MMA383 is an anti-idiotypic antibody designed as an immunogenic surrogate for the cancer specific Lewis Y antigen. Lewis Y is expressed in 70–90% of tumours of epithelial origin with limited expression in normal tissue. Five different MMA383 vaccines were prepared by mixing a MMA383 antibody solution with an Alhydrogel™ aluminium hydroxide adjuvant and tested on the biological activity in a rat model. The immunogenicity increased when: (i) the adjuvant was sterilized at 121 °C compared to no sterilization, (ii) the adjuvant was suspended in a phosphate buffer compared to water and (iii) the MMA383 solution was at a pH of 7.2. The immunogenicity of a ready-to-use MMA383 aluminium hydroxide suspension was the lowest. The in vivo data show that small differences in vaccine formulations before injection can generate significant changes in immunogenicity. Prior to mixing with the adjuvant, the physical and chemical characteristics of MMA383 antibodies were the same in all vaccines. Fluorescence and light scattering methods were developed to characterize antibodies in the presence of the adjuvant. Compared to the least active vaccines, the two most biologically active vaccines showed an increase in the antibody Trp fluorescence intensity, anisotropy, fluorescence lifetime, 90° light-scatter, sedimentation velocity and rotational correlation time. Analysis of the 90° light-scatter sedimentation kinetics indicates that stronger immune responses of vaccines can be related to the stronger binding of the antibodies to the adjuvants and the formation of more compact and condensed particles. Taken together, these results show a correlation between the in vitro fluorescence and light-scatter data and the in vivo immune response of the five MMA383 vaccines. The spectroscopic techniques described offer a new in vitro approach for the prediction of immune responses of different vaccine formulations.