Title of Invention

" A PROCESS FOR THE PREPARATION OF WESTERN BLOT STRIPS AND THE WESTERN BLOT PREPARED THEREOF"

Abstract

A process for producing Western Blot Assay for detecting the antibodies to human retrovial 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 i.e., harvesting the HIV particles from the supernants of HIV virus infected cell lines, concentrating the said harvested HIV particles for obtaining HIV-1 and HIV-2 virions concentrated to 100 X concentration, purifying and separating the said concentrated virions, transferring the said purified and separated virions to a nitrocellulose, membranes by electroblotting, treating the said membrane with a buffer solution to block unwanted sites and cutting the said dried membrane into small strips. I7

Full Text

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 Figure 1 represents the western blot in original. Figure 2 represents the immmunoblot profile. Figure 3 represents the positive control. Figure 4 represents the negative control. BRIEF DESCRIPTION OF THE IN^BMTON The present invention involves 3 stages, first the preparation of retroviral proteins preferably HIV-1 & 2 from the virus lysate obtained from tissue culture and are separated by the Western Blot, using polyacrylamide gel electrophoresis (PAGE) followed by blotting onto a solid phas6 support such as nitrocellulose mennbrance/ PolyVinylDiFlouride. Secondly, it involves the genetically engineered or chemically synthesised viral proteins/peptides directly applied to the same solid support in order to augment the profile of the electrophoretically transferred proteins. The third stage involves an immunoassay to be carried out to detect the presence of antibodies in a biological fluid such as the serum/plasma of virus infected individuals. The HIV-1 virus particles are harvested from the supernatants of virus-infected cell-lines, concentrated, purified and then separated by sodium dodecyl sulfate gel electrophoresis on a polyacrylamide slab gel. The cells infected with the HIV virus are cultured and grown in the roller bottle apparatus as explained in the co-pending application. At the high cell densities attained, the rate of delivery of nutrients (perfusion rate) and removal of toxic metabolites will support a maximum viable cell density. The large scale downstream recovery procedure involves pooling of the daily clarified harvests, chemical inactivation with f3-propiolactone (BPL) and purification of viral lysate to get the viral antigens. By this process, the production and characterisation of numerous lots of product demonstrates both consistency and comparability to material prepared by a classic method. The HiV-1 virus particles are harvested from the supernatants of HIV-1 virus infected cell lines, concentrated, purified and separated by sodium dodecyl sulphate gel electrophoresis on a polyacrylamide slab gel. The HIV-1 virus infected cell-lines are stored at -170° C in liquid Nitrogen in 1 ml vial. The vial is taken out and thawed for cell culture expansion. The cells are maintained in enriched nutrient medium containing the inorganic salts of calcium nitrate, potassium chloride, magnesium sulphate, anhydrous magnesium sulphate, magnesium chloride, sodium chloride, sodium biacrbonate, anhydrous sodium phosphate, sodium phosphate, the L-amino acids as L-arginine, L-asparagine (free base), L-asparatic acid, L-cystine,.2HCI, L-glutamic acid, L-glutamine, L-alanyl-L-glutamine, glycyl-L-glutamine, glycine, L-histadine (free base), L-Hydroxyproline, L-isoleucine, L-leucine, L-lysine.HCI, L-methionine, L-phenylalanine, L-proline, L-serine, L-thereonine, L-tryptophan, L-tyrosine, L-tyrosine.2Na.2H2O, L-valine, the vitamins as biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, niacinamide, para-aminobenzoic acid, pyridoxine-HCI, riboflavin, thiamine Hcl, Vitamin B12 and other components as D-glucose, glutathione (reduced), HEPES, phenol red and additionally the faetal bovine serum. At high cell densities attained, the rate of delivery of nutrients (perfusion rate) and removal of toxic metabolites will support a maximum viable cell density. Therefore, to maintain logarithmic growth rate, cells must be removed, along with the product, on a daily basis, and a replacement volume of fresh medium added in a bolus fashion. During the logarithmic phase of the growth or the exponential reaction, the cell mass undergoes several cell doublings and the specific growth rate of the culture remains constant. With excess substrate or nutrient supply and no inhibition of growth by a compound that is present in the the growth medium, the specific growth rate is independent of the substrate concentrtion. This operation maintains viability and growth rate at a steady level that is capable of being supported by a given rate of profusion. The inoculum is generated by thawing a vial of a manufacturer's working cell bank (MWCB) and diluting the contents into a T25 flask containing 8 -12% faetal bovine serum to initiate growth. The doubling time of the cells is 45 -51 hrs. The cells were collected by centrigugation, washed and inoculated into a T125 flask containing the medium. The culture was subsequently expanded in the smaller roller bottles of capacity 300 ml rotating at a speed of 1-3 rpm. The cultures in smaller roller bottles are kept for 70 -, 75 hrs. To increase the cell number an maintain the cell density (no. of cells/ ml), the cultures are subsequently transferred to large roller bottles rotating at a speed greater than 8 rpm. The larger roller bottles have the capacity of 2000 ml. The cultures are kept for 6-9 days. It helps to monitor the growth of the cells, thereby monitoring the requirement of nourishment to the cells the cell multiplies and gets disrupted to get the crude lysate. The roller bottles containing the crude lysate having the cells and virus are transferred to large centrifuge tubes of 500 ml capacity or centrifuge bottles of 1 litre capacity. Subsequently, the cell debris are removed by high capacity centrifugation at 6500 rpm for 1hr. The virus bein extremely light in weight does not settle down at 6500 rpm. The supernatant containing the virus is decanted into the 5 litres - 10 litres bottle provided with the magnetic stirrer. The harvests are clarified daily to pass virus particles and retain cells and debris. The daily clarified harvests representing 7-10 days of production are pooled and mixed well to ensure homogeneity. The harvests contains the live virus. The live virus needs to be inactivated. With continuous stirring, (3-propiolactone (BPL) was added in a ration of 1:2000 followed by incubation at 3 - 5°C for 18-24 hrs. The temperature of the pool was then raised to 35-38°C and held at this temperature for 3-6 hrs. to hydrolyze residual BPL into lactic acid and propionic acid. The step served to chemically reduce infectivity of the virus. The inactivated virus homogenate is centrifuged at 18,000 rpm for 3 - 4.5 hrs. and are pelleted. The lysing agent is added to the pellet to lyse the virus into different molecular weight viral proteins or viral antigens. Serological diagnosis of HIV infection rests on the detection of a host immune response to viral antigens encoded by 3 major viral structural genes as shown in fig. 2. The gag gene encodes the major viral core proteins, p17 (mol. wt. 17 kD), p24 (mol. wt. 24 kD) and their precursor, p55 (mol. wt. 55 kD.). The env. gene encodes the envelope glycoprotein precursor, gp 160 (mol. wt. 160 kD) which is cleaved into an external envelope domain, gp120 (mol. wt. 120 kD) and transmembrane region, gp 41 (mol. wt. 41 kD). The pol gene encodes major proteins such as reverse transcriptase p66 and p51 (molecular weights 66 and 51 kD) respectively and a p31 endonuclease (molecular weight 31 kD) component. Several other viral proteins, mainly having regulatory functions, are encoded and expressed by the HIV genome, but none of these gene products are sufficiently immunogenic to be used as routine markers for serological diagnosis. The Western Blot Assay is the most informative for serological confirmation of HIV infections. in the Western Blot, HIV - virus is harvested from tissue culture, lysed and the individual viral proteins separated by polyacrylamide gel electrophoresis (PAGE). The separated proteins are then transferred to nitrocellulose membranes by electroblotting ("immuno blot strips"). A test serum is incubated with the immunoblot strips and HIV - antibodies, if present, bind to the separated viral proteins. The strips are washed to remove excess unbound proteins and presence of the specific antibodies is visualised by incubation with enzyme conjugated antihuman immunoglobin antibodies and chromogenic substrates. Thus, the presence of an antibody in sufficient concentration results in the staining of the characteristic viral protein "band". The sodium dodecyl sulphate-polyacrylamide gel electrophoresis is the method used to resolve and identify individual protein from a heterogeneous mixture with the use of SDS, an anionic detergent which denatures and imparts negative charge to the protein molecules which moves and separate out in the acrylamide matrix under the influence of the electric field depending on their size or molecular weight. For polyacrylamide gel electrophoresis, the gel plates are assembled with the spacers and the sides are sealed with 1% molten agar. A protein resolving gel comprising of 10-13% acrylamide made by mixing 27-32% acrylamide, 1.5 M Tris Cl, 17-20% SDS, 9-11% APS (ammonium per sulphate), 0.03 ml TEMED and 13.4 ml of distilled water to make up the total volume to 40 ml. A stacking gel comprising of 2-4% acrylamide is made by mixing 29-32% acrylamide, 1.25 M Tris. Cl, 18-21% SDS, 10% ammonium per sulphate, 7jJ TEMED and 7.8 ml of distilled water making the total volume 10 ml. The 10-13% separating acrylamide gel solution is poured and overlayered carefully with distilled water for polymerization for 17 X 15 cm gel size. After polymerization of the separating gel, distilled water is removed and 2-4% stacking gel solution is added followed by the insertion of preparative comb. The comb is removed after the polymerization of the stacking gel. The plates are assembled to the gel tank filled with the electrode buffer. Care has to be taken to remove all air bubbles from beiow the gel. The electrophoresis of the viral proteins is done by taking a sample containing the viral antigens and protein acting as a marker. The sample and the protein standard are kept for 5 minutes in boiling water bath and loaded in the sample well and marker well respectively. The gel is run initially at 23-26 volts for 1.5-2.5 hrs and overnight at 58-62 volts till the dye front reached the lower edge of the gel. The gel was given a brief rinse in distilled water and either stained with Coomassie to evaluate the resolution profile as shown in fig-2 or kept in the transfer buffer for at least 15 minutes before transfer of the resolved proteins onto the membrane. For Coomassie staining after electrophoresis gel is washed once with distilled water and stained with 200 ml of Coomassie blue gel staining solution for 2 hours on an orbital shaker. The gel is then washed once with distilled water and then destained with destaining solution overnight to remove background. The viral proteins present on the gel are transferred onto the nitrocellulose membrane by means of electroblotting. During electroblotting, the gel membrane sandwitch is placed between two parallel electrodes completely immersed in buffer and a current is passed at right angle to the gel which results in electroelution of proteins from the acrylamide gel and subsequent transfer to the solid phase matrix. These protein blots can be used for immunodetection and aminoacid analysis. Nitrocellulose membrane are used for their purpose. These membranes are hydrophobic, mechanically strong and microporous which offers large surface area entirely accessible for the protein adsorption. Blotted proteins can be stained to determine the position of molecular weight markers which also ensure the transfer efficiency and gives a picture of the complexity of the transferred proteins. Ponceau - S staining is used for this purpose. It is rapid, reversible and compatible with most antigen visualization technique though stain is not sensitive, it gives fair idea about total composition of the proteins without distorting the membrane. The proteins from acrylamide gel are transferred on nitrocellulose membrane for 1.5-2.5 hrs. at 200 mA and for another hour at 300 mA. After transferring, the blotted membrane is washed gently for 4-7 minutes with distilled water and further stained with Ponceau-S for 3-5 minutes followed by distilled water wash to remove background. After visualization and respective labeling of the Ponceau stained marker bands the blot was destained completely with phosphate buffer saline followed by blocking with blocking with blocking agent as bovine serum albumin, gelatin, tween - 20, casein, non-fat dry milk at 37°C for 2 hours. The blocked membrane is air dried and stored at room temperature or refrigerator. The immunodetection of the HIV-1 or 2 can be done by proteins blotted on the membrane and be probed with polyclonal or monoclonal antibodies and with the use of enzyme conjugated secondary antibody and substrate system generated precipitable insoluble color reaction on the white membrane background for visual appreciation of proteins of interest. Immunoblotting permits the specific detection of protein from crude complex mixture sourced from whole cell lysates, preparation of cells, tissues or organs. For immunoblotting strips of 3 mm size are cut from the nitrocellulose sheet and reacted with different positive and negative body fluids. Different dilutions are used for different body fluids. For serum and plasma, 1:100 is used. For milk 1:10 or heat and for cerebro-spinal fluid 1:22/1:4 dilutions are used to give detectable level of intensity of the protein bands on the blot. The strips cut out from a blotted membrane are washed in 1-3 ml of western blot buffer A for 4-6 minutes at room temperature. The buffer is aspirated off. The 1-2 ml of sample buffer is added with 20 ml of test serum. The strip is then incubated at room temperature for 2-4 hours or overnight at 4°C. The sample buffer is aspirated off and the strip is washed thrice for 4-6 min. with 2 ml of wash buffer followed by two washes with 2 ml of conjugate buffer for 5 minutes. The strip is then incubated at room temperature for 1 hr. in 2 ml. of conjugate buffer containing the enzyme conjugated secondary antibody which is a Rabbit anti-human Immunoglobin (IgG) conjugated with enzyme alkaline ohosphatase. The antibody solution is aspirated off and strip is washed twice with conjugate buffer and twice with phosphate buffer saline for 5 minutes each. After the last wash 2 ml of solution containing the BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (Nitroblue tetrazolium) is added. The colour development is followed for 1 to 2 minutes till coloured bands on positive control strip appears then the reaction is stopped by washing the strip with distilled water. Almost all the virus specific bands should be visible along with serum control band as shown in figure-3. In case of negative control none of the HIV-1 and HIV-2 specific bands should be observed on the negative control strips. The band only for serum control should be visible as shown in figure-4. It is understood that modifications can be made to the invention mentioned above without departing from the scope of the invention. The subject invention is a mere statement of the invention, where various improvements and alternations are possible without deviating from the scope of the invention, hence the same should not be construed to restrict the scope of the invention. We claim: 1. 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. 2. A process for producing Western Blot Assay for detecting the antibodies to human retroviral infections as claimed in claim 1, wherein the said membrane is impregnated with HIV-1 and HIV 2 antigen base. 3. A process for producing Western Blot Assay for detecting the antibodies to human retroviral infections as claimed in claim 1, wherein the live virus contained in the said harvests as herein described are inactivated by continuous stirring, adding p-propiolactone (BPL) in the ratio of 1:2000 with respect to the total volume of the harvest, followed by incubation at 3-5°C for 18-24 hrs. 4. A process for producing Western Blot Assay for detecting the antibodies to human retroviral infections as claimed in claim 1, wherein the pooled harvest as herein described is heated to a temperature of 35-38°C and held at that temperature for 3-6 hrs. to hydrolyze residual p- propiolactone (BPL) into lactic acid and propionic acid, thus chemically reducing infectivity of the virus. 5. A process for producing Western Blot Assay for detecting the antibodies to human retroviral infections as claimed in claim 1, wherein the said inactive virus is pelleted by centrifugation at 18000 revolution per minute for 3 - 4.5 hrs. and then lysed by adding conventional lysing agent into different molecular weight viral proteins or viral antigens. 6. A process for producing Western Blot Assay for detecting the antibodies to human retrovial infections substantially as hereinbefore described with reference to the accompanying drawings.

Documents:

862-del-2002-abstract.pdf

862-del-2002-claims.pdf

862-DEL-2002-Correspondence-Others.pdf

862-del-2002-correspondence-po.pdf

862-del-2002-description (complete).pdf

862-del-2002-description (provisional).pdf

862-del-2002-drawings.pdf

862-del-2002-form-1.pdf

862-del-2002-form-13.pdf

862-del-2002-form-19.pdf

862-del-2002-form-2.pdf

862-del-2002-form-26.pdf

862-del-2002-form-3.pdf

862-del-2002-form-4.pdf

862-del-2002-form-5.pdf

862-del-2002-others-document.pdf

862-del-2002-petitiion-138.pdf