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. 2024 Nov 29;10(23):e40837. doi: 10.1016/j.heliyon.2024.e40837

Fig. 4.

Fig. 4

Isotype conversion of NP-specific antibodies with identical epitope binding properties

(A) Schematic workflow for the isotype conversion. Step 1: The variable domain of the HC was cloned into each isotype expression vectors. Step 2: HC and LC expression plasmids were co-transfected into Expi293F cells. For IgM expression, J chain expression plasmid was added to HC and LC. For secretory IgA expression, J chain and SC expression plasmids were added to HC and LC. Step 3: mAbs were purified from the culture supernatant. IgG2b, IgG2c, and IgG3 mAbs were purified by Protein G affinity purification. Histidine-tagged IgE, IgM, and IgA antibodies were purified by Ni- NTA affinity purification.

(B) NP3S2-derived γ2bHC, γ2cHC, γ3HC, or εHC were transfected into Expi293F cells with λLC expression plasmid. 3 μg of purified mAb per well was separated on 10 % SDS-PAGE either non-reducing (NR) or reducing (R) conditions.

(C) IgM multimer formation in the absence or presence of the J chain. NP3S2-derived μHC and λLC were transfected into Expi293F cells with or without J chain expression plasmid and purified using Ni-NTA agarose. 3 μg of purified mAb per well was separated on 6 % SDS-PAGE under the NR condition. The molecular weights of IgM pentamer and dimer are 890.7 kDa and 365.7 kDa (μHC 64.4 kDa, λLC 23.1 kDa, J chain 15.7 kDa), respectively. Asterisk, pentamer; dagger indicates dimer; IgM std, mouse IgM standard; H + L, HC and LC; J, J chain.

(D) IgA dimer formation in the presence of J chain and SC. The molecular weight of sIgA is 381.8 kDa (αHC 51.1 kDa, λLC 23.1 kDa, J chain 15.7 kDa, SC 69.3 kDa). IgA std, mouse IgA standard; SC, secretory component.

(E) ELISA analysis of isotype switched NP3S2 mAbs. For IgM, IgG3, IgG2b, IgG2c, and IgA, NP-binding activity was measured by ELISA using plates coated with NP21-BSA. Since the IgE standard used in this study was an anti-TNP antibody that binds to NP21-BSA but not to NP2-BSA, NP2-BSA was used for the detection of NP3S2-IgE. Closed circles are NP3S2-derived mAbs. Open circles are each isotype standard.