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PAb Anti Human IgG-XL665 HTRF®

XL665-labeled anti-human IgG antibody for capturing human IgG-tagged proteins in protein/protein interaction assays.
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  • No-wash No-wash
  • Ease-of-use Ease-of-use
  • High affinity High affinity
XL665-labeled anti-human IgG antibody for capturing human IgG-tagged proteins in protein/protein interaction assays.
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Overview

IgGs from goat anti-Human immunoserum were immunopurified and labeled with XL665. PAb Anti Human IgG-XL665 may be used to detect the binding of unlabeled human specific antibody on a target molecule or on Ig fusion proteins.

This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, protein/small molecule, receptor/ligand.

HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.

Benefits

  • LARGE COMPLEXES DETECTION
  • BATCH-TO-BATCH REPRODUCIBILITY
  • LOW TO HIGH KD DETECTION

Assay principle

In an HTRF interaction assay, one partner is labeled (directly or indirectly) with the donor, and the other with the acceptor (again, directly or indirectly). The intensity of the signal is proportional to the binding of the 2 partners. In the example shown here: PAb Anti Human IgG-XL665 binds to the Human Fc fused tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *partner B can also be biotinylated, tagged, Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with donor for the detection.
assay principle of detection assay using HTRF PAb Anti Human IgG-XL665

Assay protocol

The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a Human Fc fused-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add PAb Anti Human IgG-XL665 (5 µL) and anti-partner B labeled with donor (5 µL), incubate and read. *partner B can also be biotinylated, tagged, Fc fused or directly labeled. In these cases, use the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin) labeled with donor for the detection.
assay protocol of detection assay using HTRF PAb Anti Human IgG-XL665

Build HTRF interaction assays for your specific application

Reagents are sold by the number of tests (20 µL reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20 ng of antibody per well. The amount of active moiety per vial is also provided (as well as the number of tracers per vial - see product description sheet). The active moiety is defined as the active part of a conjugate (e.g. antibody).

How do the number of tests relate to active moiety?

The average conjugate quantity per well reflects overall biological material content. Using the active moiety amount is generally preferred to the quantity of total conjugate. For Cryptate and d2 conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. This is not the case for XL665 labeled entities for which the quantity of total conjugate will vary depending on the final molar ratio of the XL665 conjugate, however, the amount of active moiety, provided by Cisbio, is constant and based on the number of tests ordered.
description of the active moiety in an HTRF conjugate
Species Antibody Species Specificity Active Moiety 5,000 tests Europium conjugates XL665 conjugates Human immunoglobulins PAb Goat Human Fc Igs 10 µg 100 µg

Recommended quantities of Cryptate and XL665 conjugates

Cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a cryptate concentration of 1 to 5nM is appropriate, and will generate 20,000 to 80,000 cps at 620 nm depending on the HTRF compatible reader used. The XL665 conjugate must match its assay counterpart as closely as possible in order for the maximum number of biomolecules to be tagged with the XL665 acceptor. Thus, to detect a tagged molecule at an assay concentration of 20nM, the concentration of anti-Tag-XL665 should be equimolar or higher.

CD28/CD86 binding assay

CD28 provides a major costimulatory signal for CD4-positive T cells. Ligation with its natural ligands CD80 (B7-1) and CD86 (B7-2) leads to signals during activation that are required for the production of interleukin-2. HTRF reagents were used to develop an assay for antagonists of CD28 and CD86 binding. Both molecules were respectively tagged with human and rat IgG Fc fragments, and detected using the XL665-labled anti-human antibody. CD86 was expressed as a fusion protein with a rat Ig domain which is recognized by a biotinylated antibody, followed by binding of streptavidin-Eu cryptate. Mellor et al. Development of a CD28/CD86 (B7-2) binding assay for high throughput screening by homogenous time-resolved fluorescence. J Biomol Screening. 1998;3: 91-99.
CD28/CD86 binding assay using HTRF PAb Anti Human IgG-XL665 for detection

Applications of HTRF and Tag-lite-lite Assays for HTP Antibody Screening

In collaboration with Bristol Myers Squibb - Scientific Presentations

The use of HTRF in biologics discovery

In collaboration with MedImmune - Scientific Presentations

HTRF protein-protein interaction reagents

Benefit from unlimited flexibility for your assay development - Flyers

Addressing the interactome with protein-protein assays

Cover all PPIs with one approach - Brochures

HTRF -A Beneficial Tool for Lead Optimization of Biotherapeutics

In collaboration with Boehringer Ingelheim - Posters

Product Insert Anti (h) IgG-XL / 61HFCXLF-61HFCXLA-61HFCXLB

61HFCXLF-61HFCXLA-61HFCXLB - Product Insert

HTRF PPI your dream assay served on a plate

Sandwiches aren't just for eating - Infographics

Best practices for pharmacological characterization of PPI inhibitors

Easy pharmacological characterization of PPI modulators. - Technical Notes

HTRF assays handle low- to high affinity protein-protein interactions

Deciphering low- and high affinity interactions - Application Notes

Nuclear receptor ligand identification with HTRF

Monitoring nuclear receptor binding with HTRF assays - Application Notes

HTRF addresses large protein-protein interaction complexes

Challenge large complexes with HTRF assays - Application Notes

A brief history of Protein-Protein Interactions

How well do you know PPI? - Infographics

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Virology research solutions using HTRF Protein-Protein Interaction assays

See how peer researchers challenge the viral life cycle with PPI assays - Application Notes

Solutions for CAR-T research

Advance your CAR-T cell research - Flyers

Batch Information Anti (h) IgG-XL / 20240715

61HFCXLA Batch 13D - Batch Information

Batch Information Anti (h) IgG-XL / 20250209

61HFCXLA Batch 13E - Batch Information

Batch Information Anti (h) IgG-XL / 20230519

61HFCXLA Batch 13E - Batch Information

Batch Information Anti (h) IgG-XL / 20230519

61HFCXLB Batch 13A - Batch Information

Batch Information Anti (h) IgG-XL / 20230519

61HFCXLF Batch 13C - Batch Information

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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