Flow Cytometry (FCM) / FACS Antibody with Excellent Performance in Sensitivity

Antibody Affinity

Polyclonal antibodies from hyper immunized animals not only differ with regard to which epitopes they recognize on a specific antigen, but also in their affinities for the different epitopes of the antigen. The intrinsic affinity of an antibody, or the strength of antibody and antigen interaction, resides in the hyper-variable (HV) region of the antibody molecule. This is the region, or sequence of amino acids, of the molecule that determines specificity. However, to say that the greater the specificity, the stronger the affinity, is probably an oversimplification. Covalent binding between antibody and antigen does not occur. The forces that hold the antibody and antigen together include ionic interactions, hydrogen bonding and van der Waals forces. These are the major contributors to the intrinsic affinity between the antibody and the antigen.

The association constant (Ka) describes the strength of the binding between an antibody and its antigenic determinant. It is a measure of the antibody's affinity. The higher the affinity of the antibody, the lower the concentration of free antigen needed for the available binding sites of the antibody to become saturated (reach equilibrium). The Ka for antibody and antigen binding can range from 103 to 1010 liters per mole, which is the reciprocal of concentration (moles per liter).

Just as the quantity (titer) of an antibody increases with time during immunization, so does its quality (affinity). This has been called "affinity maturation". Lower doses of immunogen increase the rate of affinity maturation but result in lower titers of antibody, and vice versa.

The time required for the antibody to reach equilibrium or saturation with the cellular antigen is an important factor in determining optimum staining for flow cytometric analysis, sometimes referred to as functional affinity. If equal aliquots of two antibodies (or antisera) of identical titer are incubated for increasing periods of time with the cellular antigen, the antibody that reaches a plateau of maximum staining intensity first is of higher functional affinity. The term "avidity" is synonymous with functional affinity and has also been used to denote the strength of the binding between antibody and its antigen. Frequently, the term avidity has also been used to describe the sum total of all intrinsic affinities found in a polyclonal antibody population. 

Because antigen-antibody reactions are reversible, the simple immune complexes formed on the cell may dissociate during the washing cycles used in flow cytometry. Hydrophobicity, the ability of water to force hydrophobic groups of the complex together, appears to have a stabilizing effect on the antibody-antigen complex. Therefore, the antibody-antigen complex can be influenced by changes in salt concentrations, pH and/or temperature. The ease and degree of dissociation vary from antibody to antibody, and low salt concentrations as well as low temperatures will reduce the likelihood of weak staining due to dissociation of an already formed immune complex. Thus, high affinity antibodies are desirable and have the advantage that during washing, dissociation is less likely to occur than with low affinity antibodies. As mentioned before, a polyclonal population of antibodies contains a more or less continuous spectrum of low to high affinities against several epitopes on a given antigen.

Because antigen-antibody reactions are reversible, the simple immune complexes formed on the cell may dissociate during the washing cycles used in flow cytometry. Hydrophobicity, the ability of water to force hydrophobic groups of the complex together, appears to have a stabilizing effect on the antibody-antigen complex. Therefore, the antibody-antigen complex can be influenced by changes in salt concentrations, pH and/or temperature. The ease and degree of dissociation vary from antibody to antibody, and low salt concentrations as well as low temperatures will reduce the likelihood of weak staining due to dissociation of an already formed immune complex. Thus, high affinity antibodies are desirable and have the advantage that during washing, dissociation is less likely to occur than with low affinity antibodies. As mentioned before, a polyclonal population of antibodies contains a more or less continuous spectrum of low to high affinities against several epitopes on a given antigen.

For example,

  Recommended Concentration 1:10 1:100  
SBI Human CD14-MM06H Antigen: Human CD14
Antibody: CD14 Antibody (FITC), Mouse MAb (10073-MM06-F)
Cell: Human whole blood monocytes
Flow Cyt: Use 10µl for 10^6 cells
Instrument: BD FACSCalibur
    Monocytes    

Economic Antibody Products

The antibody products in Sino Biological Inc. have high affinity and sensitivity performance. They could be effective in very high dilution to recommended concentration in your experiments. Therefore, it's very economic for users to choose our antibody products.

Flow Cytometry / FACS Background

Flow cytometry is a method to evaluate cell membrane proteins and intracellular proteins as well as peptides and DNA. The principle behind FACS is an antigen-antibody reaction, with the antibodies being fluorescently labelled. There are three fluorescent proteins (R-PE, APC, and PerCP) conjugated to antibodies. Flow cytometry quantification is carried out with intercalating color labels (without the antibody). Flow cytometry antibodies are widely used in cell counting, cell sorting, biomarker detection and protein engineering.