Field of the Invention
The present invention relates to a production for the preparation of western blot strips for detecting the antibodies to human retrovial infections and the Western blot prepared thereof. More specifically to detect the Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2), the etiologic agents of Acquired Immuno Deficiency Syndrome (AIDS). It is intended to be used as a more specific and supplemental assay.
Prior Art:
Laboratory tests currently used to detect HIV infection are based on the observation that individuals infected with HIV will develop virus specific antibodies within a few weeks or months at the latest. Serological diagnosis of HIV infection rests on the detection of a host immune response to viral antigens encoded by 3 major viral structural genes. The gag gene encodes the mojor viral core proteins p17 (mol. wt. 17 kD), p24 (mol. wt. 2kD) and their precursor, p55 (mol. wt. 55 kD). The env gene encodes the envelope glycoprotein precursor, gp160 (mol. wt. 160 kD) which is cleaved into an external envelope domain, gp120 (mol. wt. 120kD) and the transmembrane region, gp41kD). The pol gene encloses major proteins, such as reverse transcriptase p66 and p51 (mol. wt. 66 and 51 kD respectively) and p31 endonuclease (mol. wt. 31 kD) component. Several other viral proteins, mainly having regulatory functions, are encloded and expressed by the HIV genome, but none of these gene products are sufficiently immunogenic to be used as routine markers for serological diagnosis.
Several methods for the detection of antibodies to HIV-1 have been developed over the years and as the epidemic continues to spread, it becomes important that diagnostic methods have the capability to accurately identify infected individuals.
Enzyme-linked immunosorbent assays (ELISAs) are widely used to screen for presence of virus - specific antibodies. Such a test format can be extremely sensitive but carries with it a potential for being less specific, leading to false positive reactions. A positive HIV serological diagnosis ha significant psycho-social implications for the individual being diagnosed. It is all the more pressing, therefore, that test results be accurate and reliable supplementary tests that are more specific are commonly used. A major deficiency in the art is therefore the absence of a single confirmatory immunobilot assay method that provides not only the well-characterised natural antigen reactivity profile, but also has the capability of increased sensitivity and immediate qualitative differentiation of infection by viral subtypes HIV-1 and HIV-2.
Therefore, it is an object of the present invention to overcome the deficiencies in the existing blots.
It is a further object of the invention to provide an immunoassay with significantly improved sensitivity and specificity for the serological diagnosis of viral infections, especially with human immunodeficiency virus (HIV-1 & HIV-2).
It is another object of the invention to provide an immunoassay format with significantly improved specificity for serologically distinguishing HIV-1 from HIV-2 infections.
Specifically, this invention relates to a new and enhanced method for the detectilon of antibodies to a virus, in particualr HIV-1 and/or HIV-2 by an augmented Western blot format and assay.
The new method and assay of the present invention based on augmenting the natural protein Western blot with synthetic proteins from the same virus (or a different virus) provide a distinctly sensitive and specific detection
device. Additionally use of this method of detection will provide a more complete serological profile.
SUMMARY OF THIS INVENTION
The present invention relates to a Western Blot Assay designed to detect the antibodies to human retroviral infections, more specifically the HIV-1 & HIV-2 virus which are etiologic agents of Acquired immunodeficiency Syndrome (AIDS). The Western Blot test can be used as the most informative, specific and supplemental assay on human serum or plasma specimen found, repeatedly reactive using ELISA.
The HIV-1 viral antigens are separated by gel electrophoresis, electrically transferred to nitrocellulose membrane strip which is impregnated with a specific HIV-2 antigen base. Each strip also has an internal serum inbuilt quality control band.
The HIV Western Blot is prepared from HIV-1 cell line which is lysated. It is a recovery process based on cost-effectiveness in scale-up, case of operation and automation, consisting of product quality and safety. The invention also provides a process for the manufacture of HIV-1 viral lysate which is less capital, labour and maintenance intensive. It avoids cell damage, maintains the viral production, growth rate, and cell viability. The HtV-1 viral antigen is purified and further separated by SDS gel electrophoresis.
The sodium dodecyl sulphate denatures viral components thereby yielding the proteins which migrate in the gel according to their molecular weight to producing the various bands. The low molecular weight components migrate faster and are found at the bottom of the gel, while high molecular weight proteins remain near the top. They are then transferred from SDS-PAGE gel on to the nitrocellulose membrane which is also impregnated with HIV-2
antigen and a control band. The membrane is cut and packaged as immunoblot strips.
The assay is performed by incubating the strips with the patient serum/plasma diluted in a buffer. Antibodies to HIV-1 & 2 if present, bind to viral antigens located on the strip. Unbound material is washed off and then the strip is incubated with the anti-human Immunoglobulin G (IgG) antibodies conjugated to alkaline phosphatase. After washing the unbound conjugate, substrate (BCIP/NBT) is added which results in the staining of bands. If antibodies to HIV-1 antigens are present in serum two (Envelope with GAG/POL) or more of the following bands will be seen: p17, p24, p31, gp41, p51/p55, p66, gp120, gp160.
If antibodies to HIV-2 antigens are present, HIV-2 band is also observed along with some of the other bands. If HIV specific antibodies are not present, the band pattern does not meet the required criteria.
Accordingly the present invention relates to A process for producing Western Blot Assay for detecting the antibodies to human retroviral infections, more specifically the HIV-1 and HIV-2 virus which are etiologic agents of Acquired Immuno Deficiency Syndrome (AIDS) comprising of the following steps:
a) harvesting the HIV from the supernants of HIV virus infected cell
lines.
b) concentrating the said harvested HIV particles for obtaining HIV-1
and HIV-2 virions concentrated 100 times.

c) purifying and separating by sodium dodecyl sulphate gel
electrophoresis on a polyacramide slab gel to render clear separation
between 160 to 120 bands.
d) transferring the said purified and separated virions as herein
described to a nitrocellulose membranes by electroblotting.
e) treating the said membrane with a buffer solution to block unwanted
sites as herein described.
f) cutting the said dried membrane into small strips to obtain the blot
assay.
The embodiments of the present invention is a process for producing Western Blot Assay for detecting the antibodies to human retrovial infections and the said purification and separations are carried out by sodium dodecyl sulphate gel electrophoresis on a polyacramide slab gel.
The subject invention may better be understood with reference to accompanying drawings. However, the same should not be costrued to restrict the scope of application as they are for illustrative purpose only.
BRIEF DESCRIPTION OF THE A€G®MBA-NY