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In situ vaccination with immunostimulatory compounds is a demonstrated means to treat tumors preclinically. While these therapeutic effects have been attributed to the actions of T cells or innate immune activation, characterisation of the humoral immune response is seldom performed. This study aims to identify whether the injection of immunoadjuvants, Addavax (Adda) and cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG), intratumorally can influence the antibody response. Specifically, whether intratumoral injection of immunoadjuvants can alter the tumor-specific antibody target, titre and isotype. Following this, the study aimed to investigate whether serum obtained from in situ vaccinated mice could neutralise circulating tumor cells. Serum was obtained from mice bearing B16F10-OVA-Luc-GFP tumors treated with immunoadjuvants. Antibody targets' titre and isotype were assessed by indirect ELISA. The ability of serum to neutralise circulating cancer cells was evaluated in a B16F10 pseudo-metastatic model. It was observed that tumor-bearing mice mount a specific anti-tumor antibody response. Antibody titre and target were unaffected by in situ vaccination with immunoadjuvants; however, a higher amount of IgG2c was produced in mice receiving Adda plus CpG. Serum from in situ vaccinated mice was unable to neutralise circulating B16F10 cells. Thus, this study has demonstrated that anti-tumor antibody isotype may be modified using in situ vaccination; however, this alone is not sufficient to neutralise circulating cancer cells. [ABSTRACT FROM AUTHOR]

Objectives: Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been instrumental in detecting previous exposures and analyzing vaccine‐elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS‐CoV‐2 antibodies, discriminate between natural infection‐ and vaccination‐induced responses, and assess antibody‐mediated inhibition of the spike‐angiotensin converting enzyme 2 (ACE2) interaction. Methods: We developed methods and reagents to detect SARS‐CoV‐2 antibodies by enzyme‐linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2‐spike or ‐RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results: Our single‐point IgG‐based ELISAs accurately distinguished non‐infected and infected individuals. For seroprevalence assessment (in a non‐vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti‐spike and ‐RBD (but not ‐N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. Conclusions: Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike‐ACE2 interactions in high‐throughput enables large‐scale analyses of humoral immune responses to SARS‐CoV‐2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter‐laboratory data comparison and aggregation. [ABSTRACT FROM AUTHOR]

Abstract Objectives Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been instrumental in detecting previous exposures and analyzing vaccine‐elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS‐CoV‐2 antibodies, discriminate between natural infection‐ and vaccination‐induced responses, and assess antibody‐mediated inhibition of the spike‐angiotensin converting enzyme 2 (ACE2) interaction. Methods We developed methods and reagents to detect SARS‐CoV‐2 antibodies by enzyme‐linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2‐spike or ‐RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results Our single‐point IgG‐based ELISAs accurately distinguished non‐infected and infected individuals. For seroprevalence assessment (in a non‐vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti‐spike and ‐RBD (but not ‐N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. Conclusions Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike‐ACE2 interactions in high‐throughput enables large‐scale analyses of humoral immune responses to SARS‐CoV‐2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter‐laboratory data comparison and aggregation.