Preabsorption of a polyclonal antibody raised against a peptide-keyhole limpet haemocyanin conjugate to allow analysis of molluscan protein

Keyhole limpet (Megathura crenulata) haemocyanin (KLH) is widely used in immunobiology, primarily because it is an extremely potent antigen. 1 One of the main uses of KLH is as a hapten carrier for production of polyclonal antibodies. For small, relatively nonimmunogenic target molecules, a better immune response can be elicited by conjugating these molecules to KLH. A potential disadvantage with this strategy is that polyclonal antibodies raised in this manner will not only contain antibodies against the target molecule, but will also contain antibodies against KLH. Therefore the use of antibodies raised against a hapten conjugated to KLH is restricted to situations where detection of the hapten will not be compromised by the presence of proteins to which antibodies specific to KLH moieties will also bind. We encountered this problem during an investigation into the regulation of protein synthesis in the common brown garden snail Helix aspersa. As part of this investigation we wished to analyse H. aspersa proteins by Western analysis with an antibody specific to the phosphorylated form of eukaryotic initiation factor 2� [anti-eIF2� (P) Research Genetics Inc.]. This polyclonal antibody was raised in New Zealand white rabbits against a phosphorylated 13 amino acid peptide obtained from rat eIF2� and conjugated to KLH. The phosphorylated residue is serine, and corresponds to serine51 in human eIF2� . eIF2� is a highly conserved protein in eukaryotes and the region around the regulatory phosphorylation site is completely conserved across eukaryotes. 2,3 Therefore it would be expected that this antibody could be used to detect phosphorylated eIF2� derived from any eukaryote. The manufacturers report that the serum containing the antibodies is purified by using the ‘free’ (not bound to KLH) immunizing peptide in an affinity purification step to remove non-eIF2� antibodies. Subsequently the antibody is affinity purified against the non-phosphorylated form of the protein (Research Genetics Inc. Product Data Sheet for anti-eIF2� ). However, given that the vast majority of antibodies produced will be KLH specific, even a very low level of residual contamination could manifest as confounding anti-KLH activity. While the antibody shows good specificity to eIF2� (P) in mammalian extracts we have found that there is an extremely high level of non-eIF2� (P) binding when applied to H. aspersa extracts. We report here a simple method by which non-specific antibodies raised against a KLH-hapten complex can be removed by preabsorption with H. aspersa haemolymph. Helix aspersa were collected from the grounds of the University of Western Australia and surrounding suburban gardens. Haemolymph for preabsorption of the anti-eIF2� (P) antibody was freshly prepared and removed from H. aspersa individuals by first removing a small section of the shell (the ventral side of the second largest whorl) to expose a region of the mantle directly over the pericardium. A very fine capillary tube (prepared by heating and elongating a pasteur pipette) was inserted directly into the heart and haemolymph was extracted by capillary action. To ensure that the haemolymph was cell-free, it was centrifuged at 10,000 g for 20 min at room temperature and the supernatant was removed. The anti-eIF2� (P) antibody was preabsorbed by adding it to the cell-free haemolymph (1 � l of antibody solution to 100 � l of haemolymph) and incubating the mixture at room temperature for 2 h. After preabsorption, the antibody (in haemolymph) was diluted in TBST (20 mM Tris-HCl pH 7.5, 500 mM sodium chloride, 0.05% (v/v) Tween-20) containing 1% non-fat milk to give the appropriate dilution. Tissues sectioned from H. aspersa were homogenized in 9 volumes of 1� assay/lysis buffer using an Ultra-Turrax T25 homogenizer (Janke and Kunkel IKA Labortechnik) at 20500 rpm for 30 sec on ice. 1� assay buffer consisted of 120 mM potassium