α1 acid glycoprotein

Holo-transferrin (human), β-galactosidase
(E. coli), α-1-acid glycoprotein,
GroEL, carbonic anhydrase, and ammonium acetate (99.999%, trace metal
basis) were obtained from Sigma-Aldrich (Wicklow, Ireland). The monoclonal
antibody nivolumab was obtained from the Hospital Pharmacy Unit of
the San Cecilio University Hospital (Granada, Spain). Ultrapure LC–MS
grade water was obtained from Fisher (Dublin, Ireland). Etanercept
(Enbrel) with a concentration of 50 mg/mL was commercially sourced
from Evidentic (Berlin, Germany). Micro Bio-Spin P-6 gel columns in
tris buffer were obtained from Bio-Rad (Naas, Ireland).

Two commercial mistletoe preparations, Iscador® Qu spez (VAEI; Iscador AG, Arlesheim, Switzerland) and abnobaViscum fraxini (VAEA; ABNOBA Heilmittel GmbH, Pforzheim, Germany) were used for the experiments. Mistletoe lectin was isolated and depleted from VAEI by affinity chromatography with immobilized α1-acid glycoprotein. α1-acid glycoprotein was used because of its high and non-selective affinity for all three isoforms of ML [28 (link)]. 50 mg of α1-acid glycoprotein (orosomucoid from Sigma-Aldrich, Buchs, Switzerland) was coupled to 2 ml Affi-gel 15 (Bio-Rad, Cressier, Switzerland) according to the manufacturer’s instructions. 200 ml of VAEI (20 mg/ml) was passed through 1 ml of the orosomucoid-coupled gel. This passage was carried out at 0°C to profit by the highly increased affinity of ML to the glycoprotein at cold temperatures [29 (link)]. By this procedure 85.6% of the lectins were eliminated from the VAEI. The final solution was sterilized by filtration (0.2 μm pore size). ML concentrations as measured by enzyme-linked immunosorbent assay (ELISA) [30 (link)] were 1.28 μg/ml in 20 mg VAEI and 0.18 μg/ml in 20 mg ML-depleted VAEI. According to manufacturer’s data ML concentration of VAEA was 0.89 μg/ml.

Recombinant proteins composed of the amino-terminal domains (domains 1–3) of mouse or human CD22 and the Fc region of human IgG1 (CD22-Fc) were described previously (41 (link)). Microtiter plates (96 well) were coated with 20 µg/ml α1-acid glycoprotein (Sigma). Alternatively, plates were coated with 50 µg/ml streptavidin followed by incubation with 4 µg/ml biotinylated synthetic CD22 ligand (42 (link)). Plates coated with α1-acid glycoprotein and synthetic CD22 ligand were then blocked with PBS containing 1% bovine serum albumin, followed by incubation with human or mouse CD22-Fc and compounds for 2 h, respectively. CD22-Fc bound to the plates was detected using alkaline phosphatase (AP)-conjugated goat anti-human IgG (Southern Biotechnology) and AP substrate solution (Sigma). The optical density at 405 nm was measured by a microplate reader (Molecular Devices). The concentrations of the compounds that reduce the binding of CD22-Fc to the biotinylated CD22 ligand and α1-acid glycoprotein by 50% (IC50) were determined.

Human serum albumin, fraction V, Lot No 2,742,726 (HSA), dansyl-L-phenylalanine, Lot No 8776KA (dPhe) were purchased from MP Biomedicals, Inc. (Illkirch, France). Human gamma globulin, Lot No 268129/1 32,705,352 (HGG), was obtained from Fluka Chemie AG (Buchs, Switzerland). α₁-acid glycoprotein, Lot No 049K7565V (AGP), dansyl-glycine, Lot No 9,143,321 (dGly), quinaldine red, Lot No MKBD6820 (QR) and methanol, Lot No SHBG8324V were gained from SIGMA-ALDRICH Chemie GmbH (St. Louis, MO, USA) while control normal serum (CNS), Lot 200054/724 has been obtained from Alpha Diagnostic (Warszawa, Poland). 9-fluoro-5-alkyl-12(H)-quino [3,4-b][1,4]benzothiazinium chloride (Salt2) has been synthesized in the Department of Organic Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, Poland, according to described procedure [21 (link),22 (link)].