA, MARIA, BARBIERI, UGO, BONELLI, FABRIZIO, CORNO, FEDERICO, SOLETI, ANTONIO
Applicants: BARBERO LUCA MARIA, BARBIERI UGO, BONELLI FABRIZIO, CORNO FEDERICO, DIASORIN INTERNATIONAL INC, SOLETI ANTONIO, BARBERO, LUCA, MARIA, BARBIERI, UGO, BONELLI, FABRIZIO, CORNO, FEDERICO, DIASORIN INTERNATIONAL INC., SOLETI, ANTONIO
Application year: 1999
Main classification: G01N33
IPC codes: G01N33