Table 1.
Mouse IgG reverse transcription primers.
Table 2.
Mouse IgG PCR primers.
Fig 1.
Schematic for cDNA synthesis by template-switching.
(Step 1) Primer binding and initiation of polymerization. (Step 2) MMLV reverse transcriptase adds deoxycytosines to the cDNA 3' end. (Step 3) Template-switch oligo binds the CCC overhang. (Step 4) Reverse transcriptase switches templates and continues polymerization using the template-switch oligo as the template. (Steps 5–7) The single-stranded cDNA product of reverse transcription becomes the template for second-strand synthesis primed by the universal PCR forward primer. Amplification follows using the universal PCR forward primer and nested chain-specific PCR reverse primers. Note that the lengths of the different antibody regions and primers are not drawn to scale.
Fig 2.
Comparison of primer sets for RT-PCR amplification of variable regions from 5 hybridoma mRNA samples.
K = kappa chain, L = lambda chain, H = heavy chain. (A) RT-PCR result using the same reverse primers for RT and for PCR. (B) RT-PCR result using a set of nested reverse primers for RT and for PCR.
Table 3.
Results of sequencing RT-PCR products directly and following blunt-end cloning.
Table 4.
Percent identity to IgBLAST and IMGT reference sequences.
Fig 3.
Protein sequence comparison of variable regions for 3H4 kappa and 3H4 lambda.
Blue = Frame region, Orange = Complementarity-determining region, Red = J region out-of-frame, Green = J region in-frame 3H4 kappa (top) has an early stop codon due to a frameshift mutation. 3H4 lambda (bottom) is full-length.
Fig 4.
Comparison of chimeric mAb 2D9 and mouse mAb 2D9.
R = reducing gel sample, N = non-reducing gel sample (A) SDS-PAGE gel comparing chimeric mAb 2D9 (left) to mouse mAb 2D9 (right). A reducing (R) and a non-reducing (N) sample is shown for each mAb. (B) Indirect ELISA showing that chimeric mAb 2D9 binds the Spike 8 antigen. (C) Indirect ELISA showing that mouse mAb 2D9 binds the Spike 8 antigen.
Fig 5.
RT-PCR amplification of chimeric antibody variable regions.
K = kappa chain, H = heavy chain RT-PCR result with reverse primers designed for human constant regions and using as a template the RNA extracted from HEK 293F cells transiently transfected with chimeric mAb 2D9 constructs.
Table 5.
Human IgG reverse transcription primers.
Table 6.
Human IgG PCR primers.
Table 7.
Kits and antibodies.