Humans and mice share five primary antibody (immunoglobulin) isotypes: IgM, IgD, IgG, IgA, and IgE. While they share identical foundational functions, they differ notably in structural subclasses, serum concentrations, and specific immunological functions
Human and mouse Immunoglobulin G (IgG) are structurally similar but differ significantly in their subclass classifications, receptor binding affinities, and functional profiles.
In both cases IgG is the prevalent subclass which accounts for approximately 70% of circulating antibodies but the distribution and functionality of subclasses (e.g. IgG1vs IgG2)) change a lot, which directly influences how preclinical research translates into human.
For example; while in human the IgG1 is the common subclass, highly effective at neutralizing viruses and toxins, triggering complement activation, and driving antibody-dependent cellular cytotoxicity (ADCC) and the igG4 does not activate the complement system and has limited effector functions. On the contrary in mouse the IgG1 has show functionality similar to human igG4 and the igG2a/igG2c is primary drivers of ADCC and complement activation, acting as the mouse equivalent to human IgG1.
Antibody isotyping is the process of determining the specific class (e.g., IgG, IgM, IgA, IgE, IgD) and subclass (e.g., IgG1, IgG2a, IgG2b, IgG3) of a monoclonal or polyclonal antibody based on its heavy and light chain structures.
Antinody isotyping is from long time a critical in-process validation step in the antibodyt generation required to:
- Select optimal hybridoma clones;
- Select the more appropriate downstream purification strategies;
- Choose the appropriate secondary reagents for immunoassays
More recently antibody isotyping become also a critical analitical step in Chinese Hamster Ovary (CHO) cell production iused to confirm that the recombinant antibody being produced has the correct heavy chain class (e.g., IgG1, IgG4) and light chain type (kappa or lambda ).
While often associated with characterizing hybridomas, in CHO production, a recombinant proces, isotyping is used to verify the construct design, check for proper assembly, and ensure isotype stability in stable cell line. However confirming the correct isotype (e.g., IgG1, IgG2, IgG4) is required as part of the initial structural characterization and identity testing during the drug approval process but it is not required for the routine, batch-by-batch analysis for lot release.
Several antibody isotyping kits, typically using lateral flow or ELISA, those are suggested toutilize well-characterized antibodies with negligible cross-reactivity, designed to detect specific heavy and light chain.
Recently I add the opportunity to perform some analisys using Pro-Detect human and Pro-Detect mouse kits (based on lateral flow approach) and i took the opportunity to verify their specifity by checking whether how the kits are able to distunguish a single antibody subjected to isotype switching from mouse IgG1 to human IgG1 and IgG4.
The protocola for both mouse and human kits are really simple, you need just to dilute the antibody to the suggested concentration range, transfer 150-200ul of diluted antibodies to the bottom of an 2mL eppendorf tube and insert the strip on the tube. Each kit proved to strips, a blue strip for the light chain typing and a red strips for the heavy chain typing. Wait 5-10 minutes for colored bands to appear and immediately evaluate the results.
How you can see in the following pictures:
Mouse isotyping kit show good specificity since it was able to identify the antibody produced in the mouse IgG1 form but not the same antibody in the Human IgG1 or Human IgG4.
Human isotyping kit show good specificity since it was able to identify the antibody produced in the Human IgG1 or Human IgG4 forn and not the one produced in the Mouse IgG1.
At this point I was curious to check if the Human Antibody isotyping kit may be affected by ADCC-modulating mutations that were previouly discussed in the ProteoCool Pills#31 and i tested the ability of the Pro-Detect Human kit to recognize the same clone carrying the LALA, LALAPG, LALANA mutation described to reduce or fully abrogate Fcγ binding affinity and downstream effector cell activation.
The kit perform very well with all the mutations tested. Tested mutations do not affect the ability of the kit to bind to the human IgG1 Fc.
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