Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-01-12
2003-07-15
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S007200, C435S007700, C435S007720, C435S007500, C435S007800, C435S004000, C435S007910, C435S007920, C435S007940, C435S007950, C436S543000, C436S501000
Reexamination Certificate
active
06593079
ABSTRACT:
The invention concerns a procedure for diagnosis of an HIV infection by means of an immunoassay using the specific detection of HIV antigens and HIV antibodies.
AIDS (acquired immmunodeficiency syndrome) is an acquired immunodeficiency disease caused by the HIV virus. Hitherto known pathogens are the strains HIV1 and HIV2. Both strains are similar in morphology, cell tropism, interaction with the CD4 receptor of T-cells, their in vitro cytopathic effect on CD4 cells, their general genomic structure and the capability of causing the disease AIDS (Clavel, 1987, AIDS 1, 135-140). The immunological degree of relationship is, however, only small so that generally HIV1 specific antibodies do not show any cross reactions with HIV2. Besides the most common HIV1 group M subtypes a further HIV1 subtype, the subtype 0, is known (Myers et al, Los Alamos data bank, 1994; Sharp et al. AIDS Suppl. 8, pp. 27-42, 1994). The degree of relationship between HIV1-Sub0 and HIV1 is considerably higher than that between HIV1-Sub0 and HIV2. Antibodies directed against HIV1 do, however, only partly cross react with the corresponding antigen of HIV1-Sub0. A large part of the HIV1-Sub0 specific antibodies does not react with the HIV1 group M antigens neither.
The course of an HIV infection can be divided into several diagnostically relevant phases. In the early phase of an infection HIV antigens can already be detected which does not apply to HIV antibodies. In the seroconversion phase HIV antigens can be slightly positive or negative, i.e. not detectable. HIV antibodies of the IgM class are detectable in this phase whereas HIV antibodies of the IgG class are not detectable or only slightly positive. In the next phase, which is without symptoms, mainly HIV antibodies of the IgG type are detectable whereas the HIV antigen does generally not occur. The same is true for the progressive course of disease in the clinical phase.
In the late phase of disease HIV antibodies can finally become slightly positive or negative whereas the HIV antigens remain either negative or can again be detected as positive due to the increase in viral charge with the breakdown of the patient's immune system. Thus, during these different phases of disease which can be—depending on the patient—very different in course there are always moments where antigen or antibody detections may give falsely negative results.
For the detection of HIV1, HIV2 or HIV1-Sub0 infections antibody tests (IgG and IgM) are frequently performed in the bridge test format which is for example described in EP-A-0 280 211. The antigens used are in general antigens of the so-called envelope region (env) which are gp160, gp120, gp41 for HIV1/HIV1-Sub0 and gp140, gp110, gp36 for HIV2, together with the antigen p24 (HIV1) which forms the viral nucleus and p26 (HIV2), respectively. The antigens p24 and p26 are coded by the so-called gag region. These tests give a positive signal if antibodies against the antigens mentioned are present. In the early and late phase of the disease, i.e. when free p24 or p26 antigens are present often no antibodies are detectable since either the patient's immune system has not raised enough antibodies yet or is so exhausted that the number of antibodies built is not sufficient for being detected.
Antigen tests for the detection of p24 antigens and other antibody tests are generally performed separately. The antigen titer of the patient is only increased in the early phase of infection and in the final phase of AIDS so that p24 is only reliably detectable in these phases.
A disadvantage of the hitherto known tests is that none of the tests can cover the total diagnostically relevant period of an HIV infection alone.
With combination tests antigens of a certain pathogen and antibodies directed against this pathogen can be detected simultaneously. Such a procedure for simultaneous determination of antigens and antibodies are disclosed in DE 42 36 189 A1. An approach to diagnostically cover all phases of an HIV infection completely is, however, not described.
In WO 93/21346 a combination test for the simultaneous detection of HIV p24 antigen, HIV1 gp41 antibodies and HIV2 gp36 antibodies (in both cases antibodies against proteins of the env gene) by means of a heterogeneous immunoassay is described. With this test HIV positive samples which do not contain p24 or antibodies against env proteins are, however, not detected as positive. Solely with gp41 not all antibody positive HIV1 samples can be detected since samples characterized by showing an incomplete band pattern in the western blotting and no dyeing of the gp41 band are widespread.
Hashida et al (1996, J. Clin. Lab. Anal. 10, 213-219) describe a diagnostic test for the detection of HIV1 infections. With this test the p24 antigen, IgG antibodies against p17 (from the gag region) and IgG antibodies against reverse transcriptase (RT) coded by the pol gene can be detected. This procedure allows, however, only the detection of HIV1 infections and seroconversion sera containing only low-affine IgM antibodies against the two antigens used above are not detected. Seroconversion sera containing antibodies against env proteins are not detected either. After the decrease in HIV antigen titer in the early phase of infection often only IgM antibodies against the env protein gp41 occur. In this case the Hashida et al. test shows a falsely negative result.
There is no state-of-the-art diagnostic test system which reliably enables the simultaneous detection of HIV1, HIV1-Sub0 and HIV2 completely for all infection stages.
The object of the present invention was therefore to develop an improved test for HIV infections that allows as a single test an accurate, reliable and complete detection of all diagnostically detectable phases of an HIV infection. The test should also allow the simultaneous determination of HIV1, HIV1-Sub0 and HIV2.
This object is achieved by the procedure according to the invention for diagnosis of an HIV infection by means of an immunoassay using the specific detection of p24 antigen of HIV1, HIV1-Sub0 and/or p26 antigen of HIV2, at least one antibody against the env region of HIV1, HIV1-Sub0 and/or HIV2 and at least one antibody against the pol and/or gag region from HIV1, HIV1-Sub0 and/or HIV2. With the procedure according to the invention it is possible to reliably detect an HIV infection already with the occurrence of only one of the analytes mentioned above.
Surprisingly, it has been shown that by the detection of antibodies against a gene product in particular of the pol region of HIV combined with the detection of p24 antigen of HIV1, HIV1-Sub0 and/or p26 antigen of HIV2 and the detection of at least one antibody against the HIV-env region the hitherto deplorable diagnostic gap can be closed. Surprisingly, a procedure for the detection of an HIV infection, where antibodies against the reverse transcriptase (RT) of HIV are detected, combined with the detection of p24 antigen of HIV1, HIV1-Sub0 and/or p26 antigen of HIV2 and the detection of at least one antibody against the HIV-env region has been proven to be particularly suitable.
The detection of an HIV infection by the combination test according to the invention by means of the determination of the single parameters mentioned is performed simultaneously. The term combination test means that HIV antigens and antibodies directed against HIV antigens can be detected simultaneously. With this test all HIV infection stages can be covered reliably. By the selection according to the invention of antigens or antibodies and their corresponding receptors binding specifically it is possible to perform the procedure according to the invention.
Furthermore, the detection of HIV1, HIV1-Sub0 and HIV2 can be achieved using one single test. In addition, by adequately selecting the receptors a broad detection of HIV1 subtypes of the HIV1 group M is possible. The test is preferably based on the principle of heterogeneous immunoassays. Homogeneous procedures such as turbidimetric tests with no separation of the liquid and soli
Buyse Marie-Ange
Donie Frederic
Faatz Elke
Hoess Eva
Saman Eric
Brinks Hofer Gilson & Lione
Housel James
Li Bao Qun
Roche Diagnostics GmbH
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