Immunoassays are used to quantify biomolecules (analytes) using specific antibodies produced against these particular molecules . Since 1978, when ELISA was first described , it became the gold standard antigen-antibody detection assay in basic research of which some assays were further optimized for diagnostic purposes and became commercially available. More recent point-of-care diagnostic technologies such as smart cards  and lab-on-chip technology  were also based on the same principles of ELISA. Among the different types of ELISA, sandwich ELISA is considered a more sensitive and robust assay and tends to be the most commonly used type. Sandwich ELISA targets one antigen using two different antigenic sites detected by two different antibodies in the same assay. The first is known as “the capturing antibody” that is bound to the solid phase. This antibody captures the analyte from the sample then another antibody known as “the detection antibody” is used to detect the antigen bound—now sandwiched .
Diagnostic studies rely on sandwich ELISA as a key assay in many fields. It is used in the detection of bacterial , parasitic , fungal , and viral infections in both human and animals  as well as in botany research to detect plant viral disease . In addition, it is also used in biomarker profiling [10, 11] and biomarker-based diagnostic assays for cancer diagnosis , diagnosis of autoimmune diseases , and Alzheimer’s . Moreover, it has applications in vaccine development  and conjugate vaccine assessment . Also, it is of use in discrimination of allergic cultivates for human  and even as a basic bio-approach in engineering as in early-diagnostic biosensors .
Sandwich ELISA, despite being a conventional assay, was in many cases further developed into time saving diagnostic kits. One successful example is the immunochromatographic card (ICT) for qualitative detection of circulating filariasis, Wuchereria bancrofti, antigen . The test card was further optimized into semi-quantitative card that does not only detect but also helps in grading the degree of infection . Now this rapid test-card is used as a point-of-care diagnostic assay to detect and follow-up patient’s response to therapy . Despite being a simple and direct principle, it remains a key assay due to both the sensitivity and specificity of the detection which emphasizes the importance of the development and optimization of the reaction conditions for various applications.
Sandwich ELISA development and optimization includes the selection of matrices, buffers, and detective means but most importantly the choice of a matching capturing and detection antibodies and the orientation of the binding assembly . This double antibody detection assay should be using two non-overlapping, non-interfering antigen-specific antibodies with a minimal or completely absent cross-reactivity. This raises not only a concern about the quality and specificity of the antibody, but also the source host where the antibodies were raised. For commercially available basic antibodies, this can be solved by choosing the host of the antibody from a panel of available labeled and unlabeled antibodies. On the other hand, in case of studying a specific agent analyte, the antibodies should be raised in laboratory animals.
Mice and rats remain the top chosen laboratory animals due to their feasible manipulation and breeding conditions, low cost, and the uncomplicated ethical approval. But it is well known that the two species directly relate phylogenetically to one another . This raises the questions: to which extent the chosen mouse and rat species to raise anti-sera are immunologically related? And to which extent the developed antibodies in both models might cross-react with one another if used for capturing and detection in a sandwich ELISA?
Although these questions sound very trivial, and are frequently asked, we carefully searched the available literature and found no clear experimental answers for both. What increased the confusion was the commercial availability of antibodies that cross-react and detect both mice and rat immunoglobulins, while there is also both polyclonal rat-anti-mouse and mouse-anti-rat antibodies as matched pairs for rat and mouse-based sandwich ELISA without any hint or instructions about the extent of cross-reaction among both.
Therefore, the present work evaluates both anti-mouse IgG and anti-rat IgG for their possible cross-reactivity in the specific and non-specific detection of mouse and rat serum IgG. This evaluation aims to guide on the best species-serum order that allows minimal cross-reaction when establishing a sandwich ELISA.