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Table 2 Antibodies used for immunohistochemistry and EIA; GdC15, GdQ13 and GdN20 were purchased from SantaCruz Biotechnologies, Santa Cruz, CA (SCBT)

From: Glycodelin A is a prognostic marker to predict poor outcome in advanced stage ovarian cancer patients

Immunohistochemistry

Antibody

Epitope

Source/ Clonality

Epitope retrieval

Blocking

Dilution

Incubation

Reaction system used for detection

GdC15 (SCBT)

C-terminal

goat/polyclonal

Proteinase K (30 min, RT)

UV-Block (45 min)

1:1000 (UV Block)

30 min (RT)

Vectastain elite kit (goat IgG)

GdQ13 (SCBT)

C-terminal

goat/polyclonal

citrate buffer (pH 6, 5 min, pressure cooker)

UV-Block (45 min)

1: 300 (UV Block)

o.n. (4°C)

Vectastain elite kit (goat IgG)

GdA (Jeschke et al. 2006)

Mixed glycan/protein epitope

mouse/ monoclonal

citrate buffer (pH 6, 5 min, pressure cooker)

1.5% horse serum (20 min)

1:3000 (DAKO dil.)

o.n. (4°C)

Vectastain elite kit (mouse IgG)

Competitive enzyme immuno assay (EIA)

Antibody

Epitope

Source/ Clonality

Competing peptide

Blocking

Dilution

Incubation

Reaction system used for detection

GdQ13 (SCBT)

C-terminal

goat/ polyclonal

GdQ13-P (SCBT)

1% BSA, 0.5% NaN3 in PBS

1.5 ng/ml

1 hour (RT)

POD-strepavidin

GdN20 (SCBT)

N-terminal

goat/ polyclonal

GdN20-P (SCBT)

1% BSA, 0.5% NaN3 in PBS

6 ng/ml

1 hour (RT)

POD-strepavidin

  1. rt = room temperature, BSA = bovine serum albumin, PBS = phosphate buffered salt solution, o.n. = over night, POD = horse radish peroxidase.
  2. Competing peptides came from SCBT as well and were biotinylated by the manufacturer; unmodiefied GdQ13-P, GdN20-P were used for setting up the standard curve. Preparation of GdA is published in Jeschke et al. 2006. Epitope retrieval: Proteinase K was from Quiagen (Hilden, Germany), citrate buffer contained 0.1 M citric acid and 0.1 M sodium citrate in distilled water (pH 6.0). Blocking: UV-Block was purchased from Thermo Scientific (Bonn, Germany) and horse serum was a component of the Vectastain elite kit (Vector Laboratories, Burlingame, USA).