FORM - 2
THE PATENT ACT, 1970 (39 Of 1970)
COMPLETE SPECIFICATION [SECTION 10, RULE 13]
"A Rapid One-Step Device for Detection of Typhoid"
Span Diagnostics Ltd. an Indian Company of 1?3-B, New Industrial Estate, Udhna, Surat - 394210, India
The following specification particularly describes the invention and the manner in which it is to be performed:

TECHNICAL FIELD
The present invention provides cost effective rapid detection of antibodies of infectious diseases in the human serum anc| plasma, based on immunochromatography technology. More particularly present invention relates to the detection of antibodies of Salmonella typhi antigens (j#e Lipopolysaccharide and Flagellin).
BACKGROUND
Typhoid fever caused by various strains of Salmonelia enterica serotype typhi poses serious problem in many developing countries.
Typhoid is sometimes life threatening illness caused by a bacterium Salmonella typhi. It is common in developing countries where it affects about 12.5 million people annually. The infection is acquired typically by ingestion.
In clinical laboratory the diagnosis of typhoid consist*; OF either isolation of bacilli or demonstration of antibodies from patient sample. THEisolation of bacilli is time-consuming and antibody detection is not very specific, Currently in clinical laboratory, widal test is used for diagnosis of typhoid. It is based ON the detection of antibodies.
In current practice, widal test involves long duration a,nd cumbersome procedures for detection of antibodies against Salmonella species. jnis test has long incubation period and takes time from 12 to 16 hours.
Despite being economical, widal test suffers from the fallowing disadvantages: o Time consuming (involves long incubation period); o Not user friendly; o Laborious;
o Requires incubator, waterbath; o Does not detect the early infection.

° Less specific: Salmonella typhi shares many antigens with other salmonella serotypes and cross-reacting epitopes with other Enterobacteriaceae.
o The cut-off point of titer is not universal as said cut-off points have to be determined locally.
Thus there is a need to develop rapid, single step lateral flow device for early detection of antibodies against typhoid antigen in the human serum and plasma for effective diagnosis.
Accordingly, to overcome the problem encountered in the prior art, the inventors of the present invention disclose as described herein below.
OBJECT AND SUMMARY
The principal object of the present invention is to provide a rapid one-step device for detection of typhoid bacilli in a sample.
Another object of the present invention is to provide a rapid one-step device for detection of typhoid bacilli in a sample which is simple, user friendly and does not require incubator or water bath.
Still another object of the present invention is to provide a rapid one-step device for detection of typhoid bacilli in a sample which can be used in an immunochromatographic assay.
Yet another object of the present invention is to provide a rapid one-step device for detection of typhoid bacilli in a sample which is cost effective.
The present invention relates to a rapid one-step device for detection of typhoid bacilli in a sample. The device is made up of the following:
a porous membrane having pore size in the range of 5-20 microns,
laminated on a solid support;

at least one test band on said membrane comprising native antigens, the concentration of said antigens ranging from 0.01-5mg/ml; a control band on said membrane comprising detector reagent; an absorbent and conjugate pad overlapped on the upper and lower side of said membrane respectively;
BRIEF DESCRIPTION OF DRAWINGS
Fig 1 depicts the top view of the test strip with test region T and Control region C according to the present invention.
Fig 1-A depicts the top view of the test strip with test region Tl & T2 with Control region C according to the present invention.
Fig 2 depicts the side view of the test strip according to the present invention
DETAILED DESCRIPTION
The present invention provides a device for detection of antibodies against Typhoid antigen in the human serum and plasma, which is rapid and reliable as to the conventional tests. The device can also be used for detection of antibodies against typhoid in blood and other samples as well.
The present invention relates to the rapid detection of antibodies against typhoid causing Salmonella species in human plasma and serum. The detection of typhoid antibodies in the human serum and plasma is carried out by a rapid Lateral Flow based on Immunochromatographic technology.
The present invention relates to a rapid one-step device for detection of typhoid bacilli in a sample. The device is made up of the following:
a porous membrane having pore size in the range of 5-20 microns,
laminated on a solid support;

at least one test band on said membrane comprising native antigens, the concentration of said antigens ranging from 0.01-5mg/ml; a control band on said membrane comprising detector reagent; and an absorbent and conjugate pad overlapped on the upper and lower side of said membrane respectively.
Test Strip or Lateral flow Dipstick or Immunochromatographic dipstick for carrying out the test comprises a nitrocellulose membrane laminated on a solid support made up of HIPS (high intensity polystyrene). Although one may use wide varieties of organic or inorganic material, natural or artificial or combination of polymers such as polypropylene, polyethylene, polystyrene, polymethacrylate, polyethylene terephthalate, polyvinyl chloride, polyvinyl butyrate etc. An absorbent pad is applied on the upper side on the polymer, which is slightly overlapped on to nitrocellulose membrane while a conjugate pad made of polyester is overlapped on to the other side of nitrocellulose membrane. The Absorbent pad acts as a sink pad.
Porous membranes for the Test Strip are selected from nitrocellulose membranes, nitrocellulose mixed esters, nylon membranes, polysulfonyl based membranes supported on suitable matrix such as polycarbonate filters, and the like. The porous membrane has pore size of about 5 micron to 20 micron, preferably of 10 micron. Additionally, the membrane material must allow the retaining of the antigen-antibody-gold conjugate complex. Onto this membrane, test solution of Salmonella Lipopolysaccharide antigen or Salmonella flagellin antigen or cocktail of both antigens is sprayed on region (T) or both antigen can be sprayed separately on regions Tl & T2. Control solution is sprayed on region (C). The respective solutions are sprayed as lines/bands.
The residual area of the porous membrane i.e. nitrocellulose can be saturated or blocked with blocking agents to prevent non-specific binding which typically include proteins, synthetic polymers, and surfactants or combination thereof. The other proteinaceous-blocking reagent may be casein and like. The proteinaceous-blocking reagent may also include selolaurate, TRITON X-100 (t-

octylphenoxypolyethoxyethanol, sodium dodecylsulfate, n-octyl-D-glucopyranoside, NONIDET (octylphenel ethylene oxide, sodium dioxycholate etc. in concentration of 0.01 to 1.0 % (v/v). Blocking agents are applied in a buffer solution to the membrane and include Tris(hydroxymethyl)aminomethane/ HCI (Tris/HCI), Tris/citrate, Tris/maleate, Tris/glycine, phosphate buffer, HEPES, and other biological buffers in the correct pH range.
Absorbent pad for the test strip is composed of cellulose, glass fiber or other cellulosic material like cardboard, filter paper or tissue paper. The absorbent pad should have sufficient absorption capacity so as to retain the absorbed liquid material i.e. reagents and sample through the porous membrane.
To achieve reliable results, native antigens (Flagellin and Lipopolysaccharide) extracted from Salmonella have been utilized. For the test line, native antigens (Flagellin and Lipopolysaccharide) extracted from Salmonella in Phosphate buffer of molarity in range of 5mM to 50mM and pH 6.0 to 8.0 are sprayed on to said nitrocellulose membrane. Although one may use tris(hydroxymethyl) aminomethane/HCI (Tris/HCI), carbonate buffer and other biological buffers also in the correct pH range.
The control line includes anti-human IgM, IgG or combination thereof in Phosphate buffer of molarity in range of 5mM to 50mM and pH 6.0 to 8.0 sprayed on to the nitro-cellulose membrane. Tris(hydroxymethyl) aminomethane / HCI (Tris/HCI), carbonate buffer and other biological buffers in the correct pH range can also be used for spraying. One may use Anti-human IgM/ IgG or Protein G or Protein A or Protein L or human IgM/IgG.
The conventional method for colloidal gold particles can be used. Also known non-metal colloidal particles can be used for coupling of protein as described in US 4954452. The procedure for coupling colloidal gold with proteins is described in US patent numbers 4313734, 5656503, 6534320 as well as in Romano et.al. (1974) and 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).
As a conjugate one can also use colloidal particles coupled with anti-human IgM for detection purpose. Since, as per immune response of any infection, IgM is first antibody to appear, hence detection of IgM will provide the means of early detection of Typhoid. In typhoid epidemic regions, the patient might not even produce IgM and directly switch over to the production of IgG against typhoid bacilli, hence as a conjugate one can also use colloidal particles coupled with anti-human IgG for detection of IgG antibodies against Salmonella which will also improve performance of assay for diagnosis of typhoid fever.
Conjugate pad are critical to lateral flow device. They are made of either polyester or glass fiber. They are inherently hydrophobic and require prior treatment with surfactants like Triton X-100, Tween-20 etc in appropriate buffer. The treated conjugate pad is dried and on it colloidal gold coupled with appropriate detector reagent is sprayed in specific concentration for optimum results.
Anti-human IgG and anti-human IgM are antibodies produced in goat which can be used as a detector reagent for detection of human IgG and human IgM against typhoid bacilli. Both these antibodies can be used independently or mixed for detection of antibodies.
Arrow tape is diagnostic adhesive system made of acrylic pressure sensitive adhesive and is a non-migratory, insert adhesive on the thin plastic film of different colour shades with arrow sign. This serves as the indicator as to how one should place lateral flow strip in the sample container.

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. Firstly, it reduces non-specific interactions by blocking any sites on the colloidal surface that are not occupied by the specific protein. Secondly, it helps provide a more-stable suspension.
Preservative mainly includes Thiomerosal or Sodium Azide. The subject test is stable for 2 years at room temperature.
The invention will now be described with the help of the accompanying drawings and examples.
Test Strip
Test Strip or Lateral Flow strip or Immuno-chromatography strip having Test Strip or Lateral flow Dipstick or Immunochromatographic dipstick for carrying out the method comprising Nitrocellulose membrane (3) is applied on a solid support made up of HIPS (high intensity polystyrene)(l). On bottom side of the membrane, absorbent pad (2) is applied which is slightly overlapped on nitrocellulose membrane (3) and to the other side of nitrocellulose membrane, conjugate pad (4) is applied which is slightly overlapped on nitrocellulose membrane. The Absorbant pad (2) will act as a sink pad. The adhesive arrow tape is applied over the conjugate pad.
The said nitrocellulose membrane has pore size of about 5 micron to 20 micron, preferably of 10 micron.
In one of the embodiment the porous membrane of the test strip contains two regions, marked as "C" and "T" on the top part of housing, (either of Salmonella LPS antigen or Salmonella flagellin antigen or cocktail of both antigens) is immobilized near "V region. Control line is coated near "C" region of the nitrocellulose membrane, as shown in figure 1. Appearance of test line (T) with Control line (C) indicates that the sample is REACTIVE. Appearance of only Control line (C) without test line

indicates that sample is NON REACTIVE. Non appearance of Control line (C) indicates that the test is invalid.
In another the porous membrane of the Test Strip contains three regions, marked as "C" "Tl" and "T2" on the top part of housing. Test line of Salmonella LPS antigen is immobilized near "T2" region and Salmonella Flagellin antigen is immobilized near "Tl" region. Control line is marked nearT" region of the nitrocellulose membrane, as shown in figure 1-A. Appearance of one Test line(Tl or T2) with Control lines (C) indicates that the sample is reactive. Appearance of only Control line (C) without test lines indicates that sample is non reactive. Non appearance of Control line (C) indicates that the test is invalid.
Salmonella native antigen in Phosphate buffer, pH 6 to 8.0 is sprayed onto the test line (T) on the membrane. The typical concentration of antigens ranges from O.Olmg/mL to 5mg/mL Further, the Strip has single coating of the antigens.
The Control line (C) includes Protein-A or anti-human IgM or anti-human IgG or combination thereof in Phosphate buffer of molarity in range of 5mM to 50mM and pH 6.0 to 8.0 sprayed on to the nitro-cellulose membrane.
The appearance of coloured lines [test line(s) and control line] indicates that the test sample is positive, and the presence of only control line indicates the negativity of the test results.
EXAMPLES
1. Assembly and preparation of strip
A strip (2.5 cm wide by 30 cm length) of nitrocellulose membrane (HF 75 or HF 120 or HF 180 from Millipore Inc.) was hand laminated onto a transparent backed lateral flow card. The conjugate pad, was each placed directly on bottom of the nitrocellulose chromatographic membrane, and absorbent pad was placed directly on the top of the membrane, all in a configuration as shown in FIG. 1 The assembled card is subjected

to be cut in strips with Guillotine cutting machine (Kinematics Inc) and placed in aluminum pouch with desiccant to avoid damage of strip because of humidity.
2. In this example, a device of the type shown in figure 1, 1-A & 2 "is used in which a sample flows preferentially through porous membrane chromatographically. An immunoassay device is illustrated for detection of the antibody against typhoid bacilli in serum/plasma sample. Native antigen containing Lipopolysaccharide and flagellin in optimized concentration was used for the immobilized reagent as a test line by dispenser and anti-human IgG/ IgM is sprayed as control line. Anti-human IgG/IgM colloidal gold conjugate was used as a labeled reagent and sprayed over conjugate pad. The Anti-human IgG/ IgM colloidal gold conjugate comprises a gold sol prepared in accordance with the procedure of Frens, Nature. 24:20-22 (1973).
The antigens are native antigen containing Lipopolysaccharide prepared in-house as per Westphal method and flagellin prepared in-house as per Ibrahim method. A porous membrane comprising nitrocellulose laminated in plastic surface is used. The antigen is applied to the porous membrane at a concentration of 0.5 mg/mL Anti-human IgG/IgM is sprayed as control line in concentration 0.25mg/ml. Then the membrane is subjected to drying at 37°C for 4 hours.
Conjugate pad are sprayed with colloidal gold coupled with anti-human IgG and Anti-human IgM using dispensor machine. Both the conjugates were mixed in equal proportion of optical density 10.0. Anti-human IgG and Anti-human IgM (supplied by GE life sciences) were coupled with colloidal gold in accordance with the procedure of Roth J., Histochem J. 14:791-801 (1982). The conjugate pad is dried by vacuum drying and stored in humidity free container until use.
The sprayed cards are assembled with absorbent pad and conjugate pad. The adhesive arrow tape is applied over the conjugate pad and the assembled card is subjected to cutting via Guillitone cutting machine in test strips of size 3.8 to 4.0 mm.
A lOul serum/plasma sample is dispensed onto test tube containing normal saline. The sample solution is soaked by conjugate pad to the porous membrane, through capillary action.

In this example, confirmed 179 serum/plasma specimens were tested in which 25 typhoid reactive samples and 154 typhoid non-reactive samples. Results correlated 100% with the clinical findings from the specimen source.
Although the disclosure of the device has been described in connection with the embodiment of the present disclosure illustrated in the accompanying drawings and examples, it is not limited thereto. It will be apparent to those skilled in the art that various substitutions, modifications and changes may be made thereto without departing from the scope and spirit of the disclosure.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims ave entitled. AccordiglY, the claims are not limited by the disclosure.

We Claim,
1. A rapid one-step device for detection of typhoid bacilli in a sample comprising:
a. a porous membrane having pore size in the range of 5-20 microns,
laminated on a solid support;
b. at least one test band on said membrane comprising native antigens, the
concentration of said antigens ranging from 0.01-5mg/ml;
c. a control band on said membrane comprising detector reagent; and
d. an absorbent and conjugate pad overlapped on the upper and lower side
of said membrane respectively.
2. The device as claimed in claim 1, wherein said sample is human serum or plasma.
3. The device as claimed in claim 1, wherein said membrane has a pore size of 10 microns.
4. The device as claimed in claim 1, wherein said membrane is selected from the group comprising of nitrocellulose, nitrocellulose mixed esters, nylon and polysulfonyl membranes.
5. The device as claimed in claim 1, wherein said support is selected from the group comprising of polystyrene, high intensity polystyrene, polypropylene, polyethylene, polymethacrylate, polyethylene terephthalate, polyvinyl chloride and polyvinyl butyrate.
6. The device as claimed in claim 1, wherein said native antigens are extracted from Salmonella and dispensed in a buffer at a pH of in the range of 6-8 and molarity of said buffer ranges from 5mM-50mM.
7. The device as claimed in claim 1, wherein each of said test bands comprises different antigens.

8. The device as claimed in claim 1, wherein said native antigens are selected from the group comprising of flagellin, lipopolysaccharide or combination thereof.
9. The device as claimed in claim 1, wherein said buffer is selected from the group comprising of phosphate, tris(hydroxymethyl)aminomethane/HCI and carbonate buffers.
10.The device as claimed in claim 1, wherein said detector reagent is dispensed in a buffer at a pH in the range of 6-8 and molarity of said buffer ranges from 5mM-50mM.
11.The device as claimed in claim 1, wherein said detector reagent is selected from the group comprising of anti-human IgM, anti-human IgG or combination thereof.
12.The device as claimed in claim 1, wherein said absorbent pad is selected from the group comprising of cellulose, glass fiber, cardboard, filter paper and tissue paper.
13.The device as claimed in claim 1, wherein said conjugate pad is treated with surfactant
14.The device as claimed in claim 13, wherein said conjugate pad comprises of colloidal particles coupled with anti-human IgM, IgG or combination thereof.
15.The device as claimed in claim 14, wherein said conjugate pad optionally comprises stabilizers and colloidal particles coupled with anti-human IgM, IgG or combination thereof

16.The device as claimed in claims 14 and 15, wherein said colloidal particles are selected from the group comprising of gold, sulphur, selenium, barium sulphate, iron sulphate, metal iodate, silver halide, silica, metal hydrous oxide, metal sulphide, metal selenide, metal phosphate, metal ferrite.
17.The device as claimed in claim 1, for use in an immunochromatographic assay for the detection of typhoid bacilli in human serum and plasma, said assay comprising a rapid one step dipping of said device in a test sample and observing colour change of said test band for the presence of typhoid bacilli.
18. A rapid one-step device for detection of typhoid bacilli in the human serum and plasma substantially as herein described with reference to foregoing examples and accompanying drawings.