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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Development of A Lateral Flow Immunoassay Strip for Rapid Detection of CagA Antigen of Helicobacter pylori a
Cebrail Karakus a
Department of Biology, Fatih University, Buyukcekmece, Istanbul, Turkey Accepted author version posted online: 25 Aug 2014.Published online: 20 Oct 2014.
Click for updates To cite this article: Cebrail Karakus (2015) Development of A Lateral Flow Immunoassay Strip for Rapid Detection of CagA Antigen of Helicobacter pylori, Journal of Immunoassay and Immunochemistry, 36:3, 324-333, DOI: 10.1080/15321819.2014.952440 To link to this article: http://dx.doi.org/10.1080/15321819.2014.952440
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Immunoassay and Immunochemistry, 36:324–333, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.952440
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DEVELOPMENT OF A LATERAL FLOW IMMUNOASSAY STRIP FOR RAPID DETECTION OF CagA ANTIGEN OF HELICOBACTER PYLORI
Cebrail Karakus Department of Biology, Fatih University, Buyukcekmece, Istanbul, Turkey
About half of the world populations are known to be infected with Helicobacter pylori. The CagA antigen secreting strains provoke severe mucosal damages and act as a risk factor for the development of peptic ulceration and gastric cancer. A lateral flow immunoassay (LFIA) strip was developed based on sandwich format for rapid detection of CagA antigen of H. pylori using gold conjugated monoclonal antibody. This LFIA strip will provide a good aid in the diagnosis of CagAsecreting H. pylori within 10 min instead of time consuming, expensive and laborious invasive approaches. Keywords Helicobacter pylori, lateral flow immunoassay strip, CagA antigen, colloidal gold
INTRODUCTION Approximately 50% of the world populations are known to be infected with Helicobacter pylori.[1] The CagA antigen encoded by the cytotoxinassociated gene (cagA) is considered to be one of the key virulence factors of H. pylori, since cagA-positive strains provoke severe mucosal damages and act as a risk factor for the development of peptic ulceration and gastric cancer.[2−5] Thus, diagnosis of cagA-positive H. pylori infection is a more reliable method to be carried out for treatment and for prevention of the disease. Unfortunately, the cagA gene status can only be determined by molecular techniques (PCR) that requires the use of invasive method (endoscopy) for biopsy collection. It can, however, be evaluated by detecting CagA antigen in stool sample by the ELISA (a non invasive approach) which indicates current infection with H. pylori.[6] The lateral flow immunoassay (LFIA) strip is another alternative for the ELISA that can be used in the detection of such antigen.[7,8] The available LFIA strips are Address correspondence to Cebrail Karakus, Department of Biology, Fatih University, Buyukcekmece, 34500, Istanbul, Turkey. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/ljii.
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used to detect bacteria,[9−12] viruses,[13,14] hormones,[15] pesticides,[16,17] fungal toxins,[18,19] drug residues,[20−22] and proteins such as human serum albumin.[23] The advantages of the LFIA strip are to be rapid, simple to perform, inexpensive, carriable, very sensitive, and specific. They are used without complex tools and experience at a health care unit directly, and can be stored at room temperature for 12–24 months.[24,25] The aim of this study is to develop a LFIA strip using gold conjugated monoclonal antibody (Mab) to detect the CagA antigen of H. pylori rapidly. No such test for the detection of H. pylori CagA antigen is being reported or commercially available. This test will provide a tool for the determination of pathological potential (CagA) of H. pylori strains in symptomatic subjects, determine an active status of infection, provide a predictive evaluation for the progression of the disease and eliminate the need for additional endoscopies in the follow up of eradication therapy. MATERIALS Immunoreagents Mouse B237H Mab that recognizes an epitope localized within the 580 amino acids of H. pylori CagA C-terminal end and mouse B818M Mab that recognizes an epitope localized between 562 and 795 amino acids of H. pylori CagA were purchased from Abcam (Cambridge, England). Goat anti-mouse IgG (AP124) polyclonal antibody (Pab) was purchased from Millipore (MA, USA). A recombinant CagA antigen (rCagA) prepared before in our laboratory by cloning the 5 conserved region of cagA gene of H. pylori strain isolated from a Turkish patient. Chemicals Colloidal gold (20 nm), sucrose, bovine serum albumin (BSA), sodium chloride (NaCl), tris, Tween 20, phosphate buffered saline (PBS) (pH 7,4), sodium azide (NaN3), sodium carbonate (Na2CO3), sodium borate (Na2B4O7•10H2O), EDTA, and Protein Quantification Kit-Rapid was obtained from Sigma–Aldrich (MO, USA). 5% sheep blood agar was purchased from Salubris (Istanbul, Turkey). Silica gel from the commercial products was used as desiccant. Pads and Membrane For the development of immunochromatographic assay the following materials were purchased from Millipore (MA, USA): Cellulose fiber sample and absorbent pad, glass fiber conjugate pad, and Hi-Flow Plus
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240 Membrane Card. The membrane with a nominal thickness of 135 µm have been coated on 50 µm plastic backing card (∼185 µm overall thickness) to allow cutting and handling and it has a nominal capillary flow time of 240 s/4 cm. This membrane card is provided with adhesive sections for the lamination of pads.
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METHOD Preparation of Gold Conjugated Monoclonal Antibody Both B237H and B818M anti-CagA Mabs were conjugated to 20 nm gold nanoparticle as described previously.[26] 20 nm colloidal gold solution was adjusted to pH 9.0 with 0.2 mM sodium carbonate. Then, the optimum antibody concentration for labeling was determined by the following steps: 25 µL of anti-CagA Mab solution was 2-fold diluted in double distilled water and then 25 µL of colloidal gold solution was added. Mixtures were incubated for 15 min at room temperature (RT) and then 100 µL of 10% NaCl solution was added. The color of samples gradually changed from brilliant red to blue as the concentration of Mab decreased. The optimum concentration of Mab for gold labeling was the lowest concentration of Mab solution that did not change color. 200 µL of anti-CagA Mab solution at the optimum concentration was added drop by drop to 1 mL colloidal gold solution (pH 9.0) stirred vigorously on magnetic shaker and then incubated for 45 min at room temperature. After adding 100 µL of 10% BSA solution in 20 mM sodium borate (pH 9.0), the mixture was incubated for another 15 min at RT. Labeled Mab was washed two times with 20 mM sodium borate (pH 9.0) containing 1% BSA and 0.1% sodium azide then centrifuged at 25000×g at 10◦ C for 30 min. The precipitate was resuspended in 1 mL washing buffer and stored at 4◦ C until used. Dot Blot Assay to Test Gold Conjugated Monoclonal Antibody Gold conjugated B237H and B818M anti-CagA Mabs were tested using the direct dot blot assay to confirm that these recognize our rCagA protein. 2 µL of cagA-positive H. pylori whole-cell lysate (800 µg/mL), rCagA (80 µg/mL), and goat anti-mouse Pab (500 µg/mL) which was diluted in PBS containing 0.01% Tween 20 and 0.1% BSA were spotted onto the nitrocellulose membrane. The goat anti-mouse Pab was used as internal positive control. The membrane was let to dry for 30 min at RT. Non specific sites were blocked by incubating into 5% BSA in PBS-T (Phosphate buffered saline containing 0.02% Tween 20) for 0.5–1 hr at RT. Then, the membrane was incubated with gold conjugated anti-CagA Mab solution for overnight at
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RT with shaking at low speed. The membrane was washed three times with PBS-T (3 x 5 min), then once with PBS (5 min) on the shaker. The presence of red color was an indicator of positive reaction.
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Preparation of the Conjugate Pad The conjugate pad was prepared as described previously with the minor differences.[22] The prepared 1 mL gold conjugated anti-CagA Mab-B237H was centrifuged (25000×g) at 10◦ C for 30 min to remove washing buffer. Then, the pellet was resuspended with 300 µL, 20 mM sodium borate buffer (pH 8.0) containing 8.75% (w/v) sucrose, 8.75% (w/v) BSA, 0.6 M NaCl, 10 mM EDTA, and 0.1% (w/v) NaN3. The G041 Glass Fiber conjugate pad was cut in size of 5×10 mm. It was dipped in the conjugate solution and held up to allow all excess to run back into the tube. The material was then laid flat on a nonabsorbent surface and dried for 3 hr at 37◦ C in a container with desiccant. The pad was stored with desiccant at RT to prevent rehydration. The C083 Cellulose Fiber sample pad and absorbent pad were cut in size of 5×20 mm. They were adhered directly to the adhesive backing in proper size. Immobilization of Capture Antibodies 2 µL of B818M anti-CagA Mab and 2 µL of goat anti-mouse Pab were immobilized on the Hi-Flow Plus 240 Membrane Card as Test line and Control line, respectively. After drying for 1h at 37◦ C with desiccant, non specific sites on the membrane were blocked with 4% BSA in PBS-T (0.04% Tween 20) for 1 hr. The membrane was washed once with PBS-T (0.04 % Tween 20), dried and stored at RT with desiccant. Test Strip Assembly The absorbent pad, conjugate pad and sample pad were laminated onto the Hi-Flow Plus 240 Membrane Card (5 x 60 mm) to ensure a continuous flow of sample and a proper function of the strip as illustrated in Figures 1A and 1B. The strip was housed in a plastic cassette, where only the sample application window and a reading window were exposed, for protection and easier handling. The test strip was stored in the presence of desiccant at RT. Test Principle This LFIA strip utilizes an immunochromatographic assay based on double-antibody sandwich format for the detection of CagA antigen of H.
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FIGURE 1 Test strip assembly. This figure shows the assembly of the conjugate pad, sample pad and absorbent pad on the membrane background (A). A side view of the assembled strip (B).
pylori (Figure 2). The test strip and samples were brought to RT before testing. 130 µL rCagA samples diluted in PBS were applied to sample pad of separate rapid CagA test strips to determine analytical sensitivity of the test. The sample pad allowed the diffusion of the sample into the conjugate pad that is impregnated with gold conjugated anti-CagA Mab-B237H (detector reagent) and rCagA into the sample bound to the detector reagent. The complex continued to flow by capillary action and then irreversibly bound to immobilized anti-CagA Mab-B818M (capture reagent) at the test line on the membrane and resulted in a red color line (positive result). The flow of the sample fluid continued toward the control line where the detector reagents were bound to the immobilized goat anti-mouse Pab and gave a red color line to indicate that the test has been correctly performed and the test device functioned properly. The more rCagA present in the sample, the stronger color of the test line. The absorbent pad at the end of the strip wicks the fluid through the membrane to ensure a continuous flow and thus maintains a clear background. If rCagA antigen was lower than the detection limit of the test, then only the control line was visible (negative result). If the control line shows no color, then the test is considered invalid, and similarly no results were interpreted after 30 min. Investigation of Cross Reactivity H. pylori strains and closely related microorganisms were used to test the analytical specificity of rapid CagA test strip. cagA-positive H. pylori strains from Turkish patients (722 and 13,206) which have been stored at -80◦ C
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FIGURE 2 The components of the lateral flow immunoassay strip in sandwich format for the detection of CagA is represented before (A) and after (B) the addition of the positive sample.
FIGURE 3 Identification the H. pylori by colony morphology (A), positive rapid urease test (B), positive catalase test (C), and gram staining (D).
were incubated at 37◦ C for 5 min and then inoculated onto colombia agar (5% sheep red blood cell) and incubated under microaerophilic conditions in a CO2 incubator at 37◦ C for 5–7 days. The growth culture of H. pylori was identified by colony morphology, gram stain and positive reactions to catalase and urease activities (Figure 3). Escherichia coli, Proteus vulgaris, and Staphylococcus aureus were transfered to nutrient broth media from storage separately and incubated at 37◦ C for 2 days. Bacterial cultures were centrifuged at 12000xg for 10 min and the pellet was resuspended in PBS (pH 7,4) and then disrupted by sonication at 20,000 Hz for 5 times with 45 s intervals. The supernatant was then collected (bacterium whole-cell lysate) by centrifugation.[27]
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The concentrations of the lysates were measured with Bradford assay by using Protein Quantification Kit-Rapid. 800 µg/mL concentrated lysates of bacteria were tested for cross-reactivity with rapid CagA test strips separately. RESULTS
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Preparation and Testing the Gold Conjugated Monoclonal Antibody It was found that the optimum concentration of B237H and B818M antiCagA Mabs to be conjugated with gold nanoparticle were 32 µg/mL and 64 µg/mL, respectively. The gold conjugated anti-CagA Mab-B237H and the gold conjugated anti-CagA Mab-B818M bound to the both cagA-positive H. pylori lysate and the rCagA according to red colored dots appeared on the membrane. Clear background on the membrane represented negative control and the red colored dot on the area of goat anti-mouse Pab represented positive control (Table 1). Analytical Sensitivity of the Test Strip The analytical sensitivity of the test strip was determined by testing the rCagA supplemented PBS with the concentrations of 0, 2, 4, 8, 16, 32, and 64 µg/mL. The result was obtained in approximately 10 min. The lower detection limit of the test strip by visual detection was 4 µg/mL (Figure 4). The control line was coated with secondary (anti-IgG) antibody to capture excess detector reagents regardless of the presence or absence of TABLE 1 Direct dot blot assay to test the reactivity of gold conjugated anti-CagA Mab-B237H and gold conjugated anti-CagA Mab-B818M showed that both recognized the nature CagA in H. pylori and recombinant CagA (rCagA). Red dot on the area of goat anti-mouse Pab indicated positive control and clear background indicated negative control Immobilized proteins on the nitrocellulose membrane Gold conjugated Mab incubated with membrane Anti-CagA Mab-B237H
Anti-CagA Mab-B818M
cagA-positive H. pylori lysate
rCagA
Goat anti-mouse Pab
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FIGURE 4 rCagA protein concentrations (µg/mL) was used to determine analytical sensitivity of the test strip and the minimum concentration that resulted a colored test line was 4 µg/mL. (C: Control line, T: Test line, S: Sample application window).
target CagA antigen and it was in red color for each test (Figure 4). The control line and the clear background that appeared on the reading window were the indicators of an internal positive and internal negative procedural control, respectively. Analytical Specificity of the Test Strip Closely related microorganisms were evaluated for cross reactivity with the test strip. Whole cell lysates of E. coli, P. Vulgaris, and S. aureus were supplemented into the rCagA-positive and rCagA-negative samples and tested separately. None of the microorganisms tested yielded a positive result in the negative samples or interfered with detection of the positive samples; rCagApositive sample remained positive and rCagA-negative sample remained negative with the added organisms. Both the negative and positive samples were also positive when supplemented with cagA-positive H. pylori strains 722 and 13206.
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CONCLUSION A LFIA strip to detect the CagA antigen of H. pylori qualitatively was developed using gold conjugated monoclonal antibody. It showed no crossreactivity with closely related bacteria and no interference with bacterial proteins. This LFIA strip will provide a good support and aid in the diagnosis of cagA-positive H. pylori infection instead of time consuming, expensive and laborious invasive approaches. Such a test strip will help a great deal to determine the active CagA status in H. pylori infected patients particularly those with gastritis since CagA antigen plays an important role in the progression of the disease in such patients. It can also provide a predictive evaluation whether or not to treat such patients in order to block future progression to peptic ulceration and gastric cancer, and eliminate the need for additional endoscopies in the follow up of eradication therapy. To the best of our knowledge, this is the first LFIA strip developed to detect the CagA antigen of H. pylori using gold conjugated monoclonal antibody at the world. FUNDING This research was supported by The Scientific Research Project office of the Fatih University with the project number P500211002_B. REFERENCES 1. Kimmel, B.; Bosserhoff, A.; Frank, R.; Gross, R.; Goebel, W.; Beier, D. Identification of Immunodominant Antigens from Helicobacter pylori and Evaluation of their Reactivities with Sera from Patients with Different Gastroduodenal Pathologies. Infect. Immun. 2000, 68, 915–920. 2. Ong, S.P.; Duggan, A. Eradication of Helicobacter pylori in Clinical Situations. Clin. Exp. Med. 2004, 4, 30–38. 3. Rudi, J.; Kuck, D.; Rudy, A.; Sieg, A.; Maiwald, M.; Stremmel, W. Helicobacter pylori vacA Genotypes and cagA Gene in a Series of 383 H. pylori-Positive Patients. Z. Gastroenterol. 2000, 38, 559–564. 4. Salih, B.A.; Abasiyanik, M.F.; Ahmed, N. A Preliminary Study on the Genetic Profile of cag Pathogenicity-Island and Other Virulent Gene Loci of Helicobacter pylori Strains from Turkey. Infect. Genet. Evol. 2007, 7 , 509–512. 5. Saribasak, H.; Salih, B.A.; Yamaoka, Y.; Sander, E. Analysis of Helicobacter pylori Genotypes and Correlation with Clinical Outcome in Turkey. J. Clin. Microbiol. 2004, 42, 1648–1651. 6. Ricci, C.; Holton, J.; Vaira, D. Diagnosis of Helicobacter pylori: Invasive and Non-Invasive Tests. Best. Pract. Res. Clin. Gastroenterol. 2007, 21, 299–313. 7. Karakus, C.; Salih, B.A. Comparison of the Lateral Flow Immunoassays (LFIA) for the Diagnosis of Helicobacter pylori Infection. J. Immunol. Methods. 2013, 396, 8–14. 8. Kato, S.; Ozawa, K.; Okuda, M.; Nakayama, Y.; Yoshimura, N.; Konno, M.; Minoura, T.; Iinuma, K. Multicenter Comparison of Rapid Lateral Flow Stool Antigen Immunoassay and Stool Antigen Enzyme Immunoassay for the Diagnosis of Helicobacter pylori Infection in Children. Helicobacter . 2004, 9, 669–673. 9. Kawatsu, K.; Kumeda, Y.; Taguchi, M.; Yamazaki-Matsune, W.; Kanki, M.; Inoue, K. Development and Evaluation of Immunochromatographic Assay for Simple and Rapid Detection of Campylobacter jejuni and Campylobacter coli in Human Stool Specimens. J. Clin. Microbiol. 2008, 46, 1226–1231.
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10. Nakasone, N.; Toma, C.; Lu, Y.; Iwanaga, M. Development of a Rapid Immunochromatographic Test to Identify Enteropathogenic and Enterohemorrhagic Escherichia coli by Detecting EspB. Diagn. Microbiol. Infect. Dis. 2007, 57 , 21–25. 11. Wang, Y.; Dou, H.; Chen, K.; Zhang, H.; Hu, C. Development of a Colloidal Gold-Based Immunochromatographic Test Strip for the Rapid, On-Site Detection of Pseudomonas aeruginosa in Clinical Samples. Scand. J. Infect. Dis. 2011, 43, 329–338. 12. Yu, C.Y.; Ang, G.Y.; Chua, A.L.; Tan, E.H.; Lee, S.Y.; Falero-Diaz, G.; Otero, O.; Rodriguez, I.; Reyes, F.; Acosta, A.; Sarmiento, M.E.; Ghosh, S.; Ramamurthy, T.; Yean Yean, C.; Lalitha, P.; Ravichandran, M. Dry-Reagent Gold Nanoparticle-Based Lateral Flow Biosensor for the Simultaneous Detection of Vibrio cholerae Serogroups O1 and O139. J. Microbiol. Meth. 2011, 86, 277–282. 13. Cui, S.; Zhou, S.; Chen, C.; Qi, T.; Zhang, C.; Oh, J. A Simple and Rapid Immunochromatographic Strip Test for Detecting Antibody to Porcine Reproductive and Respiratory Syndrome Virus. J. Virol. Meth. 2008, 152, 38–42. 14. Peng, F.; Wang, Z.; Zhang, S.; Wu, R.; Hu, S.; Li, Z.; Wang, X.; Bi, D. Development of an Immunochromatographic Strip for Rapid Detection of H9 Subtype Avian Influenza Viruses. Clin. Vaccine Immunol. 2008, 15, 569–574. 15. Posthuma-Trumpie, G.A.; Korf, J.; van Amerongen, A. Development of a Competitive Lateral Flow Immunoassay for Progesterone: Influence of Coating Conjugates and Buffer Components. Anal. Bioanal. Chem. 2008, 392, 1215–1223. 16. Guo, Y.R.; Liu, S.Y.; Gui, W.J.; Zhu, G.N. Gold Immunochromatographic Assay for Simultaneous Detection of Carbofuran and Triazophos in Water Samples. Anal. Biochem. 2009, 389, 32–39. 17. Zhou, P.; Lu, Y.; Zhu, J.; Hong, J.; Li, B.; Zhou, J.; Gong, D.; Montoya, A. Nanocolloidal Gold-Based Immunoassay for the Detection of the N-methylcarbamate Pesticide Carbofuran. J. Agric. Food Chem. 2004, 52, 4355–4359. 18. Kolosova, A.Y.; De Saeger, S.; Sibanda, L.; Verheijen, R.; Van Peteghem, C. Development of a Colloidal Gold-Based Lateral-Flow Immunoassay for the Rapid Simultaneous Detection of Zearalenone and Deoxynivalenol. Anal. Bioanal. Chem. 2007, 389, 2103–2107. 19. Wang, X.H.; Liu, T.; Xu, N.; Zhang, Y.; Wang, S. Enzyme-linked Immunosorbent Assay and Colloidal Gold Immunoassay for Ochratoxin A: Investigation of Analytical Conditions and Sample Matrix on Assay Performance. Anal. Bioanal. Chem. 2007, 389, 903–911. 20. Omidfar, K.; Kia, S.; Kashanian, S.; Paknejad, M.; Besharatie, A.; Larijani, B. Colloidal Nanogoldbased Immunochromatographic Strip Test for the Detection of Digoxin Toxicity. Appl. Biochem. Biotechnol. 2010, 160, 843–855. 21. Xie, H.; Ma, W.; Liu, L.; Chen, W.; Peng, C.; Xu, C.; Wang, L. Development and Validation of an Immunochromatographic Assay for Rapid Multi-Residues Detection of Cephems in Milk. Anal. Chim. Acta. 2009, 634, 129–133. 22. Zhang, G.P.; Wang, X.N.; Yang, J.F.; Yang, Y.Y.; Xing, G.X.; Li, Q.M.; Zhao, D.; Chai, S. J.; Guo, J. Q. Development of an Immunochromatographic Lateral Flow Test Strip for Detection of Beta-Adrenergic Agonist Clenbuterol Residues. J. Immunol. Methods. 2006, 312, 27–33. ˙ 23. Omidfar, K.; Kia, S.; Larijani, B. Development of a Colloidal Gold-Based Immunochromatographic Test Strip for Screening of Microalbuminuria. Hybridoma 2011, 30(2), 117–124. 24. Ngom, B.; Guo, Y.; Wang, X.; Bi, D. Development and Application of Lateral Flow Test Strip Technology for Detection of Infectious Agents and Chemical Contaminants: A Review. Anal. Bioanal. Chem. 2010, 397 , 1113–1135. ˙ ˙ Strengths, 25. Posthuma-Trumpie, G.A.; Korf, J.; van Amerongen, A. Lateral Flow (Immuno)assay: Its Weaknesses, Opportunities and Threats. A Literature Survey. Anal. Bioanal. Chem. 2009, 393, 569–582. 26. Yokota, S. Preparation of Colloidal Gold Particles and Conjugation to Protein A, IgG, F(ab’)(2), and Streptavidin. Meth. Mol. Biol. 2010, 657 , 109–119. 27. Shin, J.H.; Nam, S.W.; Kim, J.T.; Yoon, J.B.; Bang, W.G.; Roe, I.H. Identification of Immunodominant Helicobacter pylori Proteins with Reactivity to H. pylori-specific Egg-Yolk Immunoglobulin. J. Med. Microbiol. 2003, 52, 217–222.
Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Development of A Lateral Flow Immunoassay Strip for Rapid Detection of CagA Antigen of Helicobacter pylori a
Cebrail Karakus a
Department of Biology, Fatih University, Buyukcekmece, Istanbul, Turkey Accepted author version posted online: 25 Aug 2014.Published online: 20 Oct 2014.
Click for updates To cite this article: Cebrail Karakus (2015) Development of A Lateral Flow Immunoassay Strip for Rapid Detection of CagA Antigen of Helicobacter pylori, Journal of Immunoassay and Immunochemistry, 36:3, 324-333, DOI: 10.1080/15321819.2014.952440 To link to this article: http://dx.doi.org/10.1080/15321819.2014.952440
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Immunoassay and Immunochemistry, 36:324–333, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.952440
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DEVELOPMENT OF A LATERAL FLOW IMMUNOASSAY STRIP FOR RAPID DETECTION OF CagA ANTIGEN OF HELICOBACTER PYLORI
Cebrail Karakus Department of Biology, Fatih University, Buyukcekmece, Istanbul, Turkey
About half of the world populations are known to be infected with Helicobacter pylori. The CagA antigen secreting strains provoke severe mucosal damages and act as a risk factor for the development of peptic ulceration and gastric cancer. A lateral flow immunoassay (LFIA) strip was developed based on sandwich format for rapid detection of CagA antigen of H. pylori using gold conjugated monoclonal antibody. This LFIA strip will provide a good aid in the diagnosis of CagAsecreting H. pylori within 10 min instead of time consuming, expensive and laborious invasive approaches. Keywords Helicobacter pylori, lateral flow immunoassay strip, CagA antigen, colloidal gold
INTRODUCTION Approximately 50% of the world populations are known to be infected with Helicobacter pylori.[1] The CagA antigen encoded by the cytotoxinassociated gene (cagA) is considered to be one of the key virulence factors of H. pylori, since cagA-positive strains provoke severe mucosal damages and act as a risk factor for the development of peptic ulceration and gastric cancer.[2−5] Thus, diagnosis of cagA-positive H. pylori infection is a more reliable method to be carried out for treatment and for prevention of the disease. Unfortunately, the cagA gene status can only be determined by molecular techniques (PCR) that requires the use of invasive method (endoscopy) for biopsy collection. It can, however, be evaluated by detecting CagA antigen in stool sample by the ELISA (a non invasive approach) which indicates current infection with H. pylori.[6] The lateral flow immunoassay (LFIA) strip is another alternative for the ELISA that can be used in the detection of such antigen.[7,8] The available LFIA strips are Address correspondence to Cebrail Karakus, Department of Biology, Fatih University, Buyukcekmece, 34500, Istanbul, Turkey. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/ljii.
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used to detect bacteria,[9−12] viruses,[13,14] hormones,[15] pesticides,[16,17] fungal toxins,[18,19] drug residues,[20−22] and proteins such as human serum albumin.[23] The advantages of the LFIA strip are to be rapid, simple to perform, inexpensive, carriable, very sensitive, and specific. They are used without complex tools and experience at a health care unit directly, and can be stored at room temperature for 12–24 months.[24,25] The aim of this study is to develop a LFIA strip using gold conjugated monoclonal antibody (Mab) to detect the CagA antigen of H. pylori rapidly. No such test for the detection of H. pylori CagA antigen is being reported or commercially available. This test will provide a tool for the determination of pathological potential (CagA) of H. pylori strains in symptomatic subjects, determine an active status of infection, provide a predictive evaluation for the progression of the disease and eliminate the need for additional endoscopies in the follow up of eradication therapy. MATERIALS Immunoreagents Mouse B237H Mab that recognizes an epitope localized within the 580 amino acids of H. pylori CagA C-terminal end and mouse B818M Mab that recognizes an epitope localized between 562 and 795 amino acids of H. pylori CagA were purchased from Abcam (Cambridge, England). Goat anti-mouse IgG (AP124) polyclonal antibody (Pab) was purchased from Millipore (MA, USA). A recombinant CagA antigen (rCagA) prepared before in our laboratory by cloning the 5 conserved region of cagA gene of H. pylori strain isolated from a Turkish patient. Chemicals Colloidal gold (20 nm), sucrose, bovine serum albumin (BSA), sodium chloride (NaCl), tris, Tween 20, phosphate buffered saline (PBS) (pH 7,4), sodium azide (NaN3), sodium carbonate (Na2CO3), sodium borate (Na2B4O7•10H2O), EDTA, and Protein Quantification Kit-Rapid was obtained from Sigma–Aldrich (MO, USA). 5% sheep blood agar was purchased from Salubris (Istanbul, Turkey). Silica gel from the commercial products was used as desiccant. Pads and Membrane For the development of immunochromatographic assay the following materials were purchased from Millipore (MA, USA): Cellulose fiber sample and absorbent pad, glass fiber conjugate pad, and Hi-Flow Plus
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240 Membrane Card. The membrane with a nominal thickness of 135 µm have been coated on 50 µm plastic backing card (∼185 µm overall thickness) to allow cutting and handling and it has a nominal capillary flow time of 240 s/4 cm. This membrane card is provided with adhesive sections for the lamination of pads.
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METHOD Preparation of Gold Conjugated Monoclonal Antibody Both B237H and B818M anti-CagA Mabs were conjugated to 20 nm gold nanoparticle as described previously.[26] 20 nm colloidal gold solution was adjusted to pH 9.0 with 0.2 mM sodium carbonate. Then, the optimum antibody concentration for labeling was determined by the following steps: 25 µL of anti-CagA Mab solution was 2-fold diluted in double distilled water and then 25 µL of colloidal gold solution was added. Mixtures were incubated for 15 min at room temperature (RT) and then 100 µL of 10% NaCl solution was added. The color of samples gradually changed from brilliant red to blue as the concentration of Mab decreased. The optimum concentration of Mab for gold labeling was the lowest concentration of Mab solution that did not change color. 200 µL of anti-CagA Mab solution at the optimum concentration was added drop by drop to 1 mL colloidal gold solution (pH 9.0) stirred vigorously on magnetic shaker and then incubated for 45 min at room temperature. After adding 100 µL of 10% BSA solution in 20 mM sodium borate (pH 9.0), the mixture was incubated for another 15 min at RT. Labeled Mab was washed two times with 20 mM sodium borate (pH 9.0) containing 1% BSA and 0.1% sodium azide then centrifuged at 25000×g at 10◦ C for 30 min. The precipitate was resuspended in 1 mL washing buffer and stored at 4◦ C until used. Dot Blot Assay to Test Gold Conjugated Monoclonal Antibody Gold conjugated B237H and B818M anti-CagA Mabs were tested using the direct dot blot assay to confirm that these recognize our rCagA protein. 2 µL of cagA-positive H. pylori whole-cell lysate (800 µg/mL), rCagA (80 µg/mL), and goat anti-mouse Pab (500 µg/mL) which was diluted in PBS containing 0.01% Tween 20 and 0.1% BSA were spotted onto the nitrocellulose membrane. The goat anti-mouse Pab was used as internal positive control. The membrane was let to dry for 30 min at RT. Non specific sites were blocked by incubating into 5% BSA in PBS-T (Phosphate buffered saline containing 0.02% Tween 20) for 0.5–1 hr at RT. Then, the membrane was incubated with gold conjugated anti-CagA Mab solution for overnight at
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RT with shaking at low speed. The membrane was washed three times with PBS-T (3 x 5 min), then once with PBS (5 min) on the shaker. The presence of red color was an indicator of positive reaction.
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Preparation of the Conjugate Pad The conjugate pad was prepared as described previously with the minor differences.[22] The prepared 1 mL gold conjugated anti-CagA Mab-B237H was centrifuged (25000×g) at 10◦ C for 30 min to remove washing buffer. Then, the pellet was resuspended with 300 µL, 20 mM sodium borate buffer (pH 8.0) containing 8.75% (w/v) sucrose, 8.75% (w/v) BSA, 0.6 M NaCl, 10 mM EDTA, and 0.1% (w/v) NaN3. The G041 Glass Fiber conjugate pad was cut in size of 5×10 mm. It was dipped in the conjugate solution and held up to allow all excess to run back into the tube. The material was then laid flat on a nonabsorbent surface and dried for 3 hr at 37◦ C in a container with desiccant. The pad was stored with desiccant at RT to prevent rehydration. The C083 Cellulose Fiber sample pad and absorbent pad were cut in size of 5×20 mm. They were adhered directly to the adhesive backing in proper size. Immobilization of Capture Antibodies 2 µL of B818M anti-CagA Mab and 2 µL of goat anti-mouse Pab were immobilized on the Hi-Flow Plus 240 Membrane Card as Test line and Control line, respectively. After drying for 1h at 37◦ C with desiccant, non specific sites on the membrane were blocked with 4% BSA in PBS-T (0.04% Tween 20) for 1 hr. The membrane was washed once with PBS-T (0.04 % Tween 20), dried and stored at RT with desiccant. Test Strip Assembly The absorbent pad, conjugate pad and sample pad were laminated onto the Hi-Flow Plus 240 Membrane Card (5 x 60 mm) to ensure a continuous flow of sample and a proper function of the strip as illustrated in Figures 1A and 1B. The strip was housed in a plastic cassette, where only the sample application window and a reading window were exposed, for protection and easier handling. The test strip was stored in the presence of desiccant at RT. Test Principle This LFIA strip utilizes an immunochromatographic assay based on double-antibody sandwich format for the detection of CagA antigen of H.
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FIGURE 1 Test strip assembly. This figure shows the assembly of the conjugate pad, sample pad and absorbent pad on the membrane background (A). A side view of the assembled strip (B).
pylori (Figure 2). The test strip and samples were brought to RT before testing. 130 µL rCagA samples diluted in PBS were applied to sample pad of separate rapid CagA test strips to determine analytical sensitivity of the test. The sample pad allowed the diffusion of the sample into the conjugate pad that is impregnated with gold conjugated anti-CagA Mab-B237H (detector reagent) and rCagA into the sample bound to the detector reagent. The complex continued to flow by capillary action and then irreversibly bound to immobilized anti-CagA Mab-B818M (capture reagent) at the test line on the membrane and resulted in a red color line (positive result). The flow of the sample fluid continued toward the control line where the detector reagents were bound to the immobilized goat anti-mouse Pab and gave a red color line to indicate that the test has been correctly performed and the test device functioned properly. The more rCagA present in the sample, the stronger color of the test line. The absorbent pad at the end of the strip wicks the fluid through the membrane to ensure a continuous flow and thus maintains a clear background. If rCagA antigen was lower than the detection limit of the test, then only the control line was visible (negative result). If the control line shows no color, then the test is considered invalid, and similarly no results were interpreted after 30 min. Investigation of Cross Reactivity H. pylori strains and closely related microorganisms were used to test the analytical specificity of rapid CagA test strip. cagA-positive H. pylori strains from Turkish patients (722 and 13,206) which have been stored at -80◦ C
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FIGURE 2 The components of the lateral flow immunoassay strip in sandwich format for the detection of CagA is represented before (A) and after (B) the addition of the positive sample.
FIGURE 3 Identification the H. pylori by colony morphology (A), positive rapid urease test (B), positive catalase test (C), and gram staining (D).
were incubated at 37◦ C for 5 min and then inoculated onto colombia agar (5% sheep red blood cell) and incubated under microaerophilic conditions in a CO2 incubator at 37◦ C for 5–7 days. The growth culture of H. pylori was identified by colony morphology, gram stain and positive reactions to catalase and urease activities (Figure 3). Escherichia coli, Proteus vulgaris, and Staphylococcus aureus were transfered to nutrient broth media from storage separately and incubated at 37◦ C for 2 days. Bacterial cultures were centrifuged at 12000xg for 10 min and the pellet was resuspended in PBS (pH 7,4) and then disrupted by sonication at 20,000 Hz for 5 times with 45 s intervals. The supernatant was then collected (bacterium whole-cell lysate) by centrifugation.[27]
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The concentrations of the lysates were measured with Bradford assay by using Protein Quantification Kit-Rapid. 800 µg/mL concentrated lysates of bacteria were tested for cross-reactivity with rapid CagA test strips separately. RESULTS
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Preparation and Testing the Gold Conjugated Monoclonal Antibody It was found that the optimum concentration of B237H and B818M antiCagA Mabs to be conjugated with gold nanoparticle were 32 µg/mL and 64 µg/mL, respectively. The gold conjugated anti-CagA Mab-B237H and the gold conjugated anti-CagA Mab-B818M bound to the both cagA-positive H. pylori lysate and the rCagA according to red colored dots appeared on the membrane. Clear background on the membrane represented negative control and the red colored dot on the area of goat anti-mouse Pab represented positive control (Table 1). Analytical Sensitivity of the Test Strip The analytical sensitivity of the test strip was determined by testing the rCagA supplemented PBS with the concentrations of 0, 2, 4, 8, 16, 32, and 64 µg/mL. The result was obtained in approximately 10 min. The lower detection limit of the test strip by visual detection was 4 µg/mL (Figure 4). The control line was coated with secondary (anti-IgG) antibody to capture excess detector reagents regardless of the presence or absence of TABLE 1 Direct dot blot assay to test the reactivity of gold conjugated anti-CagA Mab-B237H and gold conjugated anti-CagA Mab-B818M showed that both recognized the nature CagA in H. pylori and recombinant CagA (rCagA). Red dot on the area of goat anti-mouse Pab indicated positive control and clear background indicated negative control Immobilized proteins on the nitrocellulose membrane Gold conjugated Mab incubated with membrane Anti-CagA Mab-B237H
Anti-CagA Mab-B818M
cagA-positive H. pylori lysate
rCagA
Goat anti-mouse Pab
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FIGURE 4 rCagA protein concentrations (µg/mL) was used to determine analytical sensitivity of the test strip and the minimum concentration that resulted a colored test line was 4 µg/mL. (C: Control line, T: Test line, S: Sample application window).
target CagA antigen and it was in red color for each test (Figure 4). The control line and the clear background that appeared on the reading window were the indicators of an internal positive and internal negative procedural control, respectively. Analytical Specificity of the Test Strip Closely related microorganisms were evaluated for cross reactivity with the test strip. Whole cell lysates of E. coli, P. Vulgaris, and S. aureus were supplemented into the rCagA-positive and rCagA-negative samples and tested separately. None of the microorganisms tested yielded a positive result in the negative samples or interfered with detection of the positive samples; rCagApositive sample remained positive and rCagA-negative sample remained negative with the added organisms. Both the negative and positive samples were also positive when supplemented with cagA-positive H. pylori strains 722 and 13206.
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CONCLUSION A LFIA strip to detect the CagA antigen of H. pylori qualitatively was developed using gold conjugated monoclonal antibody. It showed no crossreactivity with closely related bacteria and no interference with bacterial proteins. This LFIA strip will provide a good support and aid in the diagnosis of cagA-positive H. pylori infection instead of time consuming, expensive and laborious invasive approaches. Such a test strip will help a great deal to determine the active CagA status in H. pylori infected patients particularly those with gastritis since CagA antigen plays an important role in the progression of the disease in such patients. It can also provide a predictive evaluation whether or not to treat such patients in order to block future progression to peptic ulceration and gastric cancer, and eliminate the need for additional endoscopies in the follow up of eradication therapy. To the best of our knowledge, this is the first LFIA strip developed to detect the CagA antigen of H. pylori using gold conjugated monoclonal antibody at the world. FUNDING This research was supported by The Scientific Research Project office of the Fatih University with the project number P500211002_B. REFERENCES 1. Kimmel, B.; Bosserhoff, A.; Frank, R.; Gross, R.; Goebel, W.; Beier, D. Identification of Immunodominant Antigens from Helicobacter pylori and Evaluation of their Reactivities with Sera from Patients with Different Gastroduodenal Pathologies. Infect. Immun. 2000, 68, 915–920. 2. Ong, S.P.; Duggan, A. Eradication of Helicobacter pylori in Clinical Situations. Clin. Exp. Med. 2004, 4, 30–38. 3. Rudi, J.; Kuck, D.; Rudy, A.; Sieg, A.; Maiwald, M.; Stremmel, W. Helicobacter pylori vacA Genotypes and cagA Gene in a Series of 383 H. pylori-Positive Patients. Z. Gastroenterol. 2000, 38, 559–564. 4. Salih, B.A.; Abasiyanik, M.F.; Ahmed, N. A Preliminary Study on the Genetic Profile of cag Pathogenicity-Island and Other Virulent Gene Loci of Helicobacter pylori Strains from Turkey. Infect. Genet. Evol. 2007, 7 , 509–512. 5. Saribasak, H.; Salih, B.A.; Yamaoka, Y.; Sander, E. Analysis of Helicobacter pylori Genotypes and Correlation with Clinical Outcome in Turkey. J. Clin. Microbiol. 2004, 42, 1648–1651. 6. Ricci, C.; Holton, J.; Vaira, D. Diagnosis of Helicobacter pylori: Invasive and Non-Invasive Tests. Best. Pract. Res. Clin. Gastroenterol. 2007, 21, 299–313. 7. Karakus, C.; Salih, B.A. Comparison of the Lateral Flow Immunoassays (LFIA) for the Diagnosis of Helicobacter pylori Infection. J. Immunol. Methods. 2013, 396, 8–14. 8. Kato, S.; Ozawa, K.; Okuda, M.; Nakayama, Y.; Yoshimura, N.; Konno, M.; Minoura, T.; Iinuma, K. Multicenter Comparison of Rapid Lateral Flow Stool Antigen Immunoassay and Stool Antigen Enzyme Immunoassay for the Diagnosis of Helicobacter pylori Infection in Children. Helicobacter . 2004, 9, 669–673. 9. Kawatsu, K.; Kumeda, Y.; Taguchi, M.; Yamazaki-Matsune, W.; Kanki, M.; Inoue, K. Development and Evaluation of Immunochromatographic Assay for Simple and Rapid Detection of Campylobacter jejuni and Campylobacter coli in Human Stool Specimens. J. Clin. Microbiol. 2008, 46, 1226–1231.
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