beta Defensin-1 ELISA Assay

Additional Information

Assay Background

Defensins are 2-6 kDa, cationic, microbicidal peptides active against many Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses, containing three pairs of intramolecular disulphide bonds. On the basis of their size and pattern of disulphide bonding, mammalian defensins are classified into alpha, beta and theta categories. Every mammalian species explored thus far has beta-defensins. In cows, as many as 13 beta-defensins exist in neutrophils. However, in other species, betadefensins are more often produced by epithelial cells lining various organs (e.g. the epidermis, bronchial tree and genitourinary tract). Defensins are produced constitutively and/or in response to microbial products or proinflammatory cytokines. Some defensins are also called corticostatins (CS) because they inhibit corticotropin-stimulated corticosteroid production. The mechanism(s) by which microorganisms are killed and/or inactivated by defensins is not understood completely. However, it is generally believed that killing is a consequence of disruption of the microbial membrane. The polar topology of defensins, with spatially separated charged and hydrophobic regions, allows them to insert themselves into the phospholipid membranes so that their hydrophobic regions are buried within the lipid membrane interior and their charged (mostly cationic) regions interact with anionic phospholipid head groups and water. Subsequently, some defensins can aggregate to form `channel-like’ pores; others might bind to and cover the microbial membrane in a `carpet-like’ manner. The net outcome is the disruption of membrane integrity and function, which ultimately leads to the lysis of microorganisms. Some defensins are synthesised as propeptides which may be relevant to this process.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it. 2. Add 100 μl of standards, samples and zero controls (standard diluent buffer) into wells. Incubate for 1.5 h at 37 °C. 3. Aspirate each well and wash, repeating the process four times for a total five washes. Wash by filling each well with 1× Wash Buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels. 4. Add 100 μl 1X Antibody conjugate into each well. Cover wells and incubate for 1 hour at 37 °C. 5. Aspirate each well and wash as step 3. 6. Add 100 μl of 1X HRP-Streptavidin solution to each well. Cover wells and incubate for 30 minutes at 37 °C. 7. Aspirate each well and wash as step 3. 8. Add 100 μl of TMB Reagent to each well. Incubate for 15 minutes at 37 °C in dark. 9. Add 100 μl of Stop Solution to each well. The color of the solution should change from blue to yellow. 10. Read the OD with a microplate reader at 450nm immediately.

Typical Standard Curve