High-Resolution Epitope Mapping and Affinity Binding Analysis Comparing a New Anti-Human LAG3 Rabbit Antibody Clone to the Commonly Used Mouse 17B4 Clone.

Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applications. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare the binding characteristics of a new anti-LAG3 rabbit antibody clone, SP464, with the thirty-year old and extensively used anti-LAG3 mouse 17B4 clone. The rabbit SP464 clone exhibited between 20× to 30× greater binding to LAG3 than did the mouse 17B4 clone. Using these tools, we precisely mapped the relative locations of the epitopes of these two antibodies. The SP464 and 17B4 minimal epitopes were localized to separate, but overlapping, sub-fragments within the amino-terminal fifteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Application of this approach for quantifying the effects of alanine substitutions along the minimal SP464 epitope identified two amino acids essential for binding and four amino acids that likely contribute towards binding. Together, ACE, SPR, and IHC constitute a powerful orthologous approach for comparing antibody-binding characteristics and for fine mapping of linear epitopes within short immunogens. Our results indicate that the rabbit clone SP464 may be useful for assessing LAG3 expression.

A human cellular protein activity (OF-1), which binds herpes simplex virus type 1 origin, contains the Ku70/Ku80 heterodimer.

In an effort to identify host proteins involved in herpes simplex virus type 1 replication, monkey and human cellular protein activities (called OF-1) that bind the viral replication origin, oriS, have been described. We show by mass spectrometry that the DNA-binding component of human OF-1 contains Ku70 and Ku80 proteins.

Two-hybrid analysis of the interaction between the UL52 and UL8 subunits of the herpes simplex virus type 1 helicase-primase.

Herpes simplex virus type 1 expresses a heterotrimeric helicase-primase, the subunits of which are encoded by the viral UL5, UL8 and UL52 genes. The interactions of the UL52 protein with the UL8 and UL5 proteins were analysed by using the yeast two-hybrid system. The UL52-UL5 interaction gave a specific but weak signal in the two-hybrid system. In contrast, the UL52-UL8 interaction gave a strong signal in the two-hybrid system. Deletion analysis showed that a 548 amino acid fragment of UL52 (amino acids 366-914) retains the ability to interact with UL8 and that the N-terminal 349 amino acids are dispensable for the interaction. A fragment library screen and co-immunoprecipitation experiments confirmed the deletion analysis results.