RESUMO

Zika virus has spread around the world with rapid pace in the last five years. Although symptoms are typically mild and unspecific, Zika's major impact occurs during pregnancy, generating a congenital syndrome. Serology plays a key role in its diagnosis. However, its use is limited due to the uncertainty caused by the cross-reaction of antibodies elicited in response to other flavivirus infections when tested in direct immunoassays. Using a panel of previously generated anti-Zika non-structural protein 1 (NS1) nanobodies, a set was selected that only recognizes epitopes present in Zika and is immunogenic to humans. A proper arrangement of these nanobodies was made and conditions were optimized in order to develop a novel serology assay. This new ELISA relies on the inhibition of the binding of a set of selected nanobodies to Zika-immobilized NS1 when previously incubated with Zika convalescent sera. Using the developed blocking of binding assay, it was possible to discriminate between Zika-specific and cross-reactive antibodies in serum samples from infections with Zika and other flaviviruses.

RESUMO

Colon cancer has a high prevalence worldwide and is a serious public health problem. Early diagnosis greatly improves its prognosis and, among the existing methods, the detection of fecal occult blood is the only noninvasive test recommended for screening of the disease. To promote its massive application as a screening tool for asymptomatic populations in low-resource settings, the availability of a reliable and cost-effective method is imperative. Here, we describe the development and validation of a sensitive nanobody-based immunoassay for the detection of hemoglobin in human fecal samples. The nanobodies were selected from a library generated from a llama immunized with human hemoglobin, using a high-throughput platform that enabled the identification of the best nanobody pair. The assay allowed a sub-ng/mL limit of detection to be reached in phosphate-buffered saline, and was validated with stool samples, showing excellent reproducibility (CV% < 15 inter-day precision) and accuracy at 2 and 4 µg of hemoglobin per gram of feces, which are well below the recommended cutoff for this test (10-20 µg/g). Moreover, no cross-reactivity was observed with a panel of dietary non-human hemoglobins removing the need for pre-test dietary restrictions. Considering that the monodomain nature of nanobodies facilitates their straightforward and low-cost production by bacterial fermentation, with their provided sequences and using synthetic genes, the assay reported here could be replicated in any laboratory to perform thousands of tests for early detection of colorectal cancer at almost no cost. Graphical abstract.

Assuntos

RESUMO

With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs) has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb)] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

RESUMO

Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

Assuntos

RESUMO

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (~30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.

Assuntos