antigen&antibody selection

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Antigen:
  • The foreign target that can be recognized by antibodies is called an antigen. Antigens are usually proteins or polysaccharides. Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides. The distinct molecular surface features of an antigen capable of being bound by an antibody are epitopes, also called antigenic determinants. Numbers and properties of antigenic determinants define the antigenic specificity. Antibodies can bind to the antigenic determinants and trigger immune response which recruits immune cells.
  • To prove a protein-protein interaction, or to detect an unknown protein that interacts with your interested one, high quality protein that a natural sample (e.g. cell lysate, tissue extract, blood) can’t or can’t sufficiently supply is needed.The conformations of proteins (antigen) used in IP should be close to the natural ones. There are primarily 3 ways to obtain proteins with a conformation close to the natural: naturally extracting, prokaryotic expressed recombinant proteins and eukaryotic expressed recombinant proteins. Their advantages and disadvantages are as following:

 

Advantages

Disadvantages

Natural extracts

Natural conformation

Low antigen concentration

Hard to purify

Small amount of sample

Unknown ingredients

Prokaryotic expressed recombinant proteins

Low cost

Simple procedure

Large amount

Conformational difference

Eukaryotic expressed recombinant proteins

Close to natural conformation

Simple procedure

Large amount

 

Here we describe some kinds of recombinant proteins for immunoprecipitation and you might find your interests below (Divided by functions):

  • Cytokines

Cytokines are produced by many different cell types and function as intercellular chemical messengers. Cytokines help control and mediate haematopoiesis as well as immune and inflammatory processes. They have been implicated in many disease states including cancer, cardiovascular disease and allergic responses.

  • Chemokines

Like cytokines, chemokines are chemical messengers involved in the inflammatory response they act by sending out signals to attract white blood cells to the site of disease or injury. Chemokines are also involved in many disease states including infectious diseases and cancer.

  • Tumour Markers

Tumour Markers indicate a malignant process is present they are often used to screen for cancer or monitor the course of disease.

  • Adhesion Molecules

Adhesion molecules are complex membrane proteins that can be grouped into four major families: the selectins, the immunoglobulin (Ig) superfamily, the integrins and the cadherins. Adhesion molecules have been implicated in a wide range of physiological processes and can be found in the cell membrane or as soluble forms in circulation. Altered levels of these proteins have been found in pathological states including cardiovascular disease, stroke, cancer, diabetes and many others.

  • Proteases

Proteases occur naturally in all organisms catalysing the cleavage of peptide bonds in proteins. These enzymes are involved in a multitude of physiological reactions from simple digestion of proteins in food to highly-regulated cascades. Proteases present in the blood or serum play an important role in blood-clotting as well as lysis of the clots, and the correct action of the immune system.

Antibody:

  • For antibodies, due to a high affinity requirement, polyclonal antibodies seem to be the first choice because they can bind multiple antigenic determinant of an antigen. However, PAb can lead to non-specific binding and thus a low specificity. Monoclonal antibodies have high specificity but a relatively low affinity.
  • A simple summary of binding affinity influenced by antibody's originals:
  • For advantages and disadvantages of PAbs and MAbs:

 


Polyclonal Antibody

Monoclonal Antibody

Signal intensity of antigen-antibody reaction

Perfect

Perfect or weak (determined by its affinity)

Specificity

Good, non-specific binding sometimes

Perfect, cross-reactivity sometimes

Advantages

High affinity(due to binding of antibody to multiple antigenic determinant of target protein)

High specificity and unlimited supply

Disadvantages

Non-specific binding is hard to eliminate

High affinity antibodies need to be filtered;antigenic determinant could be sheltered by interacted proteins

IP effect

Perfect (due to high affinity)

Perfect or weak (determined by its affinity)

Co-IP effect

Good, non-specific binding leads to false positives sometimes

Perfect or weak (determined by its affinity and whether its antigenic determinant is sheltered)

ChIP effect

Good, non-specific binding leads to false positives sometimes

Perfect or weak (determined by its affinity and whether its antigenic determinant is sheltered or destructed by crosslinking experiments)