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Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody.

ABSTRACT: In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC) whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.

SUBMITTER: van Endert P

PROVIDER: S-EPMC4893942 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody.

van Endert Peter P Lawand Myriam M

F1000Research 20150522

In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-deri ...[more]

PMID: 27335633

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Project description:We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

Dataset Information

Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody.

Publications

Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody.

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