FORM-2
THE PATENT ACT, 1970
PROVISIONAL SPECIFICATION
[See Section 10]


A Rapid Comb Test for Confirmation of HIV-1 and/or HIV-2 Antibodies in the Human Blood, Serum and Plasma

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:


This invention relates to a rapid comb test for confirmation of HIV-1 and/or HIV-2 antibodies in the human blood, serum and plasma, based on immunodot technology.
Brief description of prior art
Human immunodeficiency virus was identified in 1986, as the etiological agent of AIDS.HTV 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 HIV test kits are available in the market for the detection of HIV antibodies in the human serum and plasma, but they occasionally can give a false positive results. For this reason all positive tests require confirmation by a more specific test that uses a procedure such as western blot test.
In current practice, the western blot method involves a sequence of incubation and washing steps performed on membrane bearing electrophoretically resolved antigen bands. Incubation with sample and detection reagents may take several hours; each wash step may take 5 to 10 mins each. (US Patents, patent no. 6,303,389, Lewin, et al.) Western blot is considered as the gold standard for anti-HIV testing and used as a confirmatory test for HTV infection.
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 like shaker/rocker.
The object of the invention is to overcome the aforesaid disadvantages and provide a cheaper confirmatory test of HIV-1 and/or HTV-2.
To achieve the said objective the present invention provides a diagnostic kit for rapid testing of confirmation of HIV-1 and HIV-2 antibodies in the human blood, serum and plasma comprising of a test comb, a sample

diluent, a wash buffer solution and protein-A colloidal gold conjugate wherein
a test comb having at least 8 teeth coated with different recombinant antigens and synthetic peptides, as herein described, on the whole testing zone at the bottom of said teeth,
protein A solution coated on to control zone above said test zone,
said coated comb is placed into the well of microtiter plate containing prediluted specimen in sample diluent,
the comb is washed with wash buffer to remove non-specific or unwanted binding to the control and test zone on the comb teeth,
the washed comb is again placed into the well of the microtiter plate containing colloidal gold protein A solution till coloured dot or spot appears at the coated region to interpret the test results.
Again the comb is washed with wash buffer to remove non-specific binding of colloidal gold protein-A to the control and test zone on the comb teeth.
The said antigens are HTV-l p-17 and p-24 of gag region, p-32 and p-66 of pol region, gp-120 and gp-41 of env region and HIV-2 gp-36.
The said antigens used for coating of comb are 5-500 nanograms.
The said test comb is made of polystyrene. To stabilize the comb coated with the antigens need additional steps viz. Fixing, Blocking and Stabilizing of antigen coated comb.
The fixing solution used in the coating procedure of different recombinant antigens or synthetic peptides of HTV for test comb is from the alkanol group.
The alkanol group contains methanol or ethanol or butanol.
3

After fixing of antigen coated comb the next step is to block the antigens coated comb by blocking solution. The blocking solution used in coating of different recombinant antigens or synthetic peptides of HTV for test comb is Bovine Serum Albumin or Casein or gelatin in optimal concentration.
After fixing and blocking of antigens coated comb the last step is to stabilize the antigens coated comb by stabilizing solution. The stabilizing solution used in the coating of different recombinant antigens or synthetic peptides of HTV for test comb are sugar such as sucrose or mannitol or trehalose.
The said comb is stable upto five years in tropicalized condition of temperature.
The said comb is stored at 2 to 8°C.
The present invention also provides a method for rapid testing of confirmation of HIV-1 and HIV-2 antibodies in the human blood, serum and plasma comprising the steps of:
applying different recombinant antigens and synthetic peptides, as herein described, on the whole testing zone at the bottom of teeth of polystyrene comb,
coating protein A solution on the control zone above said test zone,
placing said coated comb into the well of microtitere plate containing pre-diluted specimen in sample diluent for 5-15 minutes
washing the comb in the wash buffer, to remove non-specific or unwanted binding to the control zone on the Comb teeth,
placing washed comb into the well of microtiter plate containing Colloidal gold protein-A solution for 5-15 minutes,
repeating step 4 onward and observing the coloured dot or spot at the coated region to interpret the test results.

The invention will now be described with reference to the accompanying drawings:
Figure 1 shows a polystyrene comb having 8 teeth, according to this invention.
Figure 2 shows the polystyrene comb with the test zone coated with HTV-1 and HIV-2 recombinant antigens and synthetic peptides and protein-A solution on the control zone for the detection of HTV antibodies.
Figure 3 shows the microliter plate with wells in which test to be performed.
Detailed description:
A rapid comb test for the confirmation of HIV-1 and/or HTV-2 antibodies in the human blood, plasma and serum is based on immunodot technology. The immunogenic HTV proteins are coded by 3 genes namely gag, pol and env. To achieve 100% reliable results, two different antigens from each of the following regions are taken into account. (Val turner, June 1993.)
FromHIV-1
p17 and p24 for gag region p32 and p66 for pol region gpl20 and gp41 for env region
From HIV-2 gp36
The diagnostic kit of the invention comprises a test comb, a microtiter plate, a sample diluent, a wash buffer solution and a protein-A conjugated with colloidal gold solution. Eight different HTV-1 and HTV-2 recombinant antigens and synthetic peptides for the detection of HTV antibodies and the protein-A solution for sample addition monitoring, are coated and fixed with fixing solution of methanol or ethanol or butanol and are blocked with blocking solution of bovine serum albumin and there after combs are stabilized with stabilizing solution of sugar, sucrose or mannitol or trehalose, individually on eight different teeth of test comb.
Method of testing:
The test comb is incubated in the wells of microtitre plate containing test
sample e.g. Human blood, serum or plasma for 5-15 minutes. Then the test

comb is washed with washing buffer followed by incubation in colloidal gold protein-A conjugate for 5-15 mins and finally the comb is again washed with washing buffer. The positivity of the test is interpreted on the basis of the appearance of certain combinations of coloured spots, according to the criteria given in the table 1, and the absence of any colored spot indicates the negativity of the test results.
Below is the table for interpretation for confirmation of HTV-1 and 2 by claimed method: TABLE 1

HIV Type Immunogenic Regions Antigens Criteria 1 Criteria 2
HlV-1 GAG pl7 Anyone Any two dots from three different immunogenic regions

p24

POL p32 Anyone

p66

ENV gpl20 Anyone

gp41

HIV2 gp-36 One dot One dot
The said test comb is made up of polystyrene sheet material, but plastic sheet laminated with nylon 6-6 membrane, Biodyne A membrane, Biodyne B membrane, Biodyne plus membrane, Biodyne C membrane, Immunodyne ABC membrane, Loprodyne LP Nylon 6,6 membrane, Nitrocellulose membrane, PVDF membranes, predator membranes, ultrabind membranes etc can also be used. The said test comb has 8 teeth (1-8). The said teeth has control zone and the test zone .
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 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 protein-A/ HRPO coupled with anti-human IgG/ HRPO coupled with recombinant antigens or synthetic peptides of HIV-1 & 2 as a detection system, but here substrate like AEC ( 2-amino 9-ethyl carbazole), etc is used. One may use ALP, |3-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-l/2or cocktail of both or anti-human IgG instead of Protein-A for detection purpose.
Stabilizers like BSA, gelatin, PEG (Carbowax), or casein are commonly used as described by Chandler et.al.(2000) for stabilization of colloidal gold conjugate. 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. The subject solution is stable for 2 years at 2-8°C.
The subject invention is based on use of HlV-1 and FflV-2 antigens representing the immunodominant regions of HIV-1 and HIV-2 genome.
Dated this 2nd day of M arch 2005

of Anand And Anand, Advocates
Agents for the Applicants