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Antibodies are critical tools in protein and molecular detection systems. Although intact antibodies (IgG or IgM) are ideal for most immunoassay applications, the performances of certain procedures can be enhanced by using antibody fragments such as Fab, F(ab')2, or IgG (half antibody).
Bio-Synthesis’ antibody fragmentation services include: dialysis, digestion, concentration, purification and verification. Fragmentation can be achieved by papain or pepsin digestion, as well as IdeS Streptococcus pyogenes, which cleave human immunoglobulin (Ig)G antibodies with a unique degree of specificity, to produce half antibody (r IgG), F(ab) or F(ab')2 fragments. Both F(ab) and F(ab')2 fragments can be purified. Purity will be assessed by SDS-PAGE and Western Blot analysis. Our antibody fragmentation services include:
SDS-PAGE gel electrophoresis (optional): Analysis of antibody fragments by SDS PAGE gel electrophoresis
(Includes 2 mini gels with 1 standard each and up to 8 samples per gel and a copy of COA)
To obtain further information, contact us. Our technical staff is also available to assist you in determining the type of production system that best meets your needs
Fragmentation of IgG and IgM from a variety of species
Papain is a nonspecific, thiol-endopeptidase that has a sulfhydryl group in the active site, which must be in the reduced form for activity. Papain has been shown to produce heterogeneous fragments from IgM. Oligosaccharides in the hinge region of IgM interfere with papain digestion, causing a cleavage shift of 3-5 amino acids in either direction.
Pepsin is a nonspecific endopeptidase that is active only at an acid pH and it is irreversibly denatured at neutral or alkaline pH. It is possible to produce F(ab')2, Fab and Fv fragments using pepsin to digest IgM.
Trypsin is a serine protease that reacts optimally at pH 8.0. In general, increasing the enzyme/substrate ratio and/or temperature will increase the rate of digestion. Trypsin can generate F(ab')2, Fab, "IgG" - type and Fc5µ fragments from IgM. Trypsin digestion of several species of IgM was studied using trypsin with and without urea pretreatment. 26 Urea alters the susceptibility of the domains to digestion and produces different fragments than those digested in aqueous buffer. Many other procedures have been developed to digest IgM using trypsin.
Mild reduction can be achieved using 2-mercaptoethylamine (2-MEA), Hydrochloride (HCl). Reduction will vary among IgM species, but an "IgG" - type and/or reduced IgG ("rIgG") should be formed in varied proportions, depending upon reduction time and/or temperature fragmentation of mouse IgM also produces an inverted "IgG"-type fragment.