The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10^-10 M) and had slow off-rates (10^-4 s^-1). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.

Background: Recent studies showed that a single injection of human monoclonal allergen‐specific IgG antibodies significantly reduced allergic symptoms in birch pollen‐allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen‐specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE‐mediated responses.Methods: A cDNA‐VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope‐specificity and affinity to Bet v 1. Furthermore, cross‐reactivity to Bet v 1‐homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen‐induced basophil activation were investigated.Results: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross‐react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha‐helix at the C‐terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1‐induced basophil activation.Conclusion: We identified high‐affinity Bet v 1‐specific nanobodies that recognize an important IgE epitope and reduce allergen‐induced basophil activation revealing the first proof that allergen‐specific nanobodies are useful tools for future treatment of pollen allergy.

(1) Background: There are no reliable and widely available markers of functional iron deficiency (FID) in cancer. The aim of the study was to evaluate the role of transferrin (Tf) as a marker of cancer of the ovary (CrO) and related FID. (2) Methods: The study groups consisted of 118 patients with CrO and 69 control females. Blood serum iron status was determined on a Beckman Coulter AU (USA) analyzer. Tf quantification was performed by immunoturbidimetry. The relative contents of apo- and holo-Tf (iron-free and iron-saturated Tf, respectively) were determined in eight patients and a control female by immunochromatographic analysis based on the use of monoclonal single-domain antibodies (nanobodies). (3) Results: Four groups of patients with different iron statuses were selected according to ferritin and transferrin saturation values: absolute iron deficiency (AID) (n = 42), FID (n = 70), iron overload (n = 4), normal iron status (n = 2). The groups differed significantly in Tf values (p < 0.0001). Lower values of Tf were associated with FID. Furthermore, FID is already found in the initial stages of CrO (26%). Immunosorbents based on nanobodies revealed the accumulation of apo-Tf and the decrease in holo-Tf in patients with CrO. (4) Conclusions: Tf may be a promising tool for diagnosing both CrO and associated FID.

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.

The development of new approaches and drugs for effective control of the chronic and complicated forms of urogenital chlamydia caused by Chlamydia trachomatis, which is suspected to be one of the main causes of infertility in both women and men, is an urgent task. We used the technology of single-domain antibody (nanobody) generation both for the production of targeting anti-chlamydia molecules and for the subsequent acquisition of anti-idiotypic nanobodies (ai-Nbs) mimicking the structure of a given epitope of the pathogen (the epitope of the Chlamydial Type III Secretion System Needle Protein). In a mouse model, we have shown that the obtained ai-Nbs are able to induce a narrowly specific humoral immune response in the host, leading to the generation of intrinsic anti-Chlamydia antibodies, potentially therapeutic, specifically recognizing a given antigenic epitope of Chlamydia. The immune sera derived from mice immunized with ai-Nbs are able to suppress chlamydial infection in vitro. We hypothesize that the proposed method of the creation and use of ai-Nbs, which mimic and present to the host immune system exactly the desired region of the antigen, create a fundamentally new universal approach to generating molecular structures as a part of specific vaccine for the targeted induction of immune response, especially useful in cases where it is difficult to prepare an antigen preserving the desired epitope in its native conformation.

A generally underestimated problem of immunoassays is its susceptibility to various interferences, being the Achilles heel of these assays (interventions in the analysis). The presence of interfering substances in the patient’s specimen can cause erroneous test sample result, which may lead to incorrect diagnosis and catastrophic consequences for the patient. Hence, one should pay particular attention to identifying possible interferences in the test systems used and, when possible, develop and apply methods to overcome them. The issue of avoiding interference is particularly important for immunoassays of biomarkers in human plasma or serum. In order to reduce possible interferences, it would be desirable to have an opportunity of specific and adaptable pretreatment of blood samples for a specifically assayed marker protein, as applied to a specific test system. We assume that such pretreatment may be done by combining the immunosorbents based on the use of special single-domain antibodies (nanobodies).The nanobodies are recombinant proteins, derivatives of single-domain antigen-recognizing variable fragments of specific antibodies, consisting of a dimer of truncated heavy chains in the complete absence of light chains. Such specific antibodies are detectable in the normal samples taken from members of the Camelidae family (Camelids), and in some species of cartilaginous fishes, along with the common antibodies. The special properties of nanobodies can provide certain advantages in their use, compared with antibodies of traditional structure and their derivatives.In this paper, we have shown for the first time, that the immunosorbents based on certain combination of single-domain antibodies used as ligands able for specifically binding and removal of specific high-abundance human blood proteins, may be selected for a given marker blood antigen in such a way that they will be an effective tool for blood pretreatment, aiming to reduce possible effects of interference and increased sensitivity in the diagnostic “sandwich” enzyme immunoassay. A new method of plasma pretreatment is demonstrated with human plasma samples (at a dilution of 1:40) of two patients. A previously developed model system for detection of lactoferrin protein was used to analyze plasma samples. It is shown that a significantly increased ratio of total detected signal to the nonspecific background signal could be obtained after drastic reduction of this background by affinity removal of 3 major protein fractions, i.e., fibrinogen, IgG alpha-2-macroglobulin from blood plasma samples, using appropriate immobilized single domain antibodies.