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Biotechnology and Applied Biochemistry (2002) 36, (101–110) (Printed in Great Britain)

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Purification and characterization of lysozyme-pregnanediol glucuronide conjugates: the effect of the hapten and coupling reagent on the substitution level, sites of acylation and the consequences for the development of future immunoassays

C. Mark Smales¹ and Leonard F. Blackwell

Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand

Key words: chromatographic purification, inhibitable enzyme system, Ovarian Monitor, ovulation.

Abbreviations used: E1G, oestrone glucuronide (acid form); PdG, pregnanediol glucuronide; PdG[H], pregnanediol glucuronide (acid form).

¹To whom correspondence should be addressed, at (current address) Research School of Biosciences, University of Kent at Canterbury, Canterbury CT2 7NJ, U.K. (e-mail c.m.smales@ukc.ac.uk).

The preparation of conjugates destined for use in homogeneous enzyme immunoassays requires careful control to maximize the yield of conjugate, limit the level of substitution and obtain highly inhibitable conjugates. Furthermore, previous studies have shown that the acylation position and orientation of haptens in enzyme conjugates is important in determining the recognition and strength of binding of the hapten by anti-hapten antibodies. In this study we have prepared and purified pregnanediol glucuronide conjugates of hen egg white lysozyme by the active ester and mixed anhydride methods and compared the resulting conjugates with those obtained when conjugating with oestrone glucuronide. Conjugation of lysozyme with pregnanediol glucuronide by the mixed anhydride procedure resulted in two major derivatives monoacylated at lysine residues 33 and 97, while acylation by the active ester procedure resulted in six major derivatives acylated at one or more of lysine residues 33, 97 and 116. Comparison of these conjugates with oestrone glucuronide conjugates suggested that when using the more reactive active ester method the protein environment of the amino groups and the acylating agent directed the site(s) of acylation and level of substitution. However, in the case of the less reactive mixed anhydride coupling procedure the chemical nature of the hapten also influenced the final conjugation product makeup. These findings have implications for the design of new, highly inhibitable enzyme conjugate immunoassay systems.

Received 25 April 2002/13 June 2002; accepted 20 June 2002

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