Murine Monoclonal Antibodies
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2021 ◽  
Vol 15 (9) ◽  
pp. e0009689
Author(s):  
Leroy Versteeg ◽  
Rakesh Adhikari ◽  
Cristina Poveda ◽  
Maria Jose Villar-Mondragon ◽  
Kathryn M. Jones ◽  
...  

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi–infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.


2021 ◽  
Vol 3 (1) ◽  
pp. 97-114
Author(s):  
Albina Makio ◽  
Lillian Musila ◽  
Eddy Okoth Odari ◽  
Juliette Rose Ongus ◽  
Rosemary Sang

O’nyong-nyong virus (ONNV) and Chikungunya virus (CHIKV) are antigenically related alphaviruses responsible for febrile illnesses common to the tropics and associated with relatively high morbidity and mortality. Murine monoclonal antibodies (mAbs) targeting alphaviruses like Chikungunya have been developed and used to make commercially available kits. However, few studies have been conducted to develop antibodies specific to ONNV and no commercial kits are available for use in endemic regions where outbreak potential is high. We demonstrate the potential of in-house generated monoclonal antibodies against ONNV to detect both ONNV and CHIKV. The objective of this study was to generate mAbs using hybridoma technology, characterize the developed mAbs, determine their specificity against selected alphaviruses and check their diagnostic potential using an indirect IgG enzyme-linked immunosorbent assay (ELISA) and focus neutralization assay (FRNT50). BALB/c mice were immunized with ONNV purified proteins from ONNV infectious culture fluid. After four rounds of booster injections, the mice were sacrificed, spleen cells harvested and fused with parental myeloma cells then cultured in selective media and the successful hybrid clones with antibody-producing ability purified to yield the desired mAbs. Five monoclonal antibodies targeting the ONNV E1 protein of isotypes IgG2a/kappa, IgG2b/kappa and IgM/kappa (P1B12, P1E9, P1G11, P1B4 and P1G6) demonstrated a potential to detect both ONNV and CHIKV isolates by indirect IgG ELISA but no potential for neutralization of the viruses by FRNT50. This study demonstrates the potential efficacy of in-house serological tools as an alternative in the absence of commercial assays in screening and diagnosis of ONN and CHIK viruses which are often co-circulating. It is our recommendation that this work may be pursued further to design and optimize ELISA assays, using the developed mAbs, for the detection of both ONN and CHIK viruses in the research laboratory set-up


mBio ◽  
2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B. Ward ◽  
...  

Cross-neutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood, and very little is known about the potential protective effect of nonneutralizing antibodies in vivo . Using a panel of mouse monoclonal antibodies, we investigate both of these points.


2021 ◽  
Author(s):  
Larisa Troitskaya ◽  
Nelson Lap Shun Chan ◽  
Brendon Frank ◽  
Daniel Capon ◽  
Brian A. Zabel ◽  
...  

With the rapid spread of SARS-CoV-2 variants, including those that are resistant to antibodies authorized for emergency use, it is apparent that new antibodies may be needed to effectively protect patients against more severe disease. Differences between the murine and human antibody repertoires may allow for the isolation of murine monoclonal antibodies that recognize a different or broader range of SARS-CoV-2 variants than the human antibodies that have been characterized so far. We describe mouse antibodies B13 and O24 that demonstrate neutralizing potency against SARS-CoV-2 Wuhan (D614G) and B.1.351 variants. Such murine antibodies may have advantages in protecting against severe symptoms when individuals are exposed to new SARS-CoV-2 variants.


2021 ◽  
Vol 10 (1) ◽  
pp. 1
Author(s):  
Wenyi Liu ◽  
Zhaohua Li

<div><p>Purpose: To establish a fast, simple and accurate method and immunoassay test card for the detection of new coronavirus (nCOVID-19) antigen. Methods: In this study, colloidal gold immunochromatography technology was used to detect nCOVID-19 virus antigens through the sandwich method. At the same time, the preparation plan of colloidal gold was improved, and the application of rapid immune-diagnosis technology in other fields was developed. In this study, purified recombinant nCOVID-19 nucleocapsid protein is used as the antigen to prepare murine monoclonal antibodies. The BN02 antibody produced by the mouse is used as the detection antibody to couple with colloidal gold, forming a gold-labeled complex probe. BN9m1 is used as the coating antibody for the C-line, and ProA is used for the T-line. The polymerization of colloidal gold particles enables us to detect the new coronavirus antigen’s appearance. Thus an in vitro rapid detection kit for virus detection can be made. Results: The positive detection rate of the antigen quality control serum with this colloidal gold reagent was 100%. The specificity was 100%, and the sensitivity was 1ng/ml.  Conclusion: The nCOVID-19 antigen detection reagent (colloidal gold method) developed in this research has high specificity and sensitivity, and can be used in conjunction with nucleic acid detection. As a means of detecting nCOVID-19, it can achieve qualitative and rapid screening of samples with advantage such as accuracy, repeatability, and low cost.</p></div>


2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.


2021 ◽  
Vol 85 (2) ◽  
pp. 340-350
Author(s):  
Shinji Sakamoto ◽  
Mika Kirinashizawa ◽  
Yumi Mohara ◽  
Yoshihiro Watanabe

ABSTRACT Hepcidin regulates the quantity of ferroportin (FPN) on cellular membrane. In our cell assay expressing ferroportin labeled with green fluorescence, FPN was internalized and degraded only after treatment with hepcidin-25, not hepcidin-22 or hepcidin-20, leading to accumulation of cellular iron. Thus we generated murine monoclonal antibodies (mAbs) against hepcidin-25, and then characterized and validated their functions. Among them, several mAbs showed a neutralizing activity that may prevent ferroportin internalization induced by hepcidin-25. To measure hepcidin level in various fluids, mAbs specific for human and rat hepcidin-25 were selected. As for rat, a sandwich ELISA developed using clone rHN1 as capture antibody and biotinylated clone mHW1 as a detection reagent had high sensitivity, allowing for the detection of 1-100 ng/mL of hepcidin-25. Rat hepcidin-25 level in plasma was measured at an average concentration of 63.0 ng/mL in healthy condition, and at 218.2 ng/mL after stimulation of lipopolysaccharide.


2021 ◽  
Author(s):  
Rafael Bayarri-Olmos ◽  
Manja Idorn ◽  
Anne Rosbjerg ◽  
Laura Pérez-Alós ◽  
Cecilie Hansen ◽  
...  

Abstract Effective tools to monitor SARS-CoV-2 transmission and humoral immune responses are highly needed. Protective humoral immunity involves neutralizing antibodies and will be a hallmark for the evaluation of a vaccine response efficacy. Here we present a sensitive, fast and simple neutralization ELISA method to determine the levels of antibody-mediated virus neutralization. We can show that it is strongly correlated with the more elaborate plaque reduction neutralization test (PRNT) (ρ = 0.9231, p < 0.0001). Furthermore, we present pre-clinical vaccine models using recombinant receptor binding domain (RBD) and full-length spike antigen as immunogens showing a profound antibody neutralization capacity that exceeds the highest neutralization titers from convalescent individuals. Using a panel of novel high-affinity murine monoclonal antibodies (mAbs) we also show that majority of the RBD-raised mAbs have inhibitory properties while only a few of the spike-raised mAbs do. In conclusion, the ELISA-based viral neutralization test offers a time- and cost-effective alternative to the PRNT. The immunization results indicate that vaccine strategies focused only on the RBD region may have major advantages over those based on the full spike sequence.


2020 ◽  
Vol 16 (S4) ◽  
Author(s):  
Julia Stockmann ◽  
Léon Beyer ◽  
Sandy Galkowski ◽  
Nathalie Woitzik ◽  
Jörn Güldenhaupt ◽  
...  

2020 ◽  
Vol 24 (3-4) ◽  
pp. 6-10
Author(s):  
С.М. Карташов ◽  
Т.В. Базарінська ◽  
І.Ю. Багмут ◽  
С.М. Граматюк

An in-depth study of the biology of tumor growth will help to identify factors that allow us to understand the pathogenetic mechanisms of the development of ovarian cancer metastasis and progression, as well as to become a theoretical basis for developing new approaches to the treatment of this disease. The aim of this study was to determine immunohistochemical and endothelial criteria for ovarian cancer. The postoperative samples of ovarian tumor tissues were divided into 3 groups: comparison group - ovarian cancer; main group - borderline ovarian tumor; benign ovarian tumors. The study was conducted according to the FIGO 2009 classification. The International Histological Classification of WHO 2013 Female Genital Tumors was used for morphological characteristics. Level of growth factors - sVEGF-A was performed by ELISA using standard test systems (BenderMedSystem, Austria). IGC material studies were performed on serial paraffin sections using a standard method with murine monoclonal antibodies to p53 (clone D0-7. Dilution 1: 100. "Dako"). Ventana Medical Systems, Inc. was used as the detection system. Positive and negative control reactions were performed. The label index (MI) was used to evaluate p53 nuclear expression. WCIF ImageJ and Aperio Image Scope were used to estimate the number and degree of cell staining. Statistical analysis of the obtained data was performed using Statistica 6.0. The results of morphological studies of ovarian cancer showed that in our study patients with serous cancer predominated - 78.5%. The second most frequently diagnosed cancer was undifferentiated. In the second stage of our study, we conducted a comparative analysis of the concentration of p53 in the serum and tissue of patients in the study groups, which showed the existence of significant differences. In patients of the POY and DOY groups, both total and local p53 protein activity were significantly higher than in the comparison group, p <0.05. There was a positive correlation between p53 protein activity in serum and ovarian tissue. Serum VEGF A scores were statistically significantly correlated with the disease stage: Spearman rank correlation coefficient rho = 0.30; 95% CI = 0.02 - 0.536, p <0.05. There were no correlations with patients' age, histological subtype, and degree of tumor differentiation. Considering the results of our study, we can conclude that the criterion-important indicators of QA are serum levels of p53 and the index of serum VEGF A, which is confirmed by the results of ROC analysis p = 0.0026, and indicates a good informativeness of the method.


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