The main objective of the present study was the optimization of our current/first version of SGA through the reduction/minimization of unspecific (background) antibody binding. We explored (i) how the modification of the beads with linear PEGs of different lengths can influence both specific and unspecific antibody binding and (ii) which of these modifications reduce unspecific binding. We demonstrate that unspecific antibody binding was significantly
reduced by the direct modification of the bead surface with linear heterobifunctional PEG consisting Navitoclax of 23- and 60 PEG-units and by avoiding the employment of IgG-class antibodies. The following antibodies from the indicated suppliers were used: anti-P1 human monoclonal IgM antibody (clone P3NIL100, Immucor Gamma GmbH, Rödemark, Germany, dilutions used: 1/200; 1/500; 1/1000; 1/2000; 1/5000;
and 1/10,000); anti-Gb3 (CD77, Pk) murine IgG2b (clone BGR23, Seikagaki Biobusiness Corp., Tokyo, Japan, dilution of 1/100); anti-Gb3 (CD77, Pk) rat monoclonal IgM (AbD Serotec, Oxford, England, dilution of 1/100); biotin mouse anti-rat IgM (BD Pharmingen™, BD Biosciences, Allschwil, Switzerland, dilution of 1/1000); R-phycoerythrin (R-PE)-streptavidin (LubioScience GmbH, Luzern, Switzerland, dilution of 1/200); goat anti-human R-PE conjugated Ig (H + L), IgM buy OSI-744 or IgG antibodies (dilution of 1/1000) and goat anti-mouse Ig (H + L) antibodies (dilution of 1/1000, Southern Biotechnology Associates, Inc., Birmingham, AL, USA). Blood samples were collected prospectively from healthy women at the Department of Gynecology, University Hospital Zurich after informed consent was granted (ethical approval to V.H.S., SPUK Canton of
Zurich, Switzerland). Specimens were MycoClean Mycoplasma Removal Kit processed and stored as described previously (Pochechueva et al., 2011b and Jacob et al., 2012). Human plasma samples were used in all the experiments in the dilution of 1/40, as described previously (Pochechueva et al., 2011a). Plasma samples from healthy donors of blood groups A, B and O (five donors each group) were pooled in equal volumes and used similarly to individual plasma samples. The glycopolymers, Glyc(20)–PAA–biot1, Glyc(20)–PAA–PEG4(80)–biot1, and Glyc(20)–PAA–PEGm(5)–biot1, used for coupling to fluorescent beads were produced in-house as previously described (Chinarev et al., 2010); their chemical structures are presented in Fig. 1. The glycopolymers are composed of a polyacrylamide carrier (PAA, number of the average polymerization degree, n = 220) provided with end biotin groups and side-pendant Glyc residues, either Galα1–4Galβ1–4GlcNAcβ– (referred to as P1) or Galα1–3(Fucα1–2)Galβ– (referred to as Btri) that are statistically distributed along the polymer backbone. The content of monomer units with glycan substitution is 20 mol%.
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