New multiplexed flow cytometric assay to measure anti‐peptide antibody: a novel tool for monitoring immune responses to peptides used for immunization

Abstract

Peptide-based vaccination has a great potential for the prevention and treatment of various diseases. There is, however, no appropriate monitoring system to measure immune responses to vaccinated peptides, which hampers the development of therapeutically effective vaccine regimens to various diseases. In this study a new multiplexed flow cytometric assay using the Luminex system to monitor humoral immune responses to vaccinated peptides is described. Although the sensitivity is mostly equal to that of the traditional enzyme-linked immunosorbent assay (ELISA), the new assay has several advantages over ELISA in that it minimizes the amount of sera needed, running costs and working periods, and thus will be a novel tool for monitoring immune responses to vaccinated peptides.

Immunological evaluation of personalized peptide vaccination in combination with a 5‐fluorouracil derivative (TS‐1) for advanced gastric or colorectal carcinoma patients

“…The levels of antipeptide IgG were measured using a Luminex system as reported previously. (14) In brief, plasma was incubated with 25 µL peptide-coupled color-coded beads for 2 h at room temperature on a plate shaker. After incubation, the mixture was washed with a vacuum manifold apparatus and incubated with 100 µL biotinylated goat antihuman IgG (γ chain-specific) for 1 h at room temperature.…”

Identification of Lck-derived peptides applicable to anti-cancer vaccine for patients with human leukocyte antigen-A3 supertype alleles

“…The levels of immunoglobulin Gs (IgGs) reactive to Lck peptides were measured by the Luminext method, as reported previously (Komatsu et al, 2004). In brief, 100 ml of diluted plasma was incubated with 5 ml of colour-coded beads (Luminex Corp., Austin, TX, USA) coated with each of the Lck peptides on 96-well filter plates (MABVN1250; Millipore Corp., Bedford, MA, USA) for 2 h at room temperature on a plate shaker.…”

Personalized peptide vaccination in patients with refractory non-small cell lung cancer

“…The peptides were prepared under the conditions of good manufacturing practice (GMP) by the PolyPeptide Laboratories (San Diego, CA) and American Peptide Company (Vista, CA). Appropriate peptides for vaccination in individual patients were selected in consideration of pre-existing host immunity before vaccination, assessed from the titers of IgG specific to each of the 31 different vaccine candidates, as described previously (14). Combined chemotherapy and/or targeted therapy were allowed during the vaccination period, unless patients were unable to tolerate combined chemotherapies or declined them (Table II).…”

“…The humoral responses specific to each of the 31 candidate peptides (Table I), including those employed and not employed for vaccination, were determined by the peptide-specific IgG levels using the Luminex system (Luminex, Austin, TX), as reported previously (14). If the plasma titers of peptide-specific IgG in response to at least one of the vaccinated peptides after vaccination were >2-fold higher than those before vaccination, the changes were considered to be significant.…”

“…We developed a new approach of peptide-based vaccination, named personalized peptide vaccination (PPV), in which vaccine antigens are selected and administered based on preexisting host immunity before vaccination (6)(7)(8)(9)(10)(11)(12)(13)(14). We have shown promising results of PPV in various types of advanced cancers (6)(7)(8)(9).…”