Subclass-specific Digestion of IgA in Serum using IgaSAP™

Application

Subclass selective digestion of IgA in complex samples like serum, enabling targeted analysis of IgA1 and IgA2m1 without off-target cleavage.

IgA constitutes around 15% of the total amount of immunoglobulins in normal human serum. The ability to selectively digest IgA1 and IgA2m1 can be of great value in research and diagnostics as well as in bioprocessing and therapeutic applications.

The specificity of the IgASAP enzymes make them excellent tools for such isotype-specific digestion of IgA in human serum samples or other complex sample matrices. Here, human serum was incubated with IgASAP Sub1 Lyophilized for 1 hour at 37°C and IgASAP Sub1+2 Lyophilized overnight at 37°C, before analysis with reducing SDS-PAGE and western blot.

IgASAP enzymes are excellent tools for IgA digestion in serum

IgASAP Sub1 specifically digests IgA1 in serum while leaving IgA2 and the other components of the serum intact. IgASAP Sub1+2 elicits a higher degree of digestion in the serum samples as the IgA2m1 fraction is also hydrolyzed. Remaining intact IgA2 in the pooled human serum after IgASAP Sub1+2 treatment is from the digestion-resistant allelic forms IgA2m2 and IgA2m3.

Subclass specific digestion of IgA using IgASAP Sub1 and IgASAP Sub1+2

This shows the high subclass specificity of the IgASAP enzymes and demonstrates the possibility to use the products in complex biological samples such as serum and plasma without the risk of off-target digestion.

Efficient digestion in complex matrices without the risk of off-target digestion

IgA-specific activity of IgASAP Sub1 and IgASAP Sub1+2 in human serum. Human serum was incubated with IgASAP Sub1 for 1 hour at 37°C or IgASAP Sub1+2 overnight at 37°C. The digested samples and control samples of human IgA1 (myeloma) and IgA2m1 (recombinant) subclasses were separated on three reducing SDS-PAGE gels. One of the gels was stained with Coomassie blue (left), and the other two gels were transferred to membranes for Western blotting. After blocking of the membranes in a casein solution, alkaline phosphatase conjugated α-human IgA1 Fc antibody (middle) or alkaline phosphatase conjugated α-human IgA2 Fc antibody (right), respectively, was added to the membranes and incubated for 1 hour at room temperature. The membranes were thoroughly washed before addition of the chromogenic substrate BCIP/NBT. IgA1 in serum was digested by both IgASAP Sub1 and IgASAP Sub1+2, while IgA2m1 was specifically digested by IgASAP Sub1+2. The remaining IgA2 in the pooled serum following IgASAP Sub1+2 digestion is from the digestion-resistant IgA2m2 and IgA2m3 forms. The low anti-IgA1 response of the serum control sample in the middle membrane is likely due to interference of other proteins.

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