ELISA Protocol for Binding Peptides of Screened Target Molecules

The enzyme-linked immunosorbent assay (ELISA) for binding peptides of screened target molecules involves a technique to investigate the interaction between peptides and specific target molecules.

• The phage-spot amplified supernatant fraction is used for DNA sequencing and the rest is stored at 4°C.
• Inoculate ER2537 into 20 ml LB medium and incubate at 37°C until slightly turbid, or dilute overnight cultured ER2537 to 20 ml at 1:100.
• Add 5 ml of phage supernatant and incubate at 37°C for 4.5 h with vigorous aeration.
• Transfer the culture into a centrifuge tube and centrifuge at 10,000 rpm for 10 min. Take the supernatant transfer it into a new centrifuge tube and centrifuge again.
• Aspirate the upper 80% of the supernatant and transfer to a new centrifuge tube, add 1/6 volume of PEG/NaCl, and precipitate at 4°C for 1 h or overnight.
• Centrifuge the PEG precipitate at 4°C, 10,000 rpm for 15 min. Decant the supernatant, centrifuge again briefly, and aspirate the residual supernatant.
• Suspend the precipitate with 1 ml TBS and transfer to a microcentrifuge tube and centrifuge at 4°C for 5 min to precipitate residual cells.
• Transfer the supernatant to a new centrifuge tube and again precipitate the phage with 1/6 volume of PEG/NaCl. Incubate on ice for 15-60 min, centrifuge at 4°C for 10 min. Decant the supernatant, centrifuge again briefly, and aspirate the residual supernatant.
• Suspend the precipitate with 50 ml TBS. Determine the titer and store it at 4°C.
• Take 100-200 ml of 100 mg/ml target protein dissolved in 0.1 M NaHCO₃ (pH 8.6) to encapsulate the spiked wells of the enzyme labeling plate and incubate overnight at 4°C in a sealed wet box. One column per clone is encapsulated.
• Pour off the target solution and hold the plate upside down on absorbent paper to absorb as much as possible. Fill each well with a closure buffer. In addition, each clone requires the closure of a column of uncoated spiked wells to detect its binding to the BSA-coated plate. Closure of another enzyme labeling plate is used to dilute the phage, which ensures that the phage is not absorbed by the target proteins during the dilution process. 4°C closure for 1-2 h.
• Decant the closure solution and wash the plate 6 times with 1 × TBS/Tween, each time holding the enzyme-labeled plate upside down on absorbent paper to absorb as much as possible. The concentration of Tween should be the same as that used in the screening washing step.
• Dilute the phage with a TBS/Tween 4-fold ratio, 200 ml/well, 10¹² particles in the first well and 2×10⁵ particles in the twelfth well.
• Using a multichannel sampling gun, transfer each column of doubly diluted phage to the target protein-coated enzyme labeling plate. Incubate at room temperature with oscillation for 1-2 h.
• Wash the plate 6 times with 1 × TBS/Tween.
• Dilute HRP-labeled anti-M13 antibody with blocking buffer at a dilution of 1:5000, add 200 ml to each well, and incubate at room temperature with vibration for 1 h.
• Wash the plate 6 times with 1 × TBS/Tween.
• Prepare HRP substrate buffer. ABTS storage solution: dissolve 22 mg of ABTS in 100 ml of 50 mM sodium citrate solution (pH 4.0), filter and sterilize and store at 4°C. The use solution should be freshly prepared by adding 36 ml of 30% H₂O₂ to 21 ml of ABTS storage solution for one enzyme plate.
• Add 200 ml of substrate buffer to each well and incubate for 10-60 min at room temperature.
• The OD value at 405-415 nm is detected by an enzyme labeling instrument.

• Selecting an effective blocking agent and optimizing blocking conditions are critical for minimizing nonspecific binding of peptides to the ELISA plate. Common blocking agents include BSA (bovine serum albumin), milk, and non-fat dry milk.
• Including appropriate positive and negative controls in the assay to validate the specificity and sensitivity of the peptide binding is vital for obtaining reliable results.

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