Importance of validating antibody panels: Anti-PD-L1 clone binds AF700 fluorophore.
Abstract Researchers routinely use antibodies to assess the expression levels of proteins on the surface or intracellularly in a variety of different cell types. In this current study we highlight the importance of careful validation of antibodies for analysis of protein expression by flow cytometry and how failure to do so can significantly impact the interpretation of the data generated leading to false-positive results. There has been increasing awareness of the role the programmed death receptor 1 (PD-1) pathway plays in health and disease and a potential that programme death ligand 1 (PD-L1) may play a role i...
Source: Journal of Immunological Methods - May 25, 2020 Category: Allergy & Immunology Authors: Hughes MJ, McGettrick HM, Sapey E Tags: J Immunol Methods Source Type: research

A novel in vivo model using immunotoxin in the absence of p-glycoprotein to achieve ultra selective depletion of target cells: Applications in trogocytosis and beyond.
Abstract A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows t...
Source: Journal of Immunological Methods - May 16, 2020 Category: Allergy & Immunology Authors: Brown K, Meader L, Nowocin A, Edwards LA, Cheung LH, Smith RA, Rosenblum MG, Wong W Tags: J Immunol Methods Source Type: research

Lipid-tagged single domain antibodies for improved enzyme-linked immunosorbent assays.
Abstract Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged singl...
Source: Journal of Immunological Methods - May 15, 2020 Category: Allergy & Immunology Authors: Shriver-Lake LC, Goldman ER, Dean SN, Liu JL, Davis TM, Anderson GP Tags: J Immunol Methods Source Type: research

Rational application of nanoadjuvant for mucosal vaccine delivery system.
Abstract The surface of the mucosa is the biggest path through which pathogens enter the human body. We need an understanding of mucosal immune systems to use vaccines that generate protective mucosal and systemic immunity to regulate the outbreak of various infectious diseases. The better impact of the mucosal vaccine over traditional injectable vaccines are that not only do they induce efficient immune reactions to the mucosa but they are also comfortable in physical aspect & psychological aspect. The material of the vaccine includes pathogens antigens and adjuvants, which enable vaccination to be effective....
Source: Journal of Immunological Methods - May 6, 2020 Category: Allergy & Immunology Authors: Dewangan HK Tags: J Immunol Methods Source Type: research

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation.
Abstract BACKGROUND: Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. OBJECTIVES: This work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizi...
Source: Journal of Immunological Methods - May 6, 2020 Category: Allergy & Immunology Authors: Arif-Lusson R, Agabriel C, Carsin A, Cabon I, Senechal H, Poncet P, Vitte J, Busnel JM Tags: J Immunol Methods Source Type: research

Stabilization of blood for long-term storage can affect antibody-based recognition of cell surface markers.
ius G, Sundling C Abstract Whole-blood fixation provides a rapid and simplified method for cell preservation compared to isolation of peripheral blood mononuclear cells (PBMCs). This can be especially important for sample acquisition and storage in resource-limited settings. However, some caveats have been reported, such as reduced cell marker recognition. Here, we evaluated the whole-blood proteomic stabilizer PROT1 and compared recognition of 53 common cell markers in fixed buffy coats and cryopreserved PBMCs isolated from the same donor. Several antibodies completely lost their binding to the cells, while other...
Source: Journal of Immunological Methods - May 6, 2020 Category: Allergy & Immunology Authors: Silva MH, Lepzien R, Ols S, Dahlberg B, Grunewald J, Loré K, Smed-Sörensen A, Correia-Neves M, Empadinhas N, Färnert A, Källenius G, Sundling C Tags: J Immunol Methods Source Type: research

Adaption of a conventional ELISA to a 96-well ELISA-Array for measuring the antibody responses to influenza virus proteins and vaccines.
We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins and vaccine on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n = 9 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody...
Source: Journal of Immunological Methods - May 4, 2020 Category: Allergy & Immunology Authors: Waltari E, Carabajal E, Sanyal M, Friedland N, McCutcheon KM Tags: J Immunol Methods Source Type: research

In the search of potential epitopes for Wuhan seafood market pneumonia virus using high order nullomers.
Abstract Alarms periodically emerge for viral pneumonia infections due to coronavirus. In all cases, these are zoonoses passing the barrier between species and infect humans. The legitimate concern of the international community is due to the fact that the new identified coronavirus, named SARS-CoV-2 (previously called 2019-nCoV), has a quite high mortality rate, around 2%, and a strong ability to spread, with an estimated reproduction number higher than 2. Even though all countries are doing their utmost to stop the pandemic, the only reliable solution to tackle the infection is the rapid development of a vaccine...
Source: Journal of Immunological Methods - April 23, 2020 Category: Allergy & Immunology Authors: Santoni D, Vergni D Tags: J Immunol Methods Source Type: research

Coating M-CSF on plastic surface results in the generation of increased numbers of macrophages in vitro.
Abstract Macrophages are one of the important cell types in the innate immune system that are present in various anatomical regions of the body and promote early control of pathogens. The relative proportion of macrophages in various lymphoid and non-lymphoid regions is small, and as such it is tedious to purify these cells to homogeneity. Culture of bone marrow precursors with macrophage colony-stimulating factor (M-CSF) results in their differentiation to macrophages, however this procedure results in low numbers of differentiated macrophages. Herein we reveal a new approach of generating increased numbers of di...
Source: Journal of Immunological Methods - April 15, 2020 Category: Allergy & Immunology Authors: Sadh S, Hajjar S, Ariana A, Phuong MS, Cai D, Thakker P, Sad S Tags: J Immunol Methods Source Type: research

A new cell line for assessing HIV-1 antibody dependent cellular cytotoxicity against a broad range of variants.
Abstract Human immunodeficiency virus type 1 (HIV-1) studies suggest that antibody-dependent cellular cytotoxicity (ADCC) influences both virus acquisition and subsequent disease outcome. Technical issues with currently available assays, however, have limited the ability to comprehensively assess the impact of ADCC on transmission and disease progression. Commonly used ADCC assays use a target cell line, CEM.NKr-CCR5-Luc, that often does not support replication of relevant HIV-1 variants. Thus, the extent of ADCC responses against a large panel of HIV-1 strains often cannot be assessed using the currently availabl...
Source: Journal of Immunological Methods - March 2, 2020 Category: Allergy & Immunology Authors: Thomas AS, Ghulam-Smith M, Olson A, Coote C, Gonzales O, Sagar M Tags: J Immunol Methods Source Type: research

Laboratorial algorithm for serological diagnosis of visceral leishmaniasis using rK39-ICT, DAT-LPC and FC-Simplex IgG1.
ns-Filho OA Abstract The performance of distinct serological tests (rK39-ICT, IFAT, DAT-LPC, FC-Simplex IgG1) was assessed and a laboratorial algorithm was proposed for accurate diagnosis of VL. DAT-LPC and FC-Simplex IgG1 showed outstanding accuracy (AUC = 0.93) to identify VL patients. The use of a sequential serological algorithm (rK39-ICT screening followed by DAT-LPC or FC-Simplex IgG1) improved the global accuracy for VL (97.2%) diagnosis. An alternative approach for diagnosis of VL has been also assessed for interchangeable use of serum/whole blood lysate samples in DAT-LPC and FC-Simplex IgG1. Our data...
Source: Journal of Immunological Methods - February 28, 2020 Category: Allergy & Immunology Authors: do Vale INPC, Saliba JW, Fonseca GSF, Peruhype-Magalhães V, de Araújo FF, Pascoal-Xavier MA, Teixeira-Carvalho A, Campos FMF, Andrade MC, Lula JF, Reis AB, Lemos EM, de Carvalho SFG, Oliveira E, Martins-Filho OA Tags: J Immunol Methods Source Type: research

Reproducible quantification of IgG uptake at endogenous and overexpressed FcRn levels at pH  7.4: Comparison of a wild type IgG and a stronger FcRn binding variant.
Reproducible quantification of IgG uptake at endogenous and overexpressed FcRn levels at pH 7.4: Comparison of a wild type IgG and a stronger FcRn binding variant. J Immunol Methods. 2020 Feb 28;:112767 Authors: Zhang J, Vernes JM, Wen X, Oh E, Scales SJ, Meng YG Abstract IgG antibodies have been used to treat many diseases including cancer. IgG antibody-drug conjugates (ADCs) deliver cytotoxic drugs to target cells for cell elimination, but they have dose limiting toxicity due to target-independent uptake, including pinocytotic uptake. Neonatal Fc receptor (FcRn) recycles pinocytosed IgG in a pH-de...
Source: Journal of Immunological Methods - February 28, 2020 Category: Allergy & Immunology Authors: Zhang J, Vernes JM, Wen X, Oh E, Scales SJ, Meng YG Tags: J Immunol Methods Source Type: research

Implementation of a three-tiered approach to identify and characterize anti-drug antibodies raised against HIV-specific broadly neutralizing antibodies.
Abstract The ability to detect, quantify, and interrogate the properties of immune responses raised against biological therapeutics is not only important to our understanding of these molecules, but also to their success in the clinic. A tiered assay approach to identify the presence, specificity, and titer of anti-drug antibody (ADA) responses has been adopted as a gold standard by industry leaders, the FDA, and the EMA. In order to support pre-clinical and clinical trials, these assays must be standardized, and their performance sufficiently characterized to ensure the accuracy and reproducibility of results und...
Source: Journal of Immunological Methods - February 15, 2020 Category: Allergy & Immunology Authors: Bharadwaj P, Riekofski C, Lin S, Seaman MS, Garber DA, Montefiori D, Sarzotti-Kelsoe M, Ackerman ME, Weiner JA Tags: J Immunol Methods Source Type: research

T follicular helper and T follicular regulatory cells in colorectal cancer: A double-edged sword.
Abstract Colorectal cancer is considered to be one of the major causes of morbidity and mortality all over the world. The recent discovery of T Follicular helper (TFR) and T follicular regulatory (TFH) which are two opposite subsets of follicular T cells that have been demonstrated to be involved in the process of tumorigenesis. CXCR5+CD4+ follicular T helper cells (TFH) are specialized B cell stimulators. On the other hand, CD4+CXCR5+Foxp3+ follicular regulatory T cells (TFR) cell subset can control humoral immune response by suppressing TFH-mediated B-cell activation and antibody production via PD-1 receptor whi...
Source: Journal of Immunological Methods - February 13, 2020 Category: Allergy & Immunology Authors: Hetta HF, Elkady A, Yahia R, Meshaal AK, Saad MM, Mekky MA, Al-Kadmy IMS Tags: J Immunol Methods Source Type: research

Corrigendum to "Task-shifting of CD4 T cell count monitoring by the touchscreen-based Muse ™ Auto CD4/CD4% single-platform system for CD4 T cell numeration: Implication for decentralization in resource-constrained settings" [Journal of Immunological Methods, 455 (2018) 95-98].
Corrigendum to "Task-shifting of CD4 T cell count monitoring by the touchscreen-based Muse™ Auto CD4/CD4% single-platform system for CD4 T cell numeration: Implication for decentralization in resource-constrained settings" [Journal of Immunological Methods, 455 (2018) 95-98]. J Immunol Methods. 2020 Feb 13;:112735 Authors: Kouabosso A, Mossoro-Kpinde CD, Mboumba-Bouassa RS, Longo JD, Simaleko MM, Grésenguet G, Bélec L PMID: 32063283 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - February 13, 2020 Category: Allergy & Immunology Authors: Kouabosso A, Mossoro-Kpinde CD, Mboumba-Bouassa RS, Longo JD, Simaleko MM, Grésenguet G, Bélec L Tags: J Immunol Methods Source Type: research

Clinical value of anti-DFS70 antibodies in a cohort of patients undergoing routine antinuclear antibodies testing.
CONCLUSIONS: Although the DFS pattern cannot exclude the presence of SARD, the likelihood is lower than with other patterns. Therefore, anti-DFS70 antibodies represent an important biomarker that can aid in the interpretation of positive ANA patients and, therefore, should be included in test algorithms for ANA testing. The optimal test algorithm might be laboratory specific being dependent on referral patterns for ANA testing. PMID: 32057435 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - February 11, 2020 Category: Allergy & Immunology Authors: Yumuk Z, Demir M Tags: J Immunol Methods Source Type: research

Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFN γ+LPS), M(IL-4) and M(IL-10) phenotypes.
Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes. J Immunol Methods. 2020 Feb 04;:112721 Authors: Baxter EW, Graham AE, Re NA, Carr IM, Robinson JI, Mackie SL, Morgan AW Abstract In vitro models of differing macrophage functions are useful since human monocyte-derived macrophages are short-lived, finite and vary from donor to donor. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS....
Source: Journal of Immunological Methods - February 4, 2020 Category: Allergy & Immunology Authors: Baxter EW, Graham AE, Re NA, Carr IM, Robinson JI, Mackie SL, Morgan AW Tags: J Immunol Methods Source Type: research

openPrimeR for multiplex amplification of highly diverse templates.
n F Abstract To study the diversity of immune receptors and pathogens, multiplex PCR has become a central approach in research and diagnostics. However, insufficient primer design against highly diverse templates often prevents amplification and therefore limits the correct understanding of biological processes. Here, we present openPrimeR, an R-based tool for evaluating and designing multiplex PCR primers. openPrimeR provides a functional and intuitive interface and uses either a greedy algorithm or an integer linear program to compute the minimal set of primers that performs full target coverage. As proof of con...
Source: Journal of Immunological Methods - January 25, 2020 Category: Allergy & Immunology Authors: Kreer C, Döring M, Lehnen N, Ercanoglu MS, Gieselmann L, Luca D, Jain K, Schommers P, Pfeifer N, Klein F Tags: J Immunol Methods Source Type: research

Development of novel highly sensitive methods to detect endogenous cGAMP in cells and tissue.
Abstract Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled th...
Source: Journal of Immunological Methods - January 23, 2020 Category: Allergy & Immunology Authors: Miyakawa S, Okui T, Shiraishi T, Yoshihara T, Hirayama M, Satomi Y, Hamada T, Nishida M, Akimoto C, Sato S Tags: J Immunol Methods Source Type: research

Novel short peptide tag from a bacterial toxin for versatile applications.
Abstract The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection ...
Source: Journal of Immunological Methods - January 22, 2020 Category: Allergy & Immunology Authors: Lee TH, Kim KS, Kim JH, Jeong JH, Woo HR, Park SR, Sohn MH, Lee HJ, Rhee JH, Cha SS, Hwang JH, Chung KM Tags: J Immunol Methods Source Type: research

A method to study protein biomarkers in saliva using an automated capillary nano-immunoassay platform (Wes ™).
A method to study protein biomarkers in saliva using an automated capillary nano-immunoassay platform (Wes™). J Immunol Methods. 2020 Jan 20;:112749 Authors: Reyes-Serratos E, Marcet-Palacios M, Rosenfield D, Ritz T, Befus AD Abstract Traditional immunoprobing techniques like Western blot continue to play a crucial role in the discovery and validation of biomarkers. This technique suffers from several limitations that affect reproducibility and feasibility for large-scale studies. Modern immunoprobing techniques have addressed several of these limitations. Here we contrast the use of Western blo...
Source: Journal of Immunological Methods - January 20, 2020 Category: Allergy & Immunology Authors: Reyes-Serratos E, Marcet-Palacios M, Rosenfield D, Ritz T, Befus AD Tags: J Immunol Methods Source Type: research

A novel whole blood based method for lymphocyte transformation test in drug allergies.
Abstract Drug allergies pose a great deal of danger for the patients. It hinders effective treatment procedures in hospitalized patients. Moreover, it complicates the symptoms due to the allergic reactions of the immune system. Allergic reactions may arise against any medication including antibiotics and chemotherapeutics. Therefore, it is crucial to assess the sensitization pattern of the patients to culprit drug(s) before retreatment with the same or similar drug, or in order to confirm/exclude a suspected drug hypersensitivity reaction. In vivo and in vitro tests are performed in the evaluation of patients. Cur...
Source: Journal of Immunological Methods - January 17, 2020 Category: Allergy & Immunology Authors: Ayaz F, Arikoglu T, Demirhan A, Kuyucu S Tags: J Immunol Methods Source Type: research

A high-throughput chemotaxis detection method for CCR4+ T cell migration inhibition using image cytometry.
Abstract Chemotaxis is an important aspect of immune cell behavior within the tumor microenvironment (TME). One prominent example of chemotaxis within the TME is the migration of regulatory T cells (Tregs) in response to the chemokine ligands CCL17 and CCL22. Tregs within the TME cause the suppression of anti-tumor immunity and inhibition of the effect of immunotherapeutic treatments. Therefore, the ability to screen for therapeutic antibodies that can inhibit or stimulate the chemotaxis of various immune cell types is crucial. Traditionally, chemotaxis is studied by determining the number of cells in the bottom r...
Source: Journal of Immunological Methods - January 17, 2020 Category: Allergy & Immunology Authors: Magnotti EL, Chan LL, Zhu Q, Marasco WA Tags: J Immunol Methods Source Type: research

Establishment of an anti-hepatitis C virus IgG avidity test for dried serum/plasma spots.
Abstract Monitoring recency of infection helps to identify current transmission in vulnerable populations for effective disease control. We have established an in-house avidity based hepatitis C virus (HCV) recency assay based on the Monolisa Anti-HCV PLUS Version 3 ELISA kit for use of dried serum/plasma spots (DS/PS) in order to distinguish recent and long-term infections. A first panel of DS/PS (n = 218; genotype 1 n = 170 and non-genotype 1 n = 48) consisting of primary and at least one follow up sample was used to analyze the temporal changes of the Avidity Index (AI) over time. Sub-panels of long...
Source: Journal of Immunological Methods - January 17, 2020 Category: Allergy & Immunology Authors: Eshetu A, Hauser A, An der Heiden M, Schmidt D, Meixenberger K, Ross S, Obermeier M, Ehret R, Bock CT, Bartmeyer B, Bremer V, Bannert N Tags: J Immunol Methods Source Type: research

Normal human lymph node T follicular helper cells and germinal center B cells accessed via fine needle aspirations.
This study of 73 LNs demonstrates that human LN FNAs are a safe and feasible technique for immunological research, and suggests benchmarks for human GC biology under noninflammatory conditions. The findings indicate that assessment of the GC response via LN FNAs will have application to the study of human vaccination, allergy, and autoimmune disease. PMID: 31958451 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - January 17, 2020 Category: Allergy & Immunology Authors: Havenar-Daughton C, Newton IG, Zare SY, Reiss SM, Schwan B, Suh MJ, Hasteh F, Levi G, Crotty S Tags: J Immunol Methods Source Type: research

Culture medium used during small interfering RNA (siRNA) transfection determines the maturation status of dendritic cells.
Abstract Gene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA interference can be applied in both cell lines, as well as in primary, non-dividing cell types like dendritic cells. However, the efficacy in different cell types is variable and requires optimization. Here, we showed that the type of culture medium used during lipid-based siRNA-mediated transfection acts as a critical factor, affecting dendritic cell activation. Transfection of immature monocyte-derived dendritic cells in...
Source: Journal of Immunological Methods - January 17, 2020 Category: Allergy & Immunology Authors: van Essen MF, Schlagwein N, van Gijlswijk-Janssen DJ, Anholts JDH, Eikmans M, Ruben JM, van Kooten C, COMBAT consortium Tags: J Immunol Methods Source Type: research

Corrigendum to "IL8 and EDEM3 gene expression ratio indicates peripheral blood mononuclear cell (PBMC) quality" [Journal of Immunological Methods 465 (2019) 13-19].
Corrigendum to "IL8 and EDEM3 gene expression ratio indicates peripheral blood mononuclear cell (PBMC) quality" [Journal of Immunological Methods 465 (2019) 13-19]. J Immunol Methods. 2020 Jan 06;:112733 Authors: Kofanova O, Bellora C, Aguilar-Quesada R, Bulla A, Panadero-Fajardo S, Keipes M, Shea K, Stone M, Lescuyer P, Betsou F PMID: 31916985 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - January 6, 2020 Category: Allergy & Immunology Authors: Kofanova O, Bellora C, Aguilar-Quesada R, Bulla A, Panadero-Fajardo S, Keipes M, Shea K, Stone M, Lescuyer P, Betsou F Tags: J Immunol Methods Source Type: research

Optimization and qualification of a functional anti-drug antibody assay for HIV-1 bnAbs.
Abstract The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function...
Source: Journal of Immunological Methods - January 6, 2020 Category: Allergy & Immunology Authors: Seaman MS, Bilska M, Ghantous F, Eaton A, LaBranche CC, Greene K, Gao H, Weiner JA, Ackerman ME, Garber DA, Rosenberg YJ, Sarzotti-Kelsoe M, Montefiori DC Tags: J Immunol Methods Source Type: research

Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques.
Abstract Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires ...
Source: Journal of Immunological Methods - December 19, 2019 Category: Allergy & Immunology Authors: Wang Z, Merkenschlager J, Chen ST, Oliveira TY, Ramos V, Gordon KM, Yao KH, Jankovic M, Gazumyan A, Nussenzweig M, Escolano A Tags: J Immunol Methods Source Type: research

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY).
Abstract Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purifica...
Source: Journal of Immunological Methods - December 11, 2019 Category: Allergy & Immunology Authors: Abdool FS, Coetzer THT, Goldring JPD Tags: J Immunol Methods Source Type: research

FORSCells: 40-days fixed prepared reagent for detection of anti-Forssman in humans.
In conclusion, a novel and easy-to-produce reagent has been developed and submitted to a patent with stable FORS1 Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Abs in large populations. PMID: 31816281 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - December 6, 2019 Category: Allergy & Immunology Authors: Ferreira S, Mourato C, Corpuz A, Galvão S, Hesse C, Rocha C, Jesus C, Mendes F Tags: J Immunol Methods Source Type: research

A cocktail of polyclonal affinity enriched antibodies against melanoma mutations increases binding and inhibits tumor growth.
CONCLUSIONS: Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple doses were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free. PMID: 31812660 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - December 5, 2019 Category: Allergy & Immunology Authors: Shukla GS, Sun YJ, Pero SC, Krag DN Tags: J Immunol Methods Source Type: research

Analyzing cellular immunogenicity in vaccine clinical trials: a new statistical method including non-specific responses for accurate estimation of vaccine effect.
We describe here a new statistical approach to analyze ICS data from vaccine trials. We propose a bivariate linear regression model for estimating the non-specific and antigen-specific ICS responses. We benchmarked the performance of the model in terms of both bias and control of type-I and -II errors in comparison with conventional approaches, and applied it to simulated data as well as real pre- and post-vaccination data from two recent HIV vaccine trials (ANRS VRI01 in healthy volunteers and therapeutic VRI02 ANRS 149 LIGHT in HIV-infected participants). The model was as good as the conventional approaches (with or with...
Source: Journal of Immunological Methods - December 3, 2019 Category: Allergy & Immunology Authors: Lhomme E, Hejblum BP, Lacabaratz C, Wiedemann A, Lelièvre JD, Levy Y, Thiébaut R, Richert L Tags: J Immunol Methods Source Type: research

Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot.
Ahlborg N Abstract Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepat...
Source: Journal of Immunological Methods - December 3, 2019 Category: Allergy & Immunology Authors: Jahnmatz P, Sundling C, Zuber B, Sondén K, Färnert A, Ahlborg N Tags: J Immunol Methods Source Type: research

Analytical validation of new ELISAs for the quantitation of polyclonal free light chains and comparison to existing assays for healthy and patient samples.
CONCLUSIONS: The ELISAs displayed good analytical and technical performance. The quantification of individual κ and λ FLC appears inherently different between platforms. These differences are attenuated if using the FLC sum, which was similar between methods and provided agreement in relation to patients having normal or elevated FLCs. PMID: 31783021 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 26, 2019 Category: Allergy & Immunology Authors: Heaney JLJ, Campbell JP, Goodall M, Plant T, Shemar M, Hand C, Drayson MT Tags: J Immunol Methods Source Type: research

Antibody microarray immunoassay for screening and differential diagnosis of upper respiratory tract viral pathogens.
Abstract Upper respiratory tract infections are the world's most common infectious disease. The etiologic agents behind upper respiratory tract infections (URTIs) are, in fact, a diverse set of pathogens such as influenza, parainfluenza, adenovirus, rhinovirus, and others. More than 200 pathogens are known to be involved. Differential diagnosis of viral infections is sometimes complicated by their diversity or similarity of clinical presentation. This work is devoted to the development of a method which enables simultaneous detection of six common viral URTI pathogens: IAV; IBV; RSV; hAdV; hPIV2; and hPIV3. Antibo...
Source: Journal of Immunological Methods - November 26, 2019 Category: Allergy & Immunology Authors: Plotnikova MA, Klotchenko SA, Lebedev KI, Lozhkov AA, Taraskin AS, Gyulikhandanova NE, Ramsay ES, Vasin AV Tags: J Immunol Methods Source Type: research

Multiplex immunofluorescence staining and image analysis assay for diffuse large B cell lymphoma.
Abstract With the explosion of immuno-oncology and the approval of many immune checkpoint therapies by regulatory agencies in the last few years, understanding the tumor microenvironment (TME) in the context of patient's immune status has become essential. Among available immune profiling techniques multiplex immunofluorescence (mIF) assays offer the unique advantage of preserving the architectural features of the tumor and revealing the spatial relationships between tumor cells and immune cells. A number of mIF and image analysis assays have been described for solid tumors but most are not sufficiently suitable f...
Source: Journal of Immunological Methods - November 26, 2019 Category: Allergy & Immunology Authors: Lee CW, Ren YJ, Marella M, Wang M, Hartke J, Couto SS Tags: J Immunol Methods Source Type: research

EuroFlow and its activities: Introduction to the special EuroFlow issue of The Journal of Immunological Methods.
PMID: 31758969 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 20, 2019 Category: Allergy & Immunology Authors: van Dongen JJM, O'Gorman MRG, Orfao A Tags: J Immunol Methods Source Type: research

High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis.
Abstract Fluorescent cell barcoding (FCB) is a multiplexing technique for high-throughput flow cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique because of inter-operator variability and differences in data analysis. FCB was implemented by combining two-dye barcoding (DyLight 350 plus Pacific Orange) with five-color surface marker antibody and intracellular staining for phosphoprotein signaling analysis. We proposed a robust method to measure intra- and inter-assay variability of FCB in T/B cells and monocytes by combining range and ratio of variability to...
Source: Journal of Immunological Methods - November 11, 2019 Category: Allergy & Immunology Authors: Tsai WL, Vian L, Giudice V, Kieltyka J, Liu C, Fonseca V, Gazaniga N, Gao S, Kajigaya S, Young NS, Biancotto A, Gadina M Tags: J Immunol Methods Source Type: research

A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood.
Abstract Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of...
Source: Journal of Immunological Methods - November 8, 2019 Category: Allergy & Immunology Authors: Bedke N, Swindle EJ, Molnar C, Holt P, Strickland D, Roberts GC, Morris R, Holgate ST, Davies DE, Blume C Tags: J Immunol Methods Source Type: research

A multi-centre study for standardization of antinuclear antibody indirect immunofluorescence screening with automated system.
CONCLUSION: HELIOS could provide accurate diagnostic results, this include not only positive/negative results, but also endpoint titer, common patterns. The application of this system can help to promote standardization of ANA detection. PMID: 31705859 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 6, 2019 Category: Allergy & Immunology Authors: Li X, Pan J, Zhou H, He M, Li W, Chen Z, Dai W, Wang F, Wei Q, Lao X, Zhang L, Li L, Hu H, Li M, Qiu Y, Hou T Tags: J Immunol Methods Source Type: research

Isolation and functional characterisation of lamina propria leukocytes from helminth-infected, murine small intestine.
Abstract The use of helminth infections as tools to understand the type 2 immune response is a well-established technique and important to many areas of immunological research. The phenotype and function of immune cell populations at the site of infection is a key determinant of pathogen clearance. However, infections with helminths such as the murine nematode Heligomosmoides polygryrus cause increased mucus production and thickening of the intestinal wall, which can result in extensive cell death when isolating and analysing cells from the lamina propria (LP). Populations of larger immune cells such as macrophage...
Source: Journal of Immunological Methods - November 6, 2019 Category: Allergy & Immunology Authors: Webster HC, Andrusaite AT, Shergold AL, Milling SWF, Perona-Wright G Tags: J Immunol Methods Source Type: research

rM-CSF efficiently replaces L929 in generating mouse and rat bone marrow-derived macrophages for in vitro functional studies of immunity to intracellular bacteria.
Abstract Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as ...
Source: Journal of Immunological Methods - November 2, 2019 Category: Allergy & Immunology Authors: Rice HM, Rossi AP, Bradford MK, Elkins KL, De Pascalis R Tags: J Immunol Methods Source Type: research

An optimized five-color/seven-parameter flow cytometry panel for immunophenotyping Guinea pig peripheral blood lymphocytes.
Abstract Guinea pigs are an ideal animal model for the study of several infectious diseases, including tuberculosis, legionellosis, brucellosis, and spotted fever rickettsiosis. In comparison to the murine model, clinical signs in guinea pigs are more representative of disease in humans, the guinea pig immune system is more similar to that of the human, and their large size offers logistic advantages for sample collection while following disease progression. Unfortunately, the advantage of using guinea pigs in biomedical research, particularly in understanding the immune response to infectious agents, is limited i...
Source: Journal of Immunological Methods - November 1, 2019 Category: Allergy & Immunology Authors: Stokes JV, Crawford AE, Cross CE, Ross AL, Walker JD, Willeford BV, Varela-Stokes AS Tags: J Immunol Methods Source Type: research

The quantification of antibody elements and receptors subunit expression using qPCR: The design of VH, VL, CH, CL, FcR subunits primers for a more holistic view of the immune system.
Abstract The expression levels of Immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expressions of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all Vκ1-6, V...
Source: Journal of Immunological Methods - November 1, 2019 Category: Allergy & Immunology Authors: Ling WL, Ng YL, Wipat A, Lane DP, Gan SK Tags: J Immunol Methods Source Type: research

Antigen-independent killer cells prepared for adoptive immunotherapy: One source, divergent protocols, diverse nomenclature.
Abstract Adoptive cell therapy (ACT) using tumor antigen-independent killer cells has been widely used in clinical trials of cancer treatment. Circumventing the need for identification of a particular tumor-associated antigen on tumor cells, the approach has opened possibilities for the extension of ACT immunotherapy to patients with a wide variety of cancer types. Namely, Natural Killer (NK), Lymphokine-activated Killer (LAK) cells and Cytokine-induced killer (CIK) cells are the most commonly used cell types in antigen-independent adoptive immunotherapy of cancer. They all originate from peripheral blood mononucl...
Source: Journal of Immunological Methods - October 31, 2019 Category: Allergy & Immunology Authors: Torabi-Rahvar M, Aghayan HR, Ahmadbeigi N Tags: J Immunol Methods Source Type: research

Accuracy, precision, and consistency of methods for pathogen-specific cerebrospinal fluid/serum Q-value calculation.
n P Abstract For a CSF/serum samples pair the specific Q-value is the ratio of antibody concentrations and proportional to the pathogen-specific antibody index. It is usually extracted from optical density (OD) measurements by applying appropriate evaluation methods. Six different methods for Q-value determination, partly using parameter variation, were assessed with respect to accuracy, precision, and the methods parameter consistency. The methods are based on the Four Parameters Logistic (4PL) equation or the α-method; one method is a polygonal line composed of calibration data. We tested on OD data of 51 ...
Source: Journal of Immunological Methods - October 31, 2019 Category: Allergy & Immunology Authors: Osterman A, Böhm S, Osterman P Tags: J Immunol Methods Source Type: research

A cell-based immunogenicity assay to detect antibodies against chimeric antigen receptor expressed by tisagenlecleucel.
Abstract The use of T-cells expressing Chimeric Antigen Receptors (CARs) offers new opportunities for cancer treatment, as well as new challenges for the bioanalysis of this new class of drugs. The analysis of humoral immunogenicity (anti-drug antibodies) against CARs could be performed with a bridging ELISA, using labeled CAR fragments. However, outside of its native cell membrane environment and without potential interaction partners on the cell surface, a labeled or coated recombinant CAR fragment may structurally differ from the membrane-bound CAR expressed on CAR-T cells. Consequently, immunogenicity to CARs ...
Source: Journal of Immunological Methods - October 31, 2019 Category: Allergy & Immunology Authors: Bernd P, McBlane F, Sebastian S, Denise S Tags: J Immunol Methods Source Type: research

PromPDD, a web-based tool for the prediction, deciphering and design of promiscuous peptides that bind to HLA class I molecules.
In this study, we re-evaluated and characterized four matrix-based prediction models that have been extensively used for the prediction of HLA-binding peptides and found that the prediction models generated based on the average relative binding (ARB) matrix shared a consistent and conservative threshold for all well-studied HLA class I alleles. Evaluations performed using datasets of HLA supertype-specific peptides with various cross-binding abilities and peptide mutant analogues indicated that the ARB-based binding matrices could be used to decipher and design promiscuous peptides that bind to multiple HLA molecules. A we...
Source: Journal of Immunological Methods - October 30, 2019 Category: Allergy & Immunology Authors: Zhang S, Chen J, Hong P, Li J, Tian Y, Wu Y, Wang S Tags: J Immunol Methods Source Type: research

A novel approach to assess domain specificity of anti-drug antibodies to moxetumomab pasudotox, an immunotoxin with two functional domains.
Abstract Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determina...
Source: Journal of Immunological Methods - October 29, 2019 Category: Allergy & Immunology Authors: Vainshtein I, Sun B, Roskos LK, Liang M Tags: J Immunol Methods Source Type: research