Development of a Novel Rapid Immunodiagnostic Kit Based on Flagellar 40 kDa Antigen Epitope for the Detection of Typhoid Fever in Indian Patients

To aid the clinical diagnosis of typhoid fever in India, where most hospitals and primary health centres have no facilities for culture, we report on the development of a novel and rapid immunodiagnostic kit for the direct detection of Salmonella Typhi—specific IgG antibodies against S. Typhi flagellar H antigen. The disease often does not show a specific clinical picture, and can be confused with other febrile illness such as malaria, dengue fever and Staphylococcus aureus. To overcome the problem of cross reactivity specific epitope of the flagellar H antigen was immobilised on the testing kit strip eliminating chances of cross reactivity and false positive results thereby increasing the specificity of the test. Since the immunodiagnostic kit, uses the flagellar H antigen from bacteria present in our country, the antibodies present in the serum of patients of our country will have maximum binding affinity, enhancing the sensitivity of our test kit. The immunodiagnostic kit on analysis gave a positive result with clinically diagnosed typhoid positive patient serum and negative results were obtained with the sera of clinically diagnosed malaria, abscess of Staphylococcus aureus and Visceral leishmaniasis (Kala-azar) patients.


Introduction
Typhoid fever is an enteric fever of humans caused by infection with Salmonella enterica serovar Typhi (S. Typhi). Although the isolation of S. Typhi on blood culture remains the gold standard for diagnosing typhoid fever, this may pose a major challenge in resource-limited settings where traditional laboratory methods of diagnosing typhoid are not available. India being an agriculturist economy, most of the population is concentrated in rural areas where most hospitals and primary health centres have no laboratory facilities; the diagnosis of typhoid fever is mostly based on clinical grounds, sometimes supported by the Widal test. Although, S. Typhi is a relatively invariant pathogen to antigenic variation, many of the surface antigens, therefore, may be conserved across the genera and induce antibodies that are cross reactive. Further, with an advancing age of an individual, he/she may accumulate cross reactive antibodies to S. Typhi. It means that it is just impossible to develop a speci�c diagnostic kit for typhoid using crude or semi-puri�ed antigens.
Essentially all serological tests for typhoid are based on the detection of antibodies to lipopolysaccharide (LPS) antigens (O9 and O12) [1][2][3][4][5][6]. As shown in the lateral �ow assay for the detection of S. Typhi LPS speci�c antibodies, antibodies �rst start to develop at a time when the pathogen is already disappearing from the blood-stream [7]. e natural course and magnitude of the immune response seems to limit the sensitivity of serological testing for typhoid. Further, antigenicity of �agellar (H) proteins is higher than somatic (O) polysaccharide antigens [8]. Moreover, Brodie [9] has stated that agglutinins against �agella of S. Typhi are more frequent than O somatic (TO-9, 12) during an outbreak in Aberdeen in 1964.
Simple, reliable, point-of-care rapid diagnostic tests (RDTs) for typhoid fever have been a long-felt need of e Scienti�c �orld �ournal clinicians working in endemic areas [10]. ey should be designed to yield a simple "positive/negative" result at thresholds pre-set by the manufacturers, similar to a pregnancy test. ese results should normally be made available within 15 minutes, so that they can be used while the healthcare provider is dealing with suspected patients. Finally, such tests must be made available at low cost for use in resource-limited settings. erefore, we planned to develop an indigenous immunodiagnostic kit using most dominant antigen epitope of �agellin protein of S. Typhi of North Indian origin and to test its speci�city in different clinical specimens.

Bacterial Growth.
Peptone water (1 gm peptone, NaCl 0.5 gm, distilled water 100 mL) was adjusted to pH 7.4 autoclaved at 121 ∘ C for 15 min was prepared. 200 mL Mueller-Hinton agar media was poured in the Roux bottle and allowed to solidify for 4 h. Fiy mL of the bacterial culture in peptone water was poured on the slant formed in the Roux bottle. e Roux bottle was shaken gently for 10 min at RT near the laminar �ow and the excess peptone water was decanted. e Roux bottle containing the bacterial culture was incubated at 37 ∘ C for 48 h (without shaking). irty mL of normal saline (0.85% NaCl) was poured in the Roux bottle. It was shaken gently for 10 min near the laminar �ow and the bacterial suspension was collected in the conical �ask. Presence and purity of S. Typhi was checked by streaking on MacConkey Agar plate.

Isolation of Flagellin
Protein. e bacterial suspension was washed three times centrifuging at 12,000 rpm for 10 min by normal saline. e sediment from all the tubes was suspended in a total of 10 mL of normal saline. e suspension was then adjusted to pH 2.0 with 12 N HCl and constantly stirring for 30 min at RT. e bacterial cells, which were now devoid of �agella, were separated by centrifugation at 12,000 rpm for 30 min. e supernatant, which contained detached �agellin in monomeric form, was further centrifuged at 12,000 rpm for 1 h at 4 ∘ C. e pH of the supernatant was adjusted to 7.2 with 1 M NaOH. Ammonium sulphate was added slowly with vigorous stirring to achieve two-thirds saturation (2.67 M). e mixture was held overnight at 4 ∘ C and then centrifuged at 12,000 rpm for 15 min at 4 ∘ C. e precipitate, which contained �agellin, was dissolved in approximately one mL of dw and then transferred to dialysis tubing which had a molecular weight cutoff of 30,000 kDa (Sigma-Aldrich). Dialysis was carried out under running tap water initially for 2 h and then for 18 h at 4 ∘ C with constant stirring in 4 litres of distilled water containing 20 g of activated charcoal (Sisco Research Laboratories). e dialyzed �agellin preparations were then dissolved in 10 mM Tris and were estimated by Lowry's method [11].

SDS-Polyacrylamide Gel Electrophoresis of Flagellin.
e �agellar protein were analyzed by using SDSpolyacrylamide gel electrophoresis with slight modi�cations. Separating gel, 1.5 mm thick and 14 cm long, was prepared, consisting of 13% acrylamide, 0.325% bisacrylamide. Upon this, stacking gel of 3 cm length including wells, consisting of 5% acrylamide and 0.125% bisacrylamide, was performed. Final buffer composition in separating and stacking gels were 0.375 M Tris-HCl, pH 8.9, 0.1% SDS and 0.5 M urea and 0.125 M Tris-HCl, pH 6.7, 0.1% SDS and 0.5 M urea, respectively. ese gels were polymerized chemically by the addition of 0.025% by volume of N,N,N ′ ,N ′ -tetramethyl ethylene diamine (TEMED) and ammonium persulfate (250 g/mL). e electrophoresis buffer (pH 8.3) contained 0.025 M Tris, 0.192 M glycine, and 0.1% SDS and 0.5 M urea. e samples were mixed with 1x sample buffer to have �nally 0.06 M Tris, pH 6.7, 2% SDS, 10% glycerol, 0.001 bromophenol blue, and 0.1% by volume betamercaptoethanol just before loading. e proteins were completely dissociated by heating the samples in boiling water bath for 90 s. e electrophoresis was performed at 150 V and was stopped aer the dye eluted out of the gel. Gels were placed in 20% TCA for 30 min to �x the proteins. Gels were stained with Coomassie blue R-250 (0.3% w/v) in 50% methanol and 7.5% acetic acid overnight and destained with solution having 30% methanol, 7.5% acetic acid. Aer destaining, the gels were stored in 10% acetic acid.

Screening for Antigenicity of Isolated Flagellin Protein.
e isolated protein (3 g/5 L) w/v was immobilized to polyvinylidene �uoride sticks and blocked with 3% gelatin for 2 h at 37 ∘ C. e sticks were washed 3 times with PBS/T. e antigen coated polyvinylidene �uoride sticks were incubated in 500 L volume of diluted (1 : 100 in PBS/T) typhoid positive (culture con�rmed) and negative sera for 1 h at 37 ∘ C in small plastic vials. Sticks were washed 3 times PBS/T (for washing added PBS/T, shook gently and discarded aer 3 min). Each stick was incubated in 500 L of optimally diluted (1 : 10,000) anti-human IgG horse radish peroxidase conjugate for 1 hour at 37 ∘ C. e sticks were washed with PBS/T �ve times (as described earlier). Aer washing, the strips were incubated with diaminobenzidine substrate for 5-10 min at 37 ∘ C. e reaction was stopped using distilled water. e reaction was read by colour change.

Enzyme Hydrolysis of Flagellin Protein of S. Typhi.
One milligram amount of �agellin protein was suspended in 1.25 mL of 10 −3 M tris(hydroxymethyl)aminomethane (tris)hydrochloride buffer to give a �nal pH of 8.1 when hydrolysis by trypsin and chymotrypsin was followed. e reaction mixture was incubated at 37 ∘ C in a shaking water bath for 60 min for digestion with trypsin was and for 20 min e Scienti�c World Journal 3 for digestion with chymotrypsin (trypsin and chymotrypsin were in a 1 : 50 (w/w) ratio to protein). e enzyme trypsin was obtained from New England Bioloabs, (MA, USA) and the enzyme Chymotrypsin from Sisco Research laboratories (India). Trypsin treated with l-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) was used.

SDS-PAGE Employing Protease-Hydrolysed Flagellin
Protein. Electrophoresis was carried out on sodium dodecyl sulphate (SDS)-acrylamide gels essentially described by Weber and Osborn [12]. Gels were prepared with 10% acrylamide and 0.25% bisacrylamide. Samples of enzymatically hydrolyzed �agellin (usually 20 to 50 L) were dissociated in 10 −3 M sodium phosphate buffer (pH 7.2) containing 2% sodium dodecyl sulphate and 2% beta-mercaptoethanol by heating in a boiling water bath for 2-3 min. e electrophoresis was performed at 150 V and was stopped aer the dye eluted out of the gel. Gels were placed in 20% trichloroacetic acid for 30 min to �x the proteins. Gels were stained with Coomassie blue R-250 (0.3% w/v) in 50% methanol and 7.5% acetic acid for overnight and destained with solution having 30% methanol, 7.5% acetic acid. Aer destaining, the gels were stored in 10% acetic acid.

Western Blotting Employing Hydrolysed Flagellin Protein.
Enzymatically hydrolyzed �agellin protein were separated on 10% SDS-PAGE and were electrophoretically transferred to Polyvinylidene �uoride membranes (Pierce Biotechnology, Rockford, IL, USA or Bio-Rad Laboratories, CA, USA) by using Nova Blot semidry system (Multiphor II & EPS 600, Amersham Pharmacia Biotech, NJ, USA) as per manufacturer's instructions. e membranes were blocked with 5% bovine serum albumin in 10 mM Tris-HCl, 150 mM NaCl, pH 8.0, (Tris -buffer saline) containing 0.05% Tween-20 for 1 h at RT. Blots were then incubated for 1 h at 4 ∘ C with serum samples of patient cases suffering from typhoid fever, visceral leishmaniasis, Staphylococcus aureus abscess, and malaria (1 : 100 dilutions in 2% BSA in 1 × TBS-T) acting as primary antibodies. Following washing, the blots were incubated for 1 h with horse radish peroxidase-labeled secondary antibody (1 : 10,000 dilutions in 2% BSA in 1 × TBS-T), that is, anti-human IgG-HRP conjugate. Following washing, the enzyme activity on polyvinylidene �uoride membrane was revealed by developing the colour with freshly prepared 3,3-diaminobenzidine solution (0.05 mg dissolved in 1 mL of 50 mM citrate buffer, pH 5.6, containing 0.03% H 2 O 2 ). e reaction was stopped using distilled water.

Preparation and
Testing the Kit. e test kit prepared contained buffer well (B), sample well (A), test well (T), and the control well (C). To the test well �H� antigen �agellin protein epitope was coated, and control well had human IgG immobilised. e nitrocellulose membrane (Millipore HF Plus 135, USA) was then saturated with BSA (30%), aer coating and dried by incubation for 2 h at 40 ∘ C. Serum (∼5 L) was applied in the sample well followed by addition of �ve drops of buffer (0.01 M Tris buffer with 0.1% sodium azide) in the buffer well and buffer was allowed to run off the membrane. ereaer, colloidal Gold-conjugated antihuman IgG (∼5 L) was applied in the sample well followed by addition of 5 drops of buffer in the buffer well. Aer buffer had run off the nitrocellulose membrane, antibody binding was detected by coloured (burgundy colour) line on the test kit.

SDS-Polyacrylamide Gel Electrophoresis of Flagellin Protein.
Flagellin yielded a single stained band in polyacrylamide gel electrophoresis, indicating the probable purity of the protein isolated. e �agellar protein (�agellin) mass was determined to be ∼52 kDa as is evident from the Figure 1.

Detecting Antigenicity of Isolated Flagellin Protein by
Stick ELISA. e result of the sick ELISA demonstrates the presence antibodies to the �agellar (H) antigen protein in the serum from patient with typhoid infection (clinically positive for S. Typhi) Figure 2(a) and no antibodies were detected in serum from patient (clinically negative for S. Typhi) Figure  2(b). us, this result strongly suggests that the �agellar (H) antigen protein of S. Typhi is a strong immunogen in vivo and would be highly useful for the detection of typhoid fever. Figure  3 shows the proteolysis of isolated �agellin protein (molecular weight ∼52 kDa). On standardisation, by 60 min of trypsin treatment (Figure 3, lane L-2), the original �agellin band was no longer detected on the gels and a band with an approximate molecular weight of ∼40 kDa (antigenic epitope) was the major one seen. On the other hand, the other serine protease, chymotrypsin, on standardisation, too had a similar effect; when 20 min of the chymotrypsin treatment (Figure 3, lane L-3) was carried out on isolated �agellin protein. Lower molecular weight products of hydrolysis were formed, but they escaped detection by the Coomassie Brilliant Blue staining method.

Western
Blotting for the Antigenic Epitope. e speci�city and avidity of antibodies present in our sera of interest including the controls, for �agellar (H) antigen epitopic protein was analysed by the technique of a dot-immunobinding assay.
On analysis of the result obtained from the immunodotblot (Figure 4), the antibodies present in typhoid-positive patient serum were hardly able to recognize the lower molecular weight peptides, and this could be visualised by the nearly faint intensity of colour developed in the lower of half of the immunodotblot. On the other hand, the upper half of the immunodotblot shows markedly a very strong recognition of antibodies present in typhoid patient serum to the ∼4� k�a �agellar (H) antigen peptide band. But when the of sera of control cases (malarial, abscess due to Staphylococcus aureus, and visceral leishmaniasis) were used one could clearly �gure out from the immunodotblots (Figures 5, 6, and 7) that the sera antibodies from all the three control patients did not recognize any of the major or the minor faint �agellar (H) antigenic bands on the immunodotblot.

Result of Test Kit.
In an effort to develop a rapid, reliable, speci�c, and sensitive test for the diagnosis of typhoid fever, we attained a test kit with conspicuous results. e test kit gave a positive result with the serum of typhoid fever positive patient (clinically con�rmed) acting as "true positives"; who was admitted during the second week of illness, in the Sir Sunderlal Hospital, BHU, Varanasi where the study has been carried out and patient had no antibiotic therapy administered. e test kit gave negative results when the sera of control cases (malarial, abscess due to Staphylococcus aureus, and visceral leishmaniasis) were used "acting as controls and true negatives" as in (Figures 8, 9, and 10). e test is invalid if the control band is not visible within 15 min as in Figure 11.

Discussion
e prevalence of S. Typhi infection differs in different parts of the World. In the developing countries like India, S. Typhi infection is more frequent among general population. In this study e�planation has been done for the possible use of �agellin protein epitope to use for diagnostic kit development. For this purpose a ∼52 kDa �agellin protein from indigenous strain of S. Typhi was isolated and was found to be antigenic. Our �nding is well supported by Anuntagool et al. [13] who also could purify ∼52 kDa protein from S. Typhi �agella. Sukosol et al. [14] could raise a monoclonal antibody also to L-1 L-2 L-3 40 kDa F 7: Kala-azar-positive patient serum, L-1: standard protein molecular weight marker, L-2: trypsin digestion product, L-3: chymotrypsin digestion product.  [15] has also reported utility role of a S. Typhi �agellar protein of ∼52 kDa in serodiagnosis of typhoid fever. On hydrolysis by serine proteases trypsin and chymotrypsin, a band of ∼40 kDa protein (antigenic) epitope could be observed, while other bands were invisibly small on SDS-PAGE analysis. ere is a report showing that terminal regions of �agellin protein are very sensitive to proteolysis, resulting into small oligopeptides by tryptic digestion [16] and yielding a fragment of ∼40 kDa. Here it is interesting to mention that �agellins present in the members of family Enterobacteriaceae and Campylobacter species produce the similar ∼40 Kda protein fragment on