Vol. 11, 1992
19. Milatovie D, Braveny I, Verhoef J: Clindamycin enhances opsonization of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 1983, 24: 413417. 20. SlamaTG, Sklar S,I,MisinskiJ, Fess SW: Randomized comparison of cefamandole, cefazolin and cefuroxime prophylaxis in open-heart surgery. Antimicrobial Agents and Chemotherapy 1986, 29: 744-747. 21. Yourassowsky E, Van Der Linden MP, Crokaert F: Inoculum effect on growth-delay time of oxaeillinresistant strains of Staphylococcus aureus and Staphylococcus epidermidis exposed to cefamandole, cefazolin and cefuroxime. Antimicrobial Agents and Chemotherapy 1990, 34: 505-509. 22. Stratton CW, Liu C, Weeks IS: Activity of LY 146032 compared with that of methieillin, cefazolin, cefamandole, cefuroxime, ciprofloxacin and vancomycin against staphylococci as determined by killing-kinetic studies. Antimicrobial Agents and Chemotherapy 1987, 31: 1210-1215.
Evaluation of a Monoclonal Antibody for Detection of Helicobacterpylori in a Direct Immunofluorescence Test U. R o d e w i g 1, W. B e m b 4, D. Bitter-Suermann 1, M. Elsheikh 1, S. Fritsch 2, E. G l e n n - C a l v o 1, B. S o u d a h 2, M. Varrentrapp 3, S. Wagner 3, W. B~tr 1.
A monoclonal antibody was developed for detection of Helicobacter pylori in gastric tissue sections in a direct immunofluorescence test. On a comparison of the immunofluorescence test with standard methods for detection of HeIicobacter pylori, i.e. culture, the urease activity test and histological examination of tissue sections, using 158 biopsy specimens, 30 specimens were positive in all methods and 64 negative. In the remaining cases comparison was not possible because either immunofluorescence (29 specimens) or the standard methods (16 specimens) gave ambiguous results. The direct immunofluorescence test may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.
1Institute of Medical Microbiology,2Institute of Pathology and 3Department of Gastroenterology, Medical School Hannover, 3000 Hannover 61, Germany. 4Institute of Medical Microbiology,University Clinic, 6500 Mainz, Germany.
737
The main procedures currently used for detection of Helicobacterpylori are culture, histological examination and detection of urease activity (1). However, a disadvantage-of these techniques is that they have low sensitivity (2) so that a correct diagnosis may not be established in a number of cases. Moreover, these techniques are cumbersome to use. There is thus a need for a method for detection of Helicobacter pylori that is both convenient to use, sensitive and specific. Direct immunofluorescence using monoclonal antibodies (MABs) might seem to meet these demands. Until now there have been few reports on the production of MABs against Helicobacter pylori or the use of MABs for its identification. Indirect immunofluorescence tests (3, 4) and an immunoperoxidase method have been introduced which use MABs (5), however in all cases the epitopes are formaldehyde-sensitive so that the handling of biopsy material in these techniques has proved complicated. We therefore developed an M A B directed against Helicobacter pylori lipopolysaccharide and tested it in a direct immunofluorescence method (DIF) using gastric biopsy specimens.
Materials and Methods. Helicobacter pylori was isolated from gastric biopsies and identified using standard methods (1, 2). Monoclonal antibody was produced as described elsewhere (6). Balb/c mice were immunized i.p. with a suspension of three clinical isolates of viable Helicobacterpylori (4 x 107 bacteria per strain). Antibody titers were determined with an enzyme immunoassay (EIA) using whole bacteria (6 x 106 bacteria/well) as antigen. To determine specificity of the MAB, Helicobacter pylori and a panel of other bacteria including other Helicobacter species, Campylobacter and Wolinella species was used. The E I A was performed as described elsewhere (6). Bacterial protein was analyzed by SDS-PAGE (7) using 1.2 x 108 cells/lane. To demonstrate Helicobacter pylori lipopolysaccharide (LPS), phenol extracts were separated on SDS-PAGE using a 15 % slab gel with 4M urea (8). The gels were stained with silver (9). In a Western blot the gels were transferred electrophoretically to nitrocellulose sheets by standard methods (6) and developed with peroxidase-labelled goat anti-mouse serum (Dianova, Germany) using orthonaphthol as substrate with 3 % 1-1202. In the direct immunofluorescence (DIF) method the MAB, designated MAB 2107, was coupled
738
Eur. J. Clin. Microbiol. Infect. Dis.
with fluorescein-isothiocyanate (FITC) as described elsewhere (10). Deparaffinized tissue sections were incubated with proteinase K (1 mg/ml in 50 m M Tris-HCl o f p H 8) for 20 min at 37 oC. T h e reaction was stopped with phenylmethytsulfonylfluoride (Sigma, G e r m a n y ) and the sections counter-stained with 0.005 % Evan's blue for 20 min at 37 °C. After rinsing with PBS, the sections were incubated with F I T C - M A B for 15 min at r o o m temperature. T h e y were then rinsed for 1 h with excess PBS and observed by fluorescence microscopy.
4---"
1
T h e D I F m e t h o d was used to detect Helicobacter pylori in biopsy specimens from 158 patients (71 females, 87 males; age range 16-85 years, m e a n age 53 years) who u n d e r w e n t upper gastrointestinal tract endoscopy for dyspepsia or other reasons (e.g. preparation for liver transplantation, follow-up after gastro-intestinal surgery), including patients on antibiotic therapy. T h r e e biopsy specimens were taken from the gastric antrum and d u o d e n u m respectively for culture, the C L O test of urease activity (11) and histological examination as well as a specimen for the DIF test.
2
3
4
5
12345
1: SDS-PAGE of phenol extracted LPS of Helicobacter pylori ATCC 43504 after silver stain (left) and the corresponding Western blot (right). Lanes 1 and 2:2 and 10 lag LPS from Escherichia coli O111:B4 as control; lane 3 to 5:1 tag, 5 lag and 10 lagLPS fromHelicobacter pylori. Arrow marks corresponding major bands of Helicobacter pylori on SDS-PAGE and Western blot. Figure
In the S D S - P A G E with urea the bands showed a ladder-like pattern characteristic of LPS. T h e Western blot of the same preparation showed corresponding bands (Figure 1). T h e ladder-like distribution of the epitope is explained by LPS molecules of variable chain length linked to the detected epitope. Variation in the molecular weights of LPS preparations of different strains of Helicobacter pylori has been observed previously (12).
Results and Discussion. M A B 2107 reacted specifically with Helicobacter pylori. A crude extract of Helicobacter pylori separated by SDSP A G E and stained with Coomassie blue showed major bands of protein between 31 and 71 kDa. A Western blot of this preparation with M A B 2107 showed diffuse bands between 35 and 55 kDa, which did not correlate with protein bands.
To determine the specificity of M A B 2107, it was tested in an E I A with 27 isolates of Helicobacter pylori and a panel of other bacterial species (16
Table 1: Comparison of culture, the urease activity test and histological examination with direct immunofluorescence in the
detection of Helicobacterpylori in 158 gastric tissue sections. Results of direct immunofluorescence (%)
Results of standard methods Culture
Urease test
Histological examination
Positive
positive
positive negative
10.8 1.3
negative
positive negative
positive negative
Not evatuable
Negative
Total
1.9 0.0
5.1 1.9
17.7 3.2
0.0 0.6
0.6 1.3
0.6 1.3
1.3 3.2
positive negative
6.3 1.3
1.9 0.6
1.3 1.9
9.5 3.8
positive negative
0,0 4.4
1.9 10.1
4.4 40.5
6,3 55.1
24.7
18.4
57.0
100.0
Positive
Negative
Total
Vol. 11, I992
strains). Helicobacter pylori isolates showed 100 % reactivity, w h e r e a s the other bacteria did not react. In the DIF method FITC-coupled MAB 2107 was used to detect Helicobacter priori in tissue sections. By this means it was possible to detect coccoid and spiral shaped Helicobacter pylori directly in the crypts of the gastric mucosa. An advantage of this MAB reacting with LPS is that it is possible to treat biopsies for DIF in a manner identical to that for histological examination. For both investigations, the specimens can be stored in formaldehyde and paraffin embedded tissue sections prepared. In the DIF method specimens are thus easier to handle than in other methods using MABs (3-5). T h e standard m e t h o d o f culture, histological exa m i n a t i o n of tissue section and urease activity
tests were compared with DIF in the detection of
Helicobacter pylori in 158 biopsy specimens. The results are presented in Table I. All methods were positive in 30 of the 158 biopsy specimens, and all methods were negative in 64 of the 158 specimens. In the remaining cases either the DIF method (29 specimens) or the standard methods (16 specimens) gave ambiguous results. Variations in bacterial morphology may explain why the DIF gave positive results in seven cases (4.4 %) while the other tests were negative. In these cases coccoid forms were predominant, which were difficult to identify in the other tests, as observed by other investigators (4). Discrepancies between the different tests might also be explained by the fact that Helicobacter pylori tends to colonize the gastric mucosa in an irregular fashion (13). In addition, patients on antibiotic therapy were included in this study which might also contribute to a higher number of discrepant results in comparison to other studies (3, 4, 14,
15). Thus on comparing DIF with standard methods of detection, only a moderate rate of agreement was noted. However, it should be kept in mind that none of the other tests used represents a reference method. Therefore, the low degree of correlation might also partly reflect the insufficiency of these other tests. The total number of Helicobacter priori-positive patients was lower in our study than in other investigations (3, 4) as patients undergoing gastroscopy for dyspepsia or other reasons were included. The DIFmethod using an MAB may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.
739
References 1. Schneil GA, Schuberl TT: Usefulness of culture, histology, and urease testing in the detection of Campylobacterpylori. American Journal of Gastroenterology 1989, 84: 133-137. 2. Goodwin CS, Armstrong JA: Microbiological aspects of Helicobacter priori (Cmnpylobacter pylori). European Journal of Clinical Microbiology & Infectious Diseases 1990, 9: 1-13.
3. Engstrand L, Pahlson C, Gustavsson S, Sehwan A: Monoclonal antibodies for rapid identification of Campylobacter pyloridis. Lancet 1986, ii: 1402-1403. 4. Husson MO, Leelere H: Detection of Helicobacter pylori in stomach tissue by use of a monoclonal antibody. Journal of Clinical Microbiology 1991, 29: 28312834.
5. Negrini R, Lisalo L, Cavazzini L, Maini P, Gullini S, Basso O, Lauza G, Garofalo M, Nenci I: Monoclonal antibodies for specific immunoperoxidase detection of
Campylobacter pylori. Gastroenterology 1989, 96: 414420.
6. Bitter-Suermann D, Peters H, Jiirs M, Nehrbass R, Montenegro M, Timmis KN: Monoelonal antibody detection of IncF group plasmid-encoded TraT protein in clinical isolates of Escherichia coli. Infection and Immunity 1984, 46: 308-313. 7. Laemli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227: 680-685. 8. Westphal O, Jann K: Bacterial lipopolysaccharide: extraction with phenol-water and further application of the procedure. In: Whitler R (ed): Methods in carbohydrate chemistry. Volume 5. Academic Press, New York, 1965, p. 83-92. 9. Tsai CM, Frasch CE: A sensitive silver stain for detecting lipopolysaccharides in polyacrylamid gels. Analytical Biochemistry 1982, 119: 115-119. 10. Hum BAL, Chantler SM: Preparation of fluorochrome and enzyme-labeled antibodies. Methods in Enzymology 1980, 70A: 130--131. 11. Borromeo M, Lambert JR, Pinkard KJ: Evaluation of the 'CLO-test' to detect Campylobacter pyloridis in gastric mucosa. Journal of Clinical Pathology 1987, 40: 462-463. 12. Perez-Perez GI, Biaser MJ: Conservation and diversity of Campylobacter pyloridis major antigens. Infection and Immunity 1987, 55: 1256-1263. 13. Blaser MJ: Gastric campylobacter-like organisms,
gastritis, and peptic ulcer disease. Gastroenterology 1987, 93: 371-383.
14. Rivera E, L6pez-Vidal Y, Luquefio V, Ruiz-Palacios GM: Indirect immunofluorescence assay for detection of Helicobacter pylori in human gastric mucosal biopsies. Journal of Clinical Microbiology 1991, 29: 17481751.
15. Schaber E, Undauft F, Stoffler G, Aigner F, Paulweber B, Sandhofer F: Indirect immunofluorescence test and enzyme-linked immunosorbent assay for detection of Campylobacter pylori. Journal of Clinical Microbiology 1989, 27: 327-330.
19. Milatovie D, Braveny I, Verhoef J: Clindamycin enhances opsonization of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 1983, 24: 413417. 20. SlamaTG, Sklar S,I,MisinskiJ, Fess SW: Randomized comparison of cefamandole, cefazolin and cefuroxime prophylaxis in open-heart surgery. Antimicrobial Agents and Chemotherapy 1986, 29: 744-747. 21. Yourassowsky E, Van Der Linden MP, Crokaert F: Inoculum effect on growth-delay time of oxaeillinresistant strains of Staphylococcus aureus and Staphylococcus epidermidis exposed to cefamandole, cefazolin and cefuroxime. Antimicrobial Agents and Chemotherapy 1990, 34: 505-509. 22. Stratton CW, Liu C, Weeks IS: Activity of LY 146032 compared with that of methieillin, cefazolin, cefamandole, cefuroxime, ciprofloxacin and vancomycin against staphylococci as determined by killing-kinetic studies. Antimicrobial Agents and Chemotherapy 1987, 31: 1210-1215.
Evaluation of a Monoclonal Antibody for Detection of Helicobacterpylori in a Direct Immunofluorescence Test U. R o d e w i g 1, W. B e m b 4, D. Bitter-Suermann 1, M. Elsheikh 1, S. Fritsch 2, E. G l e n n - C a l v o 1, B. S o u d a h 2, M. Varrentrapp 3, S. Wagner 3, W. B~tr 1.
A monoclonal antibody was developed for detection of Helicobacter pylori in gastric tissue sections in a direct immunofluorescence test. On a comparison of the immunofluorescence test with standard methods for detection of HeIicobacter pylori, i.e. culture, the urease activity test and histological examination of tissue sections, using 158 biopsy specimens, 30 specimens were positive in all methods and 64 negative. In the remaining cases comparison was not possible because either immunofluorescence (29 specimens) or the standard methods (16 specimens) gave ambiguous results. The direct immunofluorescence test may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.
1Institute of Medical Microbiology,2Institute of Pathology and 3Department of Gastroenterology, Medical School Hannover, 3000 Hannover 61, Germany. 4Institute of Medical Microbiology,University Clinic, 6500 Mainz, Germany.
737
The main procedures currently used for detection of Helicobacterpylori are culture, histological examination and detection of urease activity (1). However, a disadvantage-of these techniques is that they have low sensitivity (2) so that a correct diagnosis may not be established in a number of cases. Moreover, these techniques are cumbersome to use. There is thus a need for a method for detection of Helicobacter pylori that is both convenient to use, sensitive and specific. Direct immunofluorescence using monoclonal antibodies (MABs) might seem to meet these demands. Until now there have been few reports on the production of MABs against Helicobacter pylori or the use of MABs for its identification. Indirect immunofluorescence tests (3, 4) and an immunoperoxidase method have been introduced which use MABs (5), however in all cases the epitopes are formaldehyde-sensitive so that the handling of biopsy material in these techniques has proved complicated. We therefore developed an M A B directed against Helicobacter pylori lipopolysaccharide and tested it in a direct immunofluorescence method (DIF) using gastric biopsy specimens.
Materials and Methods. Helicobacter pylori was isolated from gastric biopsies and identified using standard methods (1, 2). Monoclonal antibody was produced as described elsewhere (6). Balb/c mice were immunized i.p. with a suspension of three clinical isolates of viable Helicobacterpylori (4 x 107 bacteria per strain). Antibody titers were determined with an enzyme immunoassay (EIA) using whole bacteria (6 x 106 bacteria/well) as antigen. To determine specificity of the MAB, Helicobacter pylori and a panel of other bacteria including other Helicobacter species, Campylobacter and Wolinella species was used. The E I A was performed as described elsewhere (6). Bacterial protein was analyzed by SDS-PAGE (7) using 1.2 x 108 cells/lane. To demonstrate Helicobacter pylori lipopolysaccharide (LPS), phenol extracts were separated on SDS-PAGE using a 15 % slab gel with 4M urea (8). The gels were stained with silver (9). In a Western blot the gels were transferred electrophoretically to nitrocellulose sheets by standard methods (6) and developed with peroxidase-labelled goat anti-mouse serum (Dianova, Germany) using orthonaphthol as substrate with 3 % 1-1202. In the direct immunofluorescence (DIF) method the MAB, designated MAB 2107, was coupled
738
Eur. J. Clin. Microbiol. Infect. Dis.
with fluorescein-isothiocyanate (FITC) as described elsewhere (10). Deparaffinized tissue sections were incubated with proteinase K (1 mg/ml in 50 m M Tris-HCl o f p H 8) for 20 min at 37 oC. T h e reaction was stopped with phenylmethytsulfonylfluoride (Sigma, G e r m a n y ) and the sections counter-stained with 0.005 % Evan's blue for 20 min at 37 °C. After rinsing with PBS, the sections were incubated with F I T C - M A B for 15 min at r o o m temperature. T h e y were then rinsed for 1 h with excess PBS and observed by fluorescence microscopy.
4---"
1
T h e D I F m e t h o d was used to detect Helicobacter pylori in biopsy specimens from 158 patients (71 females, 87 males; age range 16-85 years, m e a n age 53 years) who u n d e r w e n t upper gastrointestinal tract endoscopy for dyspepsia or other reasons (e.g. preparation for liver transplantation, follow-up after gastro-intestinal surgery), including patients on antibiotic therapy. T h r e e biopsy specimens were taken from the gastric antrum and d u o d e n u m respectively for culture, the C L O test of urease activity (11) and histological examination as well as a specimen for the DIF test.
2
3
4
5
12345
1: SDS-PAGE of phenol extracted LPS of Helicobacter pylori ATCC 43504 after silver stain (left) and the corresponding Western blot (right). Lanes 1 and 2:2 and 10 lag LPS from Escherichia coli O111:B4 as control; lane 3 to 5:1 tag, 5 lag and 10 lagLPS fromHelicobacter pylori. Arrow marks corresponding major bands of Helicobacter pylori on SDS-PAGE and Western blot. Figure
In the S D S - P A G E with urea the bands showed a ladder-like pattern characteristic of LPS. T h e Western blot of the same preparation showed corresponding bands (Figure 1). T h e ladder-like distribution of the epitope is explained by LPS molecules of variable chain length linked to the detected epitope. Variation in the molecular weights of LPS preparations of different strains of Helicobacter pylori has been observed previously (12).
Results and Discussion. M A B 2107 reacted specifically with Helicobacter pylori. A crude extract of Helicobacter pylori separated by SDSP A G E and stained with Coomassie blue showed major bands of protein between 31 and 71 kDa. A Western blot of this preparation with M A B 2107 showed diffuse bands between 35 and 55 kDa, which did not correlate with protein bands.
To determine the specificity of M A B 2107, it was tested in an E I A with 27 isolates of Helicobacter pylori and a panel of other bacterial species (16
Table 1: Comparison of culture, the urease activity test and histological examination with direct immunofluorescence in the
detection of Helicobacterpylori in 158 gastric tissue sections. Results of direct immunofluorescence (%)
Results of standard methods Culture
Urease test
Histological examination
Positive
positive
positive negative
10.8 1.3
negative
positive negative
positive negative
Not evatuable
Negative
Total
1.9 0.0
5.1 1.9
17.7 3.2
0.0 0.6
0.6 1.3
0.6 1.3
1.3 3.2
positive negative
6.3 1.3
1.9 0.6
1.3 1.9
9.5 3.8
positive negative
0,0 4.4
1.9 10.1
4.4 40.5
6,3 55.1
24.7
18.4
57.0
100.0
Positive
Negative
Total
Vol. 11, I992
strains). Helicobacter pylori isolates showed 100 % reactivity, w h e r e a s the other bacteria did not react. In the DIF method FITC-coupled MAB 2107 was used to detect Helicobacter priori in tissue sections. By this means it was possible to detect coccoid and spiral shaped Helicobacter pylori directly in the crypts of the gastric mucosa. An advantage of this MAB reacting with LPS is that it is possible to treat biopsies for DIF in a manner identical to that for histological examination. For both investigations, the specimens can be stored in formaldehyde and paraffin embedded tissue sections prepared. In the DIF method specimens are thus easier to handle than in other methods using MABs (3-5). T h e standard m e t h o d o f culture, histological exa m i n a t i o n of tissue section and urease activity
tests were compared with DIF in the detection of
Helicobacter pylori in 158 biopsy specimens. The results are presented in Table I. All methods were positive in 30 of the 158 biopsy specimens, and all methods were negative in 64 of the 158 specimens. In the remaining cases either the DIF method (29 specimens) or the standard methods (16 specimens) gave ambiguous results. Variations in bacterial morphology may explain why the DIF gave positive results in seven cases (4.4 %) while the other tests were negative. In these cases coccoid forms were predominant, which were difficult to identify in the other tests, as observed by other investigators (4). Discrepancies between the different tests might also be explained by the fact that Helicobacter pylori tends to colonize the gastric mucosa in an irregular fashion (13). In addition, patients on antibiotic therapy were included in this study which might also contribute to a higher number of discrepant results in comparison to other studies (3, 4, 14,
15). Thus on comparing DIF with standard methods of detection, only a moderate rate of agreement was noted. However, it should be kept in mind that none of the other tests used represents a reference method. Therefore, the low degree of correlation might also partly reflect the insufficiency of these other tests. The total number of Helicobacter priori-positive patients was lower in our study than in other investigations (3, 4) as patients undergoing gastroscopy for dyspepsia or other reasons were included. The DIFmethod using an MAB may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.
739
References 1. Schneil GA, Schuberl TT: Usefulness of culture, histology, and urease testing in the detection of Campylobacterpylori. American Journal of Gastroenterology 1989, 84: 133-137. 2. Goodwin CS, Armstrong JA: Microbiological aspects of Helicobacter priori (Cmnpylobacter pylori). European Journal of Clinical Microbiology & Infectious Diseases 1990, 9: 1-13.
3. Engstrand L, Pahlson C, Gustavsson S, Sehwan A: Monoclonal antibodies for rapid identification of Campylobacter pyloridis. Lancet 1986, ii: 1402-1403. 4. Husson MO, Leelere H: Detection of Helicobacter pylori in stomach tissue by use of a monoclonal antibody. Journal of Clinical Microbiology 1991, 29: 28312834.
5. Negrini R, Lisalo L, Cavazzini L, Maini P, Gullini S, Basso O, Lauza G, Garofalo M, Nenci I: Monoclonal antibodies for specific immunoperoxidase detection of
Campylobacter pylori. Gastroenterology 1989, 96: 414420.
6. Bitter-Suermann D, Peters H, Jiirs M, Nehrbass R, Montenegro M, Timmis KN: Monoelonal antibody detection of IncF group plasmid-encoded TraT protein in clinical isolates of Escherichia coli. Infection and Immunity 1984, 46: 308-313. 7. Laemli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227: 680-685. 8. Westphal O, Jann K: Bacterial lipopolysaccharide: extraction with phenol-water and further application of the procedure. In: Whitler R (ed): Methods in carbohydrate chemistry. Volume 5. Academic Press, New York, 1965, p. 83-92. 9. Tsai CM, Frasch CE: A sensitive silver stain for detecting lipopolysaccharides in polyacrylamid gels. Analytical Biochemistry 1982, 119: 115-119. 10. Hum BAL, Chantler SM: Preparation of fluorochrome and enzyme-labeled antibodies. Methods in Enzymology 1980, 70A: 130--131. 11. Borromeo M, Lambert JR, Pinkard KJ: Evaluation of the 'CLO-test' to detect Campylobacter pyloridis in gastric mucosa. Journal of Clinical Pathology 1987, 40: 462-463. 12. Perez-Perez GI, Biaser MJ: Conservation and diversity of Campylobacter pyloridis major antigens. Infection and Immunity 1987, 55: 1256-1263. 13. Blaser MJ: Gastric campylobacter-like organisms,
gastritis, and peptic ulcer disease. Gastroenterology 1987, 93: 371-383.
14. Rivera E, L6pez-Vidal Y, Luquefio V, Ruiz-Palacios GM: Indirect immunofluorescence assay for detection of Helicobacter pylori in human gastric mucosal biopsies. Journal of Clinical Microbiology 1991, 29: 17481751.
15. Schaber E, Undauft F, Stoffler G, Aigner F, Paulweber B, Sandhofer F: Indirect immunofluorescence test and enzyme-linked immunosorbent assay for detection of Campylobacter pylori. Journal of Clinical Microbiology 1989, 27: 327-330.
