FORM 2
THE PATENTS ACT 1970
39 of 1970
&
The Patent Rules 2003
PROVISIONAL SPECIFICATION
(See sections 10 & rule 13)
TITLE OF THE INVENTION
RAPID DETECTION OF GRAM NEGETIVE BACTEREMIA USING NOVEL NANO BIO-PROBE

APPLICANTS (S)
NAME NATIONALITY ADDRESS
VENUS REMEDIES LTD.

IMTECH
PANJAB UNIVERSITY Indian
Indian
Indian 51-52  Industrial Area Phase-1  Panchkula  Haryana  India 134113
Sector-39-A  Chandigarh-160036

Chandigarh-160.014
PREAMBLE TO THE DESCRIPTION

PROVISIONAL

The following specification describes the invention

RAPID DETECTION OF GRAM NEGETIVE BACTEREMIA USING NOVEL NANO BIO-PROBE

FIELD OF THE INVENTION

[0001] The embodiments of the invention relate to detection of gram negative bacteremia using a novel nano bio-probe. More specifically  the embodiments of the invention relate to a dot blot immuno-probe  based on the nano bio-probe  for rapid detection of pathogen/immunogen of gram negative bacteremia including as S. typhi. The dot blot based immuno-probe makes use of the nano bio-probe that employ one or more antibody to detect presence of the pathogen/immunogen causing such bacteremia that may be present in clinical sample. The antibody is prepared based on the target pathogen/immunogen present in an analyte such as Vi polysaccharide of Salmonella Typhi and the nano bio-probe is obtained by conjugating the Vi antibody and a Gold Nano Particle. The nano bio-probe is used as reagent to rapidly measure presence of antigen/immunogen in clinical isolate using a dip-stick with minimum flase positive /flase negative results.

BACKGROUND OF THE RELATED ART
[0002] Typhoid fever caused by Salmonella enterica serovar Typhi  is a life threatening systemic infection and continues to be a major public health problem particularly in the developing countries (Parry et al.  2002). Detection of Salmonella is challenging as the infection can result into diverse clinical manifestations with symptoms that overlap with a wide spectrum of other diseases. Isolation of serovar Typhi from blood is the most reliable means of confirming an infection. However  this requires well established lab that are beyond the means of most primary health care facilities in the developing world. As a result  the diagnosis may be delayed or missed while other febrile illnesses are considered  and patients without typhoid fever may receive unnecessary and inappropriate antimicrobial therapy.
[0003] From clinical point of view  presently the blood culture is considered to be the gold standard for diagnosis of typhoid fever. However results obtained through blood culture are frequently jeopardized owing to the fact that individual often start taking antimicrobials even prior to samplingof disgnosis. Perhaps  it is observed that blood cultures are negative in 30-65% of cases with typhoid fever because of prior administration of antibiotics or low number of organisms. Accordingly  negative blood culture reports in patients with typhoid fever underestimates the actual incidence of the disease.
[0004] Further detection of somatic (O) and flagellar (H) agglutinins  as employed by Widal test  carries many shortcomings as the sensitivity  specificity and prediction values differ in different geographical areas. Hence  Widal test has been found to be non specific and difficult to interpret in areas where typhoid fever is endemic. For example  Levine et al have demonstrated (O) antibodies to be elevated in 90% of typhoid patients in Mexico but found (O) and (H) antibodies elevated in 30 and 75%  respectively  of healthy Peruvians. The Widal test (Widal’s agglutination reaction) is routinely practiced for the serodiagnosis of typhoid fever by most of the laboratories. Several workers have expressed doubt regarding the reliability of the test. Several factors have contributed to this uncertainty. In areas of the world where typhoid fever is endemic  the test has been found to be sensitive but lacking in specificity.
[0005] Even though several newer rapid methods are being developed for detection of Salmonellae  their utility as routine diagnostic test remained largly controversial. As no non-cultural test for typhoid fever has been consistently shown to be sufficiently sensitive as well as specific  most of the laboratories still rely upon blood culture Widal test.
[0006] Therefore  it observed that diagnosis of typhoid fever on clinical grounds is difficult  as the presenting symptoms are diverse and similar to those observed with other common febrile illness  such as malaria and non severe dengue fever.
[0007] For effective management of typhoid  diagnosis of the disease must be done with speed and accuracy.
[0008] The Widal test is time consuming and often times when diagnosis is reached it is too late to start an antibiotic regimen.
[0009] The Widal test should be interpreted in the light of baseline titers in a healthy local population. This is especially important when there is a high local prevalence of non-typhoid salmonellosis. The Widal test may be falsely positive in patients who have had previous vaccination or infection with raised S typhi. count.Therefore  widal test is based on poorly standardised antigens  the sharing of antigenic determinants with other Salmonellae and the effects of immunization with TAB vaccine.
[00010] Another major problem relates to the difficulty of interpreting Widal test results in areas where Salmonella typhi is endemic and where the antibody titres of the normal population are often not known. Classically  a four-fold rise of antibody in paired sera Widal test is considered diagnostic of typhoid fever. However  paired sera are often difficult to obtain and specific chemotherapy has to be instituted on the basis of a single Widal test.
[00011] Widal titers have also been reported in association with the dysgammaglobulinaemia of chronic active hepatitis and other autoimmune diseases.64 ""8 ""9 False negative results may be associated with early treatment  with "hidden organisms" in bone and joints  and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic.
[00012] Another limitation of the widal test is that it gives indirect evidence of typhoid infection.
[00013] Furthermore  in areas where fever due to infectious causes is a common occurrence the possibility exists that false positive reactions may occur as a result of non-typhoid. The Widal test is time consuming and often times when diagnosis is reached it is too late to start an antibiotic regimen.
[00014] Other serological test including slide agglutination or even polymerase chain reaction (PCR) based technique though can provide highly sensitive results for both qualitative and quantitative analysis  they are quite hard- and time consuming to perform.
[00015] It is evident that typhoid fever affects individuals from different geographical areas  ages and socio economical levels. Thus there is in consequence a great need for new  highly sensitive and specific  rapid  and easy to perform diagnostic tests.

OBJECT OF THE INVENTION

[00016] Therefore  there is pressing need in the society to develop a fast  reliable and easy to perform diagnostic test with a higher sensitivity and high specificity for a target pathogen/immunogen including S. typhi  present in any target analyte.
[00017] Accordingly  it is an object of the one of the embodiment of the present invention to develop a novel nano bio-probe for rapid detection of gram negative bacteremia.
[00018] It is an objective of the one of the embodiment of the present invention to develop a nano bio-probe for S.typhi specific analyte for the detection of typhoid.
[00019] It is another object of the one of the embodiment of the present invention to develop a dot blot immuno-probe (DBIP) based on the nano bio-probe for rapid detection of gram negative bacteremia and a method of determination thereof which is highly specific and highly sensitive to specific analyte.
[00020] Another objective of the one of the embodiment of the present invention is to determine threshold/cut-off titra for reducing false positive or false negative results with respect to presence of disease causing pathogen/immunogen including but not limited to S.typhi.
[00021] A further object of one of the embodiment of the present invention is to detection of carrier of S. typhi .

BRIEF DESCRIPTION
The embodiments described herein after would be better understood in terms of their characteristics and mode of operation from the following detailed description with reference to the figures depicting various results wherein:

[00022] FIG.1 illustrates a schematic diagram of a dot blot immuno-probe based immuno-assay technique for detection of gram negative bacteremia such as Vi antigen of Salmonella typhi using raised Vi antibody.
[00023] FIG.2 through FIG.3 iluustrat a study to determine level of expresion of pathogen of gram negative bacteremia such as Vi antigen by bacterial cell  in different NaCl concentrations and effect of pH using ELISA.
[00024] FIG.4 illustrates detection of extracted Vi antigen titre by ELISA using purified Vi antibody and commercially available Vi antiserum.
[00025] FIG. 5A through 5B illustrates a study on detection of Vi capsular polysaccharide antigen of S .typhi using anti-Vi IgG polyclonal antibody by ELISA technique and a Hyper-expressed standard strain of S. typhi Ty2 obatined from different clinical sampels.
[00026] FIG 6A through 6D illustrate a study of absorbance spectra of gold nanoparticles the (GNPS) in colloidal form and the anitbody gold nanoparticles (Ab-GNPs) conjugate and an insets show 1% agarose gel electrophoresis of GNPs and Ab-GNPs conjugate  DLS data of GNPs and GNPs-antibody conjugate  results of TEM image of old nanoparticles the (GNPS) in colloidal form and results of TEM image of Ab-GNPs conjugate.
[00027] FIG. 7 illustrates a study report on salt flocculation for stabilization of the Ab-GNPs conjugate.

DETAIL DESCRIPTION

[00028] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying figures & tables and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly  the examples should not be construed as limiting the scope of the embodiments herein and more details along with different embodiments for working this invention would be produced through a complete specification.
[00029] Further detailed embodiments  examples and experiments in the detailed description would be evident from the Complete Specification of this provisional specification which would be filed within 12 months of this filing.
[00030] It is observed that S. typhi belongs to the serotype 9  12  d  Vi  defined by the repeated sugar units (9  12) of the O antigen  that together with lipid A constitutes the lipopolysaccharide (LPS) of the outer membrane; by the H antigen (d) constituted by the flagellar protein or flagellin  and by the Vi antigen or K capsular polysaccharide (Calva  E. et al.  1988  Research opportunities in typhoid fever: Epidemiology and Molecular Biology. BioEssays 9: 173-177).
[00031] It is further observed that all strains of Salmonella typhi and S. paratyphi C  as well as a few atypical but genetically related Citrobacter and Salmonella strains  are capable of synthesizing a capsular antigen termed Vi for virulence. This galactosamine uronic acid polymer (i.e. the Vi antigen) has been associated with the virulence of S. typhi. Two separate chromosomal loci necessary for Vi antigen expression  viaA and viaB  have been identified in genetic studies of S. typhi. The viaB region appears to encode the structural genes for this antigen. Analogous and presumably allelic chromosomal sites have been identified in S. paratyphi C and in some strains of Citrobacter freundii. Although the expression of the Vi antigen is relatively stable in S. typhi  Vi-positive Citrobacter strains exhibit a rapid  reversible transition between forms that express the Vi antigen and forms that appear not to express it  referred to as non-Vi or W forms. Therefore   it is found that Vi capsular antigen appears to be essential for the intracellular survival of the bacterial host and  hence  it is an important virulence property of the bacterial cell specifically S. typhi and detection of Vi capsular antigen would decisively represent a present infection or disease condition.
[00032] Accordingly  in one aspect of this invention  a novel nano bio-probe for rapid detection of gram negative bacteremia is provided. The nano bio-probe includes one or more antibody component wherein the antibodies are designed to have high specificity to a target analyte; and one or more nano particles wherein the antibody moiety and the nano particle are conjugated to form the nano bio-probe.
[00033] For the purposes of the present invention the term "nano bio-probe" may be understood to encompass any probe designed or developed in nano scale.
[00034] In one embodiment  the analyte of interest can include Vi-antigen of S. typhi or similar immunuogen. Additionally  the analyte of interest may be any pathogen/immunogen of other gram negative bacteria.
[00035] In another embodiment  the nano particle of interest is gold nano particles (( herein after referred to as GNPs) wherein size of the particle may be in the range of 25 nm to 50 nm  preferably 30nm± 5nm.
[00036] In another aspect  GNPs based dot blot immuno-probe (DBIP) for rapid detection of gram negative bacteremia is provided. The gram negative bacteria may be S. typhi causing typhoid fever and is detected using Vi capsular polysaccharide antigen of serovar typhi. The GNPs based dot blot immuno-probe assay technique employed herein is highly suitable for the diagnosis of typhoid fever and provide an early qualitative result. The interpretation of the results obtained in dot blot immuno-probe (DBIP) by immuno-assay is based on visual detection and it can be effectively used as a qualitative test for the on-site detection of gram negative bacteremia such as typhoid fever in an endemic area.
[00037] In another aspect  the dot blot immuno-probe (DBIP) immunoassay may not only be important for detection of gram negative bacteremia in the community but may also prove to be beneficial for tracing the carriers of the disease condition on the basis of concentration of disease causing immunogen such as Vi antigen.
[00038] Similar approach could also be applied for on-site testing of other infectious disease depending on the specific antigen antibody interaction according to an embodiment herein.
[00039] According to one embodiment of the invention  Vi capsular antigen was isolated from S. typhi through over expression of the Vi capsular antigen employing different growth conditions and media. A sample strains of clinical isolates of S. typhi were obtained from Post Graduate Institute of Medical Education and research (PGIMER) and Government Medical College  Chandigarh India. All strains were identified by the routine laboratory test in the Department of Microbiology  Punjab University Chandigarh India  employing standard procedures for biochemical identification and slide agglutination test for Salmonella isolates using polyclonal Vi antisera. The strains are grown on Luria Bertiani (L.B) agar media for 24 hours at 37o C and a single colony is used to prepare a seed culture. 0.1% inoculum from the seed culture is used for growing the bacteria under different environmental conditions such as osmolarity  pH  temperature and time in order to check the bacterial yield and expression of Vi capsular antigen. The bacterial yield (whole bacterial cell) and expression of Vi antigen was studied by employing ELISA through use of a Vi antiserum and/or micro titer plates known in the art for extracted Vi capsular antigen as described hereafter according to an embodiment herein. However  other equivalent or more sensitive techniques may also be employed for checking expression of antigen responsible for virulence.
[00040] FIG.1 illustrates a schematic diagram of a dot blot immuno-probe based immuno-assay technique developed herein for detection of gram negative bacteremia such as Vi antigen of Salmonella typhi using raised Vi antibody. The Vi antigen is represented as the immunogen of interest however  other antigens derived from different pathogenic gram negative strains can also be used depending upon the interest. The Vi antibody represents the specific molecule that bind to the target pathogen/immunogen to be detected.
[00041] According to one embodiment  the Vi capsular antigen expression were observed in a broad range of NaCl concentration (10-400 mM) and in different pH levels which are illustrated in FIG 2 through FIG.3. The maximum Vi antigen expression were achieved at 200 mM NaCl concentration. However  decreasing the salt concentration below 50 mM  and increasing above 300 mM lead to the reduction of Vi antigen expression. In contrast  S. typhi was able to grow and express maximum Vi antigen at 200±100 mM  more particularly 200mM NaCl concentration. Both S. typhi CVD906 and CVD908 strains showed a marked decline in Vi antigen synthesis at and above NaCl concentrations of 0.3 M and 0.4 M respectively. Although it could still be detected at low levels at these osmolarities. This suggests that Vi may be down regulated in the gut  facilitating interactions of S. typhi with epithelial cells  while it is up regulated in the blood  where it is known that Vi capsular antigen is important for survival of the organism at this stage of the infection process. The results obtained in this experiment confirm that at high osmolarities Vi capsular antigen biosynthesis was greatly reduced whereas under low osmolarity conditions Vi capsular polysaccharide was maximally produced.
[00042] It has also been shown that presence of high concentrations of glucose at the beginning of growth inhibited the production of Vi capsular antigen  particularly during the stationary phase.
[00043] At a concentration of 400 mM sodium phosphate the synthesis of Vi capsular antigen was strongly inhibited.
[00044] It was also observed S. typhi growth at broader pH range of about 5 to about 10 according to an embodiment herein. The highest bacterial biomass and maximum Vi capsular antigen expression was obtained at pH 7±1. Bacterial growth was not observed below pH 4 and above pH 11.
[00045] Expression of Vi antigen was also checked at different time intervals of bacterial growth  using different dilutions of Vi antisera. It was found that about 6 hours to 8 hours growth of bacteria was sufficient for optimal expression of Vi antigen according to an embodiment herein.
[00046] It was also found that synthesis of Vi capsular antigen requires functional TviB  TviC  TviD and TviE polypeptides. Biosynthesis of Vi capsular antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc  followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA which serves as the building block for the formation of the Vi polysaccharide polymer. Further it was analyzed that TviB activity remains over a range of pH from about 6.0 to about 9.6 and the pH optimum was found to be at 7.2. However  a large decrease in activity was observed near pH of about 8.4. These results emphasize the need to control pH during the fermentation at the optimal pH of 7.2.
[00047] Once the growth of S. typhi and expression of Vi capsular antigen is obtained satisfactorily  Vi capsular antigen is isolated form the whole cell of S. typhi. Accordingly  Vi capsular polysaccharide antigen from S. typhi can be any method known in the art such as isolation by the acetone killed extraction method according to an embodiment herein. In brief  S. typhi are grown in LB broth culture. The culture can be treated with an equal volume of acetone following an incubation at room temperature (RT) for 2 hours. The treated bacterial cells are suspended in 10 volumes (w/v) of saline  containing 0.1 % sodium azide. The bacterial cell suspension is incubated at 37o C for 30 min on shaker (200 rpm) following a centrifugation at about 15000 rpm for 30 min and supernatant is collected. The supernatant is added with 1 N tris-chloride buffer pH of about 7.5  to obtain a final concentration of 0.05 N. The sample is then digested with deoxyribonuclease (50 µg/100 ml) and proteinase K (0.5 mg/100ml) at 37o C on shaker (150 rpm) for 6 h and 12 h respectively. After incubation  mixture is centrifuged at 15000 rpm for 30 min. Supernatant is discarded and the sediment is dissolved in saline  containing 0.1% CTAB (w/v). Mixture is centrifuged at 10000 rpm for 15 min and again supernatant is discarded and the sediment is dissolved in 1 M CaCl2  followed by filtration through 0.2 µm syringe filter. 4 volumes of cold ethanol are added and again the solution is centrifuged at 10000 rpm for 20 min. Sediment is dissolved in saline and solution is dialyzed extensively against distilled water. Dialyzed solution is concentrated and stored at -20o C for further analysis.
[00048] As mentioned herein before  the isolated Vi capsular antigen or the bacterial whole cell can be detected by ELISA employing a Vi antiserum and/or by micro-titer plate known in the art according to an embodiment herein.
[00049] In this respect Vi capsular antigen expression is observed in different environmental conditions such as osmolarity  pH  temperature and time using whole bacterial cell ELISA technique wherein ELISA plates are pre-coated with 100 µl Poly-L-lysine (0.1M) in phosphate buffer saline (PBS)  keeping pH at 7.4 which is then incubated for 1hour at room temperature. The plates are washed three times with 200 µl of PBS per well. 100 µl of different number of bacterial cells suspension (102 -107 cells /ml) in PBS is added to each well and incubated overnight at 4º C and then washed three times with PBS containing 0.05% Tween 20 (PBST). The antiserum against Vi antigen is diluted (1:100) in PBS-M (phosphate buffer containing 0.1% skimmed milk) and 100 µl is added in each well and the plates are incubated for 1 h at 37o C. After washing three times with 200 µl of PBS-T  secondary antibody (alkaline phosphatase conjugated goat anti-rabbit IgG) (Sigma) is diluted (1:10000) in PBS-M and 100 µl is added to each well and again the plates are incubated for 1 h at 37o C. The plate is washed again and subsequently 100 µl of p-nitrophenylphosphate is added to each well. The plate is incubated at 37o C for 20 min and absorbance is measured at 405 nm. Similarly  micro-titer plates (Nunc  Inter Med) are directly coated with the extracted Vi capsular antigen in 0.1 M Poly L-lysine at 4°C for overnight. The micro-titer plate is blocked with 10% skimmed milk at 37o C for 1 h. A primary antibody in the form of Vi antiserum at 0.01 to 10000   preferably 1:100 dilution is added to each well and the plate is incubated for 1±0.75 h at 30±7°C  preferably 37°C. The plate is washed with PBST and incubated with ALP-conjugated anti-rabbit IgG antibody (1:10000) for 1 h  and the rest of the steps are the same as described herein above for the whole bacterial cell ELISA according to an embodiment herein.
[00050] In another aspect of the invention  the isolated Vi capsular antigen  as described herein before  is used to raise antibody specific to Vi capsular antigen and antibody specific to S. typhi. Accordingly  two sets of young (4-6 months old) white New Zealand rabbits are bled before immunization and a pre-immune sera are collected. Vi capsular antigen of about 200 µg/ml and whole bacterial cell of S. typhi in an amount about 103 cells/ml is used with complete adjuvant in order to raise anti-sera. The rabbits are immunized subcutaneously with the whole bacterial cell or Vi capsular antigen as mentioned herein before. After 21 days  booster doses are given with incomplete adjuvant. 5 days after the first booster dose  animals are bled and blood samples are collected. Sera thus obtained are separated and kept at 40±20 °C  preferably 56°C in a water bath for 20 minutes to deactivate complement system. Serum is then diluted with saline (1:2) and is saturated with ammonium sulphate drop wise to a final concentration of 40± 15%  preferably 45%. The solution is kept on stirrer at 4o C for 1 hours followed by centrifugation at 7000 rpm for 20 min. The precipitates are then dissolved in 1/10th volume in phosphate buffer (PB  pH 7.4) and dialyzed extensively against PBS. The dialyzed antibody solution is used for IgG antibody purification. Protein-A column is used for IgG antibody purification. The antibody solution is loaded on Protein-A column  pre-equilibrated with PB (pH 7.4). The column is washed extensively with PB (pH 7.4) till O.D reached 0.01. Antibody IgG is eluted with 100mM glycine-HCL buffer (pH 2.5) and the collected fractions are immediately neutralized with 1M Tris-Cl (pH 8.0). Peak fractions are pooled and immediately dialyzed against PB followed by concentration in a vacuum concentrator. The raised antibody concentration is measured by taking absorbance at 250±100 nm  preferably 280 nm with extinction coefficient 1.5±1  preferably 1.35 for IgG.
[00051] In a subsequent aspects of the invention  a characterization of the raised antibodies is performed using Indirect Sandwich ELISA for detection of Vi capsular antigen in bacterial cell wherein ELISA plates are pre-coated with whole bacterial cell IgG antibody at 1: 10000 dilution in a phosphate buffer saline (PBS)/ well  pH 7.4 and is incubated for 1 hour at room temperature or overnight at 4º C. The plates are washed three times with 200µl of PBS per well and blocked the plate with 10 % skimmed milk   again the plate is incubated for 1 hour at 37ºC. 100µl of different number of bacterial cells suspension (102 to 107 cells/ml) in PBS is added to each well. The anti-Vi IgG antibody is diluted (0.01:10000  preferably 1:10000) in PBS-M (phosphate buffer saline containing 0.1% skimmed milk) and 100 µl is added in each well and the plates are incubated for 1 h at 37o C and the rest of the steps are the same as described herein before for the whole bacterial cell ELISA. In clinical strain the lowest detection limit using ELISA is found to be about 102 cell /ml however in standard strains in normal growth and optimized (200 mM NaCl pH 7 etc.) conditions detection limit is found to be about 104 and 103 cells /ml respectively as illustrated in FIG 4 A through FIG 4B.
[00052] Subsequently  the extracted Vi capsular antigen is detected employing direct ELISA method through the use of the raised anti-Vi IgG antibody. It this respect  micro titer plates (Nunc  Inter Med) are directly coated with the extracted Vi antigen and 0.1 M Poly L-lysine at 4°C for overnight. The micro- titer plate is blocked with 10% skimmed milk at 37o C for 1 h. A primary Vi IgG antibody at a dilution 0.01:10000  preferably 1:10000 is added to each well and the plate is incubated for 1 h at 37°C. The plate is washed with PBST and incubated with ALP-conjugated anti-rabbit IgG antibody (1:10000) for 1 h  and the rest of the steps are the same as those described above for the whole bacterial cell ELISA. It is determined and observed through ELISA that the extracted Vi capsular antigen when compared with anti-serum and purified Vi IgG antibody  that is raised herein before  has high sensitivity and high specificity. As a preferred embodiment 1: 10000 dilution of raised Vi IgG antibody and 1:100 dilution of commercial available Vi anti-serum are found to be suitable for detection of extracted Vi antigen from S. typhi as illustrated in FIG 5.
[00053] In another aspect of the invention  the nano particles are synthesized wherein aqueous gold nano particles are used according to an embodiment herein. Accordingly  gold nano particles (GNPs) are synthesized by any procedure known in the art. In the present invention  nano particle are prepared according to a procedure wherein GNPs of 30±20nm  preferably 30 nm size is obtained. A 100 ml solution of 0.01% tetrachloroauric acid in Milli-Q water is taken in an Elenmeyer flask. The solution is brought to boil on a hot plate and mixed rapidly with 2 ml of 1% trisodium citrate solution. The solution is allowed to boil further for 10 min. The reduction of the gold metal ions (Au+) to yield GNPs (Auo) is confirmed by the appearance of a dark red-wine colour according to an embodiment herein.
[00054] In a preferred aspect of the invention  the GNPs  synthesized herein before was  conjugated with the raised Vi IgG antibody to obtain a antibody-gold nano particles conjugates (referred as Ab-GNPs herein after). Accordingly the gold nano particles are used for preparing Ab-GNPs  50 µg of Vi IgG antibody is prepared in phosphate buffer (10 mM  pH 7.4). The Vi IgG antibody is added drop-wise to 1 ml colloidal gold nano particles solution under mild stirring conditions to yield a final antibody concentration 100±50 µg/ml  preferably 50 µg/ml. The pH of the GNPs solution is maintained at 7±1.5  preferably 7.4 by addition of 10 mM K2CO3 before adding the Vi IgG antibody. The mixture is incubated overnight at 4oC and centrifuged at 12000 rpm for 30 min to remove un conjugated antibody from the solution. The pellet is re suspended in phosphate buffer (10mM  pH 7.4) and stored at 4o C.
[00055] Further  in order to determine the minimum amount of Vi IgG antibody required to form a stable form of Ab-GNPs a salt flocculation assay is carried out to optimize antibody conjugation to GNPs. The minimum amount of Vi IgG antibody required to stabilize the GNPs is optimized by employing a flocculation assay and illustrated in FIG.7 . The aggregation of gold nano particles caused by NaCl could be characterized by the shift of maximum absorbance peak from 500±250nm  preferably 520 to 610 nm. The Vi IgG antibody concentration required to prevent aggregation can be determined by measuring the difference of the absorbance between 520 to 610 nm. In order to determine the critical flocculation (CFC) concentration of Ab-GNPs conjugate  increasing amount of NaCl (0 to 3.5 M) is added to Ab-GNPs conjugate and incubated for 1 hours at room temperature. The threshold NaCl concentration in the gold solution that caused the aggregation of the particles is determined as the CFC. For the purpose of the present invention  the experiments carried out showed that to obtain a minimum antibody amount for a stable gold nano particle  the pH value of about 7.5 may be chosen as this represent the median value of the acquired pH range. As a preferred embodiment the minimal antibody amount was determined to be 50 µg for every 1 ml of pH optimized GNPs. As a yet another preferred embodiment 30 nm sized was chosen because offer maximum surface area for protein binding and with highest visibility least steric hindrance. The synthesis of gold nanoparticles from HAuCl4 (tetrachloroauric acid) solution was confirmed by the absorption peak at 520 nm shown by the colloidal gold solution of dark wine-red colour. The intensity of the colour development generally indicates the size and quality of gold nanoparticles. It is also observed that concentration of sodium citrate used in its synthesis  dictates the size of GNPs. The corresponding absorbance spectra of colloidal gold solution and the GNPs conjugated with Ab-GNPs antibody  has been illustrated in FIG 6A. The conjugation of GNPs to antibody is confirmed by the spectrum analysis of the GNPs and the Ab-GNPs in the range of 400-800 nm and observed shift in the peak of about 10 nm from 521 nm to 530 nm . This observation is in compliance with the fact that nano particles can show shift in their absorption as a result of bio-conjugation due to the change in their overall structure  charge and spatial closeness of bio-molecules and nanoparticles .
[00056] Following the preparation of the Ab-GNPs conjugate  another characterization of the GNPs and the Ab-GNPs conjugates is carried out to determine the conjugation in order to study the morphology and the shape of the nano particles invloved herein. An absorbance spectra (300-700 nm) of the aqueous GNPs and the Ab-GNPs conjugate are recorded using a spectrophotometer. The average particle size of gold nano particles (GNPs) and Ab-GNPs conjugate are determined by using a transmission electron microscopy (TEM  Jeol  JEM-2100) operated at 120 kV. The TEM samples are prepared by any of the known methods  particularly by placing a drop of diluted GNPs on a carbon coated copper grid. The films on the TEM grids are allowed to dry for 5 min at room temperature before analysis. The gold –antibody complex is negatively stained with 2% PTA {sodium (Potassium) phospotungstate} stain for the visualization of protein coating on the GNPs. Further  the size of GNPs and Ab-GNPs complex are measured by a Zeta particle size analyzer. Accordingly  TEM images revealed that the GNPs are spherical in shape and uniformly distributed (monodispersity). The average GNPs"" size calculated using Dynamic Light Scattering (DLS) is found to be 50±20nm   preferably 28.9 ± 6.3 nm and poly dispersity index (P.I) 0.25±0.20  preferably 0.317 in colloidal gold solution. However in case of the Ab-GNPs  the average particles size is found to be about 100±30nm  preferably 86.2 ±23.6nm and the polydispersity index of about 0 .294. The findings clearly indicate the formation of Ab-GNPs which is illustrated in FIG 6 B.
[00057] According to one of the embodiment gel electrophoresis is carried out to confirm the conjugation of Vi antibody with carboxylated gold nanoparticles. The gel shift confirmed the binding of protein ( Vi IgG antibody) to the GNPs and formation of the Ab-GNPs.
[00058] In another preferred aspect  a dot blot based immune probe (DBIP) for detection an analyte that include one or more antigen one or more of gram negative bacteremia  is developed according to an embodiment herein. The detection may include detection of antigen such as Vi capsular antigen of S. typhi. Accordingly  the dot blot based immuno probe (DBIP) is developed by employing a gold-labelled antibody wherein the antibody has high specificity for a target analyte of one or more gram negative bacteria.
[00059] In another aspect  the antibody is anti-Vi IgG antibody for Vi antigen detection in clinical samples. PVDF membrane containing strips are immobilized with antibodies that includes IgG antibody against whole bacteria cell and purified Vi IgG antibody . It is worth mentioning that the Vi polyclonal antibody is used to detect only the Vi positive S .typhi strain and Vi positive strains bind to Vi IgG immobilized membrane strips. The strips are dried at room temperature for 10 min. The immobilized strips are blocked by 10% skimmed milk in 10 mM phosphate buffer (PB  pH 7.4) at 37º C for 1 hour followed by washing with 10 mM phosphate buffer. After this  the strips are incubated with bacterial cells (103 cells/ml) at 37º C for 1 hour  and again membrane strips are washed with 10 mM PB. Subsequently  Ab-GNPs conjugate is used for detection of Vi surface marker in S. typhi. Detection of Vi positive S. typhi strains is identified based on the colour development on the membrane containing strips. A schematic diagram of the GNPs based dot blot based immuno probe (DBIP) is illustrated in FIG. 1. In this respect  the detection of Vi antigen is based on sandwich and sandwich competitive inhibition approach  where the target bacterial cell bind to IgG antibodies against whole bacterial cell as well as Vi antigen respectively. The optimal concentrations of IgG antibodies against whole bacterial cell and anti-Vi for immobilization on strips are found to be 85 ng and 43 ng/ spot. GNPs- anti-Vi antibody of 1:5 dilutions from 50 µg /ml stocks are found optimum for developed immunoassay. The assay developed herein  could be used for detection of the Vi antigen by visual observation. Therefore  the colour intensity of the test spot on the membrane strips must be high enough to visualize.
[00060] It is one of the embodiment that  in sandwich dot blot approach  the Salmonella typhi cells (Vi expressing and non expressing cells) can bind to IgG antibody against whole bacterial cell  where the targeted Vi antigen is detected by their binding with the Ab-GNPs conjugate. By this method only Vi expressing bacterial cells is detected  and it is found to be possible to differentiate the Vi expressing and non expressing cells in sample. Subsequently  Ab-GNPs conjugate is used to detect S. enterica serovar typhi in a sandwich competitive inhibition format to evaluate the performance of the assay in comparison with the direct sandwich dot blot. The Vi antibody is first immobilized on nitrocellulose comb blot membrane  followed by the addition of S. typhi and GNPs-Vi antibody. In both developed immuno blot format  Salmonella typhimurium is used as negative control because Vi antigen is not present in S. typhimurium.
[00061] Furthermore  the data obtained from this study suggest that the DBIP developed herein  is sensitive and specific for identification for S. typhi and easier in diagnosis of typhoid fever. The DBIP can potentially be used in diagnose typhoid fever  by antibody - antigen detection using patients sample or identification of whole organism from primary plate culture.
[00062] The developed technique based on GNPs is also suitable for the detection of carriers and easily used in clinical laboratory to differentiate typhoid and non-typhoid patients. In other words  the invention proposed herein above may be used to determine a current infection as well as a past infection based on a critical ratio of “O” antigen and “H” antigen   present in the analyte of interest along with the detection of any pathogen/immunogen including the Vi capsular antigen.
[00063] It is further mentioned that similar approach could also be applied for on-site testing of other infectious disease depending on the specific antigen antibody interaction.
[00064] Above disclosure describe a manner and method of making using the invention and sets forth the best mode contemplated by the inventor for carrying out his invention but is not to be construed as limiting. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments  it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed  various modifications and equivalents of the described modes for carrying out the invention that are obvious to those skilled in formulation development or related fields are intended to be within the scope of the invention. The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can  by applying current knowledge  readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept  and  therefore  such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore  while the embodiments herein have been described in terms of preferred embodiments  those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the description given herein above.