Novel monoclonal antibodies against house dust mite allergen Der p 21 and their application to analyze allergen extracts.

Background: Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Methods: Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. Results: MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. Conclusion: HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.

Structural properties of immune complexes formed by viral antigens and specific antibodies shape the inflammatory response of macrophages.

Data on the course of viral infections revealed severe inflammation as a consequence of antiviral immune response. Despite extensive research, there are insufficient data on the role of innate immune cells in promoting inflammation mediated by immune complexes (IC) of viral antigens and their specific antibodies. Recently, we demonstrated that antigens of human polyomaviruses (PyVs) induce an inflammatory response in macrophages. Here, we investigated macrophage activation by IC. We used primary murine macrophages as a cell model, virus-like particles (VLPs) of PyV capsid protein as antigens, and a collection of murine monoclonal antibodies (mAbs) of IgG1, IgG2a, IgG2b subclasses. The inflammatory response was investigated by analysing inflammatory chemokines and activation of NLRP3 inflammasome. We observed a diverse pattern of chemokine secretion in macrophages treated with different IC compared to VLPs alone. To link IC properties with cell activation status, we characterised the IC by advanced optical and acoustic techniques. Ellipsometry provided precise real-time kinetics of mAb-antigen interactions, while quartz crystal microbalance measurements showed changes in conformation and viscoelastic properties during IC formation. These results revealed differences in mAb-antigen interaction and mAb binding parameters of the investigated IC. We found that IC-mediated cell activation depends more on IC characteristics, including mAb affinity, than on mAb affinity for the activating Fc receptor. IC formed by the highest affinity mAb showed a significant enhancement of inflammasome activation. This may explain the hyperinflammation related to viral infection and vaccination. Our findings demonstrate that IC promote the viral antigen-induced inflammatory response depending on antibody properties.

Prevalence of SARS-CoV-2-specific antibodies in a sample of the Lithuanian population-based study in Spring 2023.

Objectives: Despite positive trends in SARS-CoV-2 epidemiology, seroprevalence surveys remain an important tool for estimating the magnitude of the COVID-19 pandemic. This study aimed to investigate the prevalence of IgG antibodies against SARS-CoV-2 nucleocapsid (N) and spike (S) proteins in a sample of the Lithuanian population (N = 517) and evaluate how the pattern of seropositivity correlates with the levels of SARS-CoV-2 infection and vaccination. Methods: Study participants (aged 18-88 years) filled in the questionnaire self-reporting their demographic-social variables, health status, and SARS-CoV-2-related status. The anti-S and anti-N IgG levels were estimated using a microarray ELISA test. Results: After several pandemic waves and vaccination campaign, the seroprevalence of SARS-CoV-2-specific IgG in the analyzed sample was 97.87 % by March-May 2023. We determined the 96.91 % prevalence of anti-S and 58.03 % prevalence of anti-N IgG. The majority of study participants (71.18 %) had hybrid immunity induced by vaccination and SARS-CoV-2 infection. 20.3 % of study participants were anti-N IgG positive without reporting any previous symptoms or a positive SARS-CoV-2 test. A decline of anti-N IgG positivity within 9 months after infection was observed. Conclusions: This study demonstrates high total seroprevalence in March-May 2023 in all age groups indicating a widely established humoral immunity against SARS-CoV-2 in Lithuania.

Molecular characteristics of Neisseria meningitidis carriage strains in university students in Lithuania.

BACKGROUND: Neisseria meningitidis can be carried asymptomatically in the human oropharynx without causing symptoms. Meningococcal carriage is relevant to the epidemiology of invasive meningococcal disease (IMD). No carriage studies have been performed among the general population in Lithuania, whereas the incidence of IMD in Lithuania was among the highest in European countries from 2009 to 2019. RESULTS: We analyzed a total of 401 oropharyngeal samples collected from university students from December 2021 to February 2023 for N. meningitidis carriage using direct swab PCR assays and culture. The overall carriage prevalence based on both or either swab PCR or culture was 4.99%. PCR-based assays were used to characterize 15 carriage isolates, including detection of genogroup, multilocus sequence typing profile, and typing of antigens PorA and FetA. The most common carriage isolates were capsule null locus (cnl), accounting for 46.7%, followed by genogroups B (26.7%) and Y (13.3%). We also performed a molecular characterization of invasive N. meningitidis isolates collected during the COVID-19 pandemic and post-pandemic period to understand better the meningococcal carriage in the context of prevailing invasive strains. Despite the substantial decrease in the incidence of IMD during the 2020-2022 period, clonal complex 32 (CC32) of serogroup B continued to be the most prevalent IMD-causing CC in Lithuania. However, CC32 was not detected among carriage isolates. The most common CCs were CC269, CC198, and CC1136. The antigen peptide variants found in most carried isolates were classified as 'insufficient data' according to the MenDeVAR Index to evaluate the potential coverage by the 4CMenB vaccine. Nearly half of the isolates were potentially covered by the Men-Fhbp vaccine. Resistance to ciprofloxacin was detected only for one cnl isolate. All isolates were susceptible to penicillin and ceftriaxone. Our analysis identified frequent partying (≥ 4 times/month) as a risk factor for meningococcal carriage, whereas smoking, living in a dormitory, and previous COVID-19 illness were not associated with the carriage. CONCLUSIONS: Our study revealed a low prevalence of meningococcal carriage among university students in Lithuania. The carriage isolates showed genetic diversity, although almost half of them were identified as having a null capsule locus.

Molecular characterization of invasive Neisseria meningitidis isolates collected in Lithuania (2009-2019) and estimation of serogroup B meningococcal vaccine 4CMenB and MenB-Fhbp coverage.

Neisseria meningitidis causes invasive meningococcal disease (IMD), which is associated with significant mortality and long-term consequences, especially among young children. The incidence of IMD in Lithuania was among the highest in European Union/European Economic Area countries during the past two decades; however, the characterization of meningococcal isolates by molecular typing methods has not yet been performed. In this study, we characterized invasive meningococcal isolates (n=294) recovered in Lithuania from 2009 to 2019 by multilocus sequence typing (MLST) and typing of antigens FetA and PorA. The more recent (2017-2019) serogroup B isolates (n=60) were genotyped by analyzing vaccine-related antigens to evaluate their coverage by four-component (4CMenB) and two-component (MenB-Fhbp) vaccines using the genetic Meningococcal Antigen Typing System (gMATS) and Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index methods, respectively. The vast majority (90.5%) of isolates belonged to serogroup B. MLST revealed a predominance of clonal complex 32 (74.02%). Serogroup B strain P1.19,15: F4-28: ST-34 (cc32) accounted for 64.1% of IMD isolates. The overall level of strain coverage by the 4MenB vaccine was 94.8% (CI 85.9-98.2%). Most serogroup B isolates (87.9%) were covered by a single vaccine antigen, most commonly Fhbp peptide variant 1 (84.5% of isolates). The Fhbp peptides included in the MenB-Fhbp vaccine were not detected among the analyzed invasive isolates; however, the identified predominant variant 1 was considered cross-reactive. In total, 88.1% (CI 77.5-94.1) of isolates were predicted to be covered by the MenB-Fhbp vaccine. In conclusion, both serogroup B vaccines demonstrate potential to protect against IMD in Lithuania.

Immunogenic Properties and Antigenic Similarity of Virus-like Particles Derived from Human Polyomaviruses.

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system.

Characterization of a Panel of Cross-Reactive Hantavirus Nucleocapsid Protein-Specific Monoclonal Antibodies.

Hantaviruses are emerging pathogens with a worldwide distribution that can cause life-threatening diseases in humans. Monoclonal antibodies (MAbs) against hantavirus nucleocapsid (N) proteins are important tools in virus diagnostics, epidemiological studies and basic research studies on virus replication and pathogenesis. Here, we extend the collection of previously generated MAbs raised against a segment of Puumala orthohantavirus (PUUV) N protein harbored on virus-like particles (VLPs) and MAbs against N proteins of Sin Nombre orthohantavirus/Andes orthohantavirus by generating nine novel MAbs against N proteins of Dobrava-Belgrade orthohantavirus (DOBV), Tula orthohantavirus (TULV), Thottapalayam thottimvirus (TPMV) and PUUV. In order to have a wide collection of well-described hantavirus-specific MAbs, the cross-reactivity of novel and previously generated MAbs was determined against N proteins of 15 rodent- and shrew-borne hantaviruses by different immunological methods. We found that all MAbs, excluding TPMV-specific MAbs, demonstrated different cross-reactivity patterns with N proteins of hantaviruses and recognized native viral antigens in infected mammalian cells. This well-characterized collection of cross-reactive hantavirus-specific MAbs has a potential application in various fields of hantavirus research, diagnostics and therapy.

Cross-reactive monoclonal antibodies against fish parvalbumins as a tool for studying antigenic similarity of different parvalbumins and analysis of fish extracts.

Fish parvalbumins are heat-stable calcium-binding proteins that are highly cross-reactive in causing allergy symptoms in fish-sensitized patients. The reactivities of parvalbumin-specific monoclonal or polyclonal antibodies with parvalbumins of different fish species allowed their application for development of various immunoassays for allergen identification in fish samples. In this study, monoclonal antibodies (MAbs) were generated against two parvalbumins - natural Atlantic cod parvalbumin and recombinant common carp ß-parvalbumin expressed in E. coli. Large collections of recombinant parvalbumins and natural allergen extracts of different fish species and other animals were used to identify the specificities of these MAbs using ELISA, Western blot, and dot blot. MAbs demonstrated different patterns of cross-reactivities with recombinant parvalbumins. Their binding affinities were affected by the addition and removal of Ca2+ ions. Moreover, all MAbs showed a broad reactivity with the target antigens in natural fish, chicken, and pork extracts. The ability of two MAbs (clones 7B2 and 3F6) to identify and isolate native parvalbumins from allergen extracts was confirmed by Western blot. Epitope mapping using recombinant fragments of Atlantic cod parvalbumin (Gad m 1) and common carp parvalbumin (Cyp c 1) revealed that 4 out of 5 MAbs recognize parvalbumin regions that contain calcium binding sites. In conclusion, the generated broadly reactive well-characterized MAbs against fish ß-parvalbumins could be applied for investigation of parvalbumins of fish and other animals and their detection in allergen extracts.

Factors, associated with elevated concentration of soluble carbonic anhydrase IX in plasma of women with cervical dysplasia.

Precancerous lesions of human cervix uteri have a tendency for regression or progression. In cervical intraepithelial neoplasia grade 2 (CINII) case there is an uncertainty if a lesion will progress or regress. The carbonic anhydrase IX (CAIX) enzyme is overexpressed in cervical cancer which is more sensitive to radiotherapy. CAIX is associated with poor prognosis in solid hypoxic tumors. The aim of this study was to determine factors related to elevated soluble CAIX (s-CAIX) in high-grade intraepithelial lesion (HSIL) cases. METHODS: Patients diagnosed with HSIL (N = 77) were included into the research group whereas without HSIL (N = 72)-the control group. Concentration of the soluble CAIX (s-CAIX) in plasma was determined by the DIANA ligand-antibody-based method. C. trachomatis was detected from cervical samples by PCR. Primary outcomes were risk factors elevating s-CAIX level in HSIL group. Non-parametric statistical analysis methods were used to calculate correlations. RESULTS: The s-CAIX level in patients with HSIL was elevated among older participants (rs = 0.27, p = 0.04) and with C. trachomatis infection (p = 0.028). Among heavy smokers with HSIL, the concentration of s-CAIX was higher in older women (rs = 0.52, p = 0.005), but was not related to the age of heavy smokers' controls (τ = 0.18 p = 0.40). CONCLUSION: The concentration of s-CAIX was higher among older, heavy smoking and diagnosed with C. trachomatis patients. All these factors increased the risk for HSIL progression.

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages.

Viral antigens can activate phagocytes, inducing inflammation, but the mechanisms are barely explored. The aim of this study is to investigate how viral oligomeric proteins of different structures induce inflammatory response in macrophages. Human THP-1 cell line was used to prepare macrophages that were treated with filamentous nucleocapsid-like particles (NLPs) of paramyxoviruses and spherical virus-like particles (VLPs) of human polyomaviruses. The effects of viral proteins on cell viability, pro-inflammatory cytokines' production, and NLRP3 inflammasome activation were investigated. Filamentous NLPs did not induce inflammation while spherical VLPs mediated inflammatory response followed by NLRP3 inflammasome activation. Inhibitors of cathepsins and K+ efflux decreased IL-1ß release and cell death, indicating a complex inflammasome activation process. A similar activation pattern was observed in primary human macrophages. Single-cell RNAseq analysis of THP-1 cells revealed several cell activation states different in inflammation-related genes. This study provides new insights into the interaction of viral proteins with immune cells and suggests that structural properties of oligomeric proteins may define cell activation pathways.

Persistence of SARS-CoV-2-Specific Antibodies for 13 Months after Infection.

BACKGROUND: Dynamics of antibody responses were investigated after a SARS-CoV-2 outbreak in a private company during the first wave of the pandemic. METHODS: Workers of a sewing company (Lithuania) with known SARS-CoV-2 RT-PCR result during the outbreak (April 2020) were invited to participate in the study. Virus-specific IgG and IgM were monitored 2, 6 and 13 months after the outbreak via rapid IgG/IgM serological test and SARS-CoV-2 S protein-specific IgG ELISA. RESULTS: Six months after the outbreak, 95% (CI 86-99%) of 59 previously infected individuals had virus-specific antibodies irrespective of the severity of infection. One-third of seropositive individuals had virus-specific IgM along with IgG indicating that IgM may persist for 6 months. Serological testing 13 months after the outbreak included 47 recovered individuals that remained non-vaccinated despite a wide accessibility of COVID-19 vaccines. The seropositivity rate was 83% (CI 69-91%) excluding one case of confirmed asymptomatic reinfection in this group. Between months 6 and 13, IgG levels either declined or remained stable in 31 individual and increased in 7 individuals possibly indicating an exposure to SARS-CoV-2 during the second wave of the pandemic. CONCLUSIONS: Detectable levels of SARS-CoV-2-specific antibodies persist up to 13 months after infection for the majority of the cases.

Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9-100.0 %) and DSp to be 98.4 % (95 % CI, 96.8-99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.

Selective immunocapture and light-controlled traceless release of transiently caged proteins.

The 4,5-dimethoxy-2-nitrobenzyl (DMNB) photocaging group introduced into small biomolecules, peptides, oligonucleotides, and proteins is commonly used for spatiotemporal control of chemical and biological processes. Here, we describe the use of a DMNB-selective monoclonal antibody for non-covalent capture of chemically or biosynthetically produced proteins containing surface-exposed DMNB caging groups followed by light-controlled traceless decaging and release of the bound proteins into solution for a variety of downstream applications. For complete details on the use and execution of this protocol, please refer to Rakauskaite et al. (2020).

Papillary Thyroid Carcinoma Tissue miR-146b, -21, -221, -222, -181b Expression in Relation with Clinicopathological Features.

We analyzed miR-146b, miR-21, miR-221, miR-21, and miR-181b in formalin fixed paraffin-embedded papillary thyroid carcinoma (PTC) tissue samples of 312 individuals and evaluated their expression relationship with clinicopathological parameters. A higher expression of miR-21 was related to unifocal lesions (p < 0.011) and autoimmune thyroiditis (0.007). miR-221, miR-222 expression was higher in the PTC tissue samples with extrathyroidal extension (p = 0.049, 0.003, respectively). In a group of PTC patients with pT1a and pT1b sized tumors, the expression of miR-146b, miR-21, miR-221, and miR-222 in PTC tissue samples was lower than in patients with pT2, pT3, and pT4 (p = 0.032; 0.0044; 0.003; 0.001; 0.001, respectively). Patients with lymph node metastases had higher expression of miR-21, -221, -222, and -181b (p < 0.05). A high expression of miR-146b, miR-21, miR-221 panel was associated with decreased overall survival (OS) (Log rank p = 0.019). Univariate analysis revealed that presence of metastatic lymph nodes and high expression of miR-146b, miR-21, and miR-221 panels were associated with increased hazard of shorter OS. After multivariate analysis, only sex (male) and age (≥55 years) emerged as independent prognostic factors associated with shorter OS (HR 0.28 (95% CI 0.09-0.86) and HR 0.05 (95% CI 0.01-0.22), respectively). In conclusion, 5 analyzed miRs expression have significant relations to clinicopathologic parameters so further investigations of these molecules are expedient while searching for prognostic PTC biomarkers.

Evaluation of kinetics and thermodynamics of interaction between immobilized SARS-CoV-2 nucleoprotein and specific antibodies by total internal reflection ellipsometry.

During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔGAssoc) for SCoV-rN and anti-SCoV-rN binding was determined as -34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications.

Characterization of the GBoV1 Capsid and Its Antibody Interactions.

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.

Photocage-Selective Capture and Light-Controlled Release of Target Proteins.

Photochemical transformations enable exquisite spatiotemporal control over biochemical processes; however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. Here we created a high-affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues, which were used for raising a high-affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a variety of DMNB-caged target proteins in complex biological mixtures. This combined orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays.

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells.

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a 'wound healing' assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

Different stability of miRNAs and endogenous control genes in archival specimens of papillary thyroid carcinoma.

BACKGROUND: The most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression. METHODS: RQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years. RESULTS: The stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time. CONCLUSIONS: It is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.