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Title: The enzymeimmunoassay of progesterone in bovine milk
Author: Sauer, Maurice John
ISNI:       0000 0001 3553 641X
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1986
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The reproductive endocrinology of the cow during the oestrous cycle and early pregnancy has been reviewed, with emphasis on progesterone and how its measurement in milk may be used to improve reproductive performance. Competitive colorimetric enzyme immunoassays (EIAs) performed in microtitre plate wells were developed, enabling progesterone concentrations in unextracted milk samples to be determined. Dicyclohexylcarbodiimide/N-hydroxysuccinimide or disuccinimidyl carbonate was used to synthesize N-hydroxysuccinimide esters of progesterone derivatives: these enabled formation of enzyme-labelled steroids in a more efficient manner than conventional procedures. Poor sensitivity was observed with seven anti-progesterone 11alpha-hemisuccinate antisera when 11alpha-hemisuccinate, 11alpha-hemimaleate or 3-carboxymethyloxime bridges were used to link progesterone with the enzyme label: this problem of bridge recognition was greatly reduced when the 11alpha-glucuronide bridge was employed. Penicillinase as label provided more sensitive EIAs than alkaline phosphatase, peroxidase or beta-galactosidase but alkaline phosphatase offered practical advantages. The heterologous EIA finally adopted employed progesterone 11alpha-glucuronide-alkaline phosphatase as label, with progesterone 11alpha-hemisuccinate antibody. Assay sensitivity was similar to RIA, with a limit of detection of 5pg and 50% label displacement with 24pg progesterone. Analytical recovery,linearity of response and precision of the assay also compared favourably with RIA. Results of EIA of milk samples showed good correlation with determinations made after isolation of progesterone by HPLC (r = 0. 910) and with determinations by RIA (r = 0. 933 ). The assay enabled accurate monitoring of corpus luteum function in dairy cattle. This allowed insemination to be performed without the need for observation of behavioural oestrus or reduction of the pregnancy rate. When used for pregnancy testing, good correspondence was found with results derived from RIA performed in a commercial laboratory. Positive pregnancy tests by EIA proved 94% accurate when compared with diagnosis by palpation per rectum and in a separate study 93. 5% were confirmed as correct according to calving dates.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry