CLAMP method – A New Highly Sensitive Detection Method for Cell Surface Antigens

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– 10 – 100 times more sensitive than the conventional method.
– Applicable to various samples: live cells, PFA fixed cells, FFPE tissue
– High specificity staining: Cells must express particular antigens
– Stained sample storage: After staining, samples can be stored for 1 week in refrigerator.

Content: 10 µL x 1
Storage Condition: Store at -20oC
Shipping Condition: Ambient Temperature

The general number of usable assays per 1 set:
Floating Cells:20 tests (2-10 x 105 cells/test)
Adherent Cells:60 tests (96 well microplate)
6 tests (35 mm dish)

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DescriptionVideoReferences

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Product Description

Cell surface antigens that are specifically expressed on cancer and immune cells have been actively studied for early detection and treatment of cancer. However, many cell surface proteins have low expression levels, and most of them are difficult to detect by conventional techniques.

A method using a fluorescence-labeled antibody is widely used for a specific detection of cell surface proteins (fluorescent immunostaining method). However, this method cannot be applied for detection of low expressed surface proteins due to its low sensitivity.

Thus, we have developed a highly sensitive method (quinone methide-based Catalyzed Labeling for Signal Amplification: CLAMP) to detect surface proteins on living cells.

 

The CLAMP method can fluorescently stain cells expressing a specific antigen selectively and sensitively.

This technique involves a primary antibody designed for a particular cell surface protein, a β-galactosidase-labeled secondary antibody, and MUGF – a fluorescent substrate for β-galactosidase.

This fluorescent substrate, MUGF, is actually non-fluorescent and non-permeable to the cell membrane in its beginning state. However, if the primary antibody binds to an antigen-presenting cell, the conjugated β-galactosidase will convert MUGF near the cell surface into a compound with a quinone methide structure. This compound is then permeable to the cell membrane; it will enter the cell and react with thiol and amino groups inside, forming covalent bonds and emitting fluorescence.

Large amounts of MUGF are converted and subsequently accumulate in the cell, creating an intense signal. In this way, this mechanism is very highly sensitive, and will produce strong fluorescence, even with very low levels of antigen expression.

For more information on CLAMP method and examples, please refer to the publication below:
Noguchi, K. et al., “β-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens”, Bioconjugate Chem., 2020, 31(7), 1740–1744.


Features of CLAMP method

  1. The CLAMP method is 10 – 100 times more sensitive than the conventional method.
  2. Applicable to various samples:
    Surface antigenIntracellular antigen
    Live cells

    PFA fixed cells

    Frozen/FFPE tissue

    *FFPE: Formalin-Fixed Paraffin-Embedded
  3. High specificity staining: cells must express particular antigens
  4. Stained sample storage: After staining, samples can be stored for 1 week in refrigerator.*Live cells: please fix stained live cells with PFA before storage.

Procedure

 


Application to Live Cells

1. Fluorescent Staining of A549 Cells (Surface Antigen: CD44)

Fluorescent microscope: BZ-X700 (Keyence)
DAPI filter set: Ex 360/40 nm, Em 460/50 nm

The CLAMP method is more sensitive than the conventional method.


2. Fluorescence Image Comparison

Cell type: HeLa cell
Antigen: CD44

The CLAMP method does not provide information on antigen localization, because the cells are stained uniformly.


3. Fluorescent Staining of PD-L1-Expressing Cells (HepG2 cells)

 

“Ab” = Antibody
Fluorescent microscope: BZ-X700 (Keyence)
DAPI filter set: Ex 360/40 nm, Em 460/50 nm

 

PD-L1-expressing cells are more sensitively stained by the CLAMP method.


4. Fluorescent Staining of HepG2 Cells (Surface Antigen: PD-L1)

PD-L1-expressing cells were selectively stained by the CLAMP method.


Application to PFA-Fixed Cells and Tissue
1. Application to PFA-Fixed Cells and Tissue


CLAMP method
1’ Ab: anti-CD44
2’ Ab: βGal-labeled
dye: MUGF

CLAMP method is applicable to PFA-fixed cells.

2. Application for Frozen Tissue Section (Mouse liver) (Surface Antigen: GLUT1)


CLAMP method
1’ Ab: anti-GLUT1
2’ Ab: βGal-labeled
dye: MUGF

Frozen tissue are applicable for CLAMP method.


Application to FFPE Tissue Section (Human Small Intestine)

*Data provided by Dr. M. Hirata, Department of diagnostic pathology, Graduate School of Medicine, University of Kyoto
*TSA: Tyramide Signal Amplification

CLAMP can be : applied to FFPE tissue sections used to multiplex with other methods like TSA


Further Improvements to Flow Cytometry

MUGF3: A 405nm-Excitable Dye for FCM

Excitation spectra of the dyes
(emission wavelength460 nm)

1. Performance Comparison Between MUGF and MUGF3 (Surface Antigen: CD44)

 

Sensitivity:
Flow cytometry: MUGF3 showed 10 times higher sensitivity than MUGF in detection of CD44
Fluorescence imaging: MUGF3 and MUGF have similar sensitivity.

2. Detection of PD-1 (Low-Expression Antigen) via CLAMP Method

MUGF3-CLAMP can even detect an antigen with low expression, like PD-1.


Future plans

  • Application to clinical samples
  • Development of fluorescent dyes with different colors
  • Probes using other enzyme substrates

If you would like to try the above CLAMP method, please click here and answer the short survey!

FREE SAMPLE

*Free sample may be used only for your research of high-sensitivity fluorescence detection of cells, A material transfer agreement (MTA) must be concluded to evaluate CLAMP method.

COVID-19 Announcement

Due to rising concerns regarding the COVID-19 (Coronavirus 2019) outbreak, Dojindo Molecular Technologies, Inc will be taking the necessary steps to prioritize public health and safety.

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