Sequences of anti-rabbit-IgG-alkaline phosphatase-binding immunoscreening artefacts from a Haemonchus contortus (nematode) cDNA library
Introduction: For the screening recombinant libraries, linking molecules of certain steric characteristics to a visible marker by a cascade of interacting molecules is a technique well established since the mid-70s (#Grunstein and Hogness 1975). However, as the humoral response is usually heterogenous, this ingenious experimental design can sometimes be marred by non-specific antibody reactions. Inadvertently, five apparently positive H. contortus clones (out of some 50,000 screened) which are capable to bind directly to anti-IgG alkaline phosphatase developed in sheep (Sigma batch #) were sequenced. It was found that treating with alkaline-phophatase-coupled secondary antibody (Sigma, 1h), three 10 min washings in TBST and BCIP-NBT chromogenic solution (15min) alone gave artefacts.
Clones similar to galectin (3 clones) and tropomyosin (2 clones) were found.