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The availability of purified antibodies is a prerequisite for many applications and the appropriate choice(s) for antibody-purification is crucial. Numerous methods have been developed for the purification of antibodies from different sources with affinity chromatography-based methods being the most extensively utilized. These methods are based on high specificity, easy reversibility and biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). However, no simple techniques have yet been described to characterize and purify subclasses of immunoglobulins (Ig) from some animals of biotechnology importance such as equines, which are frequently used to produce biotherapeutic antibodies. The sera of these animals present a large number of Ig classes that have a greater complexity than other animals. The implementation of an effective protocol to purify the desired antibody class/subclasses requires meticulous planning to achieve yields at a high purity. The IgG3 subclass of equine-Ig has recently been used as antigen in a new diagnostic test for allergic responses to horse sera-based therapies. Here, we defined a simple method using Jacalin lectin immobilized on Sepharose beads to prepare highly pure equine IgG3 antibodies with a determination of the affinity constants for Jacalin lectin and horse IgG3., (Copyright © 2018. Published by Elsevier Inc.)

Lymphatic filariasis (LF) is a neglected tropical disease transmitted by mosquitoes and the second cause of permanent disability leading to a significant morbidity and mortality rate. Previously, we have identified epitopes of the filarial abundant larval transcript-2 (ALT-2) protein using a microarray mapping. In this study, one of the epitopes (Wb/ALT2-A5) was used to construct an electrochemical immunosensor. Electrochemical technique of cyclic voltammetry was performed for detecting the signal generated by the interaction between the (Wb/ALT2-A5) peptide and circulating antibodies of serum human samples. (Wb/ALT2-A5) epitope antigens were successfully immobilized on the working electrode of a screen-printed carbon electrode (SPCE) by their amine groups via chitosan film by coupling with glutaraldehyde as crosslinker. After the sensor ready, a pool of human sera infected with Wuchereria bancrofti was added to its surface. Electrochemical responses were generated by applying a potential of - 0.6 to 0.6 V, scan rate of 0.025 V/s. A detection limit of 5.0 µg mL -1 for the synthetic peptides (Wb/ALT2-A5) and 0.002 µg mL -1 for human serum, with a sensitivity of 1.86 µA. The performance of this assay was successfully tested in human serum samples from infected and healthy patients. Thus, this proposed immunosensor, which is able to identify circulating antibodies, can be applied to the diagnosis of the W. bancrofti parasitic disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Spotted fever is a rare acute and multisystemic febrile infectious disease with a mortality rate of ≥50% without adequate antibiotic treatment, and in diagnosed and treated cases, of approximately 2.5%. Currently, the applied test to diagnose this disease is the indirect immunofluorescence reaction, however two samples of paired sera are necessary to confirm the diagnosis, since using only one sample may allow for confusion with cross reactions. OmpA is an outer membrane protein present in the R. rickettsia, the etiological agent of spotted fever, able to activate dendritic and macrophage cells. It also presents immunogenicity properties, and is considered a target for the development of diagnostic tests for spotted fever. In this context, an amperometric immunosensor was developed for the identification of sera antibodies (human IgG) from patients with spotted fever aimed at improving sensitivity and minimize sample volume. The development of the immunosensor was conducted using a synthetic peptide, derivative from the H6PGA4 R. rickettsia protein, homologous to OmpA. Amperometric responses were generated at -0.6 to 0.6V, at a scan rate of 0.025Vs -1 for 20 cycles, a limit of detection of approximately 10ngmL -1 for the synthetic peptides and 0.01µgmL -1 for the humam serum, a sensitivity of 2.59µA, adequate for the detection of spotted fever antibodies. The construction of this immunosensor, capable of identifying circulating antibodies in real time, can also be applied in the diagnosis of other infectious-parasitic diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Antivenom allergy disease mediated by patient IgE is an important public health care concern. To improve detection of hypersensitive individuals prior to passive antibody therapy, an amperometric immunosensor was developed to detect reactive human IgE. Whole horse IgG3 (hoIgG3) was immobilized onto the surface of carbon or gold screen-printed electrodes through a cross-linking solution of glutaraldehyde on a chitosan film. Sera from persons with a known allergic response to hoIgG3 or non-allergic individuals was applied to the sensor. Bound human IgE (humIgE) was detected by an anti-humIgE antibody through a quantitative amperometric determination by tracking via the electrochemical reduction of the quinone generated from the hydroquinone with the application of a potential of 25 mV. The optimal immunosensor configuration detected reactive humIgE at a dilution of 1:1800 of the human sera that represent a detection limit of 0.5 pg/mL. Stability testing demonstrated that through 20 cycles of a scan, the specificity and performance remained robust. The new immunosensor successfully detected humIgE antibodies reactive against hoIgG3, which could allow the diagnosis of potential allergenic patients needing therapeutic antivenom preparations from a horse., (Copyright © 2017. Published by Elsevier Inc.)

Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 μg of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.