Specificity of antibodies: Unexpected cross-reactivity of antibodies directed against the excitatory amino acid transporter 3 (EAAT3)

Abstract

Abstract-Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479 -498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. Conclusions: Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because crossreactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

“…PMSF (phenylmethanesulfonyl fluoride) was added to inhibit proteases and NaPi (sodium phosphate) was chosen as buffer. The blot was developed with 0.2 µg/ml anti-B12 antibody (Ab#360 Holmseth et al, 2005). was detected with anti-EAAT2a antibodies (Ab#355: 0.1 µg/ml) followed by HRPconjugated secondary antibodies.…”

“…It can be due to the same antibodies as those recognizing the antigen under study (Danbolt et al, 1998;Holmseth et al, 2005;Wilson et al, 1996). Consequently, even monoclonal antibodies can display cross-reactivity.…”

“…Antibody production is straight forward. We typically immunize with synthetic peptides and prefer a "shotgun-approach": we select the hydrophilic portions of the termini (the longer the better) as well as other parts of the protein, mix the peptides together with carrier protein and glutaraldehyde, and inject this subcutaneously (for details see: Danbolt et al, 1998;Holmseth et al, 2005). We avoid intracutaneous injections as they cause unnecessary suffering.…”

“…Knockout animals are animals where a gene has been deleted and represent very powerful negative controls (e.g. Herber et al, 2004;Holmseth et al, 2005;Holmseth et al, 2012a;Li et al, 2013;Cecyre et al, 2014;Van Liefferinge et al, 2016;Baek et al, 2013;Bohmer et al, 2014). The main problem is their availability and the fact that most are mice while most immunocytochemistry is done on rat and human tissue.…”

The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS

“…Both of these were directed to a peptide (PYQLIAQDNEPEKPVADS ET-amide) representing residues 522-541 of rat GLAST (Storck et al, 1992). The anti-GLT-1 antibodies were the monoclonal 9C4 antibody (Ab#531) (Levy et al, 1993), the anti-B12 (Ab#360) (Holmseth et al, 2005) to residues 12-26 of rat GLT-1 (KQVEVRMHDSHLSSE-amide) (Pines et al, 1992), and a monoclonal antibody NCL-EAAT2 (lot 118003; Novocastra Laboratories) (Milton et al, 1997). Three synthetic peptides representing parts of EAAC1 (Kanai and Hediger, 1992;Bjørås et al, 1996) were used to produce the anti-EAAC1 antibodies used in the present report: C479 (IVNPFALEPT ILDNEDSDTK-amide), C491 (CLDNEDSDTKKSYVNGGFSVDKSDTISF TQTSQF-free acid), and C510 (VDKSDTISFTQTSQF-free acid).…”