Detection of viruses and antibodies

Details Detection of viruses and antibodies Detection of viruses and antibodies

1987-01-14

1990-06-26

Warden, Robert J.

Chemistry: analytical and immunological testing

Involving diffusion or migration of antigen or antibody

Through a gel

436518, 436547, 436548, 436825, 435 5, 435 6, 435 7, G01N 33571, G01N 33543, G01N 3353, C12Q 170

Patent

active

049371991

DESCRIPTION:

BRIEF SUMMARY
This invention relates to a method for the detection of viruses or virus antibodies and is particularly concerned with the detection of viruses of the Herpes group and their antibodies.
The Herpes group of viruses are a group of considerable clinical significance, the most important members of the group being Herpes Simplex (HSV), Cytomegalovirus (CMV), Epstein Barr virus (EBV) and Varicella-zoster virus (VZV). Infections with CMV occur frequently in the United Kingdom so that 60% of adults have evidence of past infection. Occasionally, the virus produces cases of Paul Bunnell negative glandular fever but the vast majority of infected people remain entirely asymptomatic. This virus infection is therefore primarily of medical importance in specific groups of patients which are neonates with congenital infection, and immunocompromised individuals, such as recipients of renal or bone marrow allografts or patients with the acquired immunodeficiency syndrome (AIDS). In each of these groups of patients, CMV is an important pathogen and it would be very desirable, from the clinical point of view, to have available an assay method that can reliably identify the presence of CMV and the amount of CMV present in clinical samples.
Various methods have been proposed in the past for assaying CMV in samples of blood, urine or saliva taken from patients suspected of having CMV infection. Several methods have been proposed previously for the identification of CMV involving indirect assay by cultivating the virus in tissue culture and by methods involving the use of polyclonal antibodies and monoclonal antibodies that recognise the virus. The major difficulty, at the practical level, has been that while the various assay methods have been found to work and to provide reproducible and reliable data when carried out on model experimental systems which are artificially infected with CMV, the same methods do not provide equally reliable and reproducible results when carried out on clinical samples suspected to contain or known to contain CMV.
We have investigated the possible reasons for this disparity of performance of assay methods between experimental model systems and clinical samples and we have identified what we believe to be the presence of an inhibitory material associated with CMV in the clinical environment which prevents reliable and reproducible results being obtained by the various assay systems that have been developed.
We have discovered that in the clinical environment, molecules of beta.sub.2 microglobulin become associated with the outer envelope of CMV and mask the antigenic determinants that are present on the surface of CMV so making unreliable the identification of CMV by the use of antibodies that normally recognise CMV. Beta.sub.2 microglobulin is a protein that exists in urine and other body fluids that are normally assayed for CMV and we believe that this masking effect, rendering the antigenic determinants on the outer envelope unavailable for recognition by antibodies that normally recognise such determinants, will occur with the other Herpes group viruses, HSV, EBV and VZV which all contain outer envelopes of the type possessed by CMV.
As an alternative to the direct detection of the Herpes group virus, diagnostic methods are available which involve the detection of antibody to the herpes virus. Such antibody detection methods normally involve the use of known quantities of viral antigen, often in insolubilised form, but such antibody detection assays will not necessarily be reliable since any beta.sub.2 microglobulin (hereinafter called beta.sub.2 m) associated with the clinical sample will compete with antibody for binding to the viral antigen and can become bound to any insolubilised antigen, so reducing to an unknown extent, the amount of free insolubilised antigen available for binding with the unknown antibody.
Having identified the role played by beta.sub.2 m in interactions between antibodies and antigens of viruses of the Herpes group, we have recognised that, by taking appropriate precaut

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