WesternBlots - ImmunoBlots                            

 

Helicobacter Pylori IgG/IgA

 

Immunoblot for the detection of IgG/IgA-antibodies against Helicobacter Pylori

Table of detectable antibodies against the different Helicobacter Pylori antigens and their clinical significance

120 kD (cagA генетический продукт) - высоко специфический белок H. Pylori, однако определяется не у всех штаммов H. Pylori. Его функция окончательно не установлена. СagA является важным патогенным фактором.

87 kD (vacA genetic product) - высоко специфический белок H. Pylori, так называемый вакуолизирующий цитотоксин, второй важный патогенный фактор H. Pylori.

Комментарии: Изолированное выявление антител против антигенов  cagA и vacA  свидетельствует о наличии инфекции H. Pylori в прошлом. Антитела сохраняются довольно длительное время.

67 kD (Антиген Флагеллин) - белок флагеллин имеет выраженную перекрестную реакцию с другими бактериями, также содержащими флагеллин. Иммунный ответ на этот белок встречается довольно часто и не может расцениваться как специфическая реакция.

66 kD and 29 kD (суб-единицы фермента уреаза). Эти белки являются высоко специфичными для H. Pylori. Наличие антител к этим антигенам свидетельствует об активной стадии заболевания. Исчезновение антител к антигенам фермента уреазы свидетельствует об успешности проведенного лечения (например после тройной терапии).

25 kD, 19 kD, 17 kD и 14 kD - об этих антигенах имеется мало информации, but literature reports the 19 kD (OMP) protein to be highly specific to H. Pylori.

 

Interpretation:

IgG results positive:

If 2 of the highly specific bands (120, 87, 66, 29, 19 kD) are marked - Or at least 3 of the characterised bands (without 67 kD) are marked

IgG results uncertain:

If one of the pathogenic factors (120 kD, 87 kD) or one of the highly specific urease bands (66 kD, 29 kD) is marked - Or at least 2 of the characterised bands are present (without 67 kD)

 IgA results positive:

If 2 of the following bands are marked: 120, 87, 66, 29, 25, 19, 17, 14 kD

IgA results uncertain:

If one of the highly specific bands is marked (120, 87, 66, 29, 19 kD)

Further assistance in interpretation:

The most important additional information in the westernblot is the examination of the pathogenic factors  cagA and vacA.

CagA and vacA- positive H. pylori strains are highly virulent.

Patients with antibodies against vacA (120 kD) and/ or cagA (87 kD) run a higher risk of contracting a gastric ulcer than patients infected with a vacA or cagA negative strain.

Patients with antibodies against vacA (120 kD) and/ or cagA (87 kD)  and against sub-units of the urease enzyme (66 kD and 29 kD) are candidates for eradication therapy (Triple Therapy - For Example Amoxycillin, Clarithromycin and Omeprazole)

SUMMARY AND EXPLANATION OF THE TEST

The presence of spirochetes in human gastric mucosa was described almost 100 years ago. At that time their presence was correlated with ulceration and gastric carcinoma. Periodically throughout the next century, the colonization of the human stomach with spiral organisms was redescribed, and the correlation with disease status, especially for gastritis and ulcers, was made as long ago as 1940.

In 1983, Warren and Marshall described a curved bacillus associated with gastric mucosa in cases of chronic gastritis, tentatively drawing comparisons between that organism and the genus Campylobacter. More recently, investigators have shown a correlation between colonization with this organism (now termed Helicobacter pylori) and gastric and duodenal ulcers and chronic gastritis. H. pylori infection is chronic in nature and causes histological inflammation of the gastric mucosa as the first stage of disease. When the presence of H. pylori is eliminated from the gastric mucosa, a decrease in the inflammation can be observed. If the organism returns, the inflammation increases in severity. Most recent clinical tests have proved H. pylori to be the causing agent for most cases of chronic gastritis, duodenal and peptic ulcers, thus the eradication of H. pylori has been associated with the complete elimination of chronic gastritis and ulcers. Furthermore, evidence has increased that H. pylori is associated with gastric carcinoma.

Prevalent procedures for detecting H. pylori rely upon tissue obtained from endoscopic biopsy. This tissue is subjected to histology, gram staining, culture, or testing for the enzyme urease, which is produced by the organism in abundance. In addition to the necessity for performing an invasive gastroscopy, each of these methods suffers from drawbacks. Culture of the H. pylori organisms has proven difficult and relatively insensitive. Urease testing has been described as only about 75 % sensitive, and all four methods, including histology, and gram stain, require biopsy material taken from multiple sites due to the patchy nature of the infection. The presence of H. pylori has also been detected with a urease breath test (using radioactive isotopes) and with serological methods.

A positive serological response has been described in individuals with duodenitis, chronic gastritis, and in patients with gastric or duodenal ulcer. Also, a lot of people wihout clinical symptoms are seropositive for H. pylori.

An explanation for this phenomenon is that two different types of strains exist:

bullet a highly pathogenic type which is characterized by the expression of the virulence factors cagA and vacA
bullet and a low pathogenic type which lacks these factors.

The virulence of the last type is so low, that in the case of gastritis signs it seems to be sufficient to treat them only symptomatically. If antibodies against cagA and vacA can be detected, a endoscopic examination of the stomach followed by antibiotic eradication is highly recommended.

So it seems to be useful to screen first with a cheap and simple method on antibodies against H. pylori.

If the result is positive a westernblot can be used to detect antibodies against the virulence factors. In this way the serologic results can help to find the right form of therapy and save a lot of biopsies.

references

1.      Antonescu, C. G., und Marshall, B. J.  Helicobacter pylori: A potentially curable form of peptic ulcer disease. Gastroenterology Journal Club, 1, 3-12 (1990).

2.      Barthel, J. S. und Everett, E. D. Diagnosis of Campylobacter pylori infections: the “Gold Standard“ and the alternatives. Reviews of infectious diseases, 12 (suppl. 1), 107-114 (1990).

3.      DeCross, A. J. und Peura, D. A. Role of H. pylori in peptic ulcer disease. Contemporary Gastroenterology 5, 18-28, (1990).

4.      George, L. L., Borody, T. J., Andrews, P., Devine, M., Moore-Jones, D., et. al. Cure of duodenal ulcer after eradication of H. pylori. The Medical Journal of Australia, 153 (1990).

5.      Graham, D. Y. und Klein, P. D. What you should know about the methods, problems, interpretations and uses of urea breath tests. The American J. Gastroent., 86 Nr. 9, 1118-1122 (1991).

6.      Konsunen, T. U., Seppälä, K., Sarna, S., Slipponen, P. Diagnostik value of decreasing IgG, IgA, and IgM antibody titers after eradication of Helicobacter pylori. The Lancet, 339, 893-895 (1992).

7.      Parsonnet, J., Friedman, G. D., Vandersteen, D. P., Chang, Y., Vogelman, J. H., et. al. Helicobacter pylori infection and the risk of gastric carcinoma. New Engl. J. Med., 325, 1127-1131 (1991).

8.      Rauws, E. A. J., und Tytgat, G. N. J. Cure of duodenal ulcer associated with  radication to Helicobacter pylori. The Lancet, 335, 1233-1235 (1990).

9.      Tummuru M. K. R., Cover T. L., Blaser M. J. Clonig and Expression of a High-Molekular-Mass Major Antigen of Helicobacter pylori: Evidence of Linkage to Cytotoxin Produktion. Infection and Immunity, May 1993; 6 (5): 1799-1809.

10.  Xiang Z., Censini S., Bayeli P. F., Telford J. L., Figura, N., Rappuoli R., Covacci A. Analysis of Expression of CagA and VacA Virulence Factors in 43 Strains of Helicobacter pylori Reveals that Clinical Isolates Can Be Divided into Two Major Types and that CagA Is Not Necessary for Expression of the Vacuolation Cytotoxin Infection and Immunity, January 1995; 63 (1): 94-98

11.  Cover T. L., Glupczynski Y., Lage A. P., Burette A., Tummuru M. K. R., Perez-Perez, G. I., Blaser M. J. Serologic Detection of Infection with CagA Helicobacter pylori Strains. Journal of Clinical Microbiology, June 1995; 33 (6): 1496-1500.

12.  Husson M.-D., Gottrand F., Vachee A., Dhaenens L., Martin De La Salle E., Turck D. Importance in Diagnosis of Gastritis of Detection by PCR of the cagA Gene in Helicobacter pylori Strains isolated from Children. Journal of Clinical Microbiology, December 1995; 33 (12): 3300-3303.

13.  Warren, J. R., und Marshall, B. J. Unidentified curved bacillus on gatric epithelium in active chronic gastritis. The Lancet, 1, 1273-1275 (1993).

Analysis of band pattern and interpretation of the results

 Antibodies against the following antigens could be observed:

120, 87, 67, 66, 29, 25, 19, 17, 14 kD

bullet

120 kD (cagA gene product) - The most important virulence factor (cagA = cytotoxin associated gene A). 40 – 50 % of all strains express this protein. Highly specific for H. pylori.

bullet

87 kD (vacA gene product) - The vacuolating cytotoxin, another virulence factor which is often associated with cagA. 50 % of all strains express this protein. Highly specific for H. plylori.

bullet

67 kD - The Flagellin-protein. Immunoresponse against this protein can often be observed. But on this protein the most cross reactivities to the Campylobacter species are located. Therefore all patients with antibodies against the flagellin alone must be counted as negative.

bullet

66 and 29 kD - These proteins are subunits of the urease-enzyme. They are highly specific for H. pylori. All patients having antibodies against both subunits should be counted as positive.

bullet

25, 19, 17 and 14 kD - Little is known about these proteins. It seems that the 19 kD protein is highly specific for H. pylori.

 Further Interpretation

IgG Positive - A patient should be considered to be positive for H. pylori IgG, when at least 2 of the highly specific bands (120, 87, 66, 29, 19 kD) are positive, or at least 3 of all bands (without 67 kD) are positive.

Borderline IgG positive, when one of the bands 120, 87 kD, or one of the highly specific urease bands 66, 29 kD are positive, or two of all bands (without 67 kD) are positive.

If no antibodies against cagA and/or vacA can be detected a symptomatic therapy with proton pump inhibitors could be the first step in therapy.

IgA positive - If two of the following bands detected: 120, 87, 66, 29, 25, 19, 17, 14 kD.

Borderline IgA positive - If one of the following band are detected: 120, 87, 66, 29, 19 kD.

 LIMITATIONS OF USE

·      The results of this test kit should be interpreted in the context of all available clinical and laboratory data.

·      Due to the fact that a high percentage of the population are positive for H. pylori-antibodies, this test should only be run with symptomatic patients.

·      A correlation between band pattern and intensity and the severity of the disease is not proven.

 

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