抗体功能实验方案 | 抗体分析 | 免疫学

Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement

硫醇反应探针流式细胞术检测食蟹猴疟原虫生长抑制

JO Jessica Jie Ying Ong

BR Bruce Russell

JH Jin-Hee Han

0 Q&A

4266 Views

Sep 5, 2021

The relapsing malaria species, Plasmodium vivax, is the most widely distributed and difficult-to-treat cause of human malaria. The merozoites of P. vivax preferentially invade ephemeral human CD71⁺reticulocytes (nascent reticulocytes), thereby limiting the development of a robust continuous culture in vitro. Fortunately, P. vivax’s sister species, P. cynomolgi Berok, can be cultured continuously, providing the ability to screen novel therapeutics drug and vaccine candidates in a reliable and high-throughput manner. Based on well-established growth inhibition activity (GIA) assays against P. falciparum and P. knowlesi, this protocol adopts the current flow cytometry assay methodology and investigates P. vivax inhibitory antibodies using the P. cynomolgi Berok invasion model based on the thiol-reactivity and DNA abundance of viable parasites in macaque erythrocytes. Established GIA assays screen antibodies at either a single concentration or high/low dose concentrations to provide quick insights for prioritizing potential antibodies capable of specifically interrupting parasite ligand and host receptor binding with minimal concentrations. Hence, this protocol expands on the existing GIA assay by using serially diluted antibodies and generating a dose-response curve to better quantify the inhibitory efficacy amongst selected vaccine candidates.

An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration

一种基于图像的动态高通量分析黏附细胞的迁移

MS Meng Sun

BR Bence Rethi

Ak Akilan Krishnamurthy

VJ Vijay Joshua

HW Heidi Wähämaa

SC Sergiu-Bogdan Catrina

AC Anca Catrina

0 Q&A

6668 Views

Mar 20, 2021

In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte^® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuCyte^® Cell Migration Analysis Software. Thus, our protocol allows a time-lapse analysis of treatment effects on cell migration in a highly reliable, reproducible and re-analyzable manner.

Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi

人畜共患病疟原虫——诺氏疟原虫增殖和生长抑制活性测定

FM Franziska Mohring

TR Thomas A. Rawlinson

SD Simon J. Draper

RM Robert W. Moon

0 Q&A

6857 Views

Sep 5, 2020

Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion or growth capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition activity (GIA) assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species (Pacheco et al., 2018) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions.

A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material

类风湿性关节炎患者单个B细胞中瓜氨酸特异单克隆抗体的改进鉴定方案

kA Khaled Amara

LI Lena Israelsson

RS Ragnhild Stålesen

PS Peter Sahlström

JS Johanna Steen

VM Vivianne Malmström

CG Caroline Grönwall

0 Q&A

7944 Views

Sep 5, 2019

We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs can be used for further studies including reactivity towards candidate antigens and functionality both in vitro and in vivo. This protocol can be used to generate antigen-specific mAbs from B cells derived from different tissues and compartments, including peripheral blood, synovial fluid, digested biopsies, bone marrow aspirations, and bronchoalveolar lavage fluid. Notably, although examples are given on how to identify citrulline-specific autoantibodies the general methods can also be applied to other reactivities.

Nab Escaping AAV Mutants Isolated from Mouse Muscles

从小鼠肌肉中分离Nab逃脱AAV突变体

ZC Zheng Chai

RS R. Jude Samulski

CL Chengwen Li

0 Q&A

8576 Views

May 5, 2018

Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle.

Micro Neutralization (MN) Assay of Influenza Viruses with Monoclonal Antibodies

单克隆抗体介导的流感病毒微中和(MN)测定试验

YW Ying Wu

MC MyungSam Cho

DS David Shore

MS Manki Song

JC JungAh Choi

TJ Tao Jiang [...]

RD Ruben O. Donis

+ 18 作者

0 Q&A

16816 Views

Jun 5, 2016

The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to neutralize virus infectivity. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses. This protocol is to measure the ability of human monoclonal antibody to neutralize influenza virus by microneutralization assay.

Hemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies

单克隆抗体介导的流感病毒血细胞凝集抑制(HI)试验

YW Ying Wu

MC MyungSam Cho

DS David Shore

MS Manki Song

JC JungAh Choi

TJ Tao Jiang [...]

RD Ruben O. Donis

+ 18 作者

0 Q&A

36272 Views

Jun 5, 2016

Heamagglutination is inhibited when antibodies are present because antibodies to the influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. Human monoclonal antibodies generated from single human B cells were tested to characterize their ability to inhibit hemagglutination against virus A/California/07/2009 (H1N1) and A/Brisbane/10/2007 (H3N2).

Cell-based Assays to Monitor AID Activity

基于细胞的监测AID活性的方法

0 Q&A

11159 Views

Feb 5, 2016

The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing AID-deficient primary mouse B cells. The combination of the three methods, accompanied by the necessary analysis of AID subcellular localization and protein expression levels and stability, as controls, allows detailed structure-function interrogation of AID.

Binding Affinity Measurement of Antibodies from Crude Hybridoma Samples by SPR

表面等离子共振法测定原初杂交瘤样本中抗体与抗原的结合亲和力

0 Q&A

18043 Views

Nov 5, 2014

Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the resulting complex was stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration (Pope et al., 2009). Then the interaction with the protein was monitored at several concentrations and its affinity towards the immobilized antibodies was determined with the corresponding K_D obtained from classical kinetics analysis. This set-up avoids the avidity effects of the bivalent antibodies, allows the use of non-purified analytes with unknown concentrations and the specific capture of the antibodies in a similar stable covalent-orientated manner.

Detection of Tumor Cell Surface-reactive Antibodies

肿瘤细胞膜结合性抗体的检测

BA Brian M. Andersen

Michelle R. Goulart

MO Michael R. Olin

G. Elizabeth Pluhar

0 Q&A

9468 Views

May 5, 2014

Vaccine-based immunotherapy is being used to treat dogs with primary brain tumors. The vaccines are composed of a lysate of autologous tumor cells, which stimulate an immune response producing tumor specific antibodies that are capable of inducing antibody-dependent cell-mediated cytotoxicity to allogeneic, as well as autologous, tumor cells. This protocol will describe the tumor cell serum antibody-binding assay to measure the tumor-reactive IgG antibody response. Key features of this assay are that it is performed with sera collected from the canine patient prior to and following vaccination as the source of antibodies and canine brain tumor cells used as the target cells.