Journal of immunological methods最新文献

We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the K_t (= K_s) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant K_i (= K_d) is the time required to double the slope or halve the V_max of the Lineweaver-Burk plot. The K_t values of the time courses were doubled (2 x K_t) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the K_i. The affinity constant of mAb SIM 28 was determined from ELISA data (n = 16) by three methods: i) doubling of K_t, ii) Beatty equation (K_aff = (n-1)/2 (n [HRP’]_t - [HRP]_t), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10⁻⁹ M, ii) K_aff = 0.23 ± 0.03 × 10⁹ M⁻¹ (K_d = 4.8 ± 0.81 × 10⁻⁹ M), and iii) 4.3 ± 0.57 × 10⁻⁹ M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double K_t method, is a repeatable and direct approach to mAb affinity determination.

Background

LAG3 is an immune checkpoint molecule with emerging therapeutic use. Expression of LAG3 is well studied on T cells, but the proportion of LAG3-expressing cells varies greatly by study and its comparative expression between non-T cells is lacking.

Methods/objectives

This study uses flow cytometry to compare surface LAG3 expression between T cells, NK cells, B cells, pDCs and monocytes of healthy donors. This study also compares three monoclonal LAG3 antibodies to a commonly used polyclonal LAG3 antibody on ex vivo and PHA-blasts from healthy donors and LAG3+ and LAG3- cell lines.

Results

LAG3 was most highly expressed on classical and intermediate monocytes (25 % and 32 %, respectively), while LAG3 expression on B cells, NK cells and iNKT cells was negligible. Notably, the polyclonal antibody stained a higher proportion of all cell types than the monoclonal antibodies, which had similar staining patterns to one another. Further study using LAG3+ and LAG3- cell lines showed greater specificity and similar sensitivity of the monoclonal antibody T47–530 than the polyclonal antibody.

Conclusion

Monocytes may represent an unappreciated source of LAG3 and target of LAG3 checkpoint inhibitors. Furthermore, the discrepancies between monoclonal and polyclonal LAG3 antibodies warrants consideration when designing future studies and interpreting past studies, and may explain discrepancies in the literature.

In mouse models of myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies can be quantified to monitor disease progression and treatment response. In mice, enzyme-linked immunosorbent assay (ELISA) is the gold standard to quantify these antibodies. However, this method requires antigen purification, which is both time-consuming and expensive. In humans, radioimmunoassay (RIA)—which is more sensitive than ELISA—is commonly used to quantify AChR antibodies. At present, however, no commercial RIA kits are available to quantify these antibodies in mice. The aim of this study was to compare a modified commercial human RIA kit to two ELISA methods to detect AChR antibodies in an experimental autoimmune mouse model of MG (EAMG). C57BL/6 J mice were immunized with purified AChR from Tetronarce californica (T-AChR). Serum samples were analyzed by RIA and two ELISAs (T-AChR and purified mouse AChR peptide [m-AChR]). The modified RIA showed excellent sensitivity (84.1 %) and specificity (100 %) for the detection of AChR antibodies. RIA showed a good agreement with T-AChR ELISA (κ = 0.69) but only moderate agreement with m-AChR ELISA (κ = 0.49). These results demonstrate the feasibility of modifying a commercially-available RIA kit to quantify AChR antibodies in EAMG. The advantage of this technique is that it eliminates the need to develop the entire methodology in-house and reduces inter and intra-laboratory variability.

Human monoclonal antibodies are essential diagnostic and research tools and one of the most promising therapeutics. In the past years, single B cell technologies have evolved and over-come conventional methods' limitations, enabling the isolation of scarce B cell populations with desirable characteristics. In this study, we describe a simple and efficient method to isolate anti-gen-specific plasmablasts and memory B cells from hepatitis B virus vaccinated donors' peripheral blood and consequently amplification of immunoglobulin variable region genes. Amplified immunoglobulin variable region genes were cloned into expression vectors and transfected into a human cell line to produce human recombinant monoclonal antibodies. Major challenges in this protocol were isolation of antigen-specific B cells based on surface markers, recovering mRNA from a single cell for efficient amplification, and cloning the correct insert into a desired expression vector. The essential feature of our protocol was the separation of B cells from peripheral blood mononuclear cells before sorting. We identified antigen-specific binding B cells based on the expression of surface markers CD19, CD27, IgG, and binding to hepatitis B surface antigen. Efficient single-cell reverse transcription and polymerase chain reaction (RT-PCR) were achieved using a random primer mix and Kapa Hifi Hot Start Polymerase. Amplification efficiency differed among heavy and light chain variable regions (highest at heavy chain (68 %) and lowest at lambda light chain (22 %)). After co-transfection of HEK293T/17 with successfully cloned heavy and light chain vectors, 70 % of transfected cells produced recombinant monoclonal antibodies at a concentration ∼ 4 μg/ml and 7 % of them showed binding to HBsAg. Human monoclonal antibodies from peripheral blood have advantages over antibodies of mouse origin or phage display libraries, because of their high specificity and self-tolerance. Using the described protocol, we can generate fully human monoclonal antibodies to any other antigen for application in immunotherapy or basic research.

Lateral Flow Immunoassay (LFI) is a disposable tool designed to detect target substances using minimal resources. For qualitative analysis, LFI does not require a device (i.e., reader) to interpret test results. However, various studies have been conducted to implement quantitative analysis using LFI systems, incorporating LFI along with electrical/electronic readers, to overcome the limitations associated with qualitative LFI analysis. The reader used for the quantitative analysis of LFI should ensure mobility for easy on-site diagnostics and inspections, be user-friendly in operation, and have a fast processing speed until the results are obtained. Due to these requirements, smartphones are increasingly utilized as readers in quantitative analysis of LFI. Among the various components constituting a smartphone, high-performance cameras can serve as sensors converting visual signals into electrical signals. With powerful processing units, large storage capacity, and network capabilities for transmitting analysis results, smartphones are also utilized as interfaces for quantitative analysis. Absolutely, the widespread global use of smartphones is a key advantage, leading to their utilization as diagnostic devices for acquiring, analyzing, storing, and transmitting assay test results. This paper summarizes research cases where smartphones are utilized as readers for quantitative LFI systems used in confirming contamination in food or the environment, detecting drugs, and diagnosing diseases in humans or animals. The systems are classified based on the types of label particles used in the assay, and efforts to improve the quantitative analysis performance for each are examined. Cases where smartphones were used as LFI readers for the diagnosis of the 2019 Coronavirus Disease (COVID-19), which has recently caused significant global damage, have also been investigated.

Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5′-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at −80 °C in PBS or a protein storage buffer for 3–15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.