Use of yeast-secreted in vivo biotinylated recombinant antibodies (Biobodies) in bead-based ELISA.
OBJECTIVE
To measure circulating antigens, sandwich ELISA assays require two complementary affinity reagents. Mouse monoclonal antibodies (mAb) and polyclonal antibodies (pAb) are commonly used, but because their production is lengthy and costly, recombinant antibodies are emerging as an attractive alternative.


METHODS
We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays). To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls.


RESULTS
On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38).


CONCLUSIONS
Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead-based assays for use in large validation studies.
Scholler
Nathalie
N
School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 96104-6069, USA. naths@mail.med.upenn.edu
Lowe
Kimberly A
KA
Bergan
Lindsay A
LA
Kampani
Archana V
AV
Ng
Vivian
V
Forrest
Robin M
RM
Thorpe
Jason D
JD
Gross
Jenny A
JA
Garvik
Barbara M
BM
Drapkin
Ronny
R
Anderson
Garnet L
GL
Urban
Nicole
N
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D030501:Epididymal Secretory Proteins / Q000276:immunology / Q000378:metabolism*; D005260:Female; D058851:GPI-Linked Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000097:blood* / Q000276:immunology; D000090204:Mesothelin; D008875:Middle Aged; D010051:Ovarian Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015003:Yeasts / Q000235:genetics; D023083:beta-Defensins
D000906:Antibodies; D000911:Antibodies, Monoclonal; C426184:DEFB126 protein, human; D030501:Epididymal Secretory Proteins; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; C000715528:Msln protein, mouse; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins; D000090204:Mesothelin