The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical immunochemistry assay based on the specific bond between the antigen and the antibody. The application of this test has significantly changed the practice of medical laboratories in which it is used for detection and quantification of molecules such as hormones, peptides, antibodies, and proteins. Various technical variants of this test can detect antigen (native or foreign) or antibody, determine the intensity of the immune response whether pathological or not; the type of induced immune response as well as the innate immunity potential; and much more. These capabilities, as well as the high sensitivity and robustness of the test and a small price, make it possible to quickly and reliably diagnose diseases in most laboratories. Besides, ELISA is a test that is also used in veterinary medicine, toxicology, allergology, food industry, etc. Despite the fact that it has existed for almost 50 years, different ELISA tests with different technical solutions are still being developed, which improves and expands the application of the this exceptional test. The aim of this chapter is to empower the rider to optimize, standardize and validate an enzyme linked immunosorbent assay.

Successful induction of antiphospholipid syndrome (APS) in two different non-autoimmune prone mouse strains, BALB/c and C57BL/6, was achieved by tetanus toxoid (TTd) hyperimmunization using different adjuvants (glycerol or aluminium hydroxide), and different adjuvant pretreatments (glycerol or Complete Freund’s Adjuvant (CFA)). APS had different manifestations of reproductive pathology in BALB/c and C57BL/6 mice: fetal resorption (as a consequence of extreme T-cell activation obtained in the course of pretreatment), and lowering of fecundity (as a consequence of polyclonal B-cell stimulation), respectively. In BALB/c mice fetal resorption coincided with glycerol and CFA pretreatments, while in C57BL/6 mice lowering of fecundity was most obvious in CFA-pretreated mice immunized with TTd in aluminium hydroxide. Both molecular mimicry and polyclonal B-cell activation occur in APS induction, with molecular mimicry effects being dominant in BALB/c mice, and polyclonal cell activation being dominant in C57BL/6 mice. Confirmation of molecular mimicry effects, which in the condition of T-cell stimulation generated fetal resorptions in the BALB/c strain, was achieved by passive infusion of monoclonal antibody (MoAb) T-26 specific for TTd and anti-β₂-glycoprotein I obtained after TTd hyperimunization. High polyclonal B-cell activation in C57BL/6 mice prevented fetal resorption but induced fecundity lowering, as was the case in passive administration of MoAb T-26 in this mouse strain. Passive infusion of anti-idiotypic MoAb Y7 into C57BL/6 mice induced fetal resorptions and confirmed the above suggestion on the protective role of polyclonal B-cell stimulation in fetal resorptions.

During time, both professionals and general public became aware of the importance of animal welfare. This term not only covers endangered wild animal species, animals used in food industry, pets and experimental animals, but also animals used in production of biologics. The implementation of the 3R concept (Replacement, Reduction and Refinement) is especially important in this type of production. In this article, we describe for the first time the low dose, low volume and multi-site immunization protocol, as well as appropriate ELISA we developed for production of European anti-viper ( V. ammodytes , long horned) antivenom in horses, which can help to significantly improve the welfare of the used animals.

Deep proteomic analyses identified, in total, 159 master proteins (with 1% FDR and 2 unique peptides) from 26 protein families in the venom of Vipera ammodytes ammodytes (Vaa). Data are available via ProteomeXchange with the identifier PXD056495. The relative abundance of PLA2s is 11.60% of the crude venom, of which 4.35% are neurotoxic Ammodytoxins (Atxs). The neurotoxicity of the venom of Vaa and the neutralizing effect of the antivenom were tested on the neuromuscular preparation of the diaphragm (NPD) of rats. The activity of PLA2 in the venom of Vaa and its neutralization by the antivenom were determined under in vitro conditions. The Vaa venom leads to a progressive decrease in NPD contractions. We administered pre-incubated venom/antivenom mixtures at various ratios of 1:2, 1:10 and 1:20 (w/w) and observed the effects of these mixtures on NPD contractions. The results show that the mean effective time (ET₅₀) for NPD contractions with the 1:20 mixture is highly significantly different (p < 0.001) from the ET₅₀ for the venom and the ET₅₀ for the 1:2 and 1:10 mixture ratios. We also found a highly significant (p < 0.001) reduction in Na⁺/K⁺-ATPase activity in the NPD under the influence of the venom. The reduction in the activity of this enzyme was reversible by the antivenom. Under in vitro conditions, we have achieved the complete neutralization of PLA2 by the antivenom. In conclusion, the antivenom abolished the venom-induced progressive decrease in NPD contractions in a concentration-dependent manner. Antivenom with approximately the same mass proportion almost completely restores Na⁺/K⁺-ATPase activity in the NPD and completely neutralizes the PLA2 activity of the venom in vitro.