The detection by enzyme linked immunosorbent assay of P30 and 19-OH prostaglandin F1/F2, in the presence of a range of possible contaminants.

The potential of specific human semen markers, P30 and 19-OH prostaglandin F1 alpha/F2 alpha (PG F), ELISAs were compared in a blind trial. The PG F assay was shown to be at least ten times more sensitive than the P30 technique although cross reactivity problems with spermicidal creams and other possible domestic contaminants were identified. A simple method for the removal of such contaminants using a phenyl boronate column (PBA) was evaluated in an extensive blind trial.

A survey of the concentration of 19-OH F1 alpha/F2 alpha prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains.

The levels of 19-hydroxy-prostaglandins F1 alpha/F2 alpha (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 micrograms/ml, 49.5 micrograms/ml and 59 micrograms/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4 degrees C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-microliters semen stains stored over a period of 6 weeks at room temperature.

Radioimmunoassay detection limits for 19-OH F1 alpha/F2 alpha prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects.

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F1 alpha/F2 alpha (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 microliter of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 microliter semen. The assay could be conducted over a pH range of 7.5-10.5 even after the PG has been heated to 100 degrees C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 microliters of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 microliters of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.

The development of an enzyme-linked immunosorbent assay for 19-OH PG F1 alpha/F2 alpha.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of semen-specific 19-OH prostaglandin F1 alpha/F2 alpha (PGF). The assay can reliably and reproducibly detect as little as 2 pg 19-OH PGF/100 microliters and allows for the rapid analysis of large numbers of swab and stain extracts for the presence of semen.

A comparison of the isoelectric focusing patterns of red cell phosphoglucomutase separated on an ampholine, ampholine/separator and immobilised pH gradient.

The isoelectric focusing patterns of red cell phosphoglucomutase (PGM1) separated on Ampholine, Ampholine/separator and rehydratable immobilised pH gradients have been compared. The sharpest and most intense zymograms have been observed on immobilised pH gradients provided that Ampholine was added during the rehydration of the gel. The addition of ampholine in the rehydration of Immobiline plates has been shown to improve the sharpness and intensity of the zymogram obtained and the potential of immobilised pH gradients for PGM typing has been demonstrated.

The application of a simple RIA technique for the detection of 19-OH F1 alpha/F2 alpha prostaglandin, a specific semen marker, in semen contaminated vaginal swabs: time since intercourse studies.

The specificity of the 19-OH F1 alpha/F2 alpha prostaglandin antisera for the detection of semen in seminal/vaginal mixtures, has been evaluated. Using a parallel curve test we found that the antibody showed a high specificity for these seminal prostaglandins in seminal/vaginal mixtures at concentrations of between 2 pg and 40 pg/100 microliter. The precision of the assay has been improved by the use of a donkey-anti-rabbit ferritin-bound second antibody. The application of this detection system makes it possible to complete an assay within 4.5 h. A survey of 50 semen-free vaginal swabs obtained from 3 donors, taken throughout the menstrual cycle, showed no trace of these prostaglandins. They were also not detected in the vaginal secretions of two further donors who were undergoing medication. Using only 10-microliter aliquots of a seminal/vaginal swab extract, prepared in 500 microliter, it was possible to detect semen up to approx. 80 h after one sexual act.

Evaluation of the 190H analogues of prostaglandins E1, E2, F1 alpha and F2 alpha as specific markers for the identification of human semen in body fluid mixtures.

The specificity of antisera raised against each of the prostaglandin series 190H E1/E2 and 190H F1 alpha/F2 alpha, produced in males, was evaluated by radioimmunoassay. Further, the ability of these antisera to detect semen specific prostaglandins in mixtures of body fluids was examined. Antisera directed against the 190H E1/E2 series cross-reacted with prostaglandin E1 and marginally with E2. Antisera raised to the 190H F1 alpha/F2 alpha series were, however, highly specific to the semen specific prostaglandins 190H F1 alpha/F2 alpha and 190H E1/E2. It was possible to detect picogramme quantities of contaminating 190H F1 alpha/F2 alpha on vaginal swabs taken up to 72 h after intercourse and on vaginal swabs stored at room temperature for up to 2 years. These prostaglandins were not detected on semen free vaginal swabs, in faecal material, saliva, urine or in a sample of human milk (stain). A limited study of casework material is also described. Detection of the 190H F series, as a group, has considerable potential in the identification of human semen at picogramme levels, eliminating the need for alternative chemical tests and extensive microscopic examination.

alpha-L-fucosidase polymorphism in human semen, blood, and vaginal fluid.

The diallelic enzyme alpha-L-fucosidase has been found to be highly active in semen. Eight distinct enzyme bands were observed in each homozygote and the heterozygote consisted of a combination of bands. The isoelectric points ranged between pI 4.34 and 6.65. Activity was found to be much lower in vaginal fluid, not more than three cathodal bands were visible in any sample examined. In leucocytes, 5 bands were detected in homozygotes whilst in serum only two faint anodal bands could be detected. Activity was low in vaginal fluid and could not be detected in urine or saliva. Population studies were carried out on semen samples from Oxford and Basingstoke and on lymphocyte samples from Oxford. The data were in agreement with Hardy-Weinburg equilibrium. The common allele (Fu 1) had a frequency of 0.704 in Oxford semen samples.

Identification of trout and salmon bloods by simple immunological technique and by electrofocusing patterns of red cell enzyme superoxide dismutase.

Species identification of animal bloods is readily achieved by immunological tests. Differentiation among fish species on this basis is more difficult although considerable success has been achieved on the basis of both inter- and intra-specific differences in their serum proteins. This report describes a method for the identification of the different species of fish within the Salmonidae family and some coarse fish families on the basis of an immunological test and electrofocusing patterns of the enzyme superoxide dismutase from the red cell. The immunological technique relies on the development of a specific anti-trout (Salmonidae) serum which is used initially to differentiate the blood of a Salmonidae from other freshwater fish. Further discrimination, within the Salmonidae, is made on the basis of the different polymorphic forms of the enzyme superoxide dismutase separated in a pH 2.5 to 8 gradient. Using this technique, it is possible to differentiate among salmon, sea/brown trout, char, cheetah trout, and a number of varieties of rainbow trout.