Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-07-12
2002-09-10
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C530S300000, C530S350000, C424S189100, C424S204100, C424S228100
Reexamination Certificate
active
06447992
ABSTRACT:
DESCRIPTION
The invention concerns a method for typing antibodies in a sample liquid using type-specific antigens, and in particular a method for typing antibodies to hepatitis C virus and peptide antigens that are suitable for this.
The disease referred to as non-A-non-B hepatitis is in many cases caused by the hepatitis C virus (HCV). HCV is a single-stranded encapsulated RNA virus the genome of which is composed of about 9000 to 10000 bases. Structural proteins (core and envelope proteins) and non-structural proteins are coded by this genome.
HCV is a virus that is of major clinical importance since it correlates with chronic infections and diseases which occur later in infected patients such as cryptic cirrhosis and primary carcinoma of the liver.
HCV can be transmitted by blood contact. Investigations on the occurrence of antibodies indicate that it is highly contagious.
EP-A-0 318 216 discloses a partial nucleotide sequence of a HCV. EP-A-0 450 931 discloses the complete nucleotide and amino acid sequence of a HCV. Methods are known for the diagnostic detection of a HCV infection by determining viral antibodies in body fluids using viral proteins or peptides as antigens (cf. for example Mori et al., Jpn. J. Cancer Res. 83 (1992), 264-268; WO92/11370 and DE-A-44 28 705.4).
The problem with a HCV infection is that there are different virus strains which have a considerable variability in their genome and accordingly in the polypeptides coded by this genome (cf. for example McOmish et al, Bioforum 16 (1993), 414-420.
Due to the variability of HCV it is on the one hand difficult to diagnose an infection at all and on the other hand to type the virus strain responsible for the infection. Such typing is important since there are significant differences in the virulence and response to therapy (e.g. with interferon) of the various virus strains.
One way of typing virus strains is to determine the genotype by amplifying the viral genome by means of PCR and subsequently determining the sequence (e.g. Bukh et al., Proc. Natl. Acad. Sci. USA 90 (1993), 8234-8238). A disadvantage of this determination of the genotype is, however, that the amplification and sequence determination steps are very time-consuming and can only be carried out using complicated apparatuses in laboratories that are specially equipped for this. This is all the more so in this case since there is often only an extremely small amount of viral genetic material that can be amplified in a HCV infection .
A further possibility of type determination is serotyping i.e. determination of the virus type by means of the immunological specificity of the antibody to the virus produced in the organism. Simmonds et al. (J. Clin. Microbiol. 31 (1993), 1493-1503) describe the use of type-specific peptide antigens for the serological differentiation of infections with the HCV types 1, 2 and 3. The typing was carried out by means of an indirect ELISA using peptide antigens of the amino acid regions 1691-1708 and 1710-1728 from the NS4 region of HCV. For this type-specific peptide antigens were each immobilized separately according to their type in individual wells of a microtitre plate and each was contacted with separate aliquots of a plasma sample from HCV-infected blood donors. The typing was carried out according to the reactivity of the serum sample with the individual peptide antigens. However, this method is relatively inaccurate and, moreover, does not allow the determination of individual viral subtypes i.e. individual virus strains whose immunogenicity only differs to a slight extent.
The object of the present invention was therefore to provide a new method for typing antibodies which on the one hand can be carried out routinely and without elaborate apparatus and which on the other hand enables classification between individual antibody types that is sufficiently accurate. A further object of the present invention was to identify regions from the genome of HCV which at the same time have a high immunogenicity and variability so as to be suitable for typing HCV infections.
This object is achieved by a fractional immunosorption method in which a first aliquot of a sample liquid which contains the antibodies to be typed is contacted successively with a series of type-specific antigens or antigen mixtures and optionally a second aliquot or further aliquots of the sample liquid are likewise contacted with various type-specific antigens or antigen mixtures but each time in another sequence. Furthermore new immunogenic peptide sequences from the HCV genome are provided which enable a better typing than the sequences known from the state of the art.
A first aspect of the present invention is a method for typing antibodies in a sample liquid which is characterized in that
(a) a first aliquot of the sample liquid is contacted with a first immobilized antigen which is specific for a first type of the antibodies to be examined or with a first mixture of immobilized antigens each of which is specific for a first type of the antibodies to be examined under conditions in which the antigen or antigen mixture can react with the antibodies and in which the amount of antibody in the sample liquid does not exceed the capacity of the immobilized antigen or antigen mixture,
(b) the sample liquid from step (a) is contacted with a second immobilized antigen which is specific for a second type of antibodies to be examined or with a mixture of immobilized antigens each of which is specific for a second type of antibodies to be examined under conditions as in step (a), the second antigen or antigen mixture being spatially separate from the first antigen or antigen mixture used in step (a),
(c) the measures according to step (b) are optionally repeated with one or several further antigens or antigen mixtures which are specific for one or several further types of antibodies to be examined, the further antigens or antigen mixtures each being spatially separate from the antigens or antigen mixtures used in the previous steps,
(d) a second aliquot of the sample liquid is optionally contacted with several immobilized antigens or antigen mixtures according to steps (a) to (c) in which the sequence of antigens or antigen mixtures is, however, different,
(e) the respective immunological reactivity of the immobilized antigens or antigen mixtures with the sample liquid is determined qualitatively or/and quantitatively and
(f) a typing of the antibodies present in the sample liquid is carried out based on the reactivity determination.
Any antibodies can be examined as typing objects e.g. antibodies which are directed towards pathogens or autoimmune antigens. Antibody typing in turn enables a typing of the antigens to which the organism was exposed and which have caused the formation of antibodies. It is preferable to type antibodies that are directed towards one or several pathogens, especially antibodies that are directed towards viral antigens. Examples of viruses from which viral antigens are derived are HCV, human papilloma virus (HPV), hepatitis B virus (HBV) and HIV. The method can also be used for the concurrent detection of several viral infections which are present together e.g. HCV and HIV.
The application of the method according to the invention is of particular importance for typing a HCV infection since the virulence and response to interferon therapy differs among the individual virus types and subtypes. However, the method can also be used advantageously for other viruses such as HIV in order to determine the origin of individual viral isolates or to identify subtypes (e.g. subtype O in the case of HIV).
In the method according to the invention an aliquot of sample liquid, e.g. a body fluid which is optionally diluted such as blood, plasma, serum or urine, is contacted successively with several immobilized type-specific antigens or antigen mixtures in order to enable a stepwise sorption of the antibodies capable of reacting with the respective antigens or antigen mixtures and thus to enable their stepwise removal from the sample liquid. Pre
Ihlenfeldt Hans-Georg
Jung Günther-Gerhard
Kraas Wolfgang
Schmitt Urban
Seidel Cristoph
Arent Fox Kintner Plotkin & Kahn
Roche Diagnostics Gmbh
Zeman Mary K.
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