James P. Gosling

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.

The dependence of radioimmunoassay detection limits on antibody affinity.

Competitive radioimmunoassay standard curves for progesterone were established with tritiated progesterone and six different anti-progesterone monoclonal antibodies ranging in affinity from 1.23 x 10(8) to 2.82 x 10(10) l/mol. The detection limits for these curves ranged from 133 to 6,670 pmol/l final assay concentration (16.7 to 839.1 pg/tube). Separately, a mass-action mathematical model was used to predict lower detection limits for assays run under equivalent conditions with the six antibodies. When compared to the experimental data the predictions were reliable for the higher affinity antibodies but became less accurate as the affinity decreased.

Follicular growth and corpus luteum function in women with unexplained infertility, monitored by ultrasonography and measurement of daily salivary progesterone

Ovarian function was evaluated over a minimum of 3 consecutive menstrual cycles from each of 41 women with unexplained infertility. Follicular development and ovulation were monitored using real time ultrasonography and luteal function was evaluated by daily salivary progesterone measurement. In 129 spontaneous cycles, normal single ovulations were detected in 121 (93.8%). Luteal phase insufficiency was identified in 21 (17.4%) of these 121 cycles and this was a recurrent phenomenon in the cycles of 5 of the 41 women (12.2%). A successful pregnancy was seen only in association with consistently normal salivary progesterone profiles or where the empirical use of clomiphene citrate therapy had corrected previously diagnosed luteal phase insufficiency. Basal body temperature records or mid-luteal serum progesterone measurements were less satisfactory indices of luteal function than a salivary progesterone profile.

The frequency of salivary progesterone sampling and the diagnosis of luteal phase insufficiency

A profile of salivary progesterone concentrations, based on daily samples taken over a full menstrual cycle, provides a detailed picture of changes in luteal function, at the expense of analyzing a large number of samples. Strain can be placed on analytical services by assaying daily samples instead of one or a few serum (or saliva) samples. This study sought to determine the minimum number of salivary progesterone determinations which adequately describe luteal function. Daily salivary progesterone levels from 215 cycles, of which 29 cycles had progesterone profiles indicative of luteal phase insufficiency, were analyzed to ascertain the efficiencies of various sampling patterns of reduced frequency. A single mid-luteal salivary progesterone estimation or the mid-luteal Lenton progesterone index (n = 4) satisfactorily reflected the normal luteal phase, but a frequency of one sample every 3 days over the luteal phase (n = 5-6) was necessary to allow recognition of a short luteal phase or poor progesterone surge.

13 – ENZYME IMMUNOASSAY

Enzyme immunoassay (EIA) has been the primary subject of a number of books and reviews, and an important concern of other recent books and reviews. Enzymes are the most versatile and popular class of labeling substances used for immunoassays, and they have an assured future. The invention of immunoenzymatic staining in the mid-1960s is said to have been the beginning of enzyme immunoassay (EIA), and this enabled the preparation of permanent photomicroscopic slides with visualization of specific antigens. In 1971, enzymes were introduced as alternatives to radioisotopes in immunoassays. Enzymes are perhaps the most varied class of labeling substances. About 1990, the most common enzyme label in new immunoassays was horseradish peroxidase, with alkaline phosphatase in second place. The term enzyme-mediated immunoassay encompasses, as well as all assays with enzymes as the labeling substance, assays with labels containing enzyme substrates, enzyme inhibitors, coenzymes, or enzyme cofactors. The operation of such assays depends on the use of one or more enzymes as ancillary reagents. Enzymes are also exploited in many other ways for the benefit of immunoassays. To prepare conjugates with enzyme, the method of conjugation used is particularly mild so as to retain both immuno- and enzymatic activities.

Standardization of Immunoassays for Hapten Analytes

The pace of the development and commercialization of immunoassay methodology has been so great, that the parallel development of ways to ensure comparability between the results of different immunoassays for the same substance has fallen behind. If, as is the case with many hapten analytes, the analyte is chemically defined, homogeneous and available at very high purity, the rigorous standardization of different immunoassays developed to determine the same would appear to be feasible. However, many published details of external quality assessment schemes and of studies on the comparability of commercial assays indicate that the results of immunoassays for hapten analytes are not generally and reliably comparable. Lack of comparability is most marked for assays detecting a variety of analyte subforms or metabolites. This problem is surveyed with respect to the variability of hapten immunoassays, factors related to details of immunoassays. Finally, the assessment of assay comparability and some potential strategies for improving it are discussed.