Method for the detection of H. pylori infection
10018628 ยท 2018-07-10
Assignee
Inventors
- Markus Gerhard (Munich, DE)
- Behnam Kalali (Munich, DE)
- Luca Formichella (Munich, DE)
- Mohammad Khalife-Gholi (Ghom, IR)
Cpc classification
G01N2469/20
PHYSICS
International classification
C07K1/00
CHEMISTRY; METALLURGY
Abstract
The present invention is related to a method for detecting H. pylori infection in a subject, wherein the method comprises detecting in a sample from the subject an immune response against FliD, wherein the immune response comprises an anti-FliD antibody.
Claims
1. A kit for detecting H. pylori infection in a subject, wherein detecting H. pylori infection comprises detecting in a sample from the subject an immune response against FliD, wherein the immune response comprises an anti-FliD antibody, wherein the kit comprises recombinant full-length FliD, wherein the recombinant full-length FliD is attached to a solid phase.
2. The kit of claim 1, further comprising one or more antigens selected from the group consisting of CagA, VacA, GroEL, Hp 0231, JHp 0940 and HtrA for detecting one or more antigens of Helicobacter.
3. The kit of claim 1, wherein the kit is configured to detect H. pylori infection in a sample is selected from the group consisting of blood serum, blood plasma, whole blood and stool.
4. The kit of claim 2, wherein the one or more antigens are of H. pylori.
5. The kit of claim 1, wherein the kit is configured to detect an anti-FliD antibody that is an IgG antibody, an IgA antibody, or an IgG antibody and an IgA antibody.
6. The kit of claim 1, configured for use in an ELISA, a lateral flow assay or a line assay.
7. The kit of claim 1, wherein the kit further comprises recombinant CagA.
8. The kit of claim 1, wherein FliD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.
9. The kit of claim 7, wherein CagA comprises the amino acid sequence of SEQ ID NO: 7.
10. The kit of claim 1, wherein the kit is a ready-for-use kit.
11. The kit of claim 7, wherein the recombinant CagA is attached to the solid phase.
12. The kit of claim 1, wherein the kit further comprises a buffer and/or an instruction leaflet.
13. The kit of claim 1, wherein the recombinant full-length FliD further comprises a histidine tag.
14. The kit of claim 1, wherein the recombinant full-length FliD attached to the solid phase provides a sensitivity of more than 90% and a specificity of more than 90%.
15. The kit of claim 1, wherein the recombinant full-length FliD attached to the solid phase provides a sensitivity of up to 97.4% and a specificity of up to 99%.
16. The kit of claim 1, wherein the solid phase is selected from the group consisting of an ELISA plate, nitrocellulose and gold nanoparticles.
17. The kit of claim 1, wherein the solid phase comprises gold nanoparticles.
Description
(1) The present invention is now further illustrated by the following figures and examples which are not intended to limit the scope of protection. From said figures and examples further features, embodiments and advantages may be taken, wherein
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EXAMPLE 1: CLONING OF THE H. PYLORI FLID GENE
(10) All DNA manipulations were performed under standard conditions as described by Sambrook et al. (Sambrook, et al., 1989). Briefly, the FliD gene was amplified by PCR using genomic DNA from H. pylori strain J99 as the template. Following oligonucleotides were used as primers: 5-CAT ATG GCA ATA GGT TCA TTA A-3 (SEQ ID NO: 19) and 5-CTC GAG ATT CTT TTT AGC CGC TGC-3 (SEQ ID NO: 20). Using this approach a NdeI site was introduced at the 5-end of forward primers and a XhoI site at 5-end of the reverse primers. After PCR amplification, the product (2058 bp) was ligated into the pTZ57R/T cloning vector (InsTAclone PCR Cloning Kit, MBI Fermentas, Lithuania). Subsequently, the insert was confirmed via PCR and sequencing, and was cloned into a PET-28a(+) expression vector (Qiagen, USA) using NdeI and XhoI restriction enzymes.
EXAMPLE 2: EXPRESSION, PURIFICATION AND RECOGNITION OF RECOMBINANT FLID
(11) E. coli BL21 (Qiagen, USA) competent cells were transformed with pET-28a(+)-fliD and inoculated in LB broth with antibiotic (kanamycin, 50 g/ml). Expression was induced by addition of 1 mmol/L Isopropyl -D-1-thiogalactopyranoside (IPTG) at an optical density (OD600) of 0.6. After 4 hours cells were harvested and protein analysis of whole lysate was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The soluble histidine-tagged proteins were purified using affinity chromatography (HisTrap crude, GE Healthcare). As a second polishing step and for buffer exchange, size exclusion chromatography (Superdex 75, GE Healthcare) was performed. The relevant fractions were collected and concentrated with a centrifugal filter device (Millipore) with a cut off of 10 kDa and stored at 80 C. Purified recombinant protein was evaluated by Western blot using an anti-His Tag-HRP antibody and also a mouse anti-H. pylori-HRP antibody (Pierce, Rockford, USA) and detected by ECL system (GE Healthcare, Uppsala, Sweden).
(12) Amplification of the FliD gene from H. pylori strain J99 DNA revealed a single PCR product of 2.05 kb (data not shown) which was confirmed by sequencing and ligated into the expression vector pET-28a(+). After transformation into E. coli expression strain BL21 DE3 and induction with IPTG, a clear single band could be observed on Western blot using a commercial polyclonal anti-H. pylori antiserum. The protein was purified as described in Materials and Methods to >90% purity (data not shown) and again confirmed by Western blot (data not shown).
EXAMPLE 3: PRODUCTION AND PURIFICATION OF RFLID SPECIFIC ANTIBODY
(13) A mature white New Zealand rabbit was immunized with purified protein according to the protocol of Hay et al. with light modifications (Hay, et al., 2002). Briefly, immunization was carried out by i.m. injection of 250 g purified recombinant protein (0.5 ml) with the same volume (0.5 ml) of Freund's complete adjuvant. For the recall immunizations, the rabbit was boosted with 125 g purified protein prepared in the same volume (0.5 ml) of Freund's incomplete adjuvant 4, 6, 8 and 10 weeks later. As a negative control a serum sample was taken prior to immunization. Finally, two weeks after the last immunization, blood was collected and sera separated. Polyclonal IgG antibody was purified by sepharose-4B affinity chromatography using rFliD conjugated columns prepared according to the manufacturer's protocol (Pharmacia, 1988). FliD expression of H. pylori (J99) was detected by Western blot using ultrasonic supernatant at the protein concentration of 50 g/ml. The rabbit polyclonal IgG antibody raised against rFliD protein was used as the first antibody (1:5000 dilution), HRP-labeling sheep antibody against rabbit IgG (Avicenna Research Institute, Tehran, Iran) as the second antibody (1:3000 dilution) and ECL system were used for the detection (Chen, et al., 2001).
(14) Furthermore, to test the antigenicity of the recombinant FliD and to compare it to the native protein, rabbit polyclonal antiserum was produced. Antibody titers were already determined after the third immunization and reached high levels after the fourth boost, confirming the good immunogenicity of FliD. The rabbit antiserum was able to recognize the purified rFliD and FliD in H. pylori lysate (data not shown).
EXAMPLE 4: DEVELOPMENT OF AN ELISA
(15) ELISA plates were coated with 100 l rFliD protein at a concentration of 1 g/ml in PBS and incubated overnight at 4 C. The coated wells were blocked with phosphate buffered saline (PBS) containing 2.5% bovine serum albumin (BSA, Sigma) for two hours at 37 C. All H. pylori positive and negative serologic samples used in this study were screened for antibodies against FliD by using optimal dilution of patients' sera (1:100 dilution) as the first antibody, HRP-conjugated anti-human IgG (Promega, Mannheim, Germany) (1:100 dilution) as the secondary antibody and TMB (3,3,5,5-tetra methyl benzidine) as a substrate. Moreover, wells were left uncoated as a control for each serum. The result of ELISA for a patient's serum sample was considered to be positive if its OD450 value was over the mean plus 3 SD of negative serum samples (Chen, et al., 2001).
EXAMPLE 5: DEVELOPMENT OF AN FLID LINE ASSAY
(16) A line immunoassay based on recombinant H. pylori proteins immobilized on nitrocellulose was prepared. In contrast to ELISA, the test principle allows the identification of specific antibodies against various antigens of H. pylori through separate application of different single antigens.
(17) rFliD was immobilized on nitrocellulose membrane strips together with other highly purified recombinant H. pylori antigens (CagA, VacA, GroEL, UreA (urease A), HcpC (Cysteine rich protein C) (Mittel et al., 2003) and gGT (gamma glutamyl transferase). The appropriate line conditions for rFliD were determined empirically with a selection of standard serum samples from a previously described study population comprising 20 defined H. pylori histologically positive samples and 20 defined H. pylori histologically negative samples. The optimal antigen concentration and ideal choice of additives like detergent, dithiothreitol, and NaCl was adjusted for each antigen by repeated cycles of lining and screening. The conditions with best presentation of antigen epitopes and optimal binding to the membrane, observable by perfect band appearance and best discrimination of negative and positive samples, were selected for ideal product specifications of first lots. Control bands were added on the upper end of the strip comprising rabbit anti-human IgG/IgM/IgA antibodies as incubation controls and human IgG, IgM or IgA antibodies as conjugate control as well as a cut off control that allows the assessment of the reactivity of the individual antigen bands.
(18) After scanning and densitometric analysis of the band intensities, the control was used as internal reference to calculate ratios for each band. Usually, cut off control bands are scored between 20 and 30, while strong positive bands can score up to 600 points. Every band scoring above the individual control of the each stripe is considered positive (ratio >1).
(19) The respective line assay is depicted in
EXAMPLE 6: PROTOTYPE OF A LATERAL FLOW ASSAY FOR THE DIAGNOSIS OF H. PYLORI
(20) Using the materials defined above a lateral flow assay was developed based on the principles disclosed herein related to design of a lateral flow assay.
(21) The prototype of such lateral flow assay is depicted in
(22) As may be taken from
EXAMPLE 7: ANALYSIS OF SAMPLES FROM MAN
(23) A total of six hundred and eighteen (618) human patients (308 men, 310 women) were enrolled in the study. After receiving an explanation of the purpose of the study, informed consent was obtained from each patient and a blood sample was taken at the time of endoscopy, before any therapy was initiated. Sera were separated and stored at 20 C. Diagnosis of infection was based on the histopathology as gold standard. Patients were considered H. pylori positive when the results of histopathology were positive. All patients were screened by FliD Line assay, and a subset of 246 sera was tested by FliD ELISA as described above and by line assay as described above.
(24) Table 2 shows the results of using said FliD ELISA. More specifically, Table 2 shows FliD serologic response in ELISA comparing H. pylori negative and positive human patients.
(25) TABLE-US-00002 Histology Negative Positive Total ELISA Negative 73 8 81 Positive 3 162 165 Total 76 170 246
(26) Table 3 shows the results of using said line assay for a subgroup of the group of patients. More specifically, Table 3 shows FliD serologic response in the line assay comparing H. pylori negative and positive patients.
(27) TABLE-US-00003 Histology Negative Positive Total Line Assay Negative 76 14 90 Positive 0 156 156 Total 76 170 246
(28) Using the FliD ELISA, among 170 positive reported samples, 165 positive samples were detected, whereas among 76 samples reported negative 73 were reconfirmed as negative by ELISA (Table 2). Taken together, application of FliD in ELISA based diagnosis of H. pylori infection has a specificity of 96% and a sensitivity of 97%. Interestingly, the five cases which were ELISA negative had also low but barely positive scores in the line blot which were just above the cut off (ratios ranging from 1.2 to 2.2). One of these was also regarded H. pylori negative by line blot, while the other four were line blot positive, reacting with several other antigens (data not shown). It is important to note that only one sample was negative by both tests.
(29) The entire group of 618 human patients (part of which had been screened by ELISA) was analyzed using the line assay as to antibody response against FliD. a high sensitivity of 97.4% with 310 out of 318 patients evaluated positive in histopathology being positive by line assay, whereas the line assay reaches a specificity of 99% (Table 2). The results from the patients in which discrepant results were obtained, was carefully examined. 8 sera were negative for FliD in the line assay but showed reactivity with other antigens, indicating that here, indeed, FliD was not recognized as antigen. Within these 8 samples, one had no reactivity against the FliD band at all. Seven had a weak reactivity which was barely below the cut off (ratios between 0.6 and 0.95), and four of these had weak reactivities against all other recognized antigens in general (not shown). All three samples in which FliD gave a false positive result showed reactivities with other bands as well. All these bands including FliD were relatively weak, but clearly above cut off.
(30) From said samples the prevalence of an anti-FliD antibody response was determined as a function of years after eradication. The result is shown in
(31) From said samples receiver operating characteristics (ROC) curves have been prepared for FliD, CagA and UreA. The result is shown in
EXAMPLE 8: BIOINFORMATIC ANALYSIS OF FLID SEQUENCES
(32) Using bioinformatics tools, FliD protein of H. pylori G27 strain was widely compared to other organisms, mainly prokaryotes. This analysis shows more than 97% homology between over 200 H. pylori strains.
(33) The results are shown in Table 4.
(34) TABLE-US-00004 Entry Entry name Protein names Organism Length Identity Score B5Z7B5 B5Z7B5_HELPG Putative flagellar hook-associated protein 2 Helicobacter pylori (strain G27) 685 100.0% 3412 J0KLR1 J0KLR1_HELPX Putative flagellar hook-associated protein 2 Helicobacter pylori Hp H-27 685 99.0% 3383 I9RP80 I9RP80_HELPX Flagellar capping protein Helicobacter pylori Hp A-20 685 99.0% 3381 J0MV71 J0MV71_HELPX Putative flagellar hook-associated protein 2 Helicobacter pylori Hp A-27 685 99.0% 3379 J0DL62 J0DL62_HELPX Flagellar capping protein Helicobacter pylori Hp H-11 685 99.0% 3375 J0A5P7 J0A5P7_HELPX Flagellar capping protein Helicobacter pylori Hp A-9 685 98.0% 3372 J0IU02 J0IU02_HELPX Flagellar capping protein Helicobacter pylori NQ4228 685 99.0% 3371 K2L7H4 K2L7H4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R036d 685 98.0% 3370 J0TQK4 J0TQK4_HELPX Putative flagellar hook-associated protein 2 Helicobacter pylori Hp P-30 685 99.0% 3369 M7RTJ4 M7RTJ4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori UMB_G1 685 98.0% 3367 K2L537 K2L537_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R055a 685 99.0% 3367 J0SAM4 J0SAM4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-15b 685 99.0% 3367 J0M138 J0M138_HELPX Putative flagellar hook-associated protein 2 Helicobacter pylori Hp H-45 685 99.0% 3367 I9WVW7 I9WVW7_HELPX Flagellar capping protein Helicobacter pylori Hp P-15 685 99.0% 3367 K2KUE2 K2KUE2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R030b 685 99.0% 3365 I0EHS2 I0EHS2_HELPX Flagellar capping protein Helicobacter pylori PeCan18 685 98.0% 3365 H8H4E1 H8H4E1_HELPX Flagellar capping protein Helicobacter pylori ELS37 685 98.0% 3362 K2LNG6 K2LNG6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R038b 685 98.0% 3361 D0IS88 D0IS88_HELP1 Flagellar hook-associated protein 2 Helicobacter pylori (strain 51) 685 98.0% 3360 N4T9B5 N4T9B5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-11 685 98.0% 3359 E1PZL2 E1PZL2_HELPM Flagellar capping protein Helicobacter pylori (strain SJM180) 685 98.0% 3358 J0IVU8 J0IVU8_HELPX Flagellar capping protein Helicobacter pylori NQ4099 685 98.0% 3358 J0FGE4 J0FGE4_HELPX Putative flagellar hook-associated protein 2 Helicobacter pylori Hp P-16 685 98.0% 3358 Q1CTB8 Q1CTB8_HELPH Putative flagellar hook-associated protein 2 Helicobacter pylori (strain HPAG1) 685 98.0% 3357 E8QPN8 E8QPN8_HELPR Flagellar capping protein Helicobacter pylori (strain 685 98.0% 3355 Lithuania75) K2KUX6 K2KUX6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R32b 685 98.0% 3355 K2KMU9 K2KMU9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R037c 685 98.0% 3355 J0HQJ3 J0HQJ3_HELPX Flagellar capping protein Helicobacter pylori CPY1124 685 98.0% 3355 I9XF52 I9XF52_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-74 685 98.0% 3355 I9U4H5 I9U4H5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-26 685 98.0% 3355 D7FEA9 D7FEA9_HELP3 Flagellar hook-associated protein 2 Helicobacter pylori (strain B8) 685 98.0% 3354 I9US75 I9US75_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-9 685 98.0% 3354 B9XZK1 B9XZK1_HELPX Putative uncharacterized protein Helicobacter pylori B128 685 98.0% 3354 J0J9Q0 J0J9Q0_HELPX Flagellar capping protein Helicobacter pylori NQ4076 685 98.0% 3353 I9QZB4 I9QZB4_HELPX Flagellar capping protein Helicobacter pylori NQ4110 685 98.0% 3353 G2M3P3 G2M3P3_HELPX Flagellar capping protein Helicobacter pylori Puno120 685 98.0% 3352 E1QBB7 E1QBB7_HELPC Flagellar capping protein Helicobacter pylori (strain Cuz20) 685 98.0% 3351 J0LRM5 J0LRM5_HELPX Flagellar capping protein Helicobacter pylori Hp H-43 685 98.0% 3351 E6NRT1 E6NRT1_HELPQ Flagellar capping protein Helicobacter pylori (strain F57) 685 98.0% 3350 M3MVK5 M3MVK5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM114Ai 685 98.0% 3350 K2KRX5 K2KRX5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R018c 685 98.0% 3350 K2KFQ1 K2KFQ1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R056a 685 98.0% 3350 J0IGN4 J0IGN4_HELPX Flagellar capping protein Helicobacter pylori NQ4216 685 98.0% 3349 E6S1Q8 E6S1Q8_HELPF Flagellar hook-associated protein 2 Helicobacter pylori (strain 35A) 685 98.0% 3348 I9YJR2 I9YJR2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-13b 685 98.0% 3348 I9WT57 I9WT57_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-13 685 98.0% 3348 I9U161 I9U161_HELPX Flagellar capping protein Helicobacter pylori Hp A-14 685 98.0% 3348 B6JLY6 B6JLY6_HELP2 Flagellar hook-associated protein 2 Helicobacter pylori (strain P12) 685 98.0% 3347 K2K5F9 K2K5F9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori R046Wa 685 98.0% 3347 I9XUJ1 I9XUJ1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori CPY1313 685 98.0% 3347 I9PV13 I9PV13_HELPX Flagellar hook-associated protein 2 Helicobacter pylori CPY6311 685 98.0% 3347 I9PLR1 I9PLR1_HELPX Flagellar capping protein Helicobacter pylori CPY6261 685 98.0% 3347 L8VWS3 L8VWS3_HELPX Flagellar capping protein Helicobacter pylori A45 685 98.0% 3346 K7Y5K8 K7Y5K8_HELPX Flagellar capping protein Helicobacter pylori Aklavik117 685 98.0% 3346 J0T145 J0T145_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M2 685 98.0% 3346 I9T4Z9 I9T4Z9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-44 685 98.0% 3346 E8QFQ7 E8QFQ7_HELP7 Flagellar capping protein Helicobacter pylori (strain India7) 685 98.0% 3345 C7BX84 C7BX84_HELPB Flagellar hook-associated protein 2 FliD Helicobacter pylori (strain B38) 685 98.0% 3345 I9W7Z2 I9W7Z2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-2 685 98.0% 3345 I0ZBA9 I0ZBA9_HELPX Flagellar capping protein Helicobacter pylori P79 685 98.0% 3345 F4D517 F4D517_HELPX Flagellar hook-associated protein 2 Helicobacter pylori 83 685 98.0% 3345 B9XUM1 B9XUM1_HELPX Putative uncharacterized protein Helicobacter pylori 98-10 685 98.0% 3345 P96786 FLID_HELPY Flagellar hook-associated protein 2 (HAP2) Helicobacter pylori (strain ATCC 685 98.0% 3345 (Filament cap protein) (Flagellar cap protein) 700392/26695) (Campylobacter pylori) M3SDI9 M3SDI9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAMchJs106B 685 98.0% 3344 I9XAU3 I9XAU3_HELPX Flagellar capping protein Helicobacter pylori Hp P-23 685 98.0% 3344 I9PTN1 I9PTN1_HELPX Flagellar capping protein Helicobacter pylori CPY6271 685 98.0% 3344 G2M8C7 G2M8C7_HELPX Flagellar capping protein Helicobacter pylori Puno135 685 98.0% 3344 E1Q6P5 E1Q6P5_HELPP Flagellar capping protein Helicobacter pylori (strain PeCan4) 685 98.0% 3343 M3KWT6 M3KWT6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM119Bi 685 98.0% 3343 I0ZGY9 I0ZGY9_HELPX Flagellar capping protein Helicobacter pylori NCTC 11637 = 685 98.0% 3343 CCUG 17874 I2DFT2 I2DFT2_HELPX Flagellar capping protein Helicobacter pylori XZ274 685 98.0% 3342 E6NKD5 E6NKD5_HELPL Flagellar capping protein Helicobacter pylori (strain F32) 685 98.0% 3341 E6NIS5 E6NIS5_HELPK Flagellar capping protein Helicobacter pylori (strain F30) 685 98.0% 3341 I9ZP80 I9ZP80_HELPX Flagellar hook-associated protein 2 Helicobacter pylori NQ4161 685 98.0% 3341 I9RRM1 I9RRM1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-17 685 98.0% 3341 J0A0N9 J0A0N9_HELPX Flagellar hook-associated protein Helicobacter pylori Hp P-26 685 97.0% 3340 I9QGH5 I9QGH5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori NQ4053 685 98.0% 3340 D6XPZ1 D6XPZ1_HELPV Flagellar hook-associated protein 2 Helicobacter pylori (strain v225d) 685 98.0% 3339 M5YZL4 M5YZL4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAMchJs124i 685 97.0% 3339 M5YMA1 M5YMA1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAMchJs114i 685 97.0% 3339 M4ZNA5 M4ZNA5_HELPX Flagellar capping protein Helicobacter pylori OK310 685 97.0% 3339 M3NNS0 M3NNS0_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM246Ai 685 97.0% 3339 M3MBN7 M3MBN7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM105Ai 685 97.0% 3339 I9S784 I9S784_HELPX Flagellar capping protein Helicobacter pylori Hp H-28 685 98.0% 3339 I0E4K1 I0E4K1_HELPX Flagellar capping protein Helicobacter pylori Shi417 685 98.0% 3339 J0P747 J0P747_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-23 685 97.0% 3338 J0N2H0 J0N2H0_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-4 685 97.0% 3338 I0ED42 I0ED42_HELPX Flagellar capping protein Helicobacter pylori Shi112 685 98.0% 3338 M7SSG1 M7SSG1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori CPY1662 685 97.0% 3337 M5Y955 M5Y955_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAMchJs117Ai 685 97.0% 3337 M4ZKA3 M4ZKA3_HELPX Flagellar capping protein Helicobacter pylori OK113 685 98.0% 3337 M3LA33 M3LA33_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM231Ai 685 97.0% 3337 I9Z0G2 I9Z0G2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-28b 685 97.0% 3337 I9S3M7 I9S3M7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-24 685 98.0% 3337 I0EWG9 I0EWG9_HELPX Flagellar capping protein Helicobacter pylori HUP-B14 685 97.0% 3337 M3PSG4 M3PSG4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM96Ai 685 97.0% 3336 J0U8I3 J0U8I3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-3b 685 97.0% 3336 J0RUS2 J0RUS2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-5b 685 97.0% 3336 J0Q0D5 J0Q0D5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-4 685 97.0% 3336 J0PSB5 J0PSB5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-3 685 97.0% 3336 I9Y932 I9Y932_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-4c 685 97.0% 3336 I9XWQ4 I9XWQ4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-4d 685 97.0% 3336 E6NDJ6 E6NDJ6_HELPI Flagellar capping protein Helicobacter pylori (strain F16) 685 97.0% 3334 M3QDF1 M3QDF1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM80Ai 685 97.0% 3334 M3Q5B9 M3Q5B9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM42Ai 685 97.0% 3334 M3P646 M3P646_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM245Ai 685 97.0% 3334 M3LV71 M3LV71_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM112Ai 685 97.0% 3334 M3L655 M3L655_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM101Biv 685 97.0% 3334 E1S8R1 E1S8R1_HELP9 Flagellar hook-associated protein Helicobacter pylori (strain 908) 685 97.0% 3333 E1PVI4 E1PVI4_HELPT Flagellar capping protein Helicobacter pylori (strain Sat464) 685 98.0% 3333 D0JZC3 D0JZC3_HELP5 Flagellar capping protein Helicobacter pylori (strain 52) 685 97.0% 3333 M3RS44 M3RS44_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP116Bi 685 97.0% 3333 M3R005 M3R005_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM264Ai 685 97.0% 3333 M3MJ19 M3MJ19_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM103Bi 685 97.0% 3333 J0I156 J0I156_HELPX Flagellar capping protein Helicobacter pylori CPY3281 685 98.0% 3333 J0AJS5 J0AJS5_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-16 685 97.0% 3333 I0E947 I0E947_HELPX Flagellar capping protein Helicobacter pylori Shi169 685 98.0% 3333 F2JET0 F2JET0_HELP9 Flagellar hook-associated protein Helicobacter pylori 2018 685 97.0% 3333 F2JAT7 F2JAT7_HELP9 Flagellar hook-associated protein Helicobacter pylori 2017 685 97.0% 3333 Q9ZL91 FLID_HELPJ Flagellar hook-associated protein 2 (HAP2) Helicobacter pylori (strain J99) 685 97.0% 3333 (Filament cap protein) (Flagellar cap protein) (Campylobacter pylori J99) M3NID0 M3NID0_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM270ASi 685 97.0% 3332 J0DCU5 J0DCU5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-6 685 97.0% 3332 I9V408 I9V408_HELPX Flagellar capping protein Helicobacter pylori Hp H-10 685 97.0% 3332 J0U3G8 J0U3G8_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-62 685 97.0% 3331 I9SL37 I9SL37_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-29 685 97.0% 3331 E8QM56 E8QM56_HELP4 Flagellar capping protein Helicobacter pylori (strain 685 97.0% 3330 Gambia94/24) M5YNV6 M5YNV6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAMchJs136i 685 97.0% 3330 M3TQ89 M3TQ89_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP260Bi 685 97.0% 3330 M3QIV4 M3QIV4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM260Bi 685 97.0% 3330 M3Q2L5 M3Q2L5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM263BFi 685 97.0% 3330 M3M583 M3M583_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM115Ai 685 97.0% 3330 J0SFX5 J0SFX5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-25c 685 97.0% 3330 J0HGQ0 J0HGQ0_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-25d 685 97.0% 3330 I9X9I1 I9X9I1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-25 685 97.0% 3330 I9VCT9 I9VCT9_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-19 685 97.0% 3330 M3S7G6 M3S7G6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM83T 685 97.0% 3329 M3PEV1 M3PEV1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM244Ai 685 97.0% 3329 M3P9F3 M3P9F3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM83Bi 685 97.0% 3329 M3NFC4 M3NFC4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM118Bi 685 97.0% 3329 K8GY42 K8GY42_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM100Ai 685 97.0% 3329 J0UFU9 J0UFU9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M9 685 97.0% 3329 J0T5P3 J0T5P3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M4 685 97.0% 3329 J0REL3 J0REL3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-24c 685 97.0% 3329 J0I743 J0I743_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M5 685 97.0% 3329 J0I1J2 J0I1J2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M3 685 97.0% 3329 J0HJK5 J0HJK5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M1 685 97.0% 3329 I9ZYP3 I9ZYP3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp M6 685 97.0% 3329 I9XJ16 I9XJ16_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-24b 685 97.0% 3329 M3UI84 M3UI84_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP260BFii 685 97.0% 3328 M3U8F0 M3U8F0_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250BSi 685 97.0% 3328 M3T9M6 M3T9M6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250ASi 685 97.0% 3328 M3T443 M3T443_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250ASii 685 97.0% 3328 M3T0U7 M3T0U7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250AFiV 685 97.0% 3328 M3SWF6 M3SWF6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250BFiV 685 97.0% 3328 M3SP57 M3SP57_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250AFiii 685 97.0% 3328 M3S6F4 M3S6F4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250BFiii 685 97.0% 3328 M3R7T2 M3R7T2_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250AFii 685 97.0% 3328 M3QV83 M3QV83_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM260BSi 685 97.0% 3328 M3QS41 M3QS41_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250BFii 685 97.0% 3328 M3QQ64 M3QQ64_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP250BFi 685 97.0% 3328 M3Q6I7 M3Q6I7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM250T 685 97.0% 3328 M3NV58 M3NV58_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM252Bi 685 97.0% 3328 M3NKC5 M3NKC5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM252T 685 97.0% 3328 M3LZX8 M3LZX8_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM250AFi 685 97.0% 3328 J0CLQ3 J0CLQ3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-16 685 97.0% 3328 I9XTZ6 I9XTZ6_HELPX Flagellar capping protein Helicobacter pylori CPY1962 685 98.0% 3328 B2UT80 B2UT80_HELPS Flagellar capping protein Helicobacter pylori (strain Shi470) 685 97.0% 3327 M7SW73 M7SW73_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-1 685 97.0% 3327 M3P129 M3P129_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM254Ai 685 97.0% 3327 I9P985 I9P985_HELPX Flagellar capping protein Helicobacter pylori CPY6081 685 97.0% 3326 K7YA88 K7YA88_HELPX Flagellar capping protein Helicobacter pylori Aklavik86 685 97.0% 3325 M3RIK8 M3RIK8_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM93Bi 685 97.0% 3324 J0M8U8 J0M8U8_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-6 685 97.0% 3324 M3NGP1 M3NGP1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM265BSii 685 97.0% 3323 M3KZM7 M3KZM7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM120Ai 685 97.0% 3323 M3PUQ6 M3PUQ6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM249T 685 97.0% 3322 M3PCL7 M3PCL7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM239Bi 685 97.0% 3322 M3NM23 M3NM23_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM121Aii 685 97.0% 3322 J0IWR3 J0IWR3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori NQ4200 685 97.0% 3322 J0PFP0 J0PFP0_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-1 685 97.0% 3321 I9XPS7 I9XPS7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-1b 685 97.0% 3321 J0N254 J0N254_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-3 685 97.0% 3320 M3U8B7 M3U8B7_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP260AFii 685 97.0% 3319 M3U287 M3U287_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP260AFi 685 97.0% 3319 M3RLI9 M3RLI9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori HP260ASii 685 97.0% 3319 M3Q751 M3Q751_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM268Bii 685 97.0% 3319 M3P4U3 M3P4U3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM260ASi 685 97.0% 3319 M3LLE6 M3LLE6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM201Ai 685 97.0% 3318 J0JT98 J0JT98_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-5 680 98.0% 3318 I9SDQ6 I9SDQ6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-30 677 97.0% 3314 J0BNB3 J0BNB3_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H42 680 97.0% 3311 G2MEG6 G2MEG6_HELPX Flagellar capping protein Helicobacter pylori SNT49 685 97.0% 3311 J0UBP3 J0UBP3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-2b 677 97.0% 3308 I9QNB9 I9QNB9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori NQ4044 685 96.0% 3302 I9ZZM5 I9ZZM5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-4 677 97.0% 3300 M7SHH3 M7SHH3_HELPX Flagellar hook-associated protein 2 Helicobacter pylori CCHI 33 677 97.0% 3297 I9TJA1 I9TJA1_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp A-8 677 97.0% 3297 M3NMW6 M3NMW6_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM210Bi 685 96.0% 3296 I9YL06 I9YL06_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-11b 677 97.0% 3293 I9WS67 I9WS67_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-11 677 97.0% 3293 J0PC82 J0PC82_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-34 677 97.0% 3292 I9VJH4 I9VJH4_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-21 677 97.0% 3292 J0PV65 J0PV65_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-8 677 97.0% 3291 I9YHM9 I9YHM9_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P-8b 677 97.0% 3291 J0TUW0 J0TUW0_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp P41 677 97.0% 3289 J0NSR5 J0NSR5_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-18 677 97.0% 3289 J0BAE3 J0BAE3_HELPX Flagellar hook-associated protein Helicobacter pylori Hp H-36 677 97.0% 3289 J0LI78 J0LI78_HELPX Flagellar hook-associated protein 2 Helicobacter pylori Hp H-41 677 96.0% 3278 E8QRV3 E8QRV3_HELPW Flagellar capping protein Helicobacter pylori (strain 685 95.0% 3264 SouthAfrica7) Q17Y06 Q17Y06_HELAH Flagellar hook-associated protein Helicobacter acinonychis (strain 685 94.0% 3249 Sheeba) K4NRS2 K4NRS2_HELPY Flagellar capping protein Helicobacter pylori (strain ATCC 674 97.0% 3190 700392/26695) (Campylobacter pylori) K4NL36 K4NL36_HELPX Flagellar capping protein Helicobacter pylori Rif2 674 97.0% 3190 K4NJJ9 K4NJJ9_HELPX Flagellar capping protein Helicobacter pylori Rif1 674 97.0% 3190 M3QVV4 M3QVV4_HELPX Flagellar hook-associated protein 2 Helicobacter pylori GAM71Ai 647 97.0% 3146 (Fragment) I0ETW0 I0ETW0_HELCM Flagellar capping protein Helicobacter cetorum (strain ATCC 685 88.0% 3065 BAA-540/MIT 99-5656) I0EMR1 I0EMR1_HELC0 Flagellar capping protein Helicobacter cetorum (strain ATCC 685 81.0% 2861 BAA-429/MIT 00-7128) E7ADC3 E7ADC3_HELFC Flagellar hook-associated protein Helicobacter felis (strain ATCC 684 63.0% 2190 49179/NCTC 12436/CS1) E7FYJ6 E7FYJ6_9HELI Flagellar capping protein Helicobacter suis HS1 689 61.0% 2158 F8KTH3 F8KTH3_HELBC Flagellar hook-associated protein FliD Helicobacter bizzozeronii (strain CIII- 694 59.0% 2091 1) K4RHP3 K4RHP3_HELHE Flagellar hook-associated protein FliD Helicobacter heilmannii ASB1.4 691 58.0% 2073 D3UGM5 D3UGM5_HELM1 Putative flagellar hook-associated protein Helicobacter mustelae (strain ATCC 674 52.0% 1778 43772/LMG 18044/NCTC 12198/ 12198) (Campylobacter mustelae) Q7VI19 Q7VI19_HELHP Flagellar filament capping protein FliD Helicobacter hepaticus (strain ATCC 682 51.0% 1698 51449/3B1) I2FDC5 I2FDC5_HELCP Flagellar capping protein Helicobacter cinaedi (strain 682 51.0% 1690 PAGU611) I7GZJ0 I7GZJ0_9HELI Flagellar capping protein Helicobacter cinaedi ATCC BAA-847 682 51.0% 1689 E4VHL6 E4VHL6_9HELI Flagellar hook-protein 2 Helicobacter cinaedi CCUG 18818 682 51.0% 1689 N2BQN7 N2BQN7_9HELI Uncharacterized protein Helicobacter bilis WiWa 679 45.0% 1589 C3XDT1 C3XDT1_9HELI Flagellar capping protein Helicobacter bilis ATCC 43879 679 45.0% 1583 Q7MAM3 Q7MAM3_WOLSU FLAGELLAR HOOK-ASSOCIATED Wolinella succinogenes (strain ATCC 682 45.0% 1479 PROTEIN 2 29543/DSM 1740/LMG 7466/ NCTC 11488/FDC 602W) (Vibrio succinogenes) C5EXF0 C5EXF0_9HELI Flagellar hook-protein 2 Helicobacter pullorum MIT 98-5489 685 39.0% 1272 C5ZWT4 C5ZWT4_9HELI Flagellar hook-associated protein Helicobacter canadensis MIT 98-5491 689 39.0% 1262 (Flagellar hook-protein 2) H5VEC0 H5VEC0_HELBI Flagellar hook-associated protein FliD Helicobacter bizzozeronii CCUG 458 53.0% 1185 35545 C3XLS4 C3XLS4_9HELI Flagellar hook-associated protein 2 Helicobacter winghamensis ATCC 689 37.0% 1175 BAA-430 H5VEC1 H5VEC1_HELBI Flagellar hook-associated protein FliD Helicobacter bizzozeronii CCUG 231 66.0% 807 35545 H8CS11 H8CS11_CAMJU Flagellar capping protein Campylobacter jejuni subsp. jejuni 645 27.0% 474 LMG 9872 B9KGA6 B9KGA6_CAMLR Flagellar filament cap protein FliD Campylobacter lari (strain RM2100/ 766 26.0% 471 D67/ATCC BAA-1060) C6RGG2 C6RGG2_9PROT SMR-type multidrug efflux transporter Campylobacter showae RM3277 577 29.0% 465 D2MX77 D2MX77_CAMJU Flagellar hook-associated protein Campylobacter jejuni subsp. jejuni 642 28.0% 461 414 M3I083 M3I083_9PROT Flagellar capping protein Campylobacter showae CC57C 577 28.0% 457 H7SA14 H7SA14_CAMCO Flagellar capping protein Campylobacter coli 84-2 644 26.0% 452 D2MS44 D2MS44_CAMJU Flagellar hook-associated protein FliD Campylobacter jejuni subsp. jejuni 647 26.0% 451 1336 H7XBH0 H7XBH0_CAMJU Flagellar capping protein Campylobacter jejuni subsp. jejuni 648 26.0% 451 LMG 23216 H7YRN9 H7YRN9_CAMJU Flagellar capping protein Campylobacter jejuni subsp. jejuni 648 27.0% 449 LMG 23357 Q30U48 Q30U48_SULDN Flagellar hook-associated protein Sulfurimonas denitrificans (strain 462 31.0% 441 2-like protein ATCC 33889/DSM 1251) (Thiomicrospira denitrificans (strain ATCC 33889/DSM 1251)) H8BWB9 H8BWB9_CAMJU Flagellar capping protein Campylobacter jejuni subsp. jejuni 642 27.0% 447 1213 H8AWN7 H8AWN7_CAMJU Flagellar capping protein Campylobacter jejuni subsp. jejuni 643 26.0% 447 1997-11 A3ZDR2 A3ZDR2_CAMJU Flagellar hook-associated protein FliD Campylobacter jejuni subsp. jejuni 643 26.0% 447 HB93-13 A7H4J4 A7H4J4_CAMJD Flagellar hook-associated protein FliD Campylobacter jejuni subsp. doylei 646 26.0% 447 (strain ATCC BAA-1458/RM4099/ 269.97) A3YRI3 A3YRI3_CAMJU Flagellar hook-associated protein FliD Campylobacter jejuni subsp. jejuni 642 25.0% 442 260.94 H7WEH0 H7WEH0_CAMCO Flagellar capping protein Campylobacter coli H8 637 26.0% 441 E1PLQ8 E1PLQ8_CAMJM Flagellar hook-associated protein 2 Campylobacter jejuni subsp. jejuni 643 27.0% 441 serotype HS21 (strain M1/99/308)
EXAMPLE 9: PRESENCE AND EXPRESSION OF THE FLID IN H. PYLORI
(35) Samples
(36) 81 H. pylori isolates from human patients were enrolled in the study. The samples were diagnosed as positive by conventional bacterial culture on selective plates. In such testing, bacteria were grown on Wilkins-Chalgren blood agar plates under microaerobic conditions (10% CO2, 5% O2, 8.5% N2, and 37 C.) for 36 hours, and positivity for oxidase, catalase and urease was confirmed by biochemical testing. A part of the cultured bacteria was used for DNA isolation and the remainder was applied for preparation of protein lysate for Western blot analysis.
(37) Polyclonal Mouse Anti-FliD Sera
(38) Three C57BL6 mice were immunized 3 times (weekly) with 30 g of recombinant H. pylori FliD as antigen and 10 g CT (cholera toxin) as adjuvant re-suspended in PBS. One week after the last immunization boost, mice were bleed and sera were pooled. The antigenicity and specificity of the pooled sera was tested in a Western blot analysis.
(39) Western Blot Analysis
(40) To establish the optimal conditions of the assay, different concentration of the recombinant FliD protein as well as other recombinant control proteins (Tig (Trigger factor (Tomb et al., 1997)) and gGT) generated and purified under the same conditions, were applied on 8% SDS gels. After blotting of the proteins on nitrocellulose membrane (Whatman/GE Healthcare, Freiburg, Germany), membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated overnight with different dilutions of the anti-sera as primary antibodies. After incubation of the membranes with HRP-labeled anti-mouse IgG, bands were detectable by adding of ECL Western Blotting Detection reagents.
(41) The results are shown in
(42) PCR Analysis of the Presence of the FliD's ORF in H. pylori's Genome
(43) Four PCRs were designed based on the DNA sequence of the FliD as subject to SEQ ID NO: 2. Specificity of each primer pair as indicated in Table 5 was confirmed by blast analysis against all bacterial nucleotide sequences of the gene bank. PCRs were established using H. pylori DNA as positive control and genomic DNA of 10 other microorganisms as negative controls. PCRs were performed using GoTaq polymerase master mix (Promega), annealing temperature of 56 C. and 30 seconds extension time.
(44) TABLE-US-00005 TABLE5 PrimersusedforPCRanalysis. Lengthof theamplicon Forwardprimer Reverseprimer (bp) PCR1 AGCTCATTAGGGCTT GCTCGCGCTCAACGCATC 246 GGCAG(SEQIDNO:21) (SEQIDNO:22) PCR2 ATCACGGACGCTACC AGGGACTTCATGCATGCT 288 AATGG(SEQIDNO:23) CC(SEQIDNO:24) PCR3 CACAGACGCTATCAT CCCGCTGATCACATCATT 300 TCAAGC(SEQIDNO:25) GAC(SEQIDNO:26) PCR4 CGCTAACCTCATAGA TAAGCGGCAAAGCGCTCC 150 TGGAGG(SEQIDNO:27) G(SEQIDNO:28)
Results
(45) ORF of the FliD is presented in all H. pylori patient isolates (cultured bacteria isolated from patient biopsies). Presence of the ORF of the FliD could be confirmed by all four PCRs used for this assay. PCR1, PCR2 and PCR3 performed by isolated DNA from 81 H. pylori samples were overall positive. Whereas the PCR4 was positive for 79 samples (
(46) As may be taken from
(47) As to the detection of FliD protein in samples from patients having been diagnosed as H. pylori-positive, FliD protein is detectable in 97.5% of the samples. Using Western blot analysis it could be demonstrated that the expression of the FliD protein is detectable in 79 out of 81 H. pylori protein lysates. The results are shown in
(48) The specificity of the assay was confirmed through negative results when protein lysates from other microorganisms were analyzed by Western blot analysis. The results thereof are indicated in
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(50) The features of the present invention disclosed in the specification, the sequence listing, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.