Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-07-08
2001-09-11
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C436S518000, C530S350000
Reexamination Certificate
active
06287760
ABSTRACT:
The present invention relates to a diagnostic tool for the identification in human serum of specific antibodies against Human Cytomegalovirus (HCMV); the present invention relates, in addition, to an improved form of the Western blot test for the identification of anti-HCMV antibodies which makes use of the said diagnostic tool, with the said test having a heightened level of reliability and therefore being able to be taken as an advantageous standard of reference in the serological control of the results of a first test which is carried out by means of traditional techniques, such as the Enzymatic Immune Analysis (EIA), as well as to represent a test which stands alone for the search for anti-HCMV antibodies. The invention relates, in addition, to a method of identification in the human serum of the specific IgM and/or IgG and/or IgA of Cytomegalovirus by using the said improved Western Blot test. The invention relates, finally, to the recombinant fusion proteins which are used, along with other viral antigens which are obtained from purified virions, in the said method, and to the plasmids which express the said proteins.
HCMV is a ubiquitous agent of infection in the human. It is rarely pathogenic in healthy adults, but it is associated with various disorders in immune-depressed individuals. In addition, HCMV is the most common cause of congenital infections in the human, and primary maternal infections are second only to Down's Syndrome as a known cause of mental retardation in the neonate [2].
The serological diagnosis of infections from HCMV is carried out, as a general rule, by by means of the enzymatic immune test (EIA), which uses as the antigenic material either the virus itself, purified to various levels, or else lysates of the infected cells. The various commercial kits which are currently available on the market for carrying out such tests, however, can not be referred to any standard and, for that reason, the results which they supply are frequently in little agreement with one another, with some being too specific and insufficiently sensitive, while others are too sensitive and insufficient specific [6], thus yielding, on the whole, a large number of responses which are falsely positive or falsely negative.
It is well known, in this connection, that the serological analysis which is carried out by means of the “Western blot” method, even though it is much more expensive and requires longer time in comparison with the EIA test, is still much more reliable than the latter, most particularly in regard to the specificity of the final result. This is the reason, among others, that, in the event of infections from HIV, a Western blot test (frequently referred to as “WB”) is always used to confirm a doubtful EIA test.
The most effective antigenic substance for the serological investigation of HCMV is certainly the virus itself. In the procedure in accordance with the traditional Western blot test, the virus is, after the purification of the cultures of infected cells, suspended in a denaturing mixture and its structural proteins are fractionated in preparatory gels of polyacrylamide. The typical pattern of proteins which is thus generated is then transferred to a solid support (which is, as a general rule, nitrocellulose); the spots (“blots”) which are thus obtained are successively sub-divided into various strips of a few millimeters of width: each strip is then assayed with the serum which is to be examined.
The WB test is not entirely free of disadvantages, however: in the first place, the structural antigen of the HCMV virus which is provided with the highest immunogenic capacity, against which the longest-last immunological response is produced, is a polypeptide with the molecular weight of 150 kD (coded by the gene UL32 of the virus itself, and referred to as ppUL32). This polypeptide, as a general rule, migrates in the polyacrylamide gel along with another structural component of the HCMV virus (coded by the gene UL86 and referred to as ppUL86), which has the same molecular weight and, although it is a relatively effective immunogenic substance, it is only scantily specific, because it includes antigenic epitopes in common with the homologous proteins of the other viruses of the family Herpesviridae, and is thus to be defined a “group antigen”. As the result, in the relatively frequent case in which the band of 150 kD turns out to be the only reactive band shown of a WB test, it is not possible to stabilize (at least to a sufficient extent) if the subject who is under examination has direct antibodies against the specific antigen pp150 or against the antigen of the non-specific ppUL86 group, so that the result consequently remains equivocal [11].
In the second place, the blots which are obtained from the viral proteins (vp) do not contain the non-structural viral proteins, some of which are very strong immunogenic substances. For example, the direct antibodies against such proteins, particularly those which are directed against the phosphoprotein of 52 kD (coded by the gene UL44 of the genome of HCMV) have particular relevance, because their presence allows the serological identification of the primary infection, on the condition that the presence of these antibodies is accompanied by a weak reaction against the principal structural antigen pp150.
It would be desirable, therefore, to supply a diagnostic tool which can be used in an improved Western blot test for the identification of specific antibodies of HCMV in the human serum, which is free of the disadvantages which have been described above and, in particular, is both specific and sensitive enough to be able to constitute an advantageous standard of reference (“golden reference standard”). It would be desirable, in addition, to provide a Western blot test which is improved for the identification of the IgM's which are specific for HCMV in the human serum.
It would also be desirable to provide a method for identifying the immunoglobulins IgM and/or IgG and/or IgA which are specific for HCMV in the human serum with an increased level of reliability, in such a manner that it is possible to recognize even those results which are falsely positive or negative.
It would also be desirable to supply the recombinant protein materials which are to be used in the test and in the WB method in accordance with the invention itself.
Finally, it would be further desirable to supply plasmids which are able to be inserted into prokaryotic and/or eukaryotic host organisms, in order to thereby to express such recombinant protein materials, particularly those which are fused with the protein CKS.
In accordance with the present invention, there is therefore provided a diagnostic tool for the identification of antibodies against HCMV in the human serum, with the said diagnostic tool comprising a means for solid support, a first section of which bears a plurality of viral proteins (vp) which have been obtained from purified virions, the said viral proteins being concentrated in corresponding bands and with the bands being supported by the said first section of the solid support, spaced one from the other in accordance with a predetermined pattern; with one of the said bands being formed by at least one viral protein of approximately 150 kD molecular weight; with the said diagnostic tool being characterized in that:
(i) The said means of solid support comprises, in addition, a second section which contains a plurality of bands spaced among themselves in accordance with a predetermined pattern, with at least one first band of this second section including a single recombinant protein (rp) which comprises a first region reproducing a sequence of amino acids corresponding to at least one epitope of the viral protein pp150, and a second region reproducing at least a portion of the sequence of amino acids of an exogenous protein, which may be expressed in a prokaryotic or a eukaryotic host organism;
(ii) At least one second band of the second section includes a recombinant protein which comprise
Landini Maria P.
Lazzarotto Tiziana
Maine Gregory T.
Ripalti Alessandro
Abbott Laboratories
Weinstein David L.
Wortman Donna C.
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