COMPLATE AFTER PROVISIONAL LEFT ON
FORM-2
THE PATENT ACT, 1970
COMPLETE SPECIFICATION
[See Section 10]
'Rapid comb test for the confirmation of HCV antibodies against
different individual antigens in the human blood, serum and plasma
based on immunodot technology'
Span Diagnostics Limited, an Indian Company of 173-B, New Industrial Estate, Udhna, Surat - 394210, India
The following specification particularly describes and ascertain the nature of this invention and the manner in which it is to be performed.


The subject invention is a rapid test in comb format for the detection of antibodies against individual antigens of HCV in Whole blood, serum and plasma.
BACKGROUND
The discovery of HCV genome was reported just a decade ago. HCV was first identified by molecular cloning of the virus genome in 1989(Cho et al.;Science 244:359-62; 1989).Today, applied biotechnology has strengthens the means to manage and prevent chronic HCV infectionand its complications. This accounts for about 15% actual viral hepatitis, 60-70% of chronic hepatitis and upto 50%) cirrhosis, end stage liver disease and liver cancer.(Schiff E.R. et. al., 1999)
HCV is a small (40-60 nanometer in diameter), enveloped, single stranded RNA virus of the family flaviviridae and genus hepacivirus. HCV genome is made up of 9600 nucleotides that codes various Structural and nonstructural proteins. To achieve 100% reliable results, different recombinant antigens have been selected from structural and Nonstructural regions such as Core, El, E2, NS3, NS4 and NS5 (Bradley D. W., et. al., 1993). Because the virus mutates rapidly, changes in the envelope protein may help to invade the immune system. There are at least six major genotypes and more than 50 subtypes of HCV (Lauer G. M., et.al., 2002).
The HCV test kits are available in the market for detection of HCV antibodies in the human serum/ whole blood/ plasma. They have the disadvantages of suboptimal sensitivity and specificity and abundance of false positive in low prevalance population.
In current practice, the Immuno Blot method involves a sequence of incubation and washing steps performed on membrane bearing sprayed antigen bands of various HCV genome regions. Incubation with sample and detection reagents may take several hours; each wash step may take 5 to 10 mins each. Also various ELISA tests are also available commercially.
Despite having high sensitivity and specificity, it suffers from many disadvantages like:
• Require skilled person
• Time consuming(long incubation period)
• Not user friendly.
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, • Laborious and costly
• Require equipments like shaker/rocker, ELISA reader etc.
The object of the invention is to overcome the aforesaid disadvantages.
To achieve the said objective the present invention provides an apparatus for rapid confirmatory testing of HCV in a comb format comprising:
a test comb with at least six teeth, four teeth coated with HCV antigens, and two teeth for positive and negative control, and
said teeth are coated with protein A solution for control.
The antigen is either recombinant or synthetic.
The present invention also provides a process for rapid confirmatory testing of HCV in a comb format comprising:
step 1- applying different antigens of HCV on respective teeth of the polystyrene comb,
step 2- placing coated comb into the well containing prediluted specimen (serum / plasma / blood) in sample buffer for 10 minutes,
step 3- washing the comb in the wash buffer to remove non-specific or unwanted binding to the spot on the comb teeth,
step 4- placing washed comb into the wells containing colloidal gold protein A solution or non colloidal particles / enzymatic system for 10 minutes,
step 5- repeat step 3 and observe the coloured dot or spot at the coated region to interpret the test results.
The entire process is carried out at room temperature.
The invention will now. be described with reference to the accompanying drawings.
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The said antigens of HCV are Core, NS3, NS4 and NSS at respective teeth (Figure 01) of the polystyrene comb.
Figure 2 shows the polystyrene comb with control zone and test zone coated with HCV recombinant antigens and Protein A solution control zone.
Figure 3 shows the microtitre plate with wells for testing.
Detailed Description
The present invention relates to the rapid HCV comb test for the detection of HCV antibodies against individual antigenic segments of HCV virus in Whole blood, serum and plasma in Comb format.
The diagnostic test kit of the subject invention comprises of Test comb coated with different antigens of HCV, sample diluent, a wash buffer solution and protein-A conjugated with colloidal gold solution.
Five different HCV recombinant antigens used for the detection of HCV antibodies are coated individually on five different teeth of comb.
The genome of HCV encodes for 3 structural proteins viz. Capsid protein, two envelope glycoproteins and several other non-structural (NS) proteins. To achieve 100% reliable results, different antigens from structural and nonstructural regions have been selected.
? 1 antigens from core region
? 1 antigen from NS3 region
? 1 antigen from NS4 region
? 1 antigen from NS5 region
? Negative control
? Positive control
The test procedure is quite simple and rapid i.e. the test can be completed within 30 mins. The test comb is incubated in the wells of microtiter 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 minutes and finally the comb is again washed with washing buffer. The appearance of coloured spot
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indicates that the test sample is positive, and the absence of any colored spot indicates the negativity of the test results.
The diagnostic kit of the subject invention comprises of a test comb, a sample diluent, a wash buffer solution and a protein-A conjugated with colloidal gold solution.
The subject invention relates to a rapid detection of HCV antibodies based on comb format in a very short time (within 20-30 mins) with better specificity and sensitivity. It also requires no instrumentation and no trained persons. It is also very economical.
Each tooth of the comb has a spot which comprises of respective different antigens and further more each arm is having a control spot. The function of control spot is to develop colour during the test , to confirm proper functioning of each arm & moreover it will monitor to check if the sample was added or not.
The test comb of a subject invention comprises a membrane having a plurality of coating of different recombinant antigens, thereon for the detection of HCV.
The said test comb is made up of polystyrene 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 polystyrene membrane has six teeth, each coated with different antigens of HCV and control spot.
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
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i-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 of HCV as a detection system, but here substrate like AEC ( 2-amino 9-ethyl carbazole), etc is used. 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 HCV or 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). 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 to provide a more-stable suspension.
The subject invention was based on use of HCV antigens representing the immunodominant regions of the HCV genome.
Detailed description of the Technology
The present study describes a rapid comb test for confirmation of HCV antibodies in the human blood, plasma and serum, based on immunodot technology.
Materials:-
1. Test combs:
The test comb of the subject invention as shown in figure 1 comprises of a membrane preferably polystyrene membrane having a plurality of coating of
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different recombinant antigens or synthetic peptides provided thereon for the detection of HCV antibodies.
The test comb of subject invention includes six test points, one on each tooth. There are four different antigens on four teeth and each tooth has the in-built quality control spot on the top of the each test spot. The function of the control spot is to develop the color during the test, to confirm proper functioning of each tooth of the comb, moreover it will monitor the addition of the sample.
2. Coating solution :
The composition of the said coating solution weight/volume is :
Disodium Hydrogen Phosphate : 10-100 millimolar
Sodium Dihydrogen Phosphate: 10-100 millimolar
Protein Stabilizer : 0.1-5%(w/v)
Preservative : 0.001-0.15%(w/v)
And the balance being distilled water
Although one may use Tris(hydroxymethyl)aminomethane/HCl (Tris/HCl),
carbonate buffer and other biological buffers in the correct pH range and of
optimum molarity for spotting.
3. Coating of Antigens:
The said test comb is coated with plurality of coatings of different recombinant and synthetic antigens individually on respective tooth of various region of HCV genome, such as Core, NS3, NS4 and NS5. The said antigens can be used in any of the quantity mentioned.
1. Core : 5-2000 nanogram
2. NS3 : 5-2000 nanogram
3. NS4 : 5-2000 nanogram
4. NS5 : 5-2000 nanogram
The antigens are mixed with a coating solution. The coating solution used is a buffer solution.
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In the process for the preparation of such mixture, the said antigens are mixed in coating solution by stirring in a cyclomixer for 30 seconds to 1 minute to get a homogenous solution.
2-10 ul of the said homogenous solution is coated on the respective tooth of the said test comb at following positions described below and in figure 2. For the appearance of the said control spot, the 2-10 microliter Protein A solution is coated on the said test comb membrane above the test spots on each tooth as per the attached drawing in fig.2.

Position Material to be coated
T1 HCV Core antigen
T2 HCVNS3 antigen
T3 HCV NS4 antigen
T4 HCV NS5 antigen
T5 Negative Control
T6 Positive Control
The spots are allowed to dry and then fixed with fixing solution which can be any of the organic solution preferably of group Alkanols i.e methanol or ethanol or propanol or butanol can also be used followed by drying. The combs are then subjected to blocking with blocking agent preferably Bovine Serum Albumin although Casein, Gelatin etc can also be used. After removing from blocking agent the antigen-coated combs are subjected for stabilizing with stabilizing agent preferably sucrose or mannitol or trehalose, followed by drying.
By accelerated temperature study it has been found that the stabilized comb is stable up to 5 years in tropicalised condition of temperature. However, best results are delivered if the test comb is stored at 2 to 8°C.

4. Sample diluent:
10-100 mM
1-2% (v/v)
0.6-1.5%(w/v) 0.5-2 (w/v) 0.001-0.1%(w/v)
The sample diluent buffer solution comprises:
Trizma base
Surfactant
Sodium Chloride
EDTA
Preservative
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In the process for the preparation of such buffer solution, Tris-base is mixed in the distilled water, to which non-ionic surfactant is added. Sodium Chloride is then added in the said solution followed by the addition of the preservative in the resultant solution. The pH of the said solution is adjusted between 7-8.5 by concentrated HCI..
The resultant solution is stirred to get the homogenous solution. The said homogenous solution is then filtered through a 0.22 micron filter to get the buffer solution of the subject invention to be used in the diagnostic kit for the detection of antibodies of HCV.
The subject solution is stable for 2 years at 2-8°C .
5. Wash Buffer:

The wash buffer solution comprises:
Phosphate buffer
Surfactant
Protein Stabilizer
Sodium Chloride
Preservative

10-lOOmM
1-2% (v/v)
5-1.5%(w/v) 0.6-1.5%(w/v) 0.001-0.15(w/v

In the process for the preparation of such buffer solution, Disodium Hydrogen Phosphate and Sodium Dihydrogen Phosphate is mixed in the distilled water, to get a solution. To this solution is added protein stabilizer and non-ionic surfactant. Sodium Chloride is then added in the said solution followed by the addition of the preservative in the resultant solution. The pH of the said solution is adjusted between 7-8.5 by NaOH or concentrated HC1.
The resultant solution is stirred to get the homogenous solution. The said homogenous solution is then filtered through 0.22 micron filter to get the buffer solution of the subject invention to be used in the diagnostic kit for the detection of antibodies to HCV antigens.
The subject solution is stable for 2 years at 2-8°C .
6. Preparation of the protein-A colloidal gold solution:-
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.
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4313734, 5578577, 5141850, 4775636, 4853335, 4859612, 5079172, 5202267, 5514602,5616467,5681775.
7. Protocol for the testing
The test comb is placed in microtest wells containing sample diluent buffer solution and to it test sample, positive and negative control is added into the respective wells and incubated at room temperature for 5-15 minutes, to allow the binding of the antibodies present in the blood, plasma or serum sample with the immobilized individual antigens coated on each arm of the comb. After incubation the comb is removed from the sample containing wells and immersed into the wash buffer solution, to remove unwanted antibodies therefrom and placed in microtest wells containing 200 microlitres of protein-A colloidal gold conjugate and incubated for 5-15 minutes, to allow the binding of protein-A colloidal gold conjugate with the Fc portion of the bound HCV antibodies to give distinct color at the test region. After incubation the combs are again washed with washing buffer to remove unbound protein-A colloidal gold conjugate and to see the color development at the designated spots. The positive control should show the colour dot and negative control should not show colour dot, irrespective of the test sample status, which indicates the validity of the procedure.
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We claim:
1. A device for rapid confirmatory testing of HCV in a comb format
comprising:
a test comb with at least six teeth, four teeth coated with HCV antigens, and two teeth for positive and negative control, and
said teeth are coated with protein A solution for control.
2. A device as claimed in claim 1 wherein the antigen is either recombinant or synthetic.
3. A process for rapid confirmatory testing of HCV in a comb format comprising:
step 1- applying different antigens of HCV on respective teeth of the polystyrene comb,
step 2- placing coated comb into the well containing prediluted specimen (serum / plasma / blood) in sample buffer for 10 minutes,
step 3- washing the comb in the wash buffer to remove non-specific or unwanted binding to the spot on the comb teeth,
step 4- placing washed comb into the wells containing colloidal gold protein A solution or non colloidal particles / enzymatic system for 10 minutes,
step 5- repeat step 3 and observe the coloured dot or spot at the coated region to interpret the test results.
4. A process as claimed in claim 3 wherein the entire process is carried out at room temperature.
5. A device for rapid confirmatory testing of HCV in a comb format substantially as herein described with reference to and as illustrated in the accompanying drawings.
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6 A process for rapid confimiatory testing of HCV in a comb format substantially as herein described with reference to and as illustrated in the accompanying drawings.
Dated this 28th day of February, 2006

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ABSTRACT
The present invention provides a device for rapid confirmatory testing of HCV in a comb format comprising:
a test comb with at least six teeth, four teeth coated with HCV antigens, and two teeth for positive and negative control, and
said teeth are coated with protein A solution for control.
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- 1 MAR 2006