Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols.

To develop reproducible results, it is critical that all reagents used in an experiment be validated in an alternative or independent method. We present two such independent methods for determining the specificity of antibodies: (1) "MILKSHAKE," which can be used to validate the liability and specificity of antibodies directed against post-translationally-modified epitopes, and (2) "Sundae," which is a more complete alanine-like scanning method that can be used to better understand the binding and bioactivity of specific residues of a protein. We apply both of these methods to the interaction between an antibody and its antigen.

Epivolve: A Protocol for Site-Directed Antibodies.

Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call "Epivolve," that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a "pseudo-hapten" immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.

MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation.

Knowing that an antibody's sensitivity and specificity is accurate is crucial for reliable data collection. This certainty is especially difficult to achieve for antibodies (Abs) which bind post-translationally modified proteins. Here we describe two validation methods using surrogate proteins in western blot and ELISA. The first method, which we termed "MILKSHAKE" is a modified maltose binding protein, hence the name, that is enzymatically conjugated to a peptide from the chosen target which is either modified or non-modified at the residue of interest. The surety of the residue's modification status can be used to confirm Ab specificity to the target's post-translational modification (PTM). The second method uses a set of surrogate proteins, which we termed "Sundae". Sundae consists of a set of modified maltose binding proteins with a genetically encoded target sequence, each of which contains a single amino acid substitution at one position of interest. With Sundae, Abs can be evaluated for binding specificities to all twenty amino acids at a single position. Combining MILKSHAKE and Sundae methods, Ab specificity can be determined at a single-residue resolution. These data improve evaluation of commercially available Abs and identify off-target effects for Research-Use-Only and therapeutic Abs.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Mantis shrimp identify an object by its shape rather than its color during visual recognition.

Mantis shrimp commonly inhabit seafloor environments with an abundance of visual features including conspecifics, predators, prey and landmarks used for navigation. Although these animals are capable of discriminating color and polarization, it is unknown what specific attributes of a visual object are important during recognition. Here, we show that mantis shrimp of the species Neogonodactylus oerstedii are able to learn the shape of a trained target. Further, when the shape and color of a target that they had been trained to identify were placed in conflict, N. oerstedii tended to choose the target of the trained shape over the target of the trained color. Thus, we conclude that the shape of the target was more salient than its color during recognition by N. oerstedii, suggesting that the shapes of objects, such as landmarks or other animals, are important for their identification by the species.