Comprehensive method for producing high-affinity mouse monoclonal antibodies of various isotypes against (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten

Abstract

Monoclonal antibody (mAb) technology has significantly contributed to basic research and clinical settings for various purposes, including protective and therapeutic drugs. However, a rapid and convenient method to generate high-affinity antigen-specific mAbs has not yet been reported. Here, we developed a rapid, easy, and low-cost protocol for antigen-specific mAb production from single memory B cells. Using this method, high-affinity IgG1 mAbs specific to the hapten 4-hydroxy-3-nitrophenylacetyl (NP) were established from NP-CGG immunized C57BL/6 mice within 6 days. Our mAb production system allows flexible switching of IgG1 to any other isotype with the same paratope, enabling the absolute quantification of antigen-specific serum antibody titers and affinity maturation. Additionally, we established a protocol for the production of IgM and IgA, retaining their functional pentamer and dimer structures. This method is also effective against human antigens and pathogens, making it a powerful tool for mAb development in both research and clinical settings.

Keywords: 4-Hydroxy-3-nitrophenylacetyl (NP); Affinity maturation; B cell receptor; IgA; IgE; IgG; IgM; J chain; Memory B cells; Monoclonal antibody; Secretory component; Single cell.

Fig. 1

Designing multiplexed PCR primers for single-cell Ig gene amplification (A-B) Schematic representation of HC (A) and LC (B) amplification by PCR. L-PART, leader part; UTR, untranslated region; Fw, forward primer; Rv, reverse primer (C) The number and coverage of designed forward primers. The number of alleles representing the V gene sequences retrieved from the IMGT database. V gene coverage indicates the percentage of V genes covered by the primers. (D) Efficiency of paired HC and LC amplification using designed primers. The total number represents the number of non-specific B220⁺CD19⁺IgG1⁺CD38⁺ live single memory B cells sorted from the spleen or Peyer's patch. Successful amplification means the number of amplified samples of HC and LC. The agarose gel electrophoresis of spleen-derived samples is shown in Supplementary Fig. 1. HC, heavy chain; LC, light chain.

Fig. 2

Amplification of paired immunoglobulin genes from antigen-specific single memory B cells (A) Schematic diagram of experiment. C57BL/6 mouse was immunized twice at 3-week intervals. Each immunization was performed using 100 μg/mouse NP-CGG with alum adjuvant. The mouse was analyzed on day 42. (B) Gating strategy for FACS sorting of antigen-specific memory B cells. Lymphocytes were gated by forward and side scatters. B220⁺CD19⁺IgG1⁺CD38⁺ live NP-specific memory B cells were sorted. (C) Schematic workflow of mAb production and ELISA. Step 1: a mouse was immunized twice with NP-CGG. Step 2: NP-specific memory B cells were single-cell sorted (Day 1). Step 3: RT-PCR amplification of paired HC and LC genes. PCR products were cloned in the expression vectors (Day 1–2). Step 4: Expression vector plasmids were co-transfected into Expi293F cells and cultured for 4 days for mAb production (Day 2–6). mAbs were purified from the supernatant (Day 6). Step 5: Binding affinity to NP₂-BSA and NP₂₁-BSA was determined by ELISA (Day 6). (D) Agarose gel electrophoresis of second PCR products. IgG1⁺NIP⁺ memory B cells were sorted and cDNAs were synthesized by reverse transcription. The HC and LC genes were amplified separately. The expected PCR products of HC and LC were approximately 600 and 800 base pairs (bp), respectively. (E) Efficiency of successful amplification of HC and LC genes against various antigens. Total number represents the number of antigen-specific B220⁺CD19⁺IgG1⁺CD38⁺ live single memory B cells sorted from the spleen. Successful amplification means the number of amplified samples of HC and LC. hCD80, human CD80; hCA9, human CA9; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; RBD, receptor-binding domain; SFTSV, severe fever with thrombocytopenia syndrome virus; Gc, SFSTV viral glycoprotein Gc.

Fig. 3

Development of antigen-specific mAbs from single B cells (A) Schematic workflow of mAb purification from single memory B cells. Step 1: HC and LC gene amplification by RT-PCR. Step 2: PCR products were cloned into expression vectors. Step 3: HC and LC expression plasmids were co-transfected into Expi293F cells. Step 4: mAbs were purified from culture supernatant by Protein G affinity purification. (B) Cloning of variable domain into IgG1 HC expression vector containing IgG1 CH1 (from 41Asn) to CHS domain. (C) Cloning of full-length LC gene into the expression vector. (D) γ1HC and λLC expression plasmids encoding NP3S2 IgG1 were transfected into Expi293F cells. 3 μg of purified NP3S2 IgG1 antibody per well was separated on 10 % SDS-PAGE either non-reducing (NR) or reducing (R) conditions. (E) mAbs NP3S2 and NP3S7 were produced in a combination of HC with paired LC obtained from single cells. NP3S2 and NP3S7 correspond to #2 and #7 in Fig. 2D. The NP-binding activity was measured by ELISA against NP₂₁-BSA or BSA. IgG1std, mouse IgG1 standard.

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.

Fig. 5

Electron microscopy revealed IgM pentamer and IgA dimer structures (A) Purified NP3S2-IgM antibodies were observed using TEM. Typical IgM pentamers are indicated by arrows. (B) Purified NP3S2-IgA antibodies were observed using TEM. Typical IgA dimers are indicated by arrows.

Fig. 6

Affinity quantification of antigen-specific mAbs to antigens (A) VH and VL genes, CDR3 amino acid sequence, and the number of nucleotide mutations of each NP-specific mAb clone are shown. (B–C) ELISA analysis of NP-specific mAbs (clone: NP3S1-NP3S7). The NP-binding activity was measured by ELISA using plates coated with NP₂₁-BSA (B) or NP₂-BSA (C). (D) The absolute IC50 was calculated using a nonlinear regression curve fit on Prism. The dotted line indicates the detection limit.

Fig. 7

Absolute quantification of serum antigen-specific antibody titers (A-B) Absolute quantification of NP-specific antibody concentration in serum. C57BL/6 mice were immunized with the alum-precipitated NP-CGG. ELISA plates were coated with NP₂₁-BSA (A) or NP₂-BSA (B). Antibodies bound to the plates were detected using HRP-conjugated anti-mouse isotype-specific antibodies. NP3S2-IgG1 mAb from Fig. 3 or NP3S2-IgM, IgG3, IgG1, IgG2b, IgG2c, IgE, and IgA mAbs developed from NP3S2-IgG1, which are consisted of identical V-domain recognizing NP, were used as the standard. From the four-parameter logistic (4 PL) curve fit of NP3S2-derived mAbs on Prism, NP-specific antibody concentration of each sample was determined. n.d., not detectable. (C–D) Post-immunization NP₂/NP₂₁ ratios in IgG1 (C), IgM (D), and IgG2b (D). The NP₂/NP₂₁ ratios were calculated based on the NP₂-binding antibody concentration and the NP₂₁-binding antibody concentration. Relative NP₂/NP₂₁ ratios were calculated by dividing the ratio at each time point by the average ratio at day 10. avg., average.