Journal: ACS Omega

Article Title: Phage Display-Mediated Immuno-Multiplex Quantitative PCR for the Simultaneous Quantification of IFN-γ and IL-6

doi: 10.1021/acsomega.4c09624

Figure Lengend Snippet: A: Construction of the recombinant phage displaying the anti-IFN-γ antibody. Schematic diagram of the recombinant phage plasmid. B: PD–IPCR results of M13KO7-IFN-HuZAF phage binding with the c-MYC antibody. The wild-type M13K07 phage was used as a negative control. (** p < 0.0001, n = 3). C: Western blot analysis of M13KO7-IFN-HuZAF phage binding to the c-MYC antibody. The wild-type M13K07 phage was used as a negative control.

Article Snippet: First, 50 μL each of anti-IL-6 and anti-IFN-γ capture antibodies (1:250 dilution; Sino Biological, Beijing, China; Cat. # 10395-MM14) were added to each well of the ELISA 96-well plate overnight, and the plate was blocked with 2% BSA in PBST for 2 h. One hundred microliters of gradient-diluted IL-6 and IFN-γ protein standards (CLOUD-CLONE CORP, Wuhan, China) were added to each well, and the plate was incubated at 37 °C for 2 h. Each well was blocked with 200 μL of M13KO7-blocking phage at a titer of 1 × 10 9 copies/μL for 2 h. One hundred microliters of M13KO7-blocking phage at a concentration of 5 × 10 7 copies/μL each was added.

Techniques: Recombinant, Plasmid Preparation, Binding Assay, Negative Control, Western Blot

Journal: ACS Omega

Article Title: Phage Display-Mediated Immuno-Multiplex Quantitative PCR for the Simultaneous Quantification of IFN-γ and IL-6

doi: 10.1021/acsomega.4c09624

Figure Lengend Snippet: A: Specificity of PD–IMPCR. B: Standard curve of qPCR amplification efficiency obtained with 10-fold serial dilutions of the recombinant phage, plotted with the quantification cycle (Cq) and the log10 phage concentration. The regression parameters are shown on the left. C: Standard curve of PD–IMPCR for detecting IFN-γ. D: Results of the recovery assay. E: Stability of the recombinant phage. F: ELISA results compared with the PD–IMPCR results.

Article Snippet: First, 50 μL each of anti-IL-6 and anti-IFN-γ capture antibodies (1:250 dilution; Sino Biological, Beijing, China; Cat. # 10395-MM14) were added to each well of the ELISA 96-well plate overnight, and the plate was blocked with 2% BSA in PBST for 2 h. One hundred microliters of gradient-diluted IL-6 and IFN-γ protein standards (CLOUD-CLONE CORP, Wuhan, China) were added to each well, and the plate was incubated at 37 °C for 2 h. Each well was blocked with 200 μL of M13KO7-blocking phage at a titer of 1 × 10 9 copies/μL for 2 h. One hundred microliters of M13KO7-blocking phage at a concentration of 5 × 10 7 copies/μL each was added.

Techniques: Amplification, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: ACS Omega

Article Title: Phage Display-Mediated Immuno-Multiplex Quantitative PCR for the Simultaneous Quantification of IFN-γ and IL-6

doi: 10.1021/acsomega.4c09624

Figure Lengend Snippet: Primers used in this Study

Article Snippet: First, 50 μL each of anti-IL-6 and anti-IFN-γ capture antibodies (1:250 dilution; Sino Biological, Beijing, China; Cat. # 10395-MM14) were added to each well of the ELISA 96-well plate overnight, and the plate was blocked with 2% BSA in PBST for 2 h. One hundred microliters of gradient-diluted IL-6 and IFN-γ protein standards (CLOUD-CLONE CORP, Wuhan, China) were added to each well, and the plate was incubated at 37 °C for 2 h. Each well was blocked with 200 μL of M13KO7-blocking phage at a titer of 1 × 10 9 copies/μL for 2 h. One hundred microliters of M13KO7-blocking phage at a concentration of 5 × 10 7 copies/μL each was added.

Techniques: Sequencing, Recombinant, SYBR Green Assay, Blocking Assay