Procedure used to select cancer-specific recombinant scFv antibodies.

<p>A phage-displayed antibody library was applied to immobilized IgM obtained from pooled age, smoking history matched control samples, to remove phage-displayed scFv reactive with IgM present in both controls and patients with lung cancer. The cross-absorbed phage-displayed antibody library was applied to immobilized IgM purified from pooled lung cancer patients to obtain scFv specific for lung cancer IgM. Phage-displayed antibodies bound to immobilized IgM obtained from pooled lung cancer patients were eluted and used to infect <i>E. coli</i> and plated onto selective agar plates. Bacterial colonies from two rounds of selection were picked using a colony picker from agar plates to 384 well microtiter culture plates and induced to express soluble scFv. ScFv were transferred from 384 well culture plates to separate 384 well assay plates coated with lung cancer or control antibodies. Bacterial colonies producing scFv reactive in a homogenous fluorescent assay with lung cancer but not control antibodies were identified and used to produce larger quantities of scFv for use in subsequent assays.</p

Prediction of cancer <i>versus</i> non-cancerous based on scFv seroreactivity with independent set in MSD assay (n = 22N+21Ad).

<p>Prediction of cancer <i>versus</i> non-cancerous based on scFv seroreactivity with independent set in MSD assay (n = 22N+21Ad).</p

Binding activity of scFv J4 on human serum samples in homogeneous bridging electrochemiluminescent (MSD) assay.

<p>Data points represent binding activity for scFv J4 on individual serum samples. Two outliers in control group that demonstrated high binding activity are circled. C - non-cancerous control sample, Ad1-stage 1 adenocarcinoma.</p

ScFv binding activity as determined using a novel homogeneous bridging electrochemiluminescent (MSD) assay.

<p><b>A</b> Biotinylated scFv immobilized onto streptavidin-coated microtiter wells were used to capture autoantibodies. Captured autoantibodies were detected using Sulo-Tag labeled scFv and an MSD plate reader. <b>B</b> Binding activity of scFv antibody with autoantibodies obtained from stage I lung adenocarcinoma (AdI, n = 14) and from non-cancer (C, n = 21) human serum samples. Each data point represents the assay signal intensity for individual serum samples. P = 0.031 for normal (C) vs cancer (AdI) group average (horizontal lines).</p

Approach used to determine scFv binding activity to serum IgM using fluorescent (FMAT) assay.

<p><b>A.</b> Anti-human IgM - specific antibodies immobilized onto beads were used to capture IgM from human serum samples. Recombinant scFv antibodies bound to human IgM were detected with fluorescent-labeled secondary antibodies (specific for the E- tag on the scFv) using a fluorescent (FMAT8100) plate reader. <b>B.</b> Binding activity of 384 scFv to control or cancer serum as determined using Fluorometric Microvolume Assay Technology (FMAT). Each bar represents the fluorescent signal obtained when scFv bound to beads bearing human serum IgM. ScFv demonstrating differential binding to IgM from cancer and control serum samples were selected for the further analysis (see arrows indicating the signals for scFv selected in this representative experiment: B6, J4 and E3).</p

Panel of 6 scFv demonstrated an association between assay signal intensity and survival.

<p>The prediction model presented uses assay signal intensity to generate low/high score. High score (dotted line) is associated with a lower probability of survival.</p

Discriminatory performance of single scFv in a fluorescent (FMAT) assay.

<p><b>A.</b> ScFv B6 binding activity with individual serum samples from either non-cancerous (N) or stage 1 adenocarcinoma (Ad1) patients. Each dot represents the fluorescent assay signal intensity (FMAT count) for scFv with individual serum samples (n = 15N +15Ad1). <b>B.</b> A receiver operating characteristic (ROC) curve was generated for scFv clone B6 based on fluorescent assay signal intensity for 15 control and 15 adenocarcinoma stage 1 serum samples.</p

Patients and samples clinical characteristics.

<p>Patients and samples clinical characteristics.</p

Prediction of cancer <i>versus</i> non-cancerous based on scFv seroreactivity with independent set in FMAT assay (n = 15N+15Ad).

<p>Prediction of cancer <i>versus</i> non-cancerous based on scFv seroreactivity with independent set in FMAT assay (n = 15N+15Ad).</p

Panel of six scFv antibodies discriminated NSCLC from control serum in independent validation set.

<p>ScFv binding activity was determined by a fluorescent (FMAT) assay. Each bar represents the mean fluorescent intensity (FMAT count) of scFv with 15 non-canceorus control (N) or 15 adenocarcinoma stage 1 (Ad1). P-values are presented from two-tailed student t-test for each scFv (N vs Ad1 comparison).</p