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pHAb Reactive Dyes

Measure Antibody Internalization in Real Time

  • Compatible with 96-well, plate-based assay
  • High signal-to-background ratios
  • On-Bead conjugation

Choose a conjugation option

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Catalog number selected: G9841

$ 159.00
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pHAb Reactive Dyes
Amine Reactive Dye/1 × 250µg
$ 159.00
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Screen for Antibody Internalization

pHAb Dyes are pH sensor dyes that have very low fluorescence at pH > 7 and a dramatic increase in fluorescence as the pH of the solution becomes acidic. pHAb Dyes have excitation maxima (Ex) at 532nm and emission maxima (Em) at 560nm.

pHAb Reactive Dyes are designed specifically for antibody labeling and can be used for high-throughput antibody internalization screening assays.

pHAB-Dyes-overview
47960358-Li et al Intl Immunopharm. paper on antiCD19 mAb

Development of a Novel Anti-CD19 Antibody-Drug Conjugate

B-cell lymphoma is a cancer in need of a therapy. Patients with B-cell lymphoma often respond to first line therapies, but tumor recurrence rate is high and prognosis often poor. Novel approaches to B-cell lymphoma treatment are needed. 

CD19 is a B-cell transmembrane glycoprotein that plays a key role in B-cell malignancies and autoimmune diseases and is a target of interest for new B-cell lymphoma therapies. Of the anti-CD19 therapies in development and/or clinical trials, antibody-drug conjugates (ADCs) are some of the most promising, as they are both specific and toxic. In the ADC mechanism of action, the antibody binds CD19 on the B-cell surface stimulating internalization of the antibody-receptor complex. Upon internalization toxin is released, killing the tumor cell. 

In this work, Li et al. created a novel CD19-binding monoclonal antibody, RC58 . They used pHAb Amine Reactive dyes to analyze internalization of the RC58 antibody.

Details of their work can be found in this 2018 International Immunopharmacology paper: "Development of novel anti-CD19 antibody-drug conjugates for B-cell lymphoma treatment".

Read the paper

pHAb Amine Reactive Dye

pHAb Reactive Dyes are available in two reactive forms suitable for antibody conjugation: pHAb Amine Reactive Dye and pHAb Thiol Reactive Dye.

pHAb Amine Reactive Dye has a succinimidyl ester group that reacts with primary amines available on the lysine amino acids on the antibodies.

Chemical structure of the phAb Reactive Dye
For labeling amines of lysine residues on antibodies.

pHAb Thiol Reactive Dye

pHAb Thiol Reactive Dye has a maleimide group that reacts with thiols. This maleimide group is conjugated to the antibody after the cysteine disulfide bonds in the hinge region of the antibody are reduced to thiols through the use of a reducing agent, such as DTT or TCEP. pHAb Reactive Dyes retain their fluorescent response to decreases in pH even after conjugation to antibody.


Chemical Structure of the pHAb Reactive Dye
For labeling thiols from reduced cysteines in the antibody hinge region.

Monitor Antibody Internalization

A key feature of pHAb Dyes is that they have two sulfonate groups per dye, which increases solubility and reduces the aggregation often seen with other non-sulfonated dyes. pHAb Dyes maintain their fluorescence response to pH change even after antibody conjugation.

Even though antibody conjugation is the key application, any protein containing primary amines on lysine amino acids or thiols on cysteine amino acids can be conjugated with pHAb Dyes.

 

pHAb Reactive Dyes exhibit flourescence upon internalization
Fluorescence is shown as a function of pH for trastuzumab labeled with pHAb Amine Reactive Dye or pHAb Thiol Reactive Dye compared to pHAb Amine Reactive Dye or pHAb Thiol Reactive Dye alone.

Two Options for Conjugation of pHAb Reactive Dyes to Antibodies

There are two workflow options for conjugation of pHAb Reactive Dyes to antibodies: traditional solution-based conjugation or on-bead conjugation. 

Traditional In-Solution Conjugation of Antibody

in-solution-conjugation-schematic
The traditional solution-based or in-solution chemistry workflow requires purified antibody at concentrations of 1.0–5.0mg/ml and several buffer exchange steps. 

On-Bead Conjugation of Antibody

on-bead-conjugation-schematic
On-bead conjugation uses magnetic protein A and protein G affinity beads to selectively capture antibodies from solution containing purified antibody or directly from biological samples expressing antibodies (i.e., cell media). Conjugation steps are then performed on antibodies captured on the beads, and pHAb-conjugated antibodies are subsequently eluted using low pH buffer, then quickly neutralized.

pHAb Reactive Dyes Retain Fluorescent Response to pH after Conjugation

pHAb Dye-conjugated antibodies can be used for monitoring receptor-mediated antibody internalization. When an antibody-pHAb Dye conjugate binds to its receptor on the cell membrane, it exhibits minimal fluorescence. However, upon receptor-mediated internalization, antibody-pHAb Dye conjugates traffick to the endosome and lysosomal vesicles where pH is acidic, causing the pHAb Dye to fluoresce. This fluorescence can be detected using various techniques, including cell imaging, flow cytometry and fluorescent plate-based readers with appropriate filters.

antibody internalization data
SKBR3, HER2-positive cells and MDA-MB231, HER2-negative cells were plated in a 96-well black, clear-bottom plate overnight. Trastuzumab and IgG (negative antibody control) were conjugated with pHAb Amine Reactive Dye or pHAb Thiol Reactive Dye using on-bead conjugation.

Specifications

You are viewing: G9841 Change Configuration

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pHAb Amine Reactive Dye

G984A 1 × 250μg

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

Patents and Disclaimers

Patent Pending.

Specifications

You are viewing: G9831 Change Configuration

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pHAb Thiol Reactive Dye

G983A 1 × 250μg

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

Patents and Disclaimers

Patent Pending.

Specifications

You are viewing: G9835 Change Configuration

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pHAb Thiol Reactive Dye

G983A 4 × 250μg

SDS

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

Patents and Disclaimers

Patent Pending.

Specifications

You are viewing: G9845 Change Configuration

What's in the box?

Item Part # Size

pHAb Amine Reactive Dye

G984A 4 × 250μg

SDS

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Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

Patents and Disclaimers

Patent Pending.

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