Elastase assay methods

In most of these methods some 10-20 mg of elastin is taken for each tube of the assay the elastase unit is rather large and the assay procedures are expensive in enzyme when they are used to control purification procedures. A much more sensitive method, which measures quantities of elastase of the order of one-hundredth the Banga (1952) unit, was devised by Sbarra et al. (1960). In this assay elastin-agar plates arc used and the method is based on measuring the diameter of the clear zone which is produced by elastase deposited in circular holes cut in the gel. This method of assay is also of particular value in screening microorganisms for the production of elastase. 

AAT can be quantified by all immunochemical methods, with immunoturbidimetry and immunonephelometry the most commonly used methods. Because it constitutes about 90% of the serum inhibition of trypsin or elastase activity against small substrates, such as benzoyl-DL-arginine p-nitroanilide, it can also be semiquantified by the inhibitory capacity of serum for these enzymes however, this assay is not specific for AAT. 

Samples were assayed by absorbance at 280 nm, Bradford protein assay(.27), and radial immunodiffusion plates (Helena Laboratories). Alpha-1 antitrypsin was assayed for biological activity by competitive assay with elastase (28). DNA was assayed by the diphenylamlne methods of Burton(29) and Giles and Mvers(30). with modifications due to the presence of PEG and salts (31.). Materials were also assayed by size exclusion chromatography on Superose 6 (Pharmacia FPLC), with peak integration at 280 nm. PEG was assayed by HPLC. 

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