Most often used to detect the presence of antibodies rather than antigens, this assay is capable of continuous measurements of binding interactions in real-time [84]. general IIMI screening. Herein, we review the available literature to highlight cellular and molecular mechanisms underlying IIMI-mediated inflammation and its relevance to the safety and efficacy of pharmaceutical products. We further discuss methodologies used for direct and indirect IIMI identification and quantification. amebocyte lysate (LAL) test or mass spectrometry; sensitive silver staining (and immunoblotting) of electrophoretic gels; and quantitative HCP-specific immunoassays such as ELISAs [71], all of which will be discussed below. 6. Immune-Mediated Adverse Effects to Pharmaceutical Products The combination of a strong immunostimulatory response [3,35,43] and the activation of specialized subsets of T-cells leads to target-specific destruction of pathogens and cancer cells, either by direct interaction with CD8+ T-cells and Dibutyl phthalate natural killer (NK) cells or by CD4+ T-cell activation and proliferation of B-cells to produce antigen-specific antibodies [19,23,24,78]. This IIMI-driven immunogenicity can lead to the formation of antibodies of different isotypes (e.g., IgM vs. IgG vs. P4HB IgE), allotypes (e.g., reflecting genetic differences between IgG of biologically unrelated individuals), and idiotypes (e.g., reflecting binding to specific epitopes within antibody variable sites) [19,23,79,80,81], resulting in anti-drug antibodies (ADAs) with varying impacts on drug effectiveness. Binding antibodies attach to a non-active portion of the therapeutic and therefore have little/no effect on therapeutic function, whereas cross-reactive neutralizing antibodies bind to therapeutic active sites, thereby neutralizing therapeutic function while also binding similar endogenous proteins and breaking immunological tolerance [19,23,82,83,84]. The presence of these ADAs can also have different functional consequences to the host including the HSR/anaphylaxis and autoimmune responses previously discussed [19,23,35,79,80,81]. The relationship between the occurrence of a specific antibody type and the impact on the patient are inversely related; binding antibodies are the most common but have the lowest clinical impact, while cross-reacting neutralizing antibodies are rare but have the highest clinical impact [23,79,80,81,85]. Therefore, it is important to understand, measure, and prevent this response from being induced. During the fabrication and production of drug compounds, there are many potential sources for the introduction of IIMIs into the final biotherapeutic formulation (Figure 1) [19,20]. In addition to the impurities/contaminants previously discussed, there are also several product-related and host-related factors that may have little/no impact on the function of the resulting drug product but have been shown to impact the immunotoxicity and immunogenicity of biotherapeutics [19,23,78]. Product-related factors include structural properties of the drug (sequence, epitopes, post-translational modifications), exposure to antigenic sites, solubility, formulation stability and storage, downstream processing, presence of impurities/contaminants that might be introduced during processing [19,78]. These factors can be mostly controlled through careful optimization and modification of the fabrication/purification processes. Further compounding the risk of immunogenicity are host-related factors, including host genetic predisposition, endogenous protein genetic variants, concomitant illnesses (e.g., kidney or liver Dibutyl phthalate diseases), host immune status (e.g., autoimmunity, prior exposure) as well as the treatment dose, duration, and route of administration [19,23,78]. 7. Methods for IIMI Detection 7.1. Dibutyl phthalate Direct Detection Methods The first bioassay used to measure the presence of bacterial contamination was the rabbit pyrogen Dibutyl phthalate test Dibutyl phthalate (RPT) which detected pyrogens, any contaminant that induces a histamine response, fever, chills, and other unwanted inflammatory side effects. The rabbit pyrogen test detects all pyrogens, so it is subject to high variability and low selectivity, in addition to being expensive and requiring extensive use of animals [10,31]. As an improvement, the amebocyte lysate (LAL) test detects the hemolymph coagulation of the American horseshoe crab when in the presence of bacterial endotoxin/LPS and is used as a standard for bacterial contamination [86,87]. However, this assay is specific for endotoxin, not general pyrogens [31], and has reduced specificity in the presence of fungal -glucans because the horseshoe crab lysate used for this assay contains two proteins that trigger activation of the proteolytic cascade: factor C is specific to the presence of endotoxin while factor G is specific to -glucans [88,89]. Knowing this, a modified version of the LAL assay containing glucan-blocking reagents or recombinant factor C overcomes -glucan interference during endotoxin detection [90]. While -(1,3)-d-glucans are not as immunologically potent as bacterial endotoxins, requiring g/mL concentrations as compared to the endotoxin pg/mL concentrations to elicit an immunomodulatory response, they are a common IIMI present.