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Biomedical subjects

J A Foulkes

Publications and source records attributed to J A Foulkes.

At least 19 recordsLinked to original sources
PubMed1999

A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzyme substrates for use with a screen-printed amperometric immunosensor for progesterone in cows' milk.

4-Aminophenyl phosphate (4-APP) and 1-naphthyl phosphate (1-NP) were compared as enzyme substrates for an amperometric milk progesterone biosensor utilising progesterone-conjugated alkaline phosphatase in a competitive immunoassay format. Cyclic voltammetry of the corresponding hydrolysis products, 4-aminophenol and 1-naphthol, at the surface of screen-printed carbon base transducers, uncoated or coated with anti-progesterone monoclonal antibody (mAb) showed well-defined anodic responses for both species, with the more sensitive being 4-aminophenol. Scan rate studies produced evidence that surface mAb could impede the diffusion of 4-aminophenol, but not 1-naphthol, toward the electrode surface. This was supported by computer simulation for the electrochemical rate constant (khet) using 4-aminophenol, which gave values at uncoated and mAb-coated electrodes of 6.5 x 10(-4) and 3.0 x 10(-4) cm s-1, respectively. The applied potential for oxidation of 4-aminophenol was 230 mV lower than for 1-naphthol. Nevertheless, by operating below +400 mV versus a saturated calomel reference electrode, it was possible to obtain a chronoamperometric signal for 1-naphthol in the absence of electrochemical interference from milk. Using mAb-coated SPCEs, calibration curves were obtained for progesterone in oestrus whole cow's milk spiked with standard concentrations over the range 0-50 ng/ml, using either 4-APP or 1NP as enzyme substrate. Precision values for triplicate sensors were 5.3-18.3% for 4-APP and 4.1-12.4% for 1-NP. An assay of real whole milk samples from different cows at various stages of the oestrus cycle produced correlations against a commercial EIA of r = 0.840 and 0.946 for 4-APP and 1-NP, respectively, 1-NP possesses the advantages over 4-APP of being inexpensive, easy to obtain and soluble (1-naphthol cf. 4-aminophenol) at high pH. From these observations, it is concluded that 1-NP is the preferred substrate for use with our proposed milk progesterone biosensor.

Alkaline Phosphatase↗
PubMed1990

The preparation of an ovine monoclonal antibody to progesterone.

Peripheral blood mononuclear cells were collected from a sheep immunized against progesterone-11 alpha-hemisuccinate-ovalbumin. Following fusion with NS1 mouse myeloma or heteromyeloma cells, a large number of hybrid colonies was established. These were screened for the production of sheep antibodies to progesterone. Twenty-four cell lines were cloned and one was stabilized. This cell line, O/MP.1A9.D7B2, produced a high-affinity ovine immunoglobulin G1 (dissociation constant 4.8 pmol/l) with a high degree of specificity for progesterone. The antibody was substituted into a competitive enzyme-linked immunosorbent assay for the measurement of progesterone in bovine milk, originally established using an ovine polyclonal antibody, and the results were compared. The monoclonal antibody produced an assay with a lower limit of detection and a greater degree of discrimination than the polyclonal antiserum.

Animals↗
PubMed1989

Use of an on-farm progesterone assay kit to determine pregnancy in sows.

Blood samples taken from the ear vein of 1037 sows two to three weeks after service were assayed the same day using Ovucheck 'Sowside' kits. Colour development was compared with oestrous and pregnancy controls. Reliable data on reproductive performance were obtained from 908 sows sampled 17 to 20 days after service. The accuracy of identification of 796 pregnant and 12 non-pregnant sows was 94.6 per cent and 35.7 per cent, respectively. Excluding animals which returned outside the normal range of 18 to 24 days, 52.1 per cent of 48 empty sows were identified by the test. Problems with blood sampling were reported on seven of 18 farms and this may explain the low accuracy of the kit on some farms.

Animals↗
PubMed1989

A comparison of alkaline phosphatase, beta-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtitre plate enzymeimmunoassay.

Assays for alkaline phospatase, beta-galactosidase, penicillinase and peroxidase were optimised for quantitation in microtitre plate wells. Their value as labels in microtitre plate enzymeimmunoassay (EIA) for progesterone was assessed following coupling with 11 alpha-hydroxyprogesterone 11-glucuronide using an active ester procedure. Bridge-heterologous antiserum (11 alpha-hydroxyprogesterone 11-hemisuccinate-bovine serum albumin as immunogen) was used to minimize bridge recognition. The limits of detection of the enzymes were in the order penicillinase greater than peroxidase greater than alkaline phosphatase greater than beta-galactosidase. Under appropriate conditions it was possible to achieve greater than 50% displacement of label with 50 pg of progesterone for all four labels.

Alkaline Phosphatase↗
PubMed1989

The influence of heterology, enzyme label and assay conditions on the sensitivity of microtitre plate enzymeimmunoassays for progesterone in milk.

Seven antisera raised against 11 alpha-hydroxyprogesterone 11-hemisuccinate (P11-HS) were used in microtitre plate enzymeimmunoassays (EIAs) for progesterone to identify improvements in sensitivity achievable by using various heterologous labels. EIAs using beta-galactosidase linked to P11-HS, 11 alpha-hydroxyprogesterone 11-hemimaleate (P11-HM), 11 alpha-hydroxyprogesterone 11-glucuronide (P11-Glu) or progesterone 3-(o-carboxymethyl) oxime (P3-CMO) were compared. Loss of sensitivity through bridge recognition was least evident using the P11-Glu derivative. The same seven antisera were used to evaluate assay sensitivity using beta-galactosidase, alkaline phosphatase, penicillinase and peroxidase linked to P11-HS or P11-Glu as label. Consistent improvements were achieved with the heterologous assays in the order penicillinase greater than alkaline phosphatase/peroxidase greater than beta-galactosidase: with penicillinase, sensitivity generally exceeded that of RIA. These data provide evidence for the general efficacy of the combination 11 alpha-hemisuccinate (immunogen bridge) and 11 alpha-glucuronide (label bridge) in reducing bridge recognition. EIA performed at 4 degrees C provided greater sensitivity than at ambient temperature (21 degrees C) or 40 degrees C, however, ambient temperature incubation provided a practical compromise. Equilibrium was not achieved under any of the conditions investigated.

Alkaline Phosphatase↗
PubMed1986

Seasonal variations in serum progesterone levels in pregnant sows.

In the autumn and winter of 1979 and the spring and summer of 1980 serum samples were taken from approximately 20 sows at between 40 and 90 days of gestation in each of seven commercial herds. In most of these herds progesterone concentrations were significantly lower in the autumn than in the other three seasons. Between June 1980 and June 1981 every pregnant sow in a further herd of 250 sows was sampled at 25 to 30 days and at 70 to 91 days of gestation. Seasonal differences in progesterone concentrations were again evident, with the concentrations rising from their lowest in August, September and October to a peak in March. Comparisons between the early and late pregnancy progesterone levels from sows sampled in this herd at different times of the year suggested that corpora lutea which were formed in the summer and early autumn were fully competent and responsive, but that their lower hormone production was possibly the result of reduced luteotrophic stimulation. These findings are pertinent to the pathophysiology of the autumn abortion syndrome and other seasonal reproductive problems in sows.

Animals↗
PubMed1986

Use of progesterone 11-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre-plate enzymeimmunoassay of progesterone in milk and its application to pregnancy testing in dairy cattle.

A simple direct-addition microtitre plate enzymeimmunoassay (EIA) for progesterone in whole milk is described. The assay used antiserum raised against 11 alpha-hydroxyprogesterone 11-hemisuccinate (progesterone 11-hemisuccinate) and a heterologous label prepared by conjugation of 11 alpha-hydroxyprogesterone 11-glucuronide (progesterone 11-glucuronide) with alkaline phosphatase using an active ester procedure. The sensitivity, analytical recovery, linearity of response and precision of the assay compared favourably with radioimmunoassay (RIA). Results from EIA of milk samples were compared with determinations made after isolation of progesterone by HPLC (r = 0.910). Milk samples (200) were assayed by RIA at both the Milk Marketing Board and the Cattle Breeding Centre and the results were correlated with EIA performed at the Cattle Breeding Centre (r = 0.890 and r = 0.833 respectively). Calving data were obtained from a further 110 cows for which the milk progesterone EIA had provided a pregnancy test 24 days after AI; 46 cows were correctly identified as non-pregnant and 58 as pregnant and there were 4 false positive and 2 inconclusive results.

Alkaline Phosphatase↗
PubMed1986

Semen assessment, fertility and the selection of Hereford bulls for use in AI.

Nineteen young Hereford bulls were used to study the relationship between semen characteristics and fertility in artificial insemination following 15 320 inseminations. Seven measures of sperm motility, morphological abnormalities, the release of hyaluronidase, ATP content and sperm head measurements were examined as predictors of fertility (49-day fixed-interval non-return rate). Two assessments of motility, three categories of abnormal spermatozoa, acrosomal changes and the release of hyaluronidase had predictive power. Multiple regression analysis showed that a combination of sperm motility after dilution in saline, motility after thawing and the proportion of coiled tails and proximal protoplasmic droplets provided the best prediction of fertility and allowed bulls to be ranked in order of observed non-return rate (%) with a Spearman correlation better than +0.80.

Adenosine Triphosphate↗
PubMed1984

Immunochemical investigation of the interaction of egg-yolk lipoproteins with bovine spermatozoa.

Antiserum was raised in rabbits against an egg-yolk lipoprotein fraction previously shown to have cryoprotective properties. It was used in conjunction with alkaline phosphatase-conjugated goat anti-rabbit IgG to demonstrate lipoprotein interaction with bovine spermatozoa. The lipoprotein bound firmly to spermatozoa and was not removed by extensive washing, suggesting that the lipoprotein had become irreversibly associated with the sperm membranes.

Animals↗
PubMed1983

Laser measurement of the motility of bull spermatozoa in an egg-yolk diluent.

An objective method of assessing bull sperm motility by specifying the mean swimming speed and number of motile spermatozoa in a sample is described. Laser light was conducted into diluted semen samples using a fibre-optic Doppler anemometer (FODA). The signal correlation of the back-scattered laser light was modelled using least squares computer curve fitting of the resulting data. Agreement was found between the mean swimming speeds obtained and those measured using time lapse photography.

Animals↗
PubMed1982

Direct radioimmunoassay of estradiol-17 beta in defatted bovine milk.

A radioimmunoassay was developed for rapid determination of estradiol-17 beta concentrations in unextracted defatted bovine milk. The assay was dependent on the use of a highly specific anti-estradiol-17 beta antiserum. Application of a formula to correct for the interference associated with individual milk samples and use of appropriate assay blanks facilitated interpolation on a buffer standard curve. The assay offered a high degree of sensitivity (0.6pg/ml milk) and a precision (within-assay coefficient of variation: 0.196; between-assay CV:0.191) comparable with contemporary extraction methods.

Animals↗
PubMed1981

Direct enzymeimmunoassay of progesterone in bovine milk.

A sensitive enzymeimmunoassay has been developed for measuring progesterone in unextracted bovine milk. An N-hydroxysuccinimide ester of 11 alpha -hydroxyprogesterone 11-hemisuccinate has been synthesised and used to form conjugates with beta-galactosidase in buffer at pH 7.0. The degree of incorporation of progesterone into the enzyme was demonstrated using (14C)-labelled steroid and by radioimmunoassay binding inhibition. Standard curves of comparable range and sensitivity to radioimmunoassay were obtained in the presence of whole milk taken from a cow at oestrus. These advances have allowed the development of a simple micro-titre plate enzymeimmunoassay of progesterone in whole milk and will be of particular value in determination of pregnancy, prediction of the day of oestrus and diagnosis of reproductive disorders.

Animals↗
PubMed1981

A spectrofluorometric investigation, using 1-anilino-naphthalene-8-sulphonate, of the interaction between washed bovine spermatozoa and seminal plasma or egg-yolk lipoprotein.

The dye 1-anilino-naphthalene-8-sulphonate (ANS) bound at 94.5 +/- 2.7 pmol per 10(6) spermatozoa (KD = 1.83 +/- 0.13 X 10(-5) M) and 7.30 +/- 0.17 pmol per mg seminal plasma protein (KD = 2.19 +/- 0.06 X 10(-5) M). Equilibration of excess ANS with a mixture of washed spermatozoa and seminal plasma resulted in a significant increase in fluorescence over that calculated by summation of the individual levels (P less than 0.05). A less pronounced but significant increase was seen when an egg-yolk lipoprotein, previously shown to have cryoprotective properties, was similarly added to washed spermatozoa (P less than 0.05). This increase in fluorescence was not reduced by washing spermatozoa--lipoprotein mixtures, suggesting that an interaction between the lipoprotein and the cell membrane had occurred.

Anilino Naphthalenesulfonates↗