Journal: bioRxiv

Article Title: Heritable Immunization Establishes a New Model for Pathogen Control

doi: 10.1101/2024.12.19.629026

Figure Lengend Snippet: A) Schematic representation of the CRISPR-modified Hepa 1-6 cell lines used for testing albumin expression machinery, incorporating the minimal albumin promoter with and without the albumin enhancer. B) RT-qPCR analysis showing relative mRNA levels of tdTomato and albumin in Hepa 1-6 cells transfected with the constructs described in (A). C) Leader sequences evaluated for their ability to direct LA-2 antibody secretion, including sequences from mouse albumin, alpha-fetoprotein, and fibronectin, along with their predicted cleavage efficiency. D) Schematic of the LA-2 scFv-Fc expression construct under the control of a CMV promoter, including the leader sequences tested for secretion efficiency. E) ELISA results comparing LA-2 scFv-Fc concentrations in the supernatant of Hepa 1-6 cells transfected with constructs containing two different leader sequences, measured over a 48-hour period. F) OspA binding activity of LA-2 antibodies produced using different bicistronic elements, measured by ELISA at various concentrations. G) Schematic of CRISPR-modified Hepa 1-6 cell lines expressing the full-length LA-2 antibody. H) RT-qPCR analysis showing fold change in LA-2 mRNA in engineered Hepa 1-6 cells with different CRISPR guides, normalized to β-actin and background Hepa 1-6 expression. I) Concentration of IgG2a in the supernatant of CRISPR-modified Hepa 1-6 cells, quantified by ELISA, demonstrating successful antibody secretion in cells engineered with guide 3.

Article Snippet: The following primer sets were used for amplification: LA-2 mAb 1: LA-2-1-qPCR-F: 5’-CTCCCTGTGGGTCTGAGTTT-3’ LA-2-1-qPCR-R: 5’-CCCATTGTTACATGCGTCGT-3’ LA-2 mAb 2: LA-2-2-qPCR-F: 5’-TACCTGGTTGCAGGGTTGAT-3’ LA-2-2-qPCR-R: 5’-TCTGGCTTCATGCTCAATGC-3’ Albumin 1: Alb-1-qPCR-F: 5’-CAAGAGTGAGATCGCCCATCG-3’ Alb-1-qPCR-R: 5’-TTACTTCCTGCACTAATTTGGCA-3’ Albumin 2: Alb-2-qPCR-F: 5’-TGCTTTTTCCAGGGGTGTGTT-3’ Alb-2-qPCR-R: 5’-TTACTTCCTGCACTAATTTGGCA-3’ Puromycin resistance: Puro-1-qPCR-F: 5’-CCACACCTTGCCGATGTC-3’ Puro-1-qPCR-R: 5’-CACCGAGCTGCAAGAACTC-3’ β-Actin: Bact-1-qPCR-F: 5’-GGCTGTATTCCCCTCCATCG-3’ Bact-1-qPCR-R: 5’-CCAGTTGGTAACAATGCCATGT-3’ Supernatants were collected 72 hours post-transfection, and IgG2a levels were measured using the IgG2a ELISA kit (Bethyl Laboratories, E99-107) to assess antibody secretion.

Techniques: CRISPR, Modification, Expressing, Quantitative RT-PCR, Transfection, Construct, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Produced, Concentration Assay

Journal: bioRxiv

Article Title: Heritable Immunization Establishes a New Model for Pathogen Control

doi: 10.1101/2024.12.19.629026

Figure Lengend Snippet: Binding affinity data for LA-2 IgG2a and its scFv counterpart with a Vh-(G4S)3-Vl linker. Association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (Kd) values are reported.

Article Snippet: The following primer sets were used for amplification: LA-2 mAb 1: LA-2-1-qPCR-F: 5’-CTCCCTGTGGGTCTGAGTTT-3’ LA-2-1-qPCR-R: 5’-CCCATTGTTACATGCGTCGT-3’ LA-2 mAb 2: LA-2-2-qPCR-F: 5’-TACCTGGTTGCAGGGTTGAT-3’ LA-2-2-qPCR-R: 5’-TCTGGCTTCATGCTCAATGC-3’ Albumin 1: Alb-1-qPCR-F: 5’-CAAGAGTGAGATCGCCCATCG-3’ Alb-1-qPCR-R: 5’-TTACTTCCTGCACTAATTTGGCA-3’ Albumin 2: Alb-2-qPCR-F: 5’-TGCTTTTTCCAGGGGTGTGTT-3’ Alb-2-qPCR-R: 5’-TTACTTCCTGCACTAATTTGGCA-3’ Puromycin resistance: Puro-1-qPCR-F: 5’-CCACACCTTGCCGATGTC-3’ Puro-1-qPCR-R: 5’-CACCGAGCTGCAAGAACTC-3’ β-Actin: Bact-1-qPCR-F: 5’-GGCTGTATTCCCCTCCATCG-3’ Bact-1-qPCR-R: 5’-CCAGTTGGTAACAATGCCATGT-3’ Supernatants were collected 72 hours post-transfection, and IgG2a levels were measured using the IgG2a ELISA kit (Bethyl Laboratories, E99-107) to assess antibody secretion.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Heritable Immunization Establishes a New Model for Pathogen Control

doi: 10.1101/2024.12.19.629026

Figure Lengend Snippet: A) Schematic representation of the CRISPR-modified Hepa 1-6 cell lines used for testing albumin expression machinery, incorporating the minimal albumin promoter with and without the albumin enhancer. B) RT-qPCR analysis showing relative mRNA levels of tdTomato and albumin in Hepa 1-6 cells transfected with the constructs described in (A). C) Leader sequences evaluated for their ability to direct LA-2 antibody secretion, including sequences from mouse albumin, alpha-fetoprotein, and fibronectin, along with their predicted cleavage efficiency. D) Schematic of the LA-2 scFv-Fc expression construct under the control of a CMV promoter, including the leader sequences tested for secretion efficiency. E) ELISA results comparing LA-2 scFv-Fc concentrations in the supernatant of Hepa 1-6 cells transfected with constructs containing two different leader sequences, measured over a 48-hour period. F) OspA binding activity of LA-2 antibodies produced using different bicistronic elements, measured by ELISA at various concentrations. G) Schematic of CRISPR-modified Hepa 1-6 cell lines expressing the full-length LA-2 antibody. H) RT-qPCR analysis showing fold change in LA-2 mRNA in engineered Hepa 1-6 cells with different CRISPR guides, normalized to β-actin and background Hepa 1-6 expression. I) Concentration of IgG2a in the supernatant of CRISPR-modified Hepa 1-6 cells, quantified by ELISA, demonstrating successful antibody secretion in cells engineered with guide 3.

Article Snippet: Serum samples were applied to the coated plates, and LA-2 antibody levels were detected using the IgG ELISA detection kit (Bethyl Laboratories, E99-131).

Techniques: CRISPR, Modification, Expressing, Quantitative RT-PCR, Transfection, Construct, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Produced, Concentration Assay