Goat Anti-mouse Antibody Research Articles

Immunomodulatory effects of laser-synthesized nanodiamonds on peripheral blood mononuclear cells: evaluation of unconjugated, PEGylated, and antibody-conjugated formulations

The application of laser-synthesized nanodiamonds (LNDs) is of great interest to biomedical researchers and drug developers because this emerging method of synthesis yields nanodiamonds of consistent size (<5 nm diameter) and surface chemistry that can be functionalized to perform a staggering range of highly specialized tasks. The present study assessed the threshold at which LNDs in various conjugations and concentrations triggered immune responses and cytotoxicity in peripheral mononuclear blood cells from healthy donors, as assessed by changes in ATP concentrations and induced secretion of the cytokines IFN-γ, IL-6 and TNF-α. Conjugations assessed were raw (unconjugated) NDs, PEGylated (PEG5k-NDs), and antibody conjugated to goat anti-mouse antibodies (IgG-NDs). Concentrations of each conjugation were prepared and tested at 50.0, 10.0, 2.0, 0.4, and 0.08 μg/mL. Results showed that pegylated and raw NDs were well tolerated, with the indicators of inflammation or minimal cytotoxicity emerging only at the highest concentrations tested (50.0 μg/mL). IgG-NDs showed signs of inflammatory responses at the two highest concentrations tested (10.0 and 50.0 μg/mL). There was some evidence that the dilutant vehicle used for ND suspension may have contributed to the immune response. All three ND configurations increased ATP concentration in a dose-dependent manner, up to a concentration of 10.0 μg/mL. At the highest concentration (50.0 μg/mL), the ND solutions showed minimal signs of cytotoxicity. Conclusion from this testing suggest that LNDs are likely to offer substantial utility in biomedical applications because of their capacity to evade the immune response at concentrations at least as high as 2.0 μg/mL and potentially up to 50.0 μg/mL.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species

BackgroundTimely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results.MethodsColloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera.ResultsAMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%–23.4% of these samples were found to be positive for T. gondii.ConclusionsThis study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.Graphical

Bimetallic nanozyme–bioenzyme hybrid material-mediated ultrasensitive and automatic immunoassay for the detection of aflatoxin B1 in food

Aflatoxin B1 (AFB1) is one of the most prevalent and dangerous biotoxin in crops and feedstuff, which poses a great threat to human health and also cause significant financial losses. Therefore, there is an urgent need to develop an effective method for AFB1 detection. In this work, we developed an automatic reaction equipment and nanozyme-enhanced immunosorbent assay (Auto-NEISA) for sensitive and accurate detection of AFB1 by combining the highly effective signal probes with a self-designed automated immunoreactive equipment. Wherein, polystyrene (PS) nanoparticles were used as signal carriers for loading a massive in situ-synthesized platinum and palladium bimetallic nanozyme, which could enrich horseradish peroxidase-labeled goat anti-mouse antibody (HRP-Ab2) on the nanozyme surface to form a bimetallic nanozyme–bioenzyme hybrid material for multiple signal amplification. The entire reaction could be automatically completed by the self-developed immunoreactive equipment. The Auto-NEISA method realized the sensitive detection of AFB1 with a wide linear detection range of 10–104 pg/mL, at a low limit of detection (LOD) of 5.52 pg/mL. The LOD was 65-fold lower than that of the enzyme-linked immunosorbent assay (ELISA). Additionally, Auto-NEISA was successfully applied to detect AFB1 in real food samples, demonstrating that it has considerable potential for detecting food contaminants with high accuracy and efficiency.

Overexpression of ASPH protein predicts poor outcomes in retroperitoneal liposarcoma patients.

Overexpression of ASPH protein predicts poor outcomes in retroperitoneal liposarcoma patients.

Establishment of 11-dehydro-thromboxane B2 time-resolved immunoassay and application in membranous nephropathy

Background11-Dehydro-thromboxane B2 (11-dehydro-TXB2) is the final stable metabolite of thromboxane A2 (TXA2) and is involved in thrombus formation. Patients with membranous nephropathy (MN) are prone to thromboembolism events. MethodsTime-resolved fluorescence immunoassay (TRFIA) for 11-dehydro-TXB2 was established by indirect competitive method. The coated 11-dehydro-TXB2-BSA conjugate was used to bind the 11-dehydro-TXB2 antibody competitively to the 11-dehydro-TXB2 antigen in the samples, followed by Eu3+-labeled goat anti-mouse IgG antibody, to detect 11-dehydro-TXB2. This study measured 11-dehydro-TXB2 concentrations in serum samples from healthy individuals and patients with MN. ResultsThe linear range of TRFIA was 16.38–2000 pg/mL, the sensitivity was 4.70 pg/mL, the average coefficients of variation from intra-assay and inter-assay were 3.50% and 4.95%, respectively, and the recovery was 99.38%. The serum level of 11-dehydro-TXB2 in patients with MN was significantly higher than that in healthy subjects (P < 0.05). The serum 11-dehydro-TXB2 concentration detected by TRFIA was highly consistent with that by ELISA (ρ = 0.900). DiscussionThis study successfully established a new highly sensitive method for the detection of 11-dehydro-TXB2 in serum. 11-Dehydro-TXB2 has great potential in evaluating the risk of thromboembolic events in patients with MN and is expected to be applied to other thromboembolic-related diseases.

Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection.

Canine parvovirus (CPV) is an acute and highly infectious virus causing disease in puppies and, thus, affecting the global dog industry. The current CPV detection methods are limited by their sensitivity and specificity. Hence, the current study sought to develop a rapid, sensitive, simple, and accurate immunochromatographic (ICS) test to detect and control the spread and prevalence of CPV infection. More specifically, 6A8, a monoclonal antibody (mAb) with high specificity and sensitivity, was obtained by preliminary screening. The 6A8 antibody was labelled with colloidal gold particles. Subsequently, 6A8 and goat anti-mouse antibodies were coated onto a nitrocellulose membrane (NC) as the test and control lines, respectively. Furthermore, 6A8 and rabbit IgG antibodies were labelled with fluorescent microspheres and evenly sprayed onto a glass fibre membrane. Both strips could be prepared in 15min with no noticeable cross-reactivity with other common canine intestinal pathogens. The strips were simultaneously used to detect CPV in 60 clinical samples using real-time quantitative PCR, hemagglutination, and hemagglutination inhibition assays. The colloidal gold (fluorescent) ICS test strip was stable for 6 (7) and 4 (5) months at 4 °C and room temperature (18-25 °C). Both test strips were easy to prepare and rapidly detected CPV with high sensitivity and specificity. Moreover, the results were easily interpretable. This study establishes a simple method for two CPV diseases, colloidal gold and fluorescent immunochromatographic (ICS) test strips. KEY POINTS: • CPV test strips do not exhibit cross-reactivity with other canine intestinal pathogens. • The strips are stable for months at 4 °C and at room temperature (18-25 °C). • These strips are a promising approach for the timely diagnosis and treatment of CPV.

Inducing ciliation in equine oviduct epithelial cell monolayers

Recently, we developed re-differentiated equine oviduct epithelial cell (EOEC) monolayers in Transwell inserts. EOEC monolayers demonstrated various in vivo-like morphological characteristics, but often lacked secondary cilia. The monolayers were classified as low (<1% secondary cilia) or high (>5% secondary cilia) spontaneously re-differentiated, with the latter in the minority (5-10%). Interestingly, only high re-differentiated monolayers responded to Notch inhibition by developing in vivo-like secondary ciliation rates, i.e. over 50% ciliated cells. In this study, we aimed to consistently generate high re-differentiated monolayers. We first assessed whether secondary cilia formation was affected by donor mare, cycle stage and/or experiment. For this, harvested oviduct explants were cultured for 10 days at a concentration of 10 × 106 EOECs per 9.6 cm² well, to yield a confluent de-differentiated monolayer in conventional wells. Subsequently, the monolayers were trypsinized and the EOECs were reseeded onto the microporous membrane of hanging inserts at a concentration of 0.3 × 105 EOECs per 0.33 cm² insert. Three days after seeding, the EOECs were triggered to re-differentiate by air-liquid interface introduction (ALI). After 1 and 2 months in culture, EOEC monolayers were fixed and their phenotype was assessed by combined immunofluorescence staining to detect cilia (mouse anti-acetylated α-tubulin antibody; Alexa 488-conjugated goat anti-mouse secondary antibody),cell nuclei (Hoechst 33342) and actin filaments (Alexa 568-conjugated phalloidin). Neither cell donor nor cycle stage had any effect on secondary ciliation rates, whereas experimental modifications did. In 3/15 experiments, >95% of the monolayers demonstrated high re-differentiated monolayers. To discover the source of experimental variation, the seeding concentration of the de-differentiated EOECs was varied between 0.1 and 10 × 105 EOECs per 0.33 cm² insert; this did not affect secondary ciliation rates. In a third experiment, the effect of varying the seeding concentration of the harvested oviduct explant EOECs used to establish the de-differentiated monolayers, between 1 and 30 × 106 EOECs per 9.6 cm² well, was evaluated. EOECs harvested from de-differentiated monolayers 10 days after oviduct explant seeding at 1 or 5 × 106 EOECs per well supported the formation of high re-differentiated monolayers in >95% of cases. Within 1 month after ALI, >50% and >30% of the re-differentiated EOECs showed secondary cilia after initial seeding of 1 and 5 × 106 oviduct explant EOECs per well, respectively. Notch-inhibition helped to further increase secondary ciliation rates to >70% in the re-differentiated monolayers. We conclude that initial oviduct explant EOEC seeding concentration is critical to subsequently generating monolayers that closely resemble in vivo oviduct epithelia.

An integrated immunochromatographic device for C-reactive protein detection using hierarchical dendritic gold nanostructure films

Immunochromatographic test strips typically consist of sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Even minute variations in the assembly of these components can lead to inconsistent sample-reagent interactions, thereby reducing reproducibility. In addition, the nitrocellulose membrane is susceptible to damage during assembly and handling. To address this issue, we propose to replace the sample pad, conjugate pad, and nitrocellulose membrane with hierarchical dendritic gold nanostructure (HD-nanoAu) films to develop a compact integrated immunochromatographic strip. The strip uses quantum dots as a background fluorescence signal and employs fluorescence quenching to detect C-reactive protein (CRP) in human serum. A 5.9 μm thick HD-nanoAu film was electrodeposited on an ITO conductive glass by the constant potential method. The wicking kinetics of the HD-nanoAu film was thoroughly investigated, and the results indicated that the film exhibited favorable wicking properties, with a wicking coefficient of 0.72 μm ms−0.5. The immunochromatographic device was fabricated by etching three interconnected rings on HD-nanoAu/ITO to designate sample/conjugate (S/C), test (T), and control (C) regions. The S/C region was immobilized with mouse anti-human CRP antibody (Ab1) labeled with gold nanoparticles (AuNPs), while the T region was preloaded with polystyrene microspheres decorated with CdSe@ZnS quantum dots (QDs) as background fluorescent material, followed by mouse anti-human CRP antibody (Ab2). The C region was immobilized with goat anti-mouse IgG antibody. After the samples were added to the S/C region, the excellent wicking properties of the HD-nanoAu film facilitated the lateral flow of the CRP-containing sample toward the T and C regions after binding to AuNPs labeled with CRP Ab1. In the T region, CRP-AuNPs-Ab1 formed sandwich immunocomplexes with Ab2, and the fluorescence of QDs was quenched by AuNPs. The ratio of fluorescence intensity in the T region to that in the C region was used to quantify CRP. The T/C fluorescence intensity ratio was negatively correlated with the CRP concentration in the range of 26.67–853.33 ng mL−1 (corresponding to 300-fold diluted human serum), with a correlation coefficient (R2) of 0.98. The limit of detection was 15.0 ng mL−1 (corresponding to 300-fold diluted human serum), and the range of relative standard deviation: 4.48–5.31%, with a recovery rate of 98.22–108.33%. Common interfering substances did not cause significant interference, and the range of relative standard deviation: 1.96–5.51%. This device integrates multiple components of conventional immunochromatographic strips onto a single HD-nanoAu film, resulting in a more compact structure that improves the reproducibility and robustness of detection, making it promising for point-of-care testing applications.

Adaptation of an ELISA assay for detection of IgG2a responses against therapeutic monoclonal antibodies in a mouse immunization model

Biotherapeutic monoclonal antibodies (mAb) play important roles in clinical medicine but their potential to elicit immune responses in patients remains a major issue. In a study designed to investigate the effect of aggregation on immunogenic responses, mice were immunized with two monoclonal antibodies (mAb1 and mAb2). Serum levels of total IgG, IgG1, and IgG2a were measured by ELISA. An anti-mouse IgG2a monoclonal detection antibody cross-reacted with mAb2 but not mAb1, leading to high background when the ELISA plate was coated with mAb2. The problem was solved by use of a goat anti-mouse IgG2a polyclonal antibody that demonstrated the required specificity. IgG2a responses were similar for monomer- or aggregate-coated ELISA plates. The results demonstrate the importance of assessment of the specificity of individual reagents when measuring antibody responses against therapeutic antibodies by ELISA.

Tellurium-containing polymer for mass cytometry

Mass cytometry is a powerful analytical technique for high-dimensional single-cell analysis. In this technique, cells are stained with heavy isotope-tagged antibodies and are analyzed by an inductively coupled plasma time-of-flight mass spectrometer. While the current mass cytometer has more than 100 mass channels, only about 50 are used because reagents are only available for a fraction of the useful elements. Here we describe the synthesis of a tellurium-containing polymer based on poly-l-lysine and sulfated tellurophene. The polymer carried ca. 48 tellurophene units per chain and had a terminal azide group for antibody conjugation. While the polymer could be conjugated to antibodies, the resulting conjugates gave mixed results in mass cytometry immunoassays. For example, a goat anti-mouse (secondary) antibody and an anti-CD19 primary Ab labeled with polymers gave promising results in antibody titration experiments. However, an anti-CD20 Ab labeled with a 126Te-enriched polymer no longer recognized the antigen and showed significant non-specific binding in a 7-plex assay with human PBMCs.

Development of a colloidal gold immunochromatographic strip for the rapid detection of antibodies against Fasciola gigantica in buffalo

BackgroundFasciola gigantica, a tropical liver fluke, infects buffalo in Asian and African countries, causing significant economic losses and posing public health threats. The diagnostic of buffalo fascioliasis caused by F. gigantica is vital in fascioliasis control and preventation. The 22nd gel filtration chromatography fraction of F. gigantica Excretory-Secretory Products (FgESP), namely Fasciola 22 (F22), which was used as a diagnostic antigen in indirect ELISA, has demonstrated great potential for fascioliasis diagnosing. In the absence of rapid diagnostic methods, the use of a colloidal gold immunochromatographic strip based on F22 was applied to detect F. gigantica infection in buffalo.MethodsIn the present study, the 22nd gel filtration chromatography fraction of FgESP (F22) was used as an antigen to establish the colloidal gold-based immunochromatographic strip (ICS). The nitrocellulose membrane was incubated with F22 at the test line (T line) and goat anti-mouse secondary antibody at the control line (C line). The mouse anti-buffalo secondary antibody 2G7 conjugated to colloidal gold particles was used as the detection system for line visualization. The strip was assembled and developed by optimizing reaction conditions. The sensitivity, specificity, stability, and early diagnostic value of the strip were evaluated employing buffalo-derived sera.ResultsAn immunochromatographic strip for the rapid detection of antibodies against F. gigantica-FgICS was developed. The strip demonstrated high sensitivity and specificity. Sensitivity tests confirmed positive results even when the positive reference serum was diluted 4,096 times. Except for one Schistosoma japonicum-positive serum that tested positive via FgICS, specificity tests confirmed no cross-reactivity with other positive sera of Schistosoma japonicum and Babesia bovis. The strip remained stable after storage at 4°C for up to 3 months. In infected buffalo, antibodies could be detected as early as 14–21 days post-infection. The detection of 17 positive sera yielded an 82.4% positive rate via FgICS vs. a 100.0% positive rate via ELISA based on FgESP. For FgICS, the 95% confidence interval of sensitivity was 84.8–95.4%, while specificity was 4.2–14.7%.ConclusionThe immunochromatographic strip FgICS developed in this study provides a simple and rapid method of F. gigantica antibody detection and infected buffalo monitoring in the field.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

A Novel Immunochromatographic Strip Based on Latex Microspheres for the Rapid Detection of North American-Type Porcine Reproductive and Respiratory Syndrome Virus.

A latex microsphere-based immunochromatographic strip (ICS) test was successfully developed for the rapid and sensitive detection of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV N protein-specific monoclonal antibody (mAb) 1H4 labeled with latex microspheres was dispensed on a conjugate pad for use as the detector. The same mAb and goat anti-mouse antibody were blotted on a nitrocellulose membrane to generate test and control lines, respectively. The limit of virus detection was approximately 5 × 102.0 median tissue culture infectious dose (TCID50)/ml, and the limit of N protein detection was approximately 15 ng/ml. Other common porcine viruses were tested to evaluate the specificity of the ICS, and positive results were observed for only North American-type PRRSV. A comparison of the strip with a standard diagnostic test (reverse transcriptase polymerase chain reaction, RT-PCR) was also performed, and the results showed that the ICS test exhibited relatively high specificity and sensitivity (90.32 and 73.91%, respectively) and relatively high positive predictive value (PPV) and negative predictive value (NPV; 85 and 82.35%, respectively). These results suggest that the ICS test can be used to rapidly and accurately detect PRRSV and can be suitable for diagnostic applications in the field.

Development of an Immunochromatographic Strip for Rapid Detection of Mink Enteritis Virus.

Although mink enteritis virus (MEV) is an acute, virulent, and highly contagious pathogen in minks, there is currently a lack of a quick diagnostic method. By conjugating colloidal gold nanoparticles with the MEV-specific monoclonal antibody, monoclonal antibody (MAb) 14, we developed a single-step competitive immunochromatographic strip (ICS) assay for simple determination of MEV. The optimal concentrations of the colloidal gold-coupled MAb 14 (coating antibody), the capture protein (MEV VP2 protein), and the goat anti-mouse antibody were 1.0, 0.8, and 1.0 mg/ml, respectively. The limit of detection was approximately 512 hemagglutination units/100 μl of MEV B strain. Other common viruses of mink were tested to evaluate the specificity of the ICS, and the results showed no cross-reactivity for other pathogens. In comparison with the Anigen Rapid canine parvovirus (CPV) Ag Test Kit (BioNote, Korea) in testing 289 samples, the percentage of agreement and relative sensitivity and specificity of the MEV ICS assay were 94.1, 93.2, and 97.1%, respectively. The ICS test was found to be a sufficiently sensitive and specific detection method for the convenient and rapid detection of MEV.

A centrifugal microfluidic chip for point-of-care testing of staphylococcal enterotoxin B in complex matrices.

Staphylococcal enterotoxin B (SEB) is a typical biological toxin that causes food poisoning. Currently reported SEB detection methods have the drawbacks of sophisticated sample preparation and being time-consuming and labor-intensive. Herein, we propose a strategy based on an immune sandwich structure operating on a centrifugal microfluidic chip for point-of-care testing (POCT) of SEB. The fluorescent microparticle-labeled primary antibody (CM-EUs-Ab1), capture antibody (CAb), and goat anti-mouse IgG antibody (SAb) were modified on the bond area, T-area, and C-area, respectively. When SEB was added, it first reacted with the CM-EUs-Ab1 through the specific recognition between SEB and the Ab1. Then, under capillarity, the conjugates of SEB and the CM-EUs-Ab1 were captured by the CAb when they flowed to the T-area, and the remaining CM-EUs-Ab1 bound with the SAb in the C-area. Finally, this chip was put into a dry fluorescence detection analyzer for centrifugation and on-site detection of SEB. The fluorescence intensity ratio of the T-area to the C-area was positively correlated with the concentration of SEB. The resulting linear range was 0.1-250 ng mL-1, and the limit of detection (3σ/k) was 68 pg mL-1. This POCT platform only needs 20 μL of sample and can realize the full process of detection within 12 min. This chip also exhibits good stability for 35 days. Additionally, the proposed method has been successfully utilized for the detection of SEB in urine, milk, and juice without any pre-treatment of the samples. Thus, this platform is expected to be applied to food safety testing and clinical diagnosis.

Chemiluminescence microarray immunoassay for multiple aminoglycoside antibiotics based on carbon nanotube-assisted signal amplification.

The simultaneous determination of multiple analytes has been an urgent demand in screening of antibiotic residues in food products of animal origin due to its higher analysis efficiency. Five aminoglycoside antibiotics (AGAs) have been monitored in milk, including gentamicin (GEN), kanamycin (KAN), neomycin (NEO), and streptomycin/dihydrostreptomycin (STR/diSTR). A chemiluminescence microarray immunoassay (CLMIA) based on nitrocellulose membrane had been developed for the detection of multiple AGAs, which the LODs for STR, KAN, NEO, and GEN were 4.74ng/mL, 4.97ng/mL, 2.99ng/mL, and 4.42ng/mL respectively. To improve the sensitivity of immunoassay, single-well carbon tubes (SWCNTs) were utilized as solid support for loading horseradish peroxidase-labelled goat anti-mouse antibody to obtain the multi-enzyme particles. After the optimization of usage of multi-enzyme particles and antibodies, the enhanced CLMIA was established and evaluated. The LODs were 1.25ng/mL for STR, 0.64ng/mL for KAN, 0.38ng/mL for GEN, and 0.39ng/mL for NEO, which was improved by threefold, sevenfold, 11-fold, and sevenfold compared with the conventional CLMIA developed. These methods presented higher specificity and repeatability. Finally, the enhanced CLMIA based on CNT-assisted multi-enzyme particles was utilized to analyze twenty-five milk samples from local market and dairy farm, which all the results were below the LOD. The enhanced CLMIA showed the great application potential for the detection of multiple targets simultaneously and provided efficient tool for the screening of pollutants in food.

Evaluation of New Immunohistochemical Approaches for the Study of Kidney Tumors in Geriatric.

Kidney malignancies are among the most deadly genitourinary tumors. It is more common in males and is often seen in people aged 60-70 years old. The incidence rate of kidney cancer seems to be increasing. One reason for this may be the fact that imaging techniques, such as computed tomography scans are more commonly used. These tests may lead to the accidental detection of more kidney cancers. Fortunately, kidney cancer is often detected in the early stages, when the tumor is small and confined to the kidney. The objective of this study was the development of new diagnostic immunohistochemical methods. Clinical examination material of 134 people, including 94 (70%) males and 40 (30%) females, were used in this study. Immunohistochemical staining of tryptase was carried out in compliance with the requirements using Anti-Mast Cell Tryptase antibodies. Goat anti-mouse antibodies #AS-M1-HRP were used as secondary antibodies, visualized with ImmPACTTM DAB Peroxidase Substrate Kit (#SK-4105) according to the instructions of the manufacturer. The nuclei were counterstained with Mayer's hematoxylin, and the sections were embedded in a permanent mounting medium.The immunohistochemical study showed an increase in both tryptase- and chymase-positive mast cells in the renal parenchyma, compared with the control group. The number of mast cells with tryptase expression directly in the tumor was significantly less than the peritumoral localization. A similar pattern was observed for chymase-positive mast cells as the content of the tumor was more than 10 times higher than the intratumoral arrangement. The histological and immunological characteristics did not differ in different age groups. The immunohistochemical method of research in the diagnosis of renal tumors plays an important diagnostic and prognostic value. It can assist pathologists in difficult and ambiguous cases to correctly diagnose renal tumors. This will make it possible to prescribe the correct treatment and predict the course of malignant tumor growth in patients.

Validation of PON1 as a Biomarker of Clopidogrel Resistance in Caribbean Hispanics

Background : Clopidogrel is an antiplatelet drug used to prevent heart attack and strokes in cardiovascular patients. Pharmacological studies of clopidogrel have been mainly conducted in individuals of European ancestry, therefore missing genetic diversity and enhancing disparities of care. Since admixed Caribbean Hispanics are among those often underrepresented, we want to identify predictive or risk biomarkers of the response to clopidogrel within this population. Preliminary data using Tandem Mass Tag (TMT) coupled with mass spectrometry revealed that paraoxonase-1 (PON1) is downregulated in patients treated with clopidogrel (-49.50) when compared to other cardiovascular patients. PON1 is an HDL-associated protein that protects against accumulation of oxidized lipids and prevent cardiovascular disease. Our aim is to validate the low abundance of PON1 by western blot analysis. Methods : All patients signed an informed consent approved by the Institutional Review Board (IRB-protocol number A4070417). Blood was collected and a platelet reactivity test was performed using the VerifyNow® P2Y12 assay to determine if the patient is a poor responder (PRU≥230). Plasma was separated by centrifugation and stored at -80°C. For Western Blot analysis, 25µg of plasma proteins were run on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the healthy (n=6), cardiovascular (n=6), normal responders (n=14), and poor responders (n=10) groups. Proteins were electro-transferred from the gel onto polyvinylidene membrane. The membranes were incubated with PON1 (4G8D3) and goat anti-mouse IRDye 680RD antibodies, followed by an incubation with a loading control, anti-transferrin (101) rabbit monoclonal antibody and goat anti-rabbit IRDye 680RD antibody. The membranes were scanned in the Odyssey® CLx Imager in AUTO (680 nm and 800 nm channels) to reveal the immunofluorescent bands that were quantified by the Image StudioTM Software. Statistical analysis was carried out by Graphpad Prism 6.01 for Windows. After determining the normality of the data by Shapiro Wilkins Test, a one-way ANOVA was run as a variance test. Results : The total PON1 intensity signal was detected in healthy (1.11±0.34) and cardiovascular controls (1.00±0.00), normal (1.06±0.45) and poor responders (0.89±0.34), with a lower mean value in the latter but no statistical differences among the groups (p>0.05). Conclusions : PON1 could not be validated as a downregulated protein in poor responders when compared to other cardiovascular groups by western blotting.

A new method for rapid screening of hybridoma cell clones secreting paired antibodies using sandwich cell surface fluorescence immunosorbent assay

Traditional methods of screening antibody pairs through ELISA-based methods are time-consuming and burdensome, which is not conducive for the rapid establishment of antigen detection methods. Hence, we developed a new method based on the sandwich cell surface fluorescence immunosorbent assay (SCSFIA) for rapid screening of paired antibodies. In this method, the capture antibodies were anchored to the hybridoma cells membrane through the lipid derivative Oleyl-PEG4000-NHS. Goat anti-mouse antibodies (blocking agent) were added to block the Fc fragment of the capture antibodies. The capture antibodies’ Fab fragment can specifically bind the added antigen and form the capture antibodies-antigens complex (immunocomplexes). If the antibodies secreted by hybridoma cells could recognize the immunocomplexes. A double antibody sandwich structure would form on the cell surface based on the specific binding of antigens and antibodies. The hybridoma cells would be stained with anti-mouse IgG-Fc-FITC antibodies. We first used anti-pseudorabies virus (anti-PRV) cells and anti-porcine epidemic diarrhea virus (anti-PEDV) cells to verify the new method. Then, we used this method to successfully screen 5 hybridoma cell clones secreting paired antibodies against Avian influenza A (H7N9) virus within 15 days after fusion. These results showed that this method is suitable for the screening of paired antibodies in a variety of virus. Compared with the traditional method of obtaining paired antibodies, this method can greatly shortens the time needed to screen paired antibodies and improves screening efficiency, indicating that it is a promising method for paired antibodies discovery.

Development of a Colloidal Gold Immunochromatographic Strip for the Rapid Detection of Channel Catfish Virus.

Channel catfish virus disease (CCVD) has resulted in great economic losses and has restricted the development of fisheries. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of CCVD. A colloidal gold immunochromatographic strip has been developed for the detection of CCVD. In this study, a colloidal gold immunochromatographic strip for channel catfish virus (CCV) detection was developed using the monoclonal antibody 8B6 conjugated with colloidal gold as the detector antibody. A rabbit anti-CCV antibody was used as the capture complex at the test line, and a goat anti-mouse IgG antibody was used as the capture antibody at the control line. The strip was characterized in its specificity, sensitivity, and stability. In addition, an infection experiment was performed to test the applicability of the test strip. The strip was able to detect concentrations of the virus (104 tissue culture infective dose (TCID50)/mL) and showed analytical specificity when tested against other viral pathogens. The strips were still usable after 30 days of storage at 60°C. It was possible to detect CCV experimentally in infected fish within 10-15 min of using the strip. The strip can be used as a rapid and convenient tool for on-site diagnosis to control outbreaks and the spread of CCVD. The immunochromatographic strip was the first to be developed and applied for the detection of CCVD.