Removal of cross-reactive antibody experimental steps - Database & Sql Blog Articles

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The cross-reactive antibody was carefully removed to ensure specificity in the final product. The cell lysis buffer used was prepared as 0.1 mol/L sodium acetate and 1 mol/L NaCl, which provided an optimal environment for cell disruption. After preparation, the buffer was filtered through a 0.45 µm filter to remove any particulate matter and stored at room temperature for future use. Approximately 100 ml of this buffer was required per liter of cultured bacteria to maintain consistency. For the lysis process, molecular biology grade lysozyme was employed in a solution containing 1 mol/L NaOH and Tris-buffered saline (TBS), along with TBS Triton X-100. This mixture also included 0.2% (m/V) sodium azide to prevent microbial contamination. Solid chymase was added to enhance bacterial lysis efficiency. To further degrade chromosomal DNA, pancreatic DNase I was introduced into the lysate by directly adding solid DNase I to the sample. Antibody preparation involved the use of IgG fragments obtained through protein A-Sepharose affinity chromatography, which is known to yield high-quality antibodies suitable for library screening. This method is highly recommended for its reliability and effectiveness. For more detailed procedures on protein A-Sepharose chromatography, refer to Harlow and Lane (1999). To prepare the expression libraries, appropriate E. coli strains such as Y1090 hsdR, XL1-Blue, or DH1 were used. One liter of the selected broth was inoculated and grown to a stationary phase. Cells were then harvested via centrifugation using a Sorvall GSA rotor at 4000 g for 20 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 100 ml of pre-prepared cell lysis buffer. Lysozyme (200 mg) was added, and the mixture was incubated at room temperature for 20 minutes. Next, 1 mg of trypsin and 200 µl of Triton X-100 were introduced to assist in complete lysis. The sample was then incubated at 4°C for one hour or until the supernatant became clear and less viscous. After centrifugation at 8000 g (8200 r/min using a Sorvall SS-34 rotor) for 20 minutes at 4°C, the supernatant was carefully transferred to a new beaker. The pH was adjusted to 9.0 using 1 mol/L NaOH. Protein concentration was measured using standard methods like Lowry or Bradford assays. The extract was cooled to 0°C, and the bacterial proteins were combined with hydrogen bromide-activated Sepharose 4B or Affi-Gel 10 according to the manufacturer’s instructions. Before use, the TBS solution containing 0.2% sodium azide was equilibrated with the resin and mixed with the E. coli extract. Each milligram of IgG purified via affinity chromatography required 1 ml of resin coupled to the E. coli antigen. The IgG and resin were mixed and incubated for 12–18 hours at room temperature in a rotating drum. The homogenate was then loaded onto an affinity column, and the antibody was eluted using TBS. The eluent (0.2 bed volumes per tube) was collected until the OD280 reading reached zero. Finally, the collected fractions were pooled, and the affinity-purified antibody was stored at -20°C for use in immunoscreening. This step-by-step procedure ensures high specificity and purity of the final antibody product.

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