Representative image of negative and positive results with the IA-2A LFIA.

<p>A positive signal limited to the control line is obtained with buffer alone (A) and with an IA-2A-negative sample (B). A positive signal in both the test and control line is observed with a IA-2A-positive serum (C).</p

Titer distribution of the serum samples analyzed using the IA-2A LFIA.

<p>Sera were from 35 T1D patients and 44 age-matched controls. The dotted line indicates the cut-off value (1939 a.u.) based on the control samples.</p

Comparison of IA-2A levels obtained by IA-2A LFIA and in-house IA-2A bridging ELISA.

<p>Serum samples from 35 T1D patients and 44 control subjects were analyzed with both methods and the two assays were correlated. Dotted lines indicate the cut-off values for both assay methods. mAU, milli-absorbance units.</p

PET blots of cerebellum.

<p>PET blots show prominent deposition of PrP^Sc in granular and molecular cell layers in clinically diseased vCJD and BSE inoculated animals whereas the subclinical vCJD inoculated primate only shows faint PrP^Sc in granular and molecular cell layers (scale bar in lower left image 1 mm).</p

Biochemical analysis of PrP^Sc in the CNS.

<p>A) Western Blot analysis for PrP^Sc (frontal cortex) of vCJD, BSE and sCJD infected animals. PrP^Sc could be demonstrated in the brains of preclinical vCJD and prion-diseased vCJD and BSE inoculated animals. In the vCJD cohort, PrP^Sc is detectable (using NaPTA) in subclinical state 40 weeks before onset of symptoms. sCJD inoculated macaques did not show PrP^Sc at any time point in cerebellum (data not shown) and frontal cortex in conventional as well as NaPTA enhanced Western blot. (* indicates prion-diseased animals). B) PrP^Sc-glycotype analysis demonstrates comparable glycotypes of vCJD and BSE when transmitted to primates. Densitometric measurement of relative band intensities for di-, mono- and unglycosylated form of PrP^Sc is shown in % of total signal. C) Quantification of PrP^Sc-signal shows initial exponential increase of PrP^Sc until 158 wpi when PrP^Sc levels off. Relative amounts of PrP^Sc are shown in arbitrary units as quantified in three independent experiments.</p

Competitive inhibition of IAA binding with unlabeled insulin.

<p>One IAA-positive and one IAA-negative serum sample (A) and eight IAA-positive serum samples (B) were incubated with 0, 5, 50, 500 and 5,000 ng/mL (A) or with 5,000 ng/mL (B) of unlabeled insulin together with biotinylated and GC300-labeled insulin in the first incubation step of the IAA bridging ELISA. White bars represent signals without addition of unlabeled insulin and black bars represent signals with addition of unlabeled insulin. mAU, milli-absorbance unit.</p

Principle of the IAA bridging ELISA.

<p>Serum sample or calibrator is incubated with GC300- and biotin-labeled insulin prior to transfer into anti-GC300 (MC159) mAb-coated wells. Serum IAAs form a bridge between GC300- and biotin-labeled insulin and this complex is captured on the solid phase of the MC159-coated plate. Biotin-labeled insulin bound to IAAs is detected using AChE-labeled streptavidin and the enzymatic activity is measured at 414 nm.</p

Preclinical detection of PrP^Sc in muscle of vCJD inoculated primates.

<p>A) Western Blot analysis of PrP^Sc from NaPTA precipitated arm and tongue tissue of sCJD, vCJD and BSE infected primates and controls show PrP^Sc in arm and tongue of subclinical vCJD challenged monkeys and in clinically affected vCJD and BSE inoculated animals. sCJD infected macaques did not show any detectable PrP^Sc in arm or tongue. B) Detection of PrP^Sc in arm, tongue and heart tissue from sCJD, vCJD and BSE infected primates and controls by ELISA. The scatter graph shows values (mean of two independent experiments) for arm (circle), tongue (triangle) and heart (diamond). The cut-off is indicated by grey shade and values below were considered as negative. PrP^Sc could be detected in tongue, arm and heart of healthy and clinically diseased vCJD inoculated animals. In BSE infected animals PrP^Sc could only be detected in tongue tissue and in sCJD infected animals no PrP^Sc could be detected.</p

Comparison of IAA levels obtained by IAA bridging ELISA and RIA.

<p>Serum samples from 50 newly diagnosed T1D children and 100 control subjects were analyzed with both methods and the two assays were correlated. The dotted lines indicate the cut-off values. mAU, milli-absorbance unit.</p

Primates included in the study.

<p>Primates included in the study.</p