Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGβ, hCGβn, hCGβcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGβ-, 8 hCGα-, and 13 αβ-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1 %; epitope β1), 12 with low (<1.0 %; epitopes β2/4), and 13 with high (>>1 %; epitopes β3/5) hLH cross-reactivity. The majority of hCGβ epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of β-sheet loops 1 and 3 (epitopes β2–6; 27 mAbs) and the second around the cystine knot (e.g., epitopes β1, β7, and β10; 9 mAbs). Four mAbs recognized epitopes on hCGβcf-only (e.g., epitopes β11 and β13) and six mAbs epitopes on the remote hCGβ-carboxyl-terminal peptide (epitopes β8 and β9 corresponding to amino acids 135–144 and 111–116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGβ-only, or hCG together with hCGβ or hCG together with hCGβ and hCGβcf. Sandwich assays that measure hCG plus hCGβ and eventually hCGβcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGβcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope β1 (Asp10, Asp60, and Gln89) in combination with epitopes β2 or β4 located at the top of β-sheet loops 1 + 3 of hCGβ involving aa hCGβ20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-0994-6) contains supplementary material, which is available to authorized users.
The constant need for increased sensitivity of antibody‐based assays has increased the need for high affinity molecules. Use of Fab antibody fragments can increase the specificity and sensitivity of immunoassays. We developed simple GPC, SDS‐PAGE and IEF methods that can be used to characterize Fab material produced by reduction of F(ab’)2 fragments. Specific reduction of F(ab’)2 to Fab was accomplished using cysteamine HCl. The resulting Fab fragments were analyzed with and without alkylation with iodoacetamide. Fab fragments were analyzed by gel permeation chromatography using a Waters HPLC system and multiple column types. SDS‐PAGE was performed using multiple gel types and running conditions. Scanning densitometry was performed using a Bio‐Rad GS800 and electrophoretic profiles were determined. The pI range and IEF profiles of the Fab samples were determined using a Phastsystem (GE Healthcare). The methods developed provide reproducible profiles for both relative reduced fragment content and identity. The methods were capable of detecting low percentage impurities. These methods provide quick analytical protocols that can be used to characterize Fab fragments for use in immunoassays.
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