(B) Mice surviving tumor challenge from Q-Tn +CP group are rechallenged with 5000 TA3Ha cells. control mice receiving Q only without the Tn antigen. Antibodies induced by Q-Tn recognize Tn-expressing tumor cells strongly and protect mice from tumor-induced death. The techniques for evaluating antibody titers by enzyme-linked immunosorbent assay, antibody binding to tumor cells by flow cytometry, and the protection efficacy of the vaccine in a therapeutic model of tumor are discussed in this chapter. 1. INTRODUCTION Due to their presence on many tumor types and their high expression levels on tumor cells, tumor-associated carbohydrate antigens (TACAs) are appealing for vaccine development (Buskas, Thompson, & Boons, 2009; Danishefsky & Allen, 2000; Guo & Wang, 2009; Liu & Ye, 2012; Monzavi-Karbassi, Pashov, & Kieber-Emmons, 2013; Yin & Huang, 2012). TACAs are recognized by B cell receptors (BCRs) on B cells, which are the only cell type in human bodies secreting antibodies. When administered alone, TACAs only weakly activate B cells producing low titers of IgM antibodies. Lemborexant To elicit strong and long-lasting IgG antibody responses, helper T cells need to be activated by the immunogen to provide costimulatory signals to B cells and induce antibody isotype switching from IgM to IgG (Goldsby, Kindt, & Osborne, 2000). A popular strategy to overcome the low immunogenicity of TACAs is usually to covalently conjugate TACAs to a carrier made up of epitopes for helper T cells (Goldsby et al., 2000; Yin & Huang, 2012). The most common carriers utilized are immunogenic proteins such as keyhole limpet hemocyanin (KLH) with multiple KLH-TACA constructs evaluated in clinical trials (Danishefsky & Allen, 2000; Gilewski et al., 2001; Huang et al., 2016; Livingston, 1995; Miles et al., 2011). However, for a prototypical TACA, Lemborexant the Tn antigen ((Golmohammadi et al., 1996). The inter-subunit disulfide network between cysteines at positions 74 Lemborexant and 80 makes the Q capsid stable Lemborexant toward a wide range of pH values, high temperature, and various chemical reagents. Each of the protein subunits has four amino groups, i.e., K2, K13, K16, and the N-terminus, around the external surface of the capsid, which can be potentially altered using potassium phosphate buffer (PBS), pH 7.0 Coomassie Plus Protein Reagent (Pierce) for protein concentration determination with bovine serum albumin (BSA) as the standard. DMSO 2.4 Procedure for Conjugating Q to Tn-NHS VLP Q (13.2mg, 5.1nmol of particle corresponding to 0.9mol of the subunit and 3.6mol of reactive amines) suspended in PBS (0.1PBS, Rabbit Polyclonal to Claudin 7 pH 7 to total volume 50mL. The excess Tn-NHS is usually removed by filtration through Millipore 100k MWCO centrifugal filter tube and washed thoroughly with PBS. The purity of the recovered Q conjugates is usually characterized by FPLC on Superose 6 Increase 10/300 GL column (Fig. 1B). The intact capsid is usually eluted from the column at around 11C13mL. Total protein concentration of the product is usually measured using the Coomassie Plus Protein Reagent (Pierce) with BSA as the standard. Typically, the yield of Q-Tn from this reaction is over 90%. The Q-Tn is also imaged via transmission electron microscopy showing intact particles with diameters of 28nm (Fig. 1C). The average number of conjugated Tn on each viral capsid is usually calculated from the intensities of peaks in the deconvoluted mass spectrum from LCMS analysis together with Maximum Entropy deconvolution algorithm MaxEnt? 1 (Da Ren et al., 2004). From the processed MS spectrum (Fig. 1D), the number of Tn added to each subunit is determined based on the increase of values of the peaks compared to the value of unmodified subunit (addition of one Tn to each subunit leads to an increase of 461 in value) (Table 1). The total number of Tn per capsid is usually calculated by: relative intensity of each peaknumber of Tn for each peak180 (the.