The effect of antigen-adjuvant associations on antigen uptake and antigen-specific humoral immunity is studied in detail. After formulation with a squalene-based double emulsion (referred to as PELC), the protein ovalbumin (OVA) was intramuscularly injected in mice, in either a separation (OVA-PELC_SE), a surface attachment (OVA-PELC_SA) or an encapsulation (OVA-PELC_EN) manner. As an antigen delivery system, a significant increase of OVA-loaded cells migrating into draining lymph nodes (LNs) was detected in the PELC-formulated OVA groups, attachment and encapsulation as well. Additionally, OVA-PELC_EN allowed the mice to induce a delayed but long-lasting OVA-specific antibodies production compared to OVA-PELC_SA. In the extreme case where no antigen-adjuvant association at all (i.e., OVA-PELC_SE), we found that even with the presence of PELC at the contralateral limb, an elevated level of OVA uptake was detected in ipsilateral draining CD11c⁺ LN cells, which subsequently augmented the production of OVA-specific IgG antibodies during early vaccination. The mouse study allows us to find out the optimal vaccine formulation and deepens our understandings on how antigen-adjuvant associations can govern the cellular uptake and transportation of protein antigen into the draining LNs and prolong antigen-specific humoral immunity, even if the antigen and the adjuvant are given separately.