Form 2
THE PATENTS ACT, 1970
PROVISIONAL SPECIFICATION
[See Section 10]
‘Rapid visual Assay for distinct identification of HIV-1 & 2 antibodies
and HIV p24 antigen’
Span Diagnostics Limited, an Indian company of 173-B, New Industrial Estate, Udhna, Surat-394210, India
The following specification describes the nature of this invention:

Rapid visual Assay for distinct identification of HIV-1 & 2 antibodies and HIV p24 antigen
This invention relates to a rapid Flow Through test for simultaneously detection of HIV-1 and/or HIV-2 antibodies and/or HIV p24 antigen in the human blood, serum and plasma, based on Immunofilteration technology. This product will serve as an alternative to the expensive fourth generation assay i.e. either Fluorescent or ELISA based formats, having an additional advantage of being less time consuming than the other techniques.
BACKGROUND
Human immunodeficiency virus was identified in 1986, as the etiological agent of AIDS. HIV is icosahederal RNA containing virus with a cylindrical core containing two copies of single stranded genomic RNA. It belongs to the retrovirus group. HIV pandemic began towards the end of the twentieth century and now it is one of the major causes of immunodeficiency disease in India. It is therefore, important to make a precise and accurate diagnosis.(Textbook of Biochemistry,ch: 43; Biochemistry of AIDS)
The most widely used tests are ELISAs as they are the most appropriate for screening large numbers of specimens on a daily basis, e.g. blood donations. The earliest assays used purified HIV lysates (1st generation), and often lacked sensitivity and specificity. Improved assays based on recombinant proteins and/or synthetic peptides, which also enabled the production of combined HIV-l/HIV-2 assays became rapidly available (2nd generation). The so-called 3rd generation or sandwich ELISAs, which use labeled antigen as conjugate, are extremely sensitive and have reduced the window period considerably. To further reduce the window period, enhanced ELISA assays have been developed that detect both HIV antibody and antigen (4th generation assays).
Patent WO 93/21346 teaches about the simultaneous detection of HIV antigen and HIV-1/2 antibodies. This method is based on ELISA kind of technique, which utilizes polystyrene beads or polystyrene microtiter wells coated with HIV-1 and HIV-2 antigens and antibody to HIV p24 antigen.
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Markets are flooded with HIV detection test, available in different formats i.e ELISA (Enzyme Linked Immuno Sorbant Assay), Flourescent assay, Immunodot based assay, Immunofilteration Assay and Lateral Flow Assays. Such assays have high degree of sensitivity and specificity.
Despite having high sensitivity and specificity, it suffers from many disadvantages like:
• Require skilled person.
• Time consuming (long incubation period).
• Not user friendly.
• Laborious and costly.
• Require equipment.
Summary of the invention:
The object of the invention is to overcome the aforesaid disadvantages.
To achieve the said objective, this invention provides a method for simultaneous detection of HIV p24 antigen and antibodies against HIV 1 & HIV 2 and in Flow Through Assay format:
Ø Applying antibodies to HIV p24 antigen and different antigens of HIV-1 and HIV-2 at respective position on nitrocellulose membrane of Test Device. .
Ø Applying the patient's serum or plasma on to said membrane till it is soaked,
Ø Applying wash buffer as herein described, to remove non-specific or unwanted bindings to nitrocellulose membrane or recombinant antigens,
Ø Applying conjugated colloidal gold / signal reagent on said membrane,
Ø Repeat step 3 and observe the colour dot or spot near said test zones to confirm the detection of HIV.
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Detailed description of the Technology
The present study describes a rapid Flow Through test for simultaneously detection of HIV-1 and/or HIV-2 antibodies and/or HIV p24 antigen in the human blood, serum and plasma, based on Immunofilteration technology.
Test Apparatus or Flow through device or Immuno-filtration device for carrying out the method comprising closed assembly with upper part of housing and lower part of housing made preferably from polypropylene although one may use wide variety of organic or inorganic material, natural or artificial or combination of any polymers including but not limited to polypropylene, polyethylene, polystyrene, polymethacrylate, poly ethylene terephthalate, poly vinyl chloride, poly vinyl butyrate etc. The upper part of housing contains a inlet hole/ window for dropping serum/plasma samples and Wash buffer. Inside the assembly is the porous membrane e.g. nitrocellulose supported on the absorbent pad e.g. Cellulose. The porous membrane can be visualized through the inlet hole of the upper part of housing of the Test Apparatus.
Porous membranes for the Test Apparatus may be nitrocellulose membranes, nitrocellulose mixed esters, nylon membranes, polysulfonyl based membranes supported on suitable matrix such as polycarbonate filters, etc. Manufacturers of membranes include Schleicher & Schuell, Pall/Gelman, Sartorius, Whatman, MDI and Millipore. The said porous membrane have pore size of about 0.1 micron to 1.0 micron, preferably of 0.6 micron. The pore size has to be large enough so as to allow samples and reagents to flow through the membrane. Also, the pores should not be too large so that a good volume to surface ratio can be obtained. Additionally, the membrane material must allow the retaining of the antigen-antibody-Protein-A gold conjugate complex. The porous membranes can be cut into variety of shapes, including rectangular, circular, oval, square, and the like, of limited size, to fit the dimensions of the inlet hole/window of upper part of housing of the Test Apparatus so as to allow reagents and sample to pass through porous membrane only. On this membrane Test solution and control solution is spotted.
The residual area of the porous membrane can be saturated or blocked with blocking agents to prevent non-specific binding which typically include either single or combination of any of the three compounds: proteins, synthetic polymers, and surfactants. To prevent nonspecific binding by the
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use of bovine serum albumin, as described in Towbin et al. (1979) or use of man made polymers like polyvinylpyrrolidone and polyvinylalcohol described in Bartles et al. (1984). The other proteinaceous-blocking reagent may be Casein and like. The other proteinaceous-blocking reagent may be selolaurate, TRITON X-100™ (t-octylphenoxypolyethoxyethanol), sodium dodecylsulfate, n-octyl-D-glucopyranoside, NONIDET™ (octylphenel ethylene oxide), sodium dioxycholate etc. in concentration of 0.001 to 0.005 % (v/v). Blocking agents are applied in a buffer solution to the membrane, includes Tris(hydroxymethyl)aminomethane/HCl (Tris/HCl), Tris/citrate, Tris/maleate, Tris/glycine, phosphate buffer, HEPES, and other biological buffers in the correct pH range.
Absorbent pad for the Test Apparatus may be Cellulose, glass fiber or other cellulosic material like cardboard, filter paper or tissue paper etc. Manufacturers of membranes include Schleicher & Schuell, Pall/Gelman etc. The absorbant pad should have the sufficient absorption capacity so as to retain the absorbed liquid material i.e. reagents and sample that filter through or flow through the porous membrane. The absorbent pad can be cut into variety of shapes, including rectangular, circular, oval, square, trigonal, etc., of limited size, so as to fit inside the lower part of housing of the Test Apparatus.
For the test spot, mixture/ distinctly of recombinant antigens or synthetic peptides of HIV - 1 (gpl20 and gp41) & HIV- 2 (gp36) and antibodies to HIV p24 antigen in phosphate buffer, pH 7.2 spotted on to the said nitrocellulose membrane. Although one may use Tris(hydroxymethyl) aminomethane/HCl (Tris/HCl), carbonate buffer and other biological buffers in the correct pH range for spotting. One may use other recombinant .antigens of HIV.
The Control dot includes anti-human IgG in phosphate buffer, pH 7.2 spotted on to the nitro-cellulose membrane. Although one may use Tris(hydroxymethyl)aminomethane/HCl (Tris/HCl), carbonate buffer and other biological buffers in the correct pH range for spotting. One may use protein-A or protein-G.
Colloidal gold particles may be made by any conventional method, such as the methods outlined by G. Frens, (1973) and also described in US patent no. 4313734, 5578577, 5141850, 4775636, 4853335, 4859612, 5079172, 5202267, 5514602, 5616467, 5681775. Surek, et al. (1984) described the use
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of protein A labelled colloidal gold particles for the detection of specific antigens immobilised on nitro-cellulose membranes.
One may also use non-metal colloidal particles for coupling, the preparation is described in US patent no. 4954452, the procedure for coupling colloidal gold with proteins is described in US patent no. 4313734, 5656503, 6534320 and Romano et.al. (1974), Geoghegan, et al. (1980). Examples of substances include colloidal sulphur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal iron sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; or organic polymer latex particles (US patent no. 5753517) or polymerized dye particles (US patent no. 4166105,4452886).
One may also use HRPO coupled with anti-human IgG and(or) IgM and(or) IgA/ HRPO coupled with antigens and antibody of HIV as a detection system, but here substrate like AEC ( 2-amino 9-ethyl carbazole), etc. One may use ALP, P-galactosidase instead of HRPO, but substrate should be chosen accordingly.
One can also use colloidal particles coupled with Recombinant antigens or Synthetic antigens of HIV or cocktail of both or anti-human IgG/IgM/IgA and anti-HIV-p24 coupled with colloidal particles for detection purpose.
Stabilizers like BSA, gelatin, PEG (Carbowax), or casein are commonly used as described by Chandler et.al.(2000). The purpose of the stabilizer is twofold. First, it reduces non-specific interactions by blocking any sites on the colloidal surface that are not occupied by the specific protein. Second, it helps provide a more-stable suspension.
Preservative mainly includes Thiomerosal or Sodium Azide.
Invention will now be described with reference to the accompanying drawings:
Figures 1 & 2 shows the Test apparatus
Figures 1, 3 & 4 is used for interpretation of results.
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1. Materials
1.1 Test Apparatus (shown in figures 1 & 2 of the accompanying drawings)
Test Apparatus or Flow through device or Immuno-filtration device having dimensions 30 x 30 x 6.5mm is a closed assembly with upper part (UP) of housing and lower part (LP) of housing made preferably from polypropylene. The upper part (UP) of housing contains a inlet hole/ window (H) for dropping serum/plasma samples and Wash buffer. Inside the assembly is the porous membrane (M) e.g. nitrocellulose supported on the absorbent pad (AP). The nitrocellulose membrane can be visualized through the inlet hole (H) of the upper part (UP) of housing of the Test Apparatus.
The said nitrocellulose membrane (M) have pore size of about 0.6 micron.
In one of the embodiment the porous membrane of the Test Apparatus contains three regions, marked as "1", "2" and "3" on the top part of housing, Test dot of HIV - 1 & 2 antigens is immobilized near "1" and “2” regions and anti-HIV p24 antigen is spotted on the membrane marked as "3". Control Spot is coated on the centre of the nitrocellulose membrane of the Device, as shown in figure 1.
In another embodiment the porous membrane of the Test Apparatus contains three regions, marked as "1", "2" and "C" on the top part of housing. Test dot of mixture of HIV - 1 & 2 antigens is immobilized near "1" and anti-HIV p24 antigen is spotted on the membrane marked as "2". Control Spot is coated on the top of the nitrocellulose membrane of the Device marked as "C" as shown in figure 2.
Absorbent pad for the Test Apparatus is Cellulose, rectangular in shape, having dimension 24 x 24 x 1mm, so as to fit inside the lower part of housing of the Test Apparatus.
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The Test dot(s) includes mixture or distinct coating of antigens of HIV i.e. gpl20, gp41 and gp36 in phosphate buffer, pH 7.2 spotted on to the nitro-cellulose membrane. The typical concentration of antigens ranges from 20ng to 100ng per dot.
The Control dot includes anti-human IgG in phosphate buffer, pH 7.2 spotted on to the nitro-cellulose membrane.
1.2 Wash Buffer
Wash Buffer is the isotonic phosphate buffer with protein
stabilizers, detergent and preservatives. The typical composition of
Wash buffer is as follows:
Phophate buffer : 0.5 to 1.0 Molar concentration
Detergent(nonionic : 0.25 to 2.5 (v/v)%
Protein stabilizers : 6.5 to 7.5 (w/v)%
Sodium Chloride : 0.8 to 0.9 (w/v)%
Preservative : 0.01 to 0.1 (w/v)%
pH : 7.0 to 8.0
The concentration of Disodium Hydrogen phosphate and Sodium
Dihydrogen phosphate is taken in such a way that the final total
concentration of the buffer is achieved i.e. 0.5 to 1.0 Molar. One
can use Tris-HCl buffer or other such biological buffers of correct
pH range instead of phosphate buffer. Protein stabilizers used is
Bovine Serum Albumin. Detergents includes BRIJ™
(polyoxyethyleneether), TWEEN-20™ (polyoxyethylenesorbitan
monolaurate), TRITON X-100™ (t-
octylphenoxypolyethoxyethanol), sodium dodecylsulfate, n-octyl-D-glucopyranoside, NONIDET™ (octylphenel ethylene oxide), sodium dioxycholate etc, Tween-20 and Triton X-100 is very much preferable. One can also use Chaotrophic agents e.g. Urea, Potassium thiocyanate, sodium thiocyanate, ammonium thiocyanate etc. to decrease non-specific signal if any.
Preservative mainly includes Thiomerosal or Sodium Azide.
1.3 Signal Reagent
Signal Reagent is basically colloidal gold (size 15 to 25nm)
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coupled with Protein-A with stabilizers and preservative, having optical density range between 2.0 to 10.0 at 520nm.
2.0 Method
2.1 Spotting of Test dot and Control dot on Test Apparatus
2.1.1 Test Apparatus to be spotted are arranged on clean dry area. Then the Test dots and Control dot are spotted on the specified position of the Test Device. Spotting is done either by electronic pipette with fine micro tips or Automated dispensing machine.
2.1.2 After spotting the dot are scanned for its uniformity and individuality.
2.1.3 The scanned Test Apparatus are then kept at 37°C for 36 to 48 hours for drying.
2.1.4 After drying, the Test Apparatus is properly pouched in aluminum pouch. (Adding silica to pouch is optional)
2.1.5 After pouching the Test Apparatus are stored at 2 to 8°C.
2.2 Preparation of Wash Buffer
2.2.1 Take required quantity of Distilled water in the suitable clean container.
2.2.2 One by one all the ingredients are dissolved in the distilled water.
2.2.3 The required pH is adjusted by adding either dilute hydrochloric acid or sodium hydroxide.
2.2.4 Add distilled water to set the required volume.
2.2.5 Filter the final bulk with 0.22 micron filter membrane.
2.2.6 Store the filtered wash buffer at 2 to 8°C, until use.
2.3 Preparation of Colloidal gold conjugated with Protein A.
Colloidal gold particles may be made by any conventional method, such as the methods outlined by G. Frens,(1973) and also described in US patent no. 4313734, 5578577, 5141850, 4775636, 4853335, 4859612, 5079172,5202267, 5514602, 5616467, 5681775.
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3.0 Method of testing
3.1 Place the Test Apparatus on flat clean surface and label it with appropriate identification.
3.2 Add two drops of Wash buffer in the center of the spotted Test Apparatus and allow it soak in completely.
3.3 Add two drops of sample in the center of the spotted Test Apparatus and allow it soak in completely.
3.4 Repeat Step 3.2
3.5 Add two drops of signal reagent in the center of the spotted Test Apparatus and allow it soak in completely.
3.6 Add three drops of wash buffer in the center of the spotted Test Apparatus and allow it soak in completely.
3.7 Interpret the results.
4.0 Interpretation of results
4.1 With Reference to Fiogure-1, 3 and 4
4.1.1 If coloured spot appears in Test regions 1, 2 and 3, then the test is positive for HIV p24 antigen and antibodies to HIV-1 and HIV-2.
4.1.2 If coloured spot appears in Test regions 1 and 2, then the test is positive for antibodies to HIV-1 and HIV-2.
4.1.3 If coloured spot appears in Test region 3, then the test is positive for HIV p24 antigen.
4.1.4 If coloured spot appears in Test regions 1 and 3, then the test is positive for HIV p24 antigen and antibodies to HIV-1.
4.1.5 If coloured spot appears in Test regions 2 and 3, then the test is positive for antibodies to HIV-2.
4.1.6 If coloured spot appears in Test region 1, then the test is positive for antibodies to HIV-1.
4.1.7 If coloured spot appears in Test region 2, then the test is positive for antibodies to HIV-2.
4.1.8 If coloured spot appears only in Control region, then the test is negative for HIV p24 antigen and antibodies to HIV-1 and HIV-2.
4.2 With Reference to Figure-2
4.2.1 If coloured spot appears in Test regions land 2, then the
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test is positive for HIV p24 antigen and antibodies to HIV-1 and/or HIV-2.
4.2.2 If coloured spot appears in Test regions 1, then the test is positive for antibodies to HIV-1 and/or HIV-2.
4.2.3 If coloured spot appears in Test region 2, then the test is positive for HIV p24 antigen.
4.2.4 If coloured spot appears only in Control region, then the test is negative for HIV p24 antigen and antibodies to HIV-1 and HIV-2.
Note : Control dot will always appear irrespective of the status of the sample.
Dated this 27th day of March 2007

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