Primary Antibody Research Articles

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 μL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

Distinguishable Magnetic Reporter Coordination with Buoyancy-Magnetism Separation for Immobilization-Free Dual-Target Electrochemical Immunosensing.

Simultaneous sensitive and precise determination of multibiomarkers is of great significance for improving detection efficiency, reducing diagnosis and treatment expenses, and elevating survival rates. However, the development of simple and portable biosensors for simultaneous determination of multiplexed targets in biological fluids still faces challenges. Herein, a unique and versatile immobilization-free dual-target electrochemical biosensing platform, which combines distinguishable magnetic signal reporters with buoyancy-magnetism separation, was designed and constructed for simultaneous detection of carcinoembryonic (CEA) and α-fetoprotein (AFP) in intricate biological fluids. To construct such distinguishable magnetic signal reporters with signal transduction, amplification, and output, secondary antibodies of CEA and AFP were respectively functionalized on methylene blue (MB) and 6-(ferrocenyl)hexanethiol (FeC) modified Fe3O4@Au magnetic nanocomposites. Meanwhile, a multifunctional flotation probe with dual target recognition, capture, and isolation capability was prepared by conjugating primary antibodies (Ab1-CEA, Ab1-AFP) to hollow buoyant microspheres. The target antigens of CEA and AFP can trigger a flotation-mediated sandwich-type immunoreaction and capture a certain amount of the distinguishable magnetic signal reporter, which enables the conversion of the target CEA and AFP quantities to the signal of the potential-resolved MB and FeC. Thus, the MB and FeC currents of magnetically adsorbed distinguishable magnetic reporters can be used to determine the CEA and AFP targets simultaneously and precisely. Accordingly, the proposed strategy exhibited a delightful linear response for CEA and AFP in the range of 100 fg·mL-1-100 ng·mL-1 with detection limits of 33.34 and 17.02 fg·mL-1 (S/N = 3), respectively. Meanwhile, no significant nonspecific adsorption and cross-talk were observed. The biosensing platform has shown satisfactory performance in the determination of real clinical samples. More importantly, the proposed approach can be conveniently extended to universal detection just by simply substituting biorecognition events. Thus, this work opens up a new promising perspective for dual and even multiple targets and offers promising potential applications in clinical diagnosis.

Lactylproteome analysis indicates histone H4K12 lactylation as a novel biomarker in triple-negative breast cancer

ObjectiveThe established link between posttranslational modifications of histone and non-histone lysine (K) residues in cell metabolism, and their role in cancer progression, is well-documented. However, the lactylation expression signature in triple-negative breast cancer (TNBC) remains underexplored.MethodsWe conducted a comprehensive lactylproteome profiling of eight pairs of TNBC samples and their matched adjacent tissues. This was achieved through 4-Dimensional label-free quantitative proteomics combined with lactylation analysis (4D-LFQP-LA). The expression of identified lactylated proteins in TNBC was detected using immunoblotting and immunohistochemistry (IHC) with specific primary antibodies, and their clinicopathological and prognostic significance was evaluated.ResultsOur analysis identified 58 lactylation sites on 48 proteins, delineating the protein lactylation alteration signature in TNBC. Bioinformatic and functional analyses indicated that these lactylated proteins play crucial roles in regulating key biological processes in TNBC. Notably, lactylation of lysine at position 12 (H4K12lac) in the histone H4 domain was found to be upregulated in TNBC. Further investigations showed a high prevalence of H4K12lac upregulation in TNBC, with positive rates of 93.19% (137/147) and 92.93% (92/99) in TNBC tissue chip and validation cohorts, respectively. H4K12lac expression correlated positively with Ki-67 and inversely with overall survival (OS) in TNBC (HR [hazard ratio] =2.813, 95%CI [credibility interval]: 1.242-6.371, P=0.0164), suggesting its potential as an independent prognostic marker (HR=3.477, 95%CI: 1.324-9.130, P=0.011).ConclusionsLactylation is a significant post-translational modification in TNBC proteins. H4K12lac emerges as a promising biomarker for TNBC, offering insights into the lactylation profiles of TNBC proteins and linking histone modifications to clinical implications in TNBC.

Infectious Diseases and Secondary Antibody Deficiency in Patients from a Mesoregion of São Paulo State, Brazil

Our aim was to determine the secondary antibody deficiency (SAD) profiles of patients in a mesoregion of São Paulo state, Brazil, focusing on infectious diseases. Demographic characteristics, and clinical and laboratory data were obtained from electronic files; infections were classified as organ-specific and graded as mild, moderate, life-threatening, and fatal. Non-Hodgkin’s lymphoma (NHL) accounted for 30% of patients, nephrotic syndrome (NS) 25%, chronic lymphocyte leukemia 20%, and multiple myeloma 15%. Patients with NS were younger than those in other groups, and hypo-γ-globulinemia was detected in 94.1%, IgG < 400 mg/dL in 60.0%, IgA < 40 mg/dL in 55.0%, and CD19 < 20 cells/mm3 in 30.0%. One hundred and one infections were found; 82.1% were classified as mild or moderate, 7.9% as life-threatening, and 3.0% as fatal. Respiratory tract infections were more prevalent (41.5%), and pneumonia accounted for 19.8%. Lower levels of infections were found in patients with NS compared with NHL (p = 0.0001). Most patients progressed to hypo-γ-globulinemia and SAD after treatment with immunosuppressants, and mild and moderate infections were predominant. These therapies are increasing in patients with different diseases; therefore, monitoring hypo-γ-globulinemia and infections may help to identify patients at high risk for severe complications, antibiotic prophylaxis or treatment, and immunoglobulin replacement.

Abstract PO5-18-07: A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases

Abstract Introduction: A promising novel target for breast cancer is secreted frizzled-related protein 2 (SFRP2). SFRP2 protects tumors against apoptosis, promotes T-cell exhaustion, and induces angiogenesis. SFRP2 is expressed in breast cancer but has not been specifically evaluated in triple negative breast cancer. We previously developed a humanized monoclonal antibody to SFRP2, (hSFRP2 mAb) that inhibits primary breast cancer growth in mice without toxicity. The purpose of this study is to establish the presence of SFRP2 in human TNBC, evaluate the efficacy of hSFRP2 mAb in metastatic TNBC in vivo, and in a cell line resistant to chemotherapy. Methods: Immunohistochemistry (IHC) with SFRP2 antibody on a human TNBC tissue microarray (TMA) containing 88 FFPE cores. After IHC with primary antibody to SFRP2, the TMA was scanned and staining intensity was quantified using spatial analysis. Results for SFRP2 staining were analyzed as previously described for analyses of estrogen and progesterone receptor positivity; categories are: 0-absence of staining; low positive (0-10%), and positive >10%. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb. MDA-MB-231 cells were treated with 4nM dox and increased 2nM at a time until the cells were resistant to 10µM dox after 12 months. WT and dox-resistant cells were treated for 4 hours with either hSFRP2 mAb (10uM,) or IgG control (10uM) (n=6). Apoptosis was detected with Promokine Apoptosis/Necrosis/Healthy cells kit. Cells were imaged using the EVOS FLc and counted using Imagej software. Differences between treated and control were compared with two-tailed T-test. hSFRP2 mAb treatment of mice with metastatic TNBC. PY8119 and E0771 TNBC murine cells (5 × 105/100 μL) were injected iv into 6–8-week-old C57BL/6 female mice. The next day the mice were treated with IgG1 control 8 mg/kg iv or hSFRP2 mAb 8 mg/kg iv every 3 days. After four weeks, the lungs were resected, and surface metastases were counted. Differences between treated and control were compared with Poisson distributions. FFPE sections of lungs were stained with ApopTag kit. Three metastases per lung were counted at 20 X, and the difference between IgG1 and hSFRP2 mAb groups compared with two-tailed T-test. Results: SFRP2 protein is present in human TNBC. Out of 88 cores, 83 cores (94%) were positive, 4 cores (4.5%) were minimally positive, and 1 core (1.1%) was negative for SFRP2. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb treatment in vitro. The number of apoptotic cells in IgG1 control-treated MDA-MB-231 cells was 11.5 ± 3.7, and 76.6 ± 5.0 for hSFRP2 mAb-treated tumors (p< 0.001, n=9). The mean number of apoptotic MDA-MB-231 dox-resistant cells in IgG1 control was 16.7 ± 6.0, and 68.4 ± 6.4 for hSFRP2 mAb-treated tumors, (p< 0.001, n=9). hSFRP2 mAb inhibits the growth of metastatic TNBC in vivo and increases apoptosis. For E0771, the number of surface metastases was 8.9 ±2.8 in the IgG1 control group and 4.9 ± 1.1 in the hSFRP2 mAb treated group (p< 0.05, n=15). The number of apoptotic cells/HPF was 9.8 ± 1.8 for the IgG1 control group and 21.0 ± 4.6 for the hSFRP2 mAb group (n=15, p< .01). For Py8119 tumors there were 6.4 ± 1.0 metastases in the IgG1 control group and 3.6 ± 0.9 in the hSFRP2 mAb group (p< 0.05, n=11). The number of apoptotic cells/HPF was 6 ± 1.8 for the IgG1 control group and 14 ±1.3 for the hSFRP2 mAb group (n=10, p< 0.001). Conclusions: SFRP2 protein is abundantly expressed in human TNBC. A humanized monoclonal antibody to SFRP2 reduces TNBC lung metastases growth in two in vivo models and increases tumor apoptosis. TNBC resistant to doxorubicin remains sensitive to apoptosis induced by the hSFRP2 mAb. Citation Format: Lillian Hsu, Patrick Nasarre, Julie Siegel, Eleanor Hilliard, Rupak Mukherjee, Nancy Klauber-Demore. A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-07.

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma.

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20-1000 pg mL-1, and its limit of quantitation was 20 pg mL-1. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.

A novel ratiometric electrochemical immunosensor for the detection of cancer antigen 125 based on three-dimensional carbon nanomaterial and MOFs

Accurate and sensitive detection of cancer antigen 125 (CA125) is important in early ovarian cancer diagnosis and monitoring. In the present work, a novel electrochemical ratiometric immunosensor for the determination of CA125 based on three-dimensional reduced graphene oxide/multi-walled carbon nanotube carboxylic acid- thionine (3DrGO/MWCNTs-Thi) and UIO-66-NH2/ferrocenecarboxylic acid (UiO-66-Fc) was constructed. 3DrGO/MWCNTs-Thi of immobilized primary antibody (Ab1) and UiO-66-Fc of labeled second antibody (Ab2) were used as capture probes and signaling probes for CA125, respectively. As the concentration of CA125 increases, the oxidation peak currents (IThi and IFc) generated by the two materials were changed in opposite directions, and therefore the ratio of the currents (IThi/IFc) was negatively correlated with the concentration of CA125. Thanks to the amplification of high loading of signaling molecules by the framework nanocomposites and the ratiometric strategy, the constructed electrochemical immunosensor exhibits outstanding accuracy, stability, and sensitivity. Under optimal conditions, the detection range spanned from 0.01 U mL−1 to 80 U mL−1, with a calculated limit of detection (LOD) of 0.0089 U mL−1. The immunosensor was successfully applied to real serum sample tests, indicating its potential for clinical detection applications.

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies.

The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures.

Immunohistochemistry of Lung Cancer Biomarkers.

Immunohistochemical (IHC) staining represents a comparatively inexpensive testing method that is attractive as a potential alternative to molecular sequencing methods or fluorescence in situ hybridization for pulmonary biomarker testing. While a variety of IHC tests directed at actionable genetic alterations have been developed and evaluated since the advent of targeted therapy, specific antibody clones for anaplastic lymphoma kinase, ROS-1, and potentially neurotrophic tropmyosin receptor kinase have been the primary antibodies that provide sufficiently robust results to be utilized as either a primary testing or screening method to direct targeted therapy. Antibodies for a variety of other targets such as epidermal growth factor receptors, for example, have lacked sufficient sensitivity and specificity to cover the range of mutations that may occur and are generally not recommended in lieu of molecular testing with the exception of limited resource settings. IHC is also used as a predictive marker for response to immunotherapy through evaluation of programmed death ligand 1 expression. In addition, multiple antibody-drug conjugates (ADCs) are under investigation, designed to deliver drugs directly to tumor cells through binding to specific target antigens. Some ADCs have already received accelerated FDA approval, and IHC was incorporated in many clinical trials evaluating ADC efficacy. As such, it is anticipated that ADCs may have a companion diagnostic IHC to guide patient selection.

Fenton reaction enhanced enzyme free electrochemical biosensor for rapid and highly sensitive detection of Salmonella

To fulfill the stringent demands of clinical Salmonella detection, this study employed the benefits of Ni-TCPP(Fe2+) and ConA to establish a novel approach that is culture-free, enzyme-free, secondary antibody-free, and ultra-sensitive. Our investigations revealed two key mechanisms that significantly bolster the signal-to-noise ratio (SNR) and enhance detection stability: firstly, the Fenton reaction and the mimic enzyme catalysis of Ni-TCPP(Fe2+) improved the sensitivity of the biosensor; secondly, the substitution of the secondary antibody with ConA led to increased binding of Ni-TCPP(Fe2+) due to the abundance of ConA receptors on the bacterial surface. With proper assembly, the developed biosensor exhibited high sensitivity in the range of 101–107 CFU/mL, along with a detection limit as low as 4 CFU/mL, and achieved rapid detection within a mere 2 h. Moreover, this biosensor has proven its efficacy in detecting Salmonella in clinical biological samples. Its advantages of being culture-free, secondary antibody-free, and ultra-sensitive establish a solid research foundation for the expedited detection of Salmonella in clinical settings and point-of-care testing (POCT).

Sandwich-like voltametric immunosensing of interleukin-8 based on β-cyclodextrin/carbon nanotubes and methylthionine chloride@UIO-66 framework.

The level of interleukin-8 (IL-8) in the body is an effective factor for the early diagnosis of acute tubular necrosis and oral tumor. In this work, a novel sandwich-like voltametric immunosensor (SVS) of IL-8 was constructed by preparing β-cyclodextrin/carbon nanotube (CD/CNT) to immobilize primary antibody (PAb) of IL-8 and UIO-66-NH2 MOFs structure to immobilize second antibody (SAb) and methylene blue (Mb) probe. In this designed SVS, the prepared CD/CNT nanohybrid with large surface area and conductivity can immobilize PAb via simple host-guest recognition, and UIO-66-NH2 provided an ideal platform to accommodate SAb and a large number of Mb molecules as signal-amplifier. In the existence of target IL-8, the current peak of Mb from the SVS assay increases with the increasement of IL-8 level. Through optimizing and adjusting various factors, a wide linearity (0.001-2.5ngmL-1) and low analytical limit (0.2pgmL-1) of IL-8 were realized, so it's expected the developed SVS strategy has significant applications for the detection of IL-8.

Cross-Reactivity of N6AMT1 Antibodies with Aurora Kinase A: An Example of Antibody-Specific Non-Specificity

Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of “specific” non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research.

The influence of charge on the translation of the sandwich ELISA approach to electronic biosensors

The sandwich approach, whereby an antigen is captured by a primary antibody and detected by a secondary antibody, is commonly used to improve the selectivity and sensitivity of enzyme-linked immunosorbent assays (ELISA). This work details the experimental factors that impact the reliable translation of this sandwich approach to two commonly used electronic biosensors, namely potentiometric and impedimetric biosensors. Previous studies have demonstrated the Debye screening limitations associated with potentiometric biosensors. However, the correlation between the ionic strength of the measurement buffer and the impedimetric biosensing response has not been studied. Potentiometric biosensors were able to successfully detect the primary antibody and the target antigen by decreasing the ionic strength of the phosphate buffered saline (PBS) measurement buffer from 1x PBS to 0.01x PBS. However, the secondary antibody used for the selective signal amplification was not reliably detected. Therefore, the sandwich approach is not viable for potentiometric sensing at biologically relevant ionic strengths, due to the Debye screening effect. Alternatively, decreasing the ionic strength of the measurement buffer allowed for the successful translation of the sandwich approach to impedimetric biosensors. Impedimetric biosensing literature typically attributes a measured increase in the charge transfer resistance to an increase in the thickness of the immobilized biolayer. However, this work highlights the influence that both the charge and thickness of the biolayer have on the transport of the redox couple. Decreasing the ionic strength of the measurement buffer lowers the molecular charge screening effect. This permits the transport of a positively charged redox probe through a negatively charged immobilized biolayer via migration and diffusion. The results demonstrate that the use of a buffer at a lower, yet biologically relevant ionic strength allows for the successful translation of the sandwich approach to impedimetric biosensors.

Utilization of Double-Immunofluorescence Microscopy to determine the role of FTL_1199 in erythrocyte invasion by Francisella tularensis.

Francisella tularensis is an intracellular bacterium that is the causative agent of the zoonotic disease, tularemia. F. tularensis invades host erythrocytes during infection, a phenomenon that leads to increased colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, an RNA-seq analysis was conducted to identify genes whose transcription was affected by the presence of erythrocytes. A putative transcriptional regulator, FTL_1199 was determined to be modulated in the presence of erythrocytes. We hypothesized that this gene may therefore play a role in erythrocyte invasion. Deletion of FTL_1199 resulted in a significant reduction in the invasion of rabbit erythrocytes, and complementation of FTL_1199 restored this strain’s ability to acquire the cytoplasmic space of erythrocytes. These findings were determined by a gentamicin protection assay to quantify erythrocyte invasion. To validate these findings, we conducted double-immunofluorescence microscopy. F. tularensis strains incubated with rabbit erythrocytes to allow for invasion.  Cells were probed with anti-F. tularensis antibodies followed by red fluorescent secondary antibodies.  Cells were washed and erythrocytes were permeabilized. Bacteria were probed a second time utilizing a green fluorescent secondary antibody. Therefore, intracellular bacteria were only labeled with a singular secondary antibody, while extracellular bacteria were tagged with two secondary antibodies. Intracellular bacterial enumeration showed that deletion of FTL_1199 resulted in a significant reduction of invasion (p = 0.0253), while FTL_1199 complementation to the deletion mutant restored invasion to that of wild type (p = 0.3258). These data demonstrate that FTL_1199 plays an important role in erythrocyte invasion.   (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).

Molecular characteristics of non-small cell lung cancer tissue based on quantitative indicators of progesterone receptors expression

Background. Progesterone receptors (PR) are regulators of cell proliferation and therefore can be considered as an aim for targeted medications in the treatment of oncological diseases. At the same time, a quantitative assessment of PR expression in the tissue of non-small cell lung cancer (NSCLC), which has not yet been carried out in other studies, will determine the possibility of using PR modulators for the treatment of this disease and identify the potential category of patients most susceptible to these drugs.Purpose. To characterize NSCLC by quantitative indicators of PR expression and to determine the correlation of clinically significant characteristics of patients and clinical and morphological parameters of a NSCLC tumor with the PR expression to assess the possibility of using PR modulators in the treatment of this disease.Methods. The PR expression in 130 surgical samples of NSCLC was quantified using an immunofluorescence method associated with flow cytometry. Primary antibodies to PR (NBP2-46388) and secondary antibodies conjugated with DyLight650 (ab98729) were used.Results. The expression of progesterone receptors was detected in all the studied tumors; an abnormal distribution of the marker expression level was noted (P=0.01). The mean expression level was 55.3±16.2%, and the median was 57% with a range of 70%, which indicates heterogeneity of PR expression in tumors of different patients. There were no statistically significant differences in the level of PR expression depending on the histotype and stage of NSCLC, as well as on the sex of patients. At the same time, the level of expression and the frequency of overexpression of PR (>67%) in tumors in non-smoking patients are higher than in smokers (P⩽0.02).Conclusion. The high frequency of occurrence and level of PR expression in NSCLC indicate the possible effectiveness of the use of their modulators in the treatment of this disease, especially in non-smoking patients.

Secondary reagents for immunohistochemical research of rat brain

BACKGROUND: The critical factor when working with immunohistochemistry in laboratory animals is to select appropriate secondary antibodies, that allow clear and specific visualization of tissue antigens. Many reliable secondary reagents are currently not available for purchase, which determines the high relevance of replacing them with other detection systems.AIM: To verify the effectiveness of available secondary reagents for immunohistochemical research of the rat brain.METHODS: Brain samples from Wistar and SHR rats (n=4) were used for the study. Iba-1, GFAP and vimentin immunohistochemistry was carried out using various polymer-based detection systems, namely UltraVision Quanto, N-Histofine Simple stain MAX PO ® and UnoVue Rabbit HRP.RESULTS: All three studied polymer systems demonstrated visualisation of target proteins in brain tissues and cells corresponding to the general understanding of structures containing Iba-1, GFAP and vimentin. The UnoVue Rabbit HRP and Quanto Detection System HRP kits showed good and similarly specific immunohistochemical reaction. However, the UnoVue Rabbit HRP kit was less sensitive compared to the Quanto Detection System HRP. The reaction with N-Histofine® Simple Stain MAX PO was not optimal due to the presence of non-specific background staining that was not present with other reagents.CONCLUSION: Two secondary antibody kits from the three studied showed optimal efficiency of immunohistochemical reaction and minimal background staining. N-Histofine® Simple Stain MAX PO is not suitable for immunohistochemical research of rat brain tissue.

Trial of Cardiovascular Risk Factor Assessment and Transthoracic Echocardiography Results in Patients with Primary Antibody Deficiency

The life expectancy and the risk of developing cardiovascular diseases in patients with inborn errors of immunity are systematically increasing.
 The aim of the study was to assess cardiovascular risk factors and to evaluate the heart in echocardiography in patients with primary antibody deficiency (PAD). Cardiac echography and selected cardiovascular risk factors, including body mass index, sedentary lifestyle, nicotine, glucose, C-reactive protein, lipid profile, uric acid level, certain chronic diseases, and glucocorticoid use, were analyzed in 94 patients >18 years of age with PAD.
 Of the patients,25.5% had a cardiovascular disease (mostly hypertension, 18%), 10.5% smoked, 17% were overweight, 14% were obese, and 15% were underweight. Abnormal blood pressure was found in 6.5% of the patients. Lipid metabolism disorders were found in 72.5% of in the studied cohort, increased total cholesterol (45.5%), non-high-density lipoprotein (HDL) (51%),
 low-density lipoprotein (LDL) (47%), and triglycerides (32%) were observed. Furthermore, 28.5% had a decrease in HDL and 9.5% had a history of hyperuricemia. The average number of risk factors was 5 ± 3 for the entire population and 4 ± 2 for those under 40 years of age. Elevated uric acid levels were found de novo in 4% of participants. In particular, 74.5% of the patients had never undergone an echocardiogram with a successful completion rate of 87% among those tested. Among them, 30% showed parameters within normal limits, primarily regurgitation (92.5%). New pathologies were identified in 28% of patients.
 Prevention in patients with PAD, aimed at reducing cardiovascular risk, should be a priority.
 low-density lipoprotein (LDL) (47%), and triglycerides (32%) were observed. Furthermore, 28.5% had a decrease in HDL and 9.5% had a history of hyperuricemia. The average number of risk factors was 5 ± 3 for the entire population and 4 ± 2 for those under 40 years of age. Elevated uric acid levels were found de novo in 4% of participants. In particular, 74.5% of the patients had never undergone an echocardiogram with a successful completion rate of 87% among those tested. Among them, 30% showed parameters within normal limits, primarily regurgitation (92.5%). New pathologies were identified in 28% of patients.
 Prevention in patients with PAD, aimed at reducing cardiovascular risk, should be a priority.

[Primary antibody deficiencies and respiratory manifestations in adults].

Primary antibody deficiencies (PAD) are a group of congenital disorders caused by genetic defects that affect the development and function of the body's immune defence mechanisms. Patients with PAD may present with recurrent infections, lymphoproliferation, autoimmune diseases, autoinflammation, or malignancies. Respiratory system manifestations may include bronchiectasis, bronchial asthma, and interstitial lung disease, among others. A comprehensive understanding of PADs will help to distinguish these covert cases from more common respiratory diseases.