Methods

Sera Collection

Sera samples were collected from 62 CD patients. The patients were confirmed to have documented CD based on clinical, endoscopic, radiologic, and/or histologic evidence. Samples collected for ASCA evaluation were sent to Prometheus Laboratories in San Diego, California. These samples were collected and sent in accordance with the guidelines outlined in the Prometheus IBD Diagnostic Serum Specimen Transportation Kits. Sera obtained for p35 and p36 evaluation were immediately refrigerated and shipped on ice to the University of Central Florida for further analysis.

ASCA Analysis

Prometheus Laboratories measured the IgA and IgG ASCA antibody titer levels through their CD Diagnostic System. Measurements were defined in ELISA Units with a standard calibrated ELISA microplate assay. Respectively, the cutoff for positivity was set at 20 and 40 EU/ml for IgA and IgG ASCA. Prometheus previously established these cutoff values based upon the results obtained in prior studies looking at well-defined patients with CD.

p35 and p36 Analysis

The Department of Molecular and Microbiology at the University of Central Florida performed sera analysis to determine the positivity of p35 and p36. The sera samples were immunoblotted with the following protocol - 
1)  100 ul of serum sample was absorbed with non-recombiant E. coli sonicate-lysate mixture by incubating at room temperature overnight.
2) The supernates were filtered and used in Immunoblot (1:30 dilution).
3) Rabbit anti-M. paratuberculosis sera was also adsorbed with E. coli and used as 1:500 dilution.
4)  Appropriate conjugate labeled with IgG-peroxidase were used at a final concentration of 1:1000 for rabbit sera and 1:500 for human sera.
5) Reactivity of p35 and p36 with individual serum were recorded as weak, moderate, strong or very strong. The presence of reactivity was recorded as a positive result. 

Objectives

OBJECTIVE

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

PURPOSE

A) Evaluate the utility of these serologic markers for the diagnosis of CD

B) Provide insights into the combined use of these serologic markers for the possible diagnosis and stratification of CD patients.

APPROACH

Analyze the sera of 60 confirmed CD patients for ASCA IgA and IgG antibody titers through Prometheus Laboratories and for antibody to the antigens p35 and p36 recombinant clones through the University of Central Florida.

Results

ASCA Serology Results

	Number	Percent (%)
Positve	36	60.0
Both Markers Positive	24	40.0
Single Marker Positive	12	20.0
One Marker Positive (IgA)	6	10.0
One Marker Positive (IgG)	6	10.0
Negative	24	40.0