Isotype and Subclass Specificity of IgASAP™

Performance Activity

Highly specific digestion of IgA, with no activity on IgG or IgM, enabling targeted middle-level characterization of IgA1 and IgA2 subclasses.

To establish the specificity of the IgASAP enzymes, all four subclasses of human IgG along with IgM and IgA from different sources have been incubated with IgASAP Sub1 Lyophilized and IgASAP Sub1+2 Lyophilized, before analysis with reducing SDS-PAGE.

All IgA substrates were specifically hydrolyzed, with high specificity of IgASAP Sub1 towards IgA1 and the high specificity of IgASAP Sub1+2 towards both IgA1 and IgA2m1 subclasses. No digestion was observed for any of the IgG subclasses or IgM.

IgASAP specifically digests IgA with no digestion of IgG or IgM

IgA1 constitutes 80-90% of total IgA in human serum while IgA2 makes up for the remaining percentage. This corresponds well with the observed digestion levels as the majority of IgA was digested with both IgASAP Sub1 and IgASAP Sub1+2. Some undigested IgA2 of the pooled serum IgA is expected from the IgASAP Sub1+2 digestion-resistant allelic forms A2m2 and A2m3.

Digestion-resistant forms of IgA2 are not digested by IgASAP Sub1+2

IgA1 in mucosal secretions (e.g. colostrum) only accounts for approximately 40% of total IgA which can be observed following digestion with IgASAP Sub1. IgA purified from colostrum is clearly from donors carrying A2m1 genetic variant as all IgA2 is completely hydrolyzed by IgASAP Sub1+2. The slightly larger IgA1 Fc fragments from IgASAP Sub1+2 digestion as compared to IgASAP Sub1 digestion results from the six amino acids difference in digestion site.

IgA2 subclass can be determined by digestion using IgASAP Sub1+2

Isotype and subclass specificity of IgASAP Sub1 and IgASAP Sub1+2. The activity of the enzymes was assessed using human antibodies of various isotypes and subclasses. a) IgA substrates were digested as follows: IgA1 components were digested by both IgASAP Sub1 and IgASAP Sub1+2, IgA2m1 components were digested by IgASAP Sub1+2 only, and other IgA2 components (IgA2m2, IgA2m3) were not digested by either enzyme. b) The IgASAP enzymes did not show activity on either IgG or IgM components. All samples were treated at 1U/µg and incubated with IgASAP Sub1 Lyophilized for 1 hour at 37°C, or with IgASAP Sub1+2 Lyophilized overnight at 37°C, before analysis on reducing SDS-PAGE.

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