A Comparative Analysis of Anti-Saccharomyces cervisiae Antibodies (ASCA) and p35 and p36 Recombinant Clones for the Diagnosis of Crohn's Disease 

I. Shafran, M.D., C. Piromalli, F. El-Zaatari, Ph.D.*, S. A. Naser, Ph.D.; Dept. Molecular Biology, UCF, Orlando, FL;  *Baylor College of Medicine, Houston, TX. 

(submitted for publication)
Abstract

BACKGROUND: Numerous studies have confirmed the utility of ASCA as a serological marker for CD. Recent investigations have identified the utility of another potential CD diagnostic serum marker, p35 and p36 recombinant clones specific for Mycobacterium avium ss. paratuberculosis. Our lab is currently investigating the utility of these serologic markers for the diagnosis of CD.

AIM: To evaluate the diagnostic accuracy of ASCA to both p35 and p36 serologic markers for the diagnosis of CD patients.

METHODS: Serum samples were obtained from 60 patients with confirmed and documented CD. The sera samples were sent to Prometheus Laboratories to determine the IgA and IgG ASCA titers with the Prometheus IBD Diagnostic System. The serologic response to p35 and p36 were analyzed by Western blotting at the University of Central Florida.

RESULTS: The ASCA titer levels for IgA and/or IgG were positive for 61.7% (36/60) of CD patients. The p35 and/or p36 recombinant clones were positive for 86.7% (52/60) of CD patients. The 24 CD patients not detected by ASCA were identified as positive by the p35 and/or p36 recombinant clones in 87.5% (21/24) of those CD patients missed by ASCA. The 9 CD patients not detected by p35 and/or p36 were identified by the ASCA serologic markers in 62.5%(5/8) of those CD patients missed by p35 and p36.

CONCLUSION: This investigation further establishes the utility of p35 and p36 recombinant clones for the diagnosis of CD. These results also reveal added evidence to demonstrate the complimentary role that ASCA may have with p35 and p36 for increasing the sensitivity and specificity of these serologic tests. Larger studies are needed to investigate the combined use of these serologic markers for the diagnosis of CD and as predictive markers to facilitate the development of treatment strategies.

Background
Development of Serological Markers for Crohn’s Disease

• The differentiation of CD from other Inflammatory Bowel Diseases, such as Ulcerative colitis, can be quite challenging especially in cases where the disease is limited to the large bowel. 
• Serological markers for the diagnosis of CD are still not fully reliable and require clinical, radiological, endoscopic and histopathologic evidence to verify their diagnostic accuracy. 
• Currently, traditional diagnostic tools appear to be more reliable and cost effective in the diagnosis of CD when compared to current serologic markers.
• The clinically available serologic tests for CD do not help in establishing the possible etiological factors underlying their disease.

• Saccharomyces cerevisiae (SC), known as baker's yeast, is a common dietary antigen. 
• Studies in the late 1980’s demonstrated higher IgG and IgA antibody titer levels towards this yeast in CD patients versus controls. There has not been any direct evidence to suggest that SC plays a primary or partial role in the etiology of CD since it is not a common pathogen. The elevated titer levels may correlate with a hypersensitivity of CD patients to SC antigens or the possibility that an infection of an unknown agent may cross react antigenically with SC.1
• Past studies have identified the sensitivity (61%) and specificity (88%) of ASCA in patients confirmed with CD.2
• ASCA has demonstrated some utility in identifying IBD patients and assisting physicians in differentiating between CD and UC.3

• The recombinant clones p35 and p36 express 35- and 36 kDa proteins, respectively, and were identified from a previously constructed expression genomic library of MAP.4
• p35 encoding gene expresses a 35K protein that reacts well with sera from Johne’s animals5 while the p36 encoding gene has been sequenced (AF048899) and expresses a protein that is considered a Mycobacterium- genus specific antigen.6
• MAP recombinant antigens (p35 and p36) individually and combined have been shown to have a statistically significant difference in reactivity between Crohn’s disease-positive serum and serum samples from controls (Ulcerative colitis and healthy controls). [See Table Below]7
• MAP is the etiologic agent that causes Johne’s disease, a chronic inflammatory bowel condition of ruminants and primates.
• MAP has been suspected as a possible etiological agent of CD.8 A number of studies have isolated MAP from CD tissue9,10,11 and lymph nodes12. A recent study has isolated and cultured MAP from CD resected tissue and CD breast milk. 13, 14
• The p35 and p36 recombinant clones may be useful as both a diagnostic tool for identifying CD patients and as a potential tool to determine the presence of an active immune response to MAP.
• The combination of p35 and p36 with ASCA may increase the utility of these serologic markers as diagnostic tools for the identification and stratification of CD patients.