Title of Invention	A PROCESS FOR PREPARING ANTIBODY LABELLED MICROSPHERES USING ANTI CD4/ANTI CD8 ANTIBODY FOR DETECTING CD4/CD8 POSITIVE CELLS IN THE BLOOD
Abstract	A process for preparing antibody labelled microspheres using anti CD4 /anti CD8 antibody for detecting CD4 and CD8 positive cells in the blood which comprises the steps of: Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and washing twice with sufficient carbonate buffer; resuspending the pellet in phosphate buffer and washing two times in phosphate buffer; the pellet is then resuspended in a 0.625 ml of the 0.02M sodium phosphate buffer, pH 4.5; Adding drop wise 0.625 ml of the 2% carbodiimide solution; Mixing 3-4 hours at room temperature; Centrifuging for 5-6 minutes and removing and discarding supernatant; Resuspending pellet in phosphate buffer; Centrifuging for 5-6 minutes and removing and discarding supernatant; remove unreacted carbodiimide; Resuspending pellet in 1.2ml of borate buffer; Adding 200-400 ug of Anti CD4 / Anti CD8 antibody; mixing gently overnight at room temperature on an end-to-end mixer; Adding 50ul 0.25M ethanolamine(2-aminoethanol) and mixing gently for 30 minutes for blocking unreacted sites on the microparticles; Centrifuging for 10 minutes and saving supernatant for protein determination; Resuspending pellet in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex; Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites; Centrifuging for 5-6 minutes and removing and discarding supernatant; Resuspending pellet in 0.5 ml PHS, pH 7.4 containing 10mg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer); storing at 4°C.

Title of Invention

A PROCESS FOR PREPARING ANTIBODY LABELLED MICROSPHERES USING ANTI CD4/ANTI CD8 ANTIBODY FOR DETECTING CD4/CD8 POSITIVE CELLS IN THE BLOOD

Abstract

A process for preparing antibody labelled microspheres using anti CD4 /anti CD8 antibody for detecting CD4 and CD8 positive cells in the blood which comprises the steps of: Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and washing twice with sufficient carbonate buffer; resuspending the pellet in phosphate buffer and washing two times in phosphate buffer; the pellet is then resuspended in a 0.625 ml of the 0.02M sodium phosphate buffer, pH 4.5; Adding drop wise 0.625 ml of the 2% carbodiimide solution; Mixing 3-4 hours at room temperature; Centrifuging for 5-6 minutes and removing and discarding supernatant; Resuspending pellet in phosphate buffer; Centrifuging for 5-6 minutes and removing and discarding supernatant; remove unreacted carbodiimide; Resuspending pellet in 1.2ml of borate buffer; Adding 200-400 ug of Anti CD4 / Anti CD8 antibody; mixing gently overnight at room temperature on an end-to-end mixer; Adding 50ul 0.25M ethanolamine(2-aminoethanol) and mixing gently for 30 minutes for blocking unreacted sites on the microparticles; Centrifuging for 10 minutes and saving supernatant for protein determination; Resuspending pellet in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex; Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites; Centrifuging for 5-6 minutes and removing and discarding supernatant; Resuspending pellet in 0.5 ml PHS, pH 7.4 containing 10mg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer); storing at 4°C.

Full Text	The present invention relates to a process for preparing antibody labelled microspheres using anti CD4 / anti CD8 antibody for detecting CD4/CD8 cells in the blood. PRIOR ART Introduction HIV and HAART: Recent developments in the field of HIV/AIDS therapy in resource-poor settings are spectacular and raise hope that Highly Active Anti-Retroviral Therapy (HAART) will soon become a reality for substantially more HIV-infected people. The very significant drawback to HAART is its cost; however, this impediment is quickly disappearing: prices of drugs have decreased considerably. The Accelerated Access Initiative (AAI), a collaborative effort of UNAIDS and the major pharmaceutical industries, has achieved important price reductions of branded HAART drugs (down to 5-10%). Manufacturers of generic drugs are continuously offering further price reductions, the latest breakthrough being the one provided by the Thai Government Pharmaceutical Organization (GPO), which will allow drugs to be sold at Rs 40 per day. In March 2002, the World Health Organization (WHO) officially released a list of manufacturers of safe HAART drugs, approved for UN purchase, which is further strengthening the option of generic HAART drugs for resource-poor countries. There are a multitude of issues that still hamper access to HAART for the developing world. This project was conceived to instead, concentrate on solving one of the next hurdles to be taken; simplifying and reducing the costs of laboratory monitoring of HAART. Laboratory monitoring of HAART: Today, the costs of HAART laboratory monitoring equal or even surpass the costs of treatment. Prices for measuring the most important HAART monitoring markers are high: Rs 1250/- for a CD4 T-cell test and Rs 7500/- for a viral load measurement (conservative estimate) are rather rule than exception in Africa or Asia. This is partly because the manufacturers of laboratory equipment and assays for HAART monitoring have not yet been pressured to reduce prices and partly because the market for these industries is too small, mostly since there are still very few people in developing countries actually receiving HAART and thus in need of lab monitoring. With small markets, there is obviously little interest to invest in the development of more affordable technology. WHO Initiative: In light of this reality, various initiatives have been launched to come up with recommendations for affordable lab technologies for the developing world. The WHO organized technical working groups on diagnostic support of HAART in August and November 2001 and in February 2002, specifically on CD4 T-cell enumeration. It is important to mention that the absence of laboratory facilities should not be a reason to refrain from implementing HAART in a resource-poor setting. In principle, good clinical follow-up is sufficient, provided it is combined with an extra effort in guaranteeing compliance, for example through direct observed therapy (DOT), as applied in tuberculosis control programs. It should be emphasized that HAART does not cure the patient and constant alertness is required from both the patient (compliance) and the clinician (monitoring) to avoid sub-optimal use of the medication from resulting in potentially catastrophic, massive drug resistance in the developing world. Importance of CD4 counts: CD4+ T lymphocytes are currently the most common surrogate marker indicating disease progression in individuals infected with human immunodeficiency virus (HIV). The enumeration of CD4+ T cells from persons infected with the human immunodeficiency virus (HIV) is important. Traditionally this measurement has been calculated by multiplying the percent of lymphocytes that are CD4+ T cells (from flow cytometry) and the number of lymphocytes per microlitre of blood (from hematology measures). Quantification of CD4 +ive T Cells: Quantification of CD4 T-cells is especially important to help clinicians in making a decision on the initiation of HAART. Although there is no consensus for application in the developing world, the start of HAART is usually recommended when CD4 T-cell counts are between 200-350 per microlitre of whole blood. The cut-off point for starting HAART is an important issue, not only because it has implications for the incidence of opportunistic infections, but also regarding costs. Preliminary data suggest that, as a rule of thumb, 50% of all HIV-infected patients qualify for HAART when 350 cells/^iL is taken as a cut-off, but only 25% qualifies with the lower cut-off (200/µL). Flowcytometry: The gold standard for CD4 enumeration remains flow cytometry. The easiest instrument to use is a single-platform volumetric flow cytometer, like the B&D FACScount. However, this equipment is very expensive ($35,000) and the maintenance contracts ($6000/year) and reagents ($ 12/test, cold-chain dependent) are not affordable for most resource poor settings. There are other flow cytometers, like the B&D FACScan and FACScalibur, the Coulter XL and the Ortho Cytoron, which are even more expensive and either need an additional blood cell counter (dual platform technology) or very expensive beads for CD4 T-cell quantification. Alternative flow cytometry-based CD4 T-cell enumeration methods: Alternative flow cytometry-based CD4 T-cell enumeration methods are all in various stages of development. First of all, there is the possibility of using simpler protocols on already existing flow cytometers. These protocols use innovative ways of gating (CD45 pan-leukogating, CD4-gating) and as a consequence need only one or two generic monoclonal antibodies, which are substantially less expensive than commercial kits. However, these protocols cannot be performed on a FACScount which excludes the far majority of those privileged labs in India that do have a flow cytometer. Currently, these pan-leukogating and CD4-gating technologies are under multi-center evaluation. Low cost flowcytometers: Another alternative is the use of flow cytometers with low energy red-diode lasers. These machines are smaller, less expensive and need considerably less electrical power (a car battery of 12V is sufficient). A new instrument has been launched by Partec (the CyFlow), which is basically a half-price FACScount with half-price kits, but needs multi-center validation before it can be recommended. The alternative approach of undressing" the complex Luminex 100 to a simpler red diode laser-based cytometer has met little enthusiasm from industry, even though multiplex technology will be the future in laboratory diagnostics. Flowcytometry assays: CDC and WHO developed guidelines for laboratories who perform immunophenotyping for detection and enumeration of CD4+ T-cells and other lymphocyte subsets in persons infected with human immunodeficiency virus (HIV) (6). The previous report MMWR 1997; 46 [No. RR-2]) described a dual-platform technology, a method in which absolute counts are derived from measurements obtained from two instruments—a flow cytometer and hematology analyzer. The guidelines describe single-platform technology (SPT), a process in which absolute counts of lymphocyte subsets are measured from a single tube by a single instrument. SPT incorporates internal calibrator beads of known quantity in the analysis of specimens by three- or four-color flow cytometry. With CD45 gating, the relative numbers of beads and lymphocyte subsets are enumerated, and their absolute numbers and percentage values are calculated. Recent technologic advances have allowed for the development of a system for the determination of CD4+ T-lymphocyte counts by simultaneous 4-color flow7 cytometry. Kutok et al (7) evaluated a new 4-color 2-tube flow cytometric method for analyzing CD4+ T-lymphocyte subsets in whole blood was compared with a standard 2-color 5-tube method. The new method provides results almost identical to those of the well-established 2-color method used in our clinical laboratory. Statistical analyses indicate very low variability in CD4+ counts between the 2 methods, strongly supporting the usefulness of this new procedure. In addition, the 4-color procedure provides a 15% reduction in the materials cost per test compared with the 2-color method, as well as a marked reduction in the time expenditure of flow cytometry technologists. Sherman et al (8) examined absolute CD4 counts as recommended by the Centers for Disease Control (CDC) by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively and compared it to the CDC recommended 6 tube 2 colour panel [6(2)]. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision. Advantages of flowcytometry: The advantages of using flow cytometry for CD4 T-cell enumeration are clear: the automated methodology is easier to use, more reproducible, statistically more reliable and features a higher throughput (up to 300 samples/day). Using pan-leukogating, blood samples of more than one week old can be analyzed and blood fixatives can be applied without problems significantly increasing the action radius of central laboratories. Moreover data can be stored on computers, reanalyzed and a good quality control system for flow cytometry is in place, including the NEQAS standards with 200, 400 and 800 CD4 cells/µL blood. Newer technologies for CD4 counts Several manufacturers have developed new techniques for determining absolute numbers of CD4+ and CD8+ cells without the need for the flow cytometer and hematology analyzer (9). ELISA based methods: Saah et al (10), Moss et al (11) and Cordiali et al (12) evaluated a recently developed enzyme-linked immunosorbent assay system for measuring helper T-lymphocyte count (the TRAx enzyme-linked immunosorbent assay) and compared it with flow cytometry and concluded that the TRAx enzyme-linked immunosorbent assay system is an acceptable method for measuring helper T-lymphocyte count, but should be recalibrated for better performance at helper T-cell counts below 200 per µL. However the TRAx system is no longer marketed. Lisse et al (13) tested an alternative method for CD4 and CD8 T lymphocytes enumeration, the immunoalkaline phosphatase method (IA), in three African countries and in Denmark. The authors found good correspondence between the two methods for measurements of CD4 and CD8 T lymphocytes independent of serological status and geographical site. IA compared to flow cytometry consistently gives higher percentage of CD4 T lymphocytes, and lower percentage of CD8 T lymphocytes. The IA method is therefore suitable for use in areas with limited resources and laboratory facilities where there is a need for immunological surveillance in hospital or community studies. Magnetic separation and ELISA based techniques: Carriere et al (14) evaluated the Whole Blood Capcellia(R) CD4/CD8, an immunoenzymatic method that provides absolute counts of CD4+ and CD8+ T cells in peripheral blood. The assay is based on the separation of T cells by use of an anti-CD2 magnetic bead suspension, followed by reaction of the CD4 or CD8 molecules with the corresponding monoclonal antibody coupled to peroxidase. CD4-positive monocytes were excluded from the assay. Freeze-dried magnetic bead-T-cell complexes were used as calibrators. Capcellia counts from H1V-1-infected patients were compared with those obtained by flow cytometry as the comparison method. The results by Capcellia correlated well with those by flowcytometric analysis and indicate that this new ELISA technique for lymphocyte immunophenotyping is an efficient alternative to flow cytometry. The kit was evaluated in India by Kannangai et al (15) who concluded that the assay system has certain limitations inherent to ELISA techniques. Automated Volumetric capillary cytometry: Volumetric capillary cytometry (VCC) is a new technology that involves the detection and enumeration of dually fluorochrome-labeled cells in a precise volume. Gorman et al (16) compared the accuracy and precision of VCC with the accuracy and precision of flow cytometry and hematology (F&H) for the measurement of the absolute numbers of CD4 and CD8 T cells in the whole blood of patients infected with human immunodeficiency virus, and showed that it may represent a viable alternative to flow cytometry for obtaining absolute T-cell subset counts. Fluorescence immuno assay: Denny et al (17) evaluated a new alternative fluorescence immunoassay method (Zymmune CD4/CD8 Cell Monitoring Kit; Zynaxis, Inc., Malvern, Pa.) for determining the absolute CD4+ and CD8+ T-lymphocyte concentrations in whole blood. The investigation was performed as a two-site comparison of the reference whole blood flow cytometric method with the Zymmune method, and concluded that the Zymmune CD4/CD8 Cell Monitoring Kit method provides absolute CD4+ and CD8+ T-lymphocyte concentrations which are equivalent to those given by the reference flow cytometric method. Non-flow cytometry-based devices: Recent developments on non-flow cytometry-based devices, like the Easy Count (developed at Twente University) based on magnetic sorting and optical image analysis of CD4 T-cells. Major advantages are: compact stand-alone, volumetric device, short sample measurement time, good linearity, low power consumption (10W, can be run on a laptop battery) and potentially low costs. However, the instrument still has a long way to go: it is in its proof of concept phase and a second prototype is just being constructed Comparable methods: Bead based methods: Bead based methods available are of two types: The antibody coated latex bead assay (18) and the immunomagnetic bead (Dynal) (1, 19, and 20). Antibody coated Bead based Technologies: The most affordable way of quantifying CD4 T-cells is by manual technologies, using synthetic beads and microscopy for read-out. Currently, the most efficient methods for low-volume applications are Dynabeads and Cytospheres. At least nine studies, several involving African populations, have correlated CD4 counts assessed by Cyto-Spheres or Dynabeads with counts measured by flow cytometry. In most (but not all) studies, the correlation has been excellent. Cyto-Spheres have been studied more thoroughly. Both assays involve the physical counting of cells, which can be done under a light microscope. Dynabeads: Dynabeads cost approximately $3 per test and need an investment of $750 for a magnet and mixer. The original technology needs an additional expensive immuno-fluorescence microscope ($7500), (Costs available on Dynal Website) but there are ways to get around this, either by using alternative staining techniques that are compatible with light microscopy or purchasing a device, which converts a light microscope into a fluorescent microscope ($1800). Cytospheres: Cytospheres are easier to use but more expensive ($9/test) (actual landed costs of a dollar worth of material in India after taxes and freight work to about Rs 85 for 1$), although investments are low: only a light microscope and a hemacytometer. Both Dynabeads and Cytospheres are widely used in laboratories in the developing world. SUMMARY OF THE INVENTION According to the present invention there is provided a process for preparing antibody labelled microspheres using Anti CD4 / Anti CDS antibody for detecting HIV in the blood which comprises the steps of: i) Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and adding 1 ml of carbonate bufferas herein described to fill tube; the microparticles form the substrate on which the antibodies are bound; ii) Suspending the reactants of step (i) which comprises of the following steps: a. Centrifuging the reactants for 5-6 minutes in a micro-centrifuge for packing the microparticles; b. Carefully removing supernatant using a Pasteur pipette which helps to concentrate the microparticles for a wash procedure; c. Discarding supernatant for removing the fluid in which the microparticles were suspended; iii) Repeating the above procedure of step (ii) for resuspending the pellet, the said resuspension is carried out by: a. Filling tube halfway and capping; b. Filling tube till vortex; c. Filling tube to capacity; iv) Resuspending pellet in phosphate buffer as herein described and centrifuging for 5-6 minutes ensures completeness of wash procedure; v) Carefully removing supernatant using a Pasteur pipette and discarding supernatant ensures completeness of wash procedure; vi) Repeating step (ii) above two more times ensures completeness of wash procedure and resuspends in phosphate buffer as herein described; vii) Resuspending pellet as herein described in a 0.625 ml of the 0.02M sodium phosphate buffer, pH 4.5; viii) Adding drop wise 0.625 ml of the 2% carbodiimide solution for activating the carboxylated groups on the microparticles; ix) Mixing 3-4 hours at room temperature ensures complete activation; x) Centrifuging for 5-6 minutes and removing and discarding supernatant for removing the activator; xi) Resuspending pellet (as herein described) in phosphate buffer as herein described for preparing the microparticles for binding with antibody; xii) Centrifuging for 5-6 minutes and removing and discarding supernatant; Prepares the microparticles for binding with antibody xiii) Repeating steps (xi) and (xii) two more times for getting rid of unreacted carbodiimide for cleaning up the procedure; xiv) Resuspending pellet (as herein described) in 1.2ml of borate buffer wherein the buffer acts to prepare the milieu interior of the reaction mix for antibody binding to the activated microparticles; xv) Adding 200-400 g of Anti CD4 / Anti CD8 antibody for binding to the activated microparticles; xvi) Mixing gently overnight at room temperature on an end-to-end mixer wherein the microparticles bind completely with antibody and the antibody is coated evenly over the microparticles; xvii) Adding 50(xl 0.25M ethanolamine(2-aminoethanol) and mixing gently for 30 minutes for blocking unreacted sites on the microparticles, the unreacted sites on the microparticles are blocked to prevent nonspecific binding with any other amine bearing chemical compound xviii) Centrifuging for 10 minutes and saving supernatant for protein determination, wherein the centrifuging helps in concentrating the preparation; xix) Resuspending pellet (as herein described) in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex, wherein the resuspension helps in storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage; xx) Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites, wherein the BSA reacts with any unblocked sites still open; xxi) Centrifuging for 5-6 minutes and removing and discarding supernatant for concentrating the preparation; xxii) Repeating steps xix and xxi once; xxiii) Resuspending pellet (as herein described) in 0.5 ml PBS, pH 7.4 containing lOmg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer), wherein resuspension helps storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage. NaN3 is a preservative and prevents growth of contaminants; xxiv) Storage at 4 °C enhances the useful life of the product DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Making antibody coated beads Material Required i) Carboxyl-Modified Microspheres (often supplied at 10% solids) ii) Water Soluble Carbodiimide, (EDAC: l-Ethyl-3-(3- dimethylaminopropyl) carbodiimide) iii) Quenching Solution with ethanolamine iv) Blocking molecule (Bovine serum albumin) Preparation of reagents: Prepare using distilled or deionized water unless otherwise indicated. Carbonate Buffer: 0.1 M Carbonate buffer, pH 9.6-prepare by adding 0.02 M Sodium Carbonate (Na2C03) to 0.1 M NaHC03 until pH = 9.6. Phosphate Buffer: 0.02 M Sodium phosphate buffer, pH 4.5-prepare by adding 0.02 M Sodium hydro phosphate (NaHP04 ) to 0.02 M Sodium dihydro Phosphate (NaH2P04 ) until pH = 4.5. 2% Carbodiimide: 2% l-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydochloride dissolved in phosphate buffer (see above). This has to be prepared within 15 minutes of using. Borate Buffer: 0.2 M borate buffer, pH 8.5-use Boric acid to prepare this buffer. Adjust pH to 8.5 using 1 M Sodium Hydroxide (NaOH). 0.25 Methanolamine: Add 20µl of ethanolamine (2-aminoethanol) to 1.3ml of borate buffer. Storage Buffer: First prepare 0.1 M phosphate buffer, pH 7.4, by adding 0.1 M Sodium dihydrophosphate (NaH2PO4) to 0.1 Sodium hydrophosphate (Na2HP04 ), until pH becomes 7.4. Take 20 ml of the 0.1 M phosphate buffer, pH 7.4, in a 100 ml graduated cylinder. Add 0.88g Sodium Chloride (NaCl), Ig bovine serum albumin (BSA), 5 ml glycerol, and O.lg NaN3, and make up the volume to 100 ml. Check the pH of the final solution. If necessary, adjust the pH to 7.4 by adding dilute Hydrochloric acid (HCl) or Sodium hydroxide (NaOH). Making RBC lysis solution: To make RBC lysis solution take 2 ml of glacial acetic acid, add 98 ml of water and 25 mg of crystal violet. Shake. Store at 4°C. Process for preparing antibody labelled microspheres The process for preparing antibody labelled microspheres using anti CD4 antibody for detecting HIV in the blood, which comprises the steps of: (i) Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and adding 1 ml of carbonate bufferas herein described to fill tube; the microparticles form the substrate on which the antibodies are bound; (ii) Suspending the reactants of step (i) which comprises of the following steps: (a) Centrifuging the reactants for 5-6 minutes in a micro-centrifuge for packing the microparticles; (b) Carefully removing supernatant using a Pasteur pipette which helps to concentrate the microparticles for a wash procedure; (c) Discarding supernatant for removing the fluid in which the microparticles were suspended; (iii) Repeating the above procedure of step (ii) for resuspending the pellet, the said resuspension is carried out by: (a) Filling tube halfway and capping; (b) Filling tube till vortex; (c) Filling tube to capacity; (iv) Resuspending pellet in phosphate buffer as herein described and centrifuging for 5-6 minutes ensures completeness of wash procedure ; (v) (v Carefully removing supernatant using a Pasteur pipette and discarding supernatant ensures completeness of wash procedure; (vi) Repeating step (ii) above two more times ensures completeness of wash procedure and resuspends in phosphate buffer as herein described; (vii) Resuspending pellet as herein described in a 0.625 ml of the 0.02M sodium phosphate buffer, pH 4.5; (viii) Adding drop wise 0.625 ml of the 2% carbodiimide solution for activating the carboxylated groups on the microparticles; (ix) Mixing 3-4 hours at room temperature ensures complete activation; (x) Centrifuging for 5-6 minutes and removing and discarding supernatant for removing the activator; (xi) Resuspending pellet (as herein described) in phosphate buffer as herein described for preparing the microparticles for binding with antibody; (xii) Centrifuging for 5-6 minutes and removing and discarding supernatant; Prepares the microparticles for binding with antibody; (xiii) Repeating steps xi and xii two more times for getting rid of unreacted carbodiimide for cleaning up the procedure; (xiv) Resuspending pellet (as herein described) in 1.2ml of borate buffer wherein the buffer acts to prepare the milieu interior of the reaction mix for antibody binding to the activated microparticles; (xv) Adding 200-400 g of Anti CD4 / Anti CD8 antibody for binding to the activated microparticles; (xvi) Mixing gently overnight at room temperature on an end-to-end mixer wherein the microparticles bind completely with antibody and the antibody is coated evenly over the microparticles; (xvii) Adding 50(µl 0.25M ethanolamine(2-aminoethanol) and mixing gently for 30 minutes for blocking unreacted sites on the microparticles, the unreacted sites on the microparticles are blocked to prevent nonspecific binding with any other amine bearing chemical compound; (xviii) Centrifuging for 10 minutes and saving supernatant for protein determination, wherein the centrifuging helps in concentrating the preparation; (xix) Resuspending pellet (as herein described) in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex, wherein the resuspension helps in storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage; (xx) Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites, wherein the BSA reacts with any unblocked sites still open; (xxi) Centrifuging for 5-6 minutes and removing and discarding supernatant for concentrating the preparation; (xxii) Repeating steps xix and xxi once; (xxiii) Resuspending pellet (as herein described) in 0.5 ml PBS, pH 7.4 containing lOmg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer), wherein resuspension helps storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage. NaN3 is a preservative and prevents growth of contaminants; (xxiv) Storage at 4 °C enhances the useful life of the product MANUAL COUNTING PROCEDURES AND CALCULATIONS FOR DILUTIONS Requirements for manual counting: Micropipette: 0-lOµl, 10-200µl Naeubers Chamber Manual counting procedures: For the assay Blood was collected in an EDTA bottle. I. 100 (µL of whole blood is taken in a clean test tube. II. 10 µL of antibody labelled microspheres as obtained in the above process is added. III. The mixture was gently shaken for 2 minutes and then the beads allowed to adhere for a further 3 minutes. IV. lOµL from step III (the blood-latex bead mixture) is added to a RBC lysis solution, shaken for 30 seconds, and pipetted onto both sides of a 0.1-mm-deep hemacytometer (18 mm ). V. With a light microscope at 40X magnification, lymphocytes with two or more beads attached to their surface are counted in the two 9 mm , of the hemacytometer. VI. The number obtained is multiplied by 6.72 to obtain the absolute count of CD4/CD8 cells per cumm of blood (Derivation of dilution factor given below). VII. The test was carried out every week with both high and low standards to determine CD4/CD8 counts obtained with the standards. Calculations for dilutions: Area counted in haemocytometer: 18 mm2 Corrections for dilution: Primary dilution: 100 µL of whole blood and 10 µL of reagent = 100/110 Secondary dilution: 10µL of whole blood of 100/110 dilution to 100 µL of RBC lysis solution 10 100 110 110 — 0.0826 Hence correction for sample dilution is 1/0.0826=12.1 Correction for sample depth of .01 mm= 1/.01 = 10 Hence correction factor to be multiplied with so as to obtain total number of CD4/CD8 + T eels in 1 cumm of blood is (10xl2.1)/18 = 6.72 Quality control. It is recommended that 1. Kit be stored at 4°C 2. Every Monday the Kit be run against the Immunotrol reagents (both high and low) and results be counted If the test results are within ± 5% of the value given for the standards the kit performance is considered satisfactory. I Claim: 1. The process for preparing antibody labelled microspheres using anti CD4 antibody for detecting HIV in the blood, which comprises the steps of: i) Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and adding 1 ml of carbonate bufferas herein described to fill tube; the microparticles form the substrate on which the antibodies are bound; ii) Suspending the reactants of step (i) which comprises of the following steps: a) Centrifuging the reactants for 5-6 minutes in a micro-centrifuge for packing the microparticles; b) Carefully removing supernatant using a Pasteur pipette which helps to concentrate the microparticles for a wash procedure; c) Discarding supernatant for removing the fluid in which the microparticles were suspended; iii) Repeating the above procedure of step (ii) for resuspending the pellet, the said resuspension is carried out by: a) Filling tube hallway and capping; b) Filling tube till vortex; c) Filling tube to capacity; iv) Resuspending pellet in phosphate buffer as herein described and centrifuging for 5-6 minutes ensures completeness of wash procedure ; v) Carefully removing supernatant using a Pasteur pipette and discarding supernatant ensures completeness of wash procedure; vi) Repeating step (ii) above two more times ensures completeness of wash procedure and resuspends in phosphate buffer as herein described; vii) Resuspending pellet as herein described in a 0.625 ml of the 0.02M sodium phosphate buffer, pH 4.5; viii) Adding drop wise 0.625 ml of the 2% carbodiimide solution for activating the carboxylated groups on the microparticles; ix) Mixing 3-4 hours at room temperature ensures complete activation; x) Centrifuging for 5-6 minutes and removing and discarding supernatant for removing the activator; xi) Resuspending pellet (as herein described) in phosphate buffer as herein described for preparing the microparticles for binding with antibody; xii) Centrifuging for 5-6 minutes and removing and discarding supernatant; Prepares the microparticles for binding with antibody xiii) Repeating steps xi and xii two more times for getting rid of unreacted carbodiimide for cleaning up the procedure; xiv) Resuspending pellet (as herein described) in 1.2ml of borate buffer wherein the buffer acts to prepare the milieu interior of the reaction mix for antibody binding to the activated microparticles; xv) Adding 200-400 g of Anti CD4 / Anti CDS antibody for binding to the activated microparticles; xvi) Mixing gently overnight at room temperature on an end-to-end mixer wherein the microparticles bind completely with antibody and the antibody is coated evenly over the microparticles; xvii) Adding 50µl 0.25M ethanolamine(2-aminoethanol) and mixing gently for 30 minutes for blocking unreacted sites on the microparticles, the unreacted sites on the microparticles are blocked to prevent nonspecific binding with any other amine bearing chemical compound xviii) Centrifuging for 10 minutes and saving supernatant for protein determination, wherein the centrifuging helps in concentrating the preparation; xix) Resuspending pellet (as herein described) in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex, wherein the resuspension helps in storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage; xx) Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites, wherein the BSA reacts with any unblocked sites still open; xxi) Centrifuging for 5-6 minutes and removing and discarding supernatant for concentrating the preparation; xxii) Repeating steps xix and xxi once; xxiii) Resuspending pellet (as herein described) in 0.5 ml PBS, pH 7.4 containing lOmg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer), wherein resuspension helps storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage. NaN3 is a preservative and prevents growth of contaminants; xxiv) Storage at 4 °C enhances the useful life of the product 2. The process as claimed in claim 1, wherein the carbonate buffer is prepared by adding 0.02 M Na2C03 to 0.1 M Sodium Hydrocarbonate (NaHC03) until pH = 9.6. 3. The process as claimed in claim 1, wherein the phosphate buffer is prepared by adding 0.02 M Sodium hydrophosphate (NaHP04) to 0.02 M Sodium dihydrophosphate (NaH2P04) until pH = 4.5 4. The process as claimed in claim 1, wherein the 2% carbodiimide is prepared by dissolving 2% l-(3-Dimethylaminopropyl)-3-ethyI carbodiimide hydochloride in said phosphate buffer, which has to be prepared within 15 minutes of using. 5. The process as claimed in claim 1, wherein borate buffer is prepared by using Boric acid and adjusting pH to 8.5 by using 1 M Sodium hydroxide (NaOH). 0.25 M Methanolamine and adding 20µ1 of ethanolamine (2-aminoethanol) to 1.3 ml of borate buffer. 6. The process as claimed in claim 1, wherein the storage buffer is prepared by adding 0.1 M Sodium dihydrophosphate (NaH2P04) to 0.1 Sodium hydrophosphate (Na2HP04), until pH becomes 7.4, taking 20 ml of the 0.1 M phosphate buffer, in a 100 ml graduated cylinder, adding 0.88g Sodium chloride (NaCl), Ig bovine serum albumin (BSA), 5 ml glycerol, and O.lg NaN3, and making up the volume to 100 ml checking the pH of the final solution and, adjusting the pH to 7.4 by adding dilute Hydrochloric acid (HO) or Sodium hydroxide (NaOH). 7. The process as claimed in claim 1, wherein the pH of carbonate buffer is maintained at pH 9.6. 8. The process as claimed in claim 1, wherein the pH of Phosphate buffer is maintained at pH 4.5. 9. The process as claimed in claim 1, wherein the pH of borate buffer is maintained at pH 8.5. 10. The process as claimed in claim 1, wherein the pH of storage buffer is maintained at pH 7.4.

Full Text

The present invention relates to a process for preparing antibody labelled microspheres using anti CD4 / anti CD8 antibody for detecting CD4/CD8 cells in the blood.
PRIOR ART Introduction
HIV and HAART: Recent developments in the field of HIV/AIDS therapy in resource-poor settings are spectacular and raise hope that Highly Active Anti-Retroviral Therapy (HAART) will soon become a reality for substantially more HIV-infected people. The very significant drawback to HAART is its cost; however, this impediment is quickly disappearing: prices of drugs have decreased considerably. The Accelerated Access Initiative (AAI), a collaborative effort of UNAIDS and the major pharmaceutical industries, has achieved important price reductions of branded HAART drugs (down to 5-10%). Manufacturers of generic drugs are continuously offering further price reductions, the latest breakthrough being the one provided by the Thai Government Pharmaceutical Organization (GPO), which will allow drugs to be sold at Rs 40 per day. In March 2002, the World Health Organization (WHO) officially released a list of manufacturers of safe HAART drugs, approved for UN purchase, which is further strengthening the option of generic HAART drugs for resource-poor countries. There are a multitude of issues that still hamper access to HAART for the developing world. This project was conceived to instead, concentrate on solving one of the next hurdles to be taken; simplifying and reducing the costs of laboratory monitoring of HAART.
Laboratory monitoring of HAART: Today, the costs of HAART laboratory monitoring equal or even surpass the costs of treatment. Prices

for measuring the most important HAART monitoring markers are high: Rs 1250/- for a CD4 T-cell test and Rs 7500/- for a viral load measurement (conservative estimate) are rather rule than exception in Africa or Asia. This is partly because the manufacturers of laboratory equipment and assays for HAART monitoring have not yet been pressured to reduce prices and partly because the market for these industries is too small, mostly since there are still very few people in developing countries actually receiving HAART and thus in need of lab monitoring. With small markets, there is obviously little interest to invest in the development of more affordable technology.
WHO Initiative: In light of this reality, various initiatives have been launched to come up with recommendations for affordable lab technologies for the developing world. The WHO organized technical working groups on diagnostic support of HAART in August and November 2001 and in February 2002, specifically on CD4 T-cell enumeration. It is important to mention that the absence of laboratory facilities should not be a reason to refrain from implementing HAART in a resource-poor setting. In principle, good clinical follow-up is sufficient, provided it is combined with an extra effort in guaranteeing compliance, for example through direct observed therapy (DOT), as applied in tuberculosis control programs. It should be emphasized that HAART does not cure the patient and constant alertness is required from both the patient (compliance) and the clinician (monitoring) to avoid sub-optimal use of the medication from resulting in potentially catastrophic, massive drug resistance in the developing world.
Importance of CD4 counts: CD4+ T lymphocytes are currently the most common surrogate marker indicating disease progression in individuals infected with human immunodeficiency virus (HIV). The

enumeration of CD4+ T cells from persons infected with the human immunodeficiency virus (HIV) is important. Traditionally this measurement has been calculated by multiplying the percent of lymphocytes that are CD4+ T cells (from flow cytometry) and the number of lymphocytes per microlitre of blood (from hematology measures).
Quantification of CD4 +ive T Cells: Quantification of CD4 T-cells is especially important to help clinicians in making a decision on the initiation of HAART. Although there is no consensus for application in the developing world, the start of HAART is usually recommended when CD4 T-cell counts are between 200-350 per microlitre of whole blood. The cut-off point for starting HAART is an important issue, not only because it has implications for the incidence of opportunistic infections, but also regarding costs. Preliminary data suggest that, as a rule of thumb, 50% of all HIV-infected patients qualify for HAART when 350 cells/^iL is taken as a cut-off, but only 25% qualifies with the lower cut-off (200/µL).
Flowcytometry: The gold standard for CD4 enumeration remains flow cytometry. The easiest instrument to use is a single-platform volumetric flow cytometer, like the B&D FACScount. However, this equipment is very expensive ($35,000) and the maintenance contracts ($6000/year) and reagents ($ 12/test, cold-chain dependent) are not affordable for most resource poor settings. There are other flow cytometers, like the B&D FACScan and FACScalibur, the Coulter XL and the Ortho Cytoron, which are even more expensive and either need an additional blood cell counter (dual platform technology) or very expensive beads for CD4 T-cell quantification.

Alternative flow cytometry-based CD4 T-cell enumeration methods: Alternative flow cytometry-based CD4 T-cell enumeration methods are all in various stages of development. First of all, there is the possibility of using simpler protocols on already existing flow cytometers. These protocols use innovative ways of gating (CD45 pan-leukogating, CD4-gating) and as a consequence need only one or two generic monoclonal antibodies, which are substantially less expensive than commercial kits. However, these protocols cannot be performed on a FACScount which excludes the far majority of those privileged labs in India that do have a flow cytometer. Currently, these pan-leukogating and CD4-gating technologies are under multi-center evaluation.
Low cost flowcytometers: Another alternative is the use of flow cytometers with low energy red-diode lasers. These machines are smaller, less expensive and need considerably less electrical power (a car battery of 12V is sufficient). A new instrument has been launched by Partec (the CyFlow), which is basically a half-price FACScount with half-price kits, but needs multi-center validation before it can be recommended. The alternative approach of undressing" the complex Luminex 100 to a simpler red diode laser-based cytometer has met little enthusiasm from industry, even though multiplex technology will be the future in laboratory diagnostics.
Flowcytometry assays: CDC and WHO developed guidelines for laboratories who perform immunophenotyping for detection and enumeration of CD4+ T-cells and other lymphocyte subsets in persons infected with human immunodeficiency virus (HIV) (6). The previous report MMWR 1997; 46 [No. RR-2]) described a dual-platform technology, a method in which absolute counts are derived from measurements obtained from two instruments—a flow cytometer and

hematology analyzer. The guidelines describe single-platform technology (SPT), a process in which absolute counts of lymphocyte subsets are measured from a single tube by a single instrument. SPT incorporates internal calibrator beads of known quantity in the analysis of specimens by three- or four-color flow cytometry. With CD45 gating, the relative numbers of beads and lymphocyte subsets are enumerated, and their absolute numbers and percentage values are calculated. Recent technologic advances have allowed for the development of a system for the determination of CD4+ T-lymphocyte counts by simultaneous 4-color flow7 cytometry. Kutok et al (7) evaluated a new 4-color 2-tube flow cytometric method for analyzing CD4+ T-lymphocyte subsets in whole blood was compared with a standard 2-color 5-tube method. The new method provides results almost identical to those of the well-established 2-color method used in our clinical laboratory. Statistical analyses indicate very low variability in CD4+ counts between the 2 methods, strongly supporting the usefulness of this new procedure. In addition, the 4-color procedure provides a 15% reduction in the materials cost per test compared with the 2-color method, as well as a marked reduction in the time expenditure of flow cytometry technologists. Sherman et al (8) examined absolute CD4 counts as recommended by the Centers for Disease Control (CDC) by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively and compared it to the CDC recommended 6 tube 2 colour panel [6(2)]. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision.

Advantages of flowcytometry: The advantages of using flow cytometry for CD4 T-cell enumeration are clear: the automated methodology is easier to use, more reproducible, statistically more reliable and features a higher throughput (up to 300 samples/day). Using pan-leukogating, blood samples of more than one week old can be analyzed and blood fixatives can be applied without problems significantly increasing the action radius of central laboratories. Moreover data can be stored on computers, reanalyzed and a good quality control system for flow cytometry is in place, including the NEQAS standards with 200, 400 and 800 CD4 cells/µL blood.
Newer technologies for CD4 counts Several manufacturers have developed new techniques for determining absolute numbers of CD4+ and CD8+ cells without the need for the flow cytometer and hematology analyzer (9).
ELISA based methods: Saah et al (10), Moss et al (11) and Cordiali et al (12) evaluated a recently developed enzyme-linked immunosorbent assay system for measuring helper T-lymphocyte count (the TRAx enzyme-linked immunosorbent assay) and compared it with flow cytometry and concluded that the TRAx enzyme-linked immunosorbent assay system is an acceptable method for measuring helper T-lymphocyte count, but should be recalibrated for better performance at helper T-cell counts below 200 per µL. However the TRAx system is no longer marketed. Lisse et al (13) tested an alternative method for CD4 and CD8 T lymphocytes enumeration, the immunoalkaline phosphatase method (IA), in three African countries and in Denmark. The authors found good correspondence between the two methods for measurements of CD4 and CD8 T lymphocytes independent

of serological status and geographical site. IA compared to flow cytometry consistently gives higher percentage of CD4 T lymphocytes, and lower percentage of CD8 T lymphocytes. The IA method is therefore suitable for use in areas with limited resources and laboratory facilities where there is a need for immunological surveillance in hospital or community studies.
Magnetic separation and ELISA based techniques: Carriere et al (14) evaluated the Whole Blood Capcellia(R) CD4/CD8, an immunoenzymatic method that provides absolute counts of CD4+ and CD8+ T cells in peripheral blood. The assay is based on the separation of T cells by use of an anti-CD2 magnetic bead suspension, followed by reaction of the CD4 or CD8 molecules with the corresponding monoclonal antibody coupled to peroxidase. CD4-positive monocytes were excluded from the assay. Freeze-dried magnetic bead-T-cell complexes were used as calibrators. Capcellia counts from H1V-1-infected patients were compared with those obtained by flow cytometry as the comparison method. The results by Capcellia correlated well with those by flowcytometric analysis and indicate that this new ELISA technique for lymphocyte immunophenotyping is an efficient alternative to flow cytometry. The kit was evaluated in India by Kannangai et al (15) who concluded that the assay system has certain limitations inherent to ELISA techniques.
Automated Volumetric capillary cytometry: Volumetric capillary cytometry (VCC) is a new technology that involves the detection and enumeration of dually fluorochrome-labeled cells in a precise volume. Gorman et al (16) compared the accuracy and precision of VCC with the accuracy and precision of flow cytometry and hematology (F&H) for the measurement of the absolute numbers of CD4

and CD8 T cells in the whole blood of patients infected with human immunodeficiency virus, and showed that it may represent a viable alternative to flow cytometry for obtaining absolute T-cell subset counts.
Fluorescence immuno assay: Denny et al (17) evaluated a new alternative fluorescence immunoassay method (Zymmune CD4/CD8 Cell Monitoring Kit; Zynaxis, Inc., Malvern, Pa.) for determining the absolute CD4+ and CD8+ T-lymphocyte concentrations in whole blood. The investigation was performed as a two-site comparison of the reference whole blood flow cytometric method with the Zymmune method, and concluded that the Zymmune CD4/CD8 Cell Monitoring Kit method provides absolute CD4+ and CD8+ T-lymphocyte concentrations which are equivalent to those given by the reference flow cytometric method.
Non-flow cytometry-based devices: Recent developments on non-flow cytometry-based devices, like the Easy Count (developed at Twente University) based on magnetic sorting and optical image analysis of CD4 T-cells. Major advantages are: compact stand-alone, volumetric device, short sample measurement time, good linearity, low power consumption (10W, can be run on a laptop battery) and potentially low costs. However, the instrument still has a long way to go: it is in its proof of concept phase and a second prototype is just being constructed
Comparable methods:
Bead based methods: Bead based methods available are of two types: The antibody coated latex bead assay (18) and the immunomagnetic bead (Dynal) (1, 19, and 20).

Antibody coated Bead based Technologies: The most affordable way of quantifying CD4 T-cells is by manual technologies, using synthetic beads and microscopy for read-out. Currently, the most efficient methods for low-volume applications are Dynabeads and Cytospheres. At least nine studies, several involving African populations, have correlated CD4 counts assessed by Cyto-Spheres or Dynabeads with counts measured by flow cytometry. In most (but not all) studies, the correlation has been excellent. Cyto-Spheres have been studied more thoroughly. Both assays involve the physical counting of cells, which can be done under a light microscope.
Dynabeads: Dynabeads cost approximately $3 per test and need an investment of $750 for a magnet and mixer. The original technology needs an additional expensive immuno-fluorescence microscope ($7500), (Costs available on Dynal Website) but there are ways to get around this, either by using alternative staining techniques that are compatible with light microscopy or purchasing a device, which converts a light microscope into a fluorescent microscope ($1800).
Cytospheres: Cytospheres are easier to use but more expensive ($9/test) (actual landed costs of a dollar worth of material in India after taxes and freight work to about Rs 85 for 1$), although investments are low: only a light microscope and a hemacytometer. Both Dynabeads and Cytospheres are widely used in laboratories in the developing world.
SUMMARY OF THE INVENTION
According to the present invention there is provided a process for preparing antibody labelled microspheres using Anti CD4 / Anti CDS

antibody for detecting HIV in the blood which comprises the steps of:
i) Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and adding 1 ml of carbonate bufferas herein described to fill tube; the microparticles form the substrate on which the antibodies are bound;
ii) Suspending the reactants of step (i) which comprises of the following steps:
a. Centrifuging the reactants for 5-6 minutes in a micro-centrifuge
for packing the microparticles;
b. Carefully removing supernatant using a Pasteur pipette which
helps to concentrate the microparticles for a wash procedure;
c. Discarding supernatant for removing the fluid in which the
microparticles were suspended;
iii) Repeating the above procedure of step (ii) for resuspending the pellet, the said resuspension is carried out by:
a. Filling tube halfway and capping;
b. Filling tube till vortex;
c. Filling tube to capacity;
iv) Resuspending pellet in phosphate buffer as herein described and
centrifuging for 5-6 minutes ensures completeness of wash
procedure; v) Carefully removing supernatant using a Pasteur pipette and
discarding supernatant ensures completeness of wash procedure; vi) Repeating step (ii) above two more times ensures completeness of
wash procedure and resuspends in phosphate buffer as herein
described;

vii) Resuspending pellet as herein described in a 0.625 ml of the 0.02M
sodium phosphate buffer, pH 4.5; viii) Adding drop wise 0.625 ml of the 2% carbodiimide solution for
activating the carboxylated groups on the microparticles; ix) Mixing 3-4 hours at room temperature ensures complete activation; x) Centrifuging for 5-6 minutes and removing and discarding
supernatant for removing the activator; xi) Resuspending pellet (as herein described) in phosphate buffer as
herein described for preparing the microparticles for binding with
antibody; xii) Centrifuging for 5-6 minutes and removing and discarding
supernatant; Prepares the microparticles for binding with antibody xiii) Repeating steps (xi) and (xii) two more times for getting rid of
unreacted carbodiimide for cleaning up the procedure; xiv) Resuspending pellet (as herein described) in 1.2ml of borate buffer
wherein the buffer acts to prepare the milieu interior of the reaction
mix for antibody binding to the activated microparticles; xv) Adding 200-400 g of Anti CD4 / Anti CD8 antibody for binding to
the activated microparticles; xvi) Mixing gently overnight at room temperature on an end-to-end mixer
wherein the microparticles bind completely with antibody and the
antibody is coated evenly over the microparticles; xvii) Adding 50(xl 0.25M ethanolamine(2-aminoethanol) and mixing
gently for 30 minutes for blocking unreacted sites on the
microparticles, the unreacted sites on the microparticles are blocked
to prevent nonspecific binding with any other amine bearing
chemical compound xviii) Centrifuging for 10 minutes and saving supernatant for protein
determination, wherein the centrifuging helps in concentrating the
preparation;

xix) Resuspending pellet (as herein described) in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex, wherein the resuspension helps in storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage;
xx) Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites, wherein the BSA reacts with any unblocked sites still open;
xxi) Centrifuging for 5-6 minutes and removing and discarding supernatant for concentrating the preparation;
xxii) Repeating steps xix and xxi once;
xxiii) Resuspending pellet (as herein described) in 0.5 ml PBS, pH 7.4 containing lOmg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer), wherein resuspension helps storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage. NaN3 is a preservative and prevents growth of contaminants;
xxiv) Storage at 4 °C enhances the useful life of the product
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Making antibody coated beads Material Required
i) Carboxyl-Modified Microspheres (often supplied at 10% solids)
ii) Water Soluble Carbodiimide, (EDAC: l-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide)
iii) Quenching Solution with ethanolamine
iv) Blocking molecule (Bovine serum albumin)
Preparation of reagents:

Prepare using distilled or deionized water unless otherwise indicated.
Carbonate Buffer: 0.1 M Carbonate buffer, pH 9.6-prepare by adding 0.02 M Sodium Carbonate (Na2C03) to 0.1 M NaHC03 until pH = 9.6.
Phosphate Buffer: 0.02 M Sodium phosphate buffer, pH 4.5-prepare by adding 0.02 M Sodium hydro phosphate (NaHP04 ) to 0.02 M Sodium dihydro Phosphate (NaH2P04 ) until pH = 4.5.
2% Carbodiimide: 2% l-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydochloride dissolved in phosphate buffer (see above). This has to be prepared within 15 minutes of using.
Borate Buffer: 0.2 M borate buffer, pH 8.5-use Boric acid to prepare this buffer. Adjust pH to 8.5 using 1 M Sodium Hydroxide (NaOH). 0.25 Methanolamine: Add 20µl of ethanolamine (2-aminoethanol) to 1.3ml of borate buffer.
Storage Buffer: First prepare 0.1 M phosphate buffer, pH 7.4, by adding 0.1 M Sodium dihydrophosphate (NaH2PO4) to 0.1 Sodium hydrophosphate (Na2HP04 ), until pH becomes 7.4. Take 20 ml of the 0.1 M phosphate buffer, pH 7.4, in a 100 ml graduated cylinder. Add 0.88g Sodium Chloride (NaCl), Ig bovine serum albumin (BSA), 5 ml glycerol, and O.lg NaN3, and make up the volume to 100 ml. Check the pH of the final solution. If necessary, adjust the pH to 7.4 by adding dilute Hydrochloric acid (HCl) or Sodium hydroxide (NaOH).
Making RBC lysis solution: To make RBC lysis solution take 2 ml of glacial acetic acid, add 98 ml of water and 25 mg of crystal violet. Shake. Store at 4°C.

Process for preparing antibody labelled microspheres
The process for preparing antibody labelled microspheres using anti CD4 antibody for detecting HIV in the blood, which comprises the steps of:
(i) Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and adding 1 ml of carbonate bufferas herein described to fill tube; the microparticles form the substrate on which the antibodies are bound;
(ii) Suspending the reactants of step (i) which comprises of the following steps:
(a) Centrifuging the reactants for 5-6 minutes in a micro-centrifuge for packing the microparticles;
(b) Carefully removing supernatant using a Pasteur pipette which helps to concentrate the microparticles for a wash procedure;
(c) Discarding supernatant for removing the fluid in which the microparticles were suspended;
(iii) Repeating the above procedure of step (ii) for resuspending the pellet, the said resuspension is carried out by:
(a) Filling tube halfway and capping;
(b) Filling tube till vortex;
(c) Filling tube to capacity;
(iv) Resuspending pellet in phosphate buffer as herein described and centrifuging for 5-6 minutes ensures completeness of wash procedure ; (v) (v Carefully removing supernatant using a Pasteur pipette and discarding supernatant ensures completeness of wash procedure;
(vi) Repeating step (ii) above two more times ensures completeness of wash procedure and resuspends in phosphate buffer as herein described;

(vii) Resuspending pellet as herein described in a 0.625 ml of the 0.02M
sodium phosphate buffer, pH 4.5;
(viii) Adding drop wise 0.625 ml of the 2% carbodiimide solution for
activating the carboxylated groups on the microparticles;
(ix) Mixing 3-4 hours at room temperature ensures complete activation;
(x) Centrifuging for 5-6 minutes and removing and discarding supernatant
for removing the activator;
(xi) Resuspending pellet (as herein described) in phosphate buffer as herein
described for preparing the microparticles for binding with antibody;
(xii) Centrifuging for 5-6 minutes and removing and discarding supernatant;
Prepares the microparticles for binding with antibody;
(xiii) Repeating steps xi and xii two more times for getting rid of unreacted
carbodiimide for cleaning up the procedure;
(xiv) Resuspending pellet (as herein described) in 1.2ml of borate buffer
wherein the buffer acts to prepare the milieu interior of the reaction mix for
antibody binding to the activated microparticles;
(xv) Adding 200-400 g of Anti CD4 / Anti CD8 antibody for binding to the
activated microparticles;
(xvi) Mixing gently overnight at room temperature on an end-to-end mixer
wherein the microparticles bind completely with antibody and the antibody is
coated evenly over the microparticles;
(xvii) Adding 50(µl 0.25M ethanolamine(2-aminoethanol) and mixing gently
for 30 minutes for blocking unreacted sites on the microparticles, the unreacted
sites on the microparticles are blocked to prevent nonspecific binding with any
other amine bearing chemical compound;
(xviii) Centrifuging for 10 minutes and saving supernatant for protein
determination, wherein the centrifuging helps in concentrating the preparation;
(xix) Resuspending pellet (as herein described) in 1 ml of 10 mg/ml BSA
solution in borate buffer; Cap and vortex, wherein the resuspension helps in
storage of the prepared beads and the BSA provides the stabilization and matrix
effect to stabilize the preparation and helps in long term storage;

(xx) Mixing gently for 30 minutes at room temperature for blocking any
remaining non-specific protein binding sites, wherein the BSA reacts with any
unblocked sites still open;
(xxi) Centrifuging for 5-6 minutes and removing and discarding supernatant
for concentrating the preparation;
(xxii) Repeating steps xix and xxi once;
(xxiii) Resuspending pellet (as herein described) in 0.5 ml PBS, pH 7.4
containing lOmg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer),
wherein resuspension helps storage of the prepared beads and the BSA
provides the stabilization and matrix effect to stabilize the preparation and
helps in long term storage. NaN3 is a preservative and prevents growth of
contaminants;
(xxiv) Storage at 4 °C enhances the useful life of the product
MANUAL COUNTING PROCEDURES AND CALCULATIONS FOR DILUTIONS
Requirements for manual counting:
Micropipette: 0-lOµl, 10-200µl Naeubers Chamber
Manual counting procedures:
For the assay Blood was collected in an EDTA bottle. I. 100 (µL of whole blood is taken in a clean test tube. II. 10 µL of antibody labelled microspheres as obtained in the above process is added.
III. The mixture was gently shaken for 2 minutes and then the beads allowed to adhere for a further 3 minutes.
IV. lOµL from step III (the blood-latex bead mixture) is added to a RBC lysis solution, shaken for 30 seconds, and pipetted onto both sides of a 0.1-mm-deep hemacytometer (18 mm ).

V. With a light microscope at 40X magnification, lymphocytes with two or more beads attached to their surface are counted in the two 9 mm , of the hemacytometer. VI. The number obtained is multiplied by 6.72 to obtain the absolute count of CD4/CD8 cells per cumm of blood (Derivation of dilution factor given below). VII. The test was carried out every week with both high and low standards to determine CD4/CD8 counts obtained with the standards.
Calculations for dilutions:
Area counted in haemocytometer: 18 mm2
Corrections for dilution:
Primary dilution: 100 µL of whole blood and 10 µL of reagent = 100/110
Secondary dilution: 10µL of whole blood of 100/110 dilution to 100 µL of
RBC lysis solution

10

100

110
110

— 0.0826

Hence correction for sample dilution is 1/0.0826=12.1
Correction for sample depth of .01 mm= 1/.01 = 10
Hence correction factor to be multiplied with so as to obtain total number of
CD4/CD8 + T eels in 1 cumm of blood is
(10xl2.1)/18
= 6.72
Quality control. It is recommended that
1. Kit be stored at 4°C
2. Every Monday the Kit be run against the Immunotrol reagents (both high and low) and results be counted

If the test results are within ± 5% of the value given for the standards the kit performance is considered satisfactory.

I Claim:
1. The process for preparing antibody labelled microspheres using anti CD4 antibody for detecting HIV in the blood, which comprises the steps of:
i) Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and adding 1 ml of carbonate bufferas herein described to fill tube; the microparticles form the substrate on which the antibodies are bound;
ii) Suspending the reactants of step (i) which comprises of the following steps:
a) Centrifuging the reactants for 5-6 minutes in a micro-centrifuge for packing the microparticles;
b) Carefully removing supernatant using a Pasteur pipette which helps to concentrate the microparticles for a wash procedure;
c) Discarding supernatant for removing the fluid in which the microparticles were suspended;
iii) Repeating the above procedure of step (ii) for resuspending the pellet, the said resuspension is carried out by:
a) Filling tube hallway and capping;
b) Filling tube till vortex;
c) Filling tube to capacity;
iv) Resuspending pellet in phosphate buffer as herein described and centrifuging for 5-6 minutes ensures completeness of wash procedure ;
v) Carefully removing supernatant using a Pasteur pipette and discarding supernatant ensures completeness of wash procedure;
vi) Repeating step (ii) above two more times ensures completeness of wash procedure and resuspends in phosphate buffer as herein described;

vii) Resuspending pellet as herein described in a 0.625 ml of the 0.02M sodium
phosphate buffer, pH 4.5; viii) Adding drop wise 0.625 ml of the 2% carbodiimide solution for activating
the carboxylated groups on the microparticles; ix) Mixing 3-4 hours at room temperature ensures complete activation; x) Centrifuging for 5-6 minutes and removing and discarding supernatant for
removing the activator; xi) Resuspending pellet (as herein described) in phosphate buffer as herein
described for preparing the microparticles for binding with antibody; xii) Centrifuging for 5-6 minutes and removing and discarding supernatant;
Prepares the microparticles for binding with antibody xiii) Repeating steps xi and xii two more times for getting rid of unreacted
carbodiimide for cleaning up the procedure; xiv) Resuspending pellet (as herein described) in 1.2ml of borate buffer wherein
the buffer acts to prepare the milieu interior of the reaction mix for antibody
binding to the activated microparticles; xv) Adding 200-400 g of Anti CD4 / Anti CDS antibody for binding to the
activated microparticles; xvi) Mixing gently overnight at room temperature on an end-to-end mixer
wherein the microparticles bind completely with antibody and the antibody
is coated evenly over the microparticles; xvii) Adding 50µl 0.25M ethanolamine(2-aminoethanol) and mixing gently for
30 minutes for blocking unreacted sites on the microparticles, the unreacted
sites on the microparticles are blocked to prevent nonspecific binding with
any other amine bearing chemical compound xviii) Centrifuging for 10 minutes and saving supernatant for protein
determination, wherein the centrifuging helps in concentrating the
preparation; xix) Resuspending pellet (as herein described) in 1 ml of 10 mg/ml BSA
solution in borate buffer; Cap and vortex, wherein the resuspension helps in

storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage;
xx) Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites, wherein the BSA reacts with any unblocked sites still open;
xxi) Centrifuging for 5-6 minutes and removing and discarding supernatant for concentrating the preparation;
xxii) Repeating steps xix and xxi once;
xxiii) Resuspending pellet (as herein described) in 0.5 ml PBS, pH 7.4 containing lOmg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer), wherein resuspension helps storage of the prepared beads and the BSA provides the stabilization and matrix effect to stabilize the preparation and helps in long term storage. NaN3 is a preservative and prevents growth of contaminants;
xxiv) Storage at 4 °C enhances the useful life of the product
2. The process as claimed in claim 1, wherein the carbonate buffer is prepared by adding 0.02 M Na2C03 to 0.1 M Sodium Hydrocarbonate (NaHC03) until pH = 9.6.
3. The process as claimed in claim 1, wherein the phosphate buffer is prepared by adding 0.02 M Sodium hydrophosphate (NaHP04) to 0.02 M Sodium dihydrophosphate (NaH2P04) until pH = 4.5
4. The process as claimed in claim 1, wherein the 2% carbodiimide is prepared by dissolving 2% l-(3-Dimethylaminopropyl)-3-ethyI carbodiimide hydochloride in said phosphate buffer, which has to be prepared within 15 minutes of using.
5. The process as claimed in claim 1, wherein borate buffer is prepared by using Boric acid and adjusting pH to 8.5 by using 1 M Sodium hydroxide (NaOH). 0.25 M Methanolamine and adding 20µ1 of ethanolamine (2-aminoethanol) to 1.3 ml of borate buffer.

6. The process as claimed in claim 1, wherein the storage buffer is prepared by adding 0.1 M Sodium dihydrophosphate (NaH2P04) to 0.1 Sodium hydrophosphate (Na2HP04), until pH becomes 7.4, taking 20 ml of the 0.1 M phosphate buffer, in a 100 ml graduated cylinder, adding 0.88g Sodium chloride (NaCl), Ig bovine serum albumin (BSA), 5 ml glycerol, and O.lg NaN3, and making up the volume to 100 ml checking the pH of the final solution and, adjusting the pH to 7.4 by adding dilute Hydrochloric acid (HO) or Sodium hydroxide (NaOH).
7. The process as claimed in claim 1, wherein the pH of carbonate buffer is maintained at pH 9.6.
8. The process as claimed in claim 1, wherein the pH of Phosphate buffer is maintained at pH 4.5.
9. The process as claimed in claim 1, wherein the pH of borate buffer is
maintained at pH 8.5.
10. The process as claimed in claim 1, wherein the pH of storage buffer is
maintained at pH 7.4.

Documents:

1354-che-2004-abstract.pdf

1354-che-2004-claims.pdf

1354-che-2004-correspondnece-others.pdf

1354-che-2004-correspondnece-po.pdf

1354-che-2004-description(complete).pdf

1354-che-2004-form 1.pdf

1354-che-2004-form 19.pdf

1354-che-2004-form 26.pdf

1354-che-2004-form 3.pdf

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Patent Number

219701

Indian Patent Application Number

1354/CHE/2004

PG Journal Number

27/2008

Publication Date

04-Jul-2008

Grant Date

13-May-2008

Date of Filing

13-Dec-2004

Name of Patentee

WG. CDR. PALAT KRISHNA MENON (RETD)

Applicant Address

Inventors:

#	Inventor's Name	Inventor's Address
1	WG. CDR. PALAT KRISHNA MENON (RETD)

PCT International Classification Number

B32B 5/16

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA