Use of biotinylated 17beta-estradiol in enzyme-immunoassay development: spacer length and chemical structure of the bridge are the main determinants in simultaneous streptavidin-antibody binding.

17beta-estradiol (E2) concentrations are in the low pg/ml range in plasma. To develop a sensitive enzyme immunoassay (EIA) for E2-determination a highly specific antibody raised against a 6-carboxymethyl (CMO)-E2-bovine serum albumine conjugate was used. Based on 6-CMO-E2 and 6-amino-E2, four biotinylated tracers with two different spacer lengths between E2 and biotin were synthesized using biotinylation reagents in one step reactions. All amino-based tracers were unsuitable for assay development because the antibody binding was too weak compared to the analyte E2. For 6-CMO-based tracers the simultaneous binding of the tracer to the antibody and streptavidin seems to be the determining step in the procedure depending on incubation temperature and spacer lengths. While a short spacer of 9 carbon atoms was susceptible to room temperature, a longer spacer of 16 carbon atoms showed nearly the same results for incubation at 4 degrees C or at room temperature. The absolute detection limit of this system was 0.63 pg/well. For sample clean-up, porcine plasma was solvent-extracted and depending on the initial plasma volume further purified by solvent partition. Determination of reproducibility resulted in intraassay coefficients of variation of 13% and 5.3% for samples with E2-levels of 15 pg/ml and 236 pg/ml, respectively. Measurement of E2-spiked blood plasma revealed recoveries of 83% up to 100% for E2 concentrations between 50 pg/ml and 1000 pg/ml. Only for the lowest concentration (20 pg/ml) a recovery of 58% was observed. Correlation of the EIA with an established radio immunoassay resulted in r=0.991 using the same antibody.

Effects of resistant potato starch on odor emission from feces in Swine production units.

Odor emission from swine facilities is determined by microbial breakdown of amino acids or carbohydrates in the pig colon. It was the aim to influence apoptosis and thus amino acid availability for odor formation by feeding resistant starch (300 g kg(-1) feed) over the whole fattening period to 40 pigs. Concentrations of 12 key components (indoles, volatile fatty acids, methanethiol) were measured in feces and headspace over the slurry duct and compared to 40 normally fed controls in a separate compartment. Concentrations of substances resulting from amino acids were reduced in feces by 70% (indoles) and 8% (branched chain fatty acids) and in the headspace by 72% and 20%. Resistant starch only led to minor increases of straight chain fatty acid concentration. Maximal reduction occurred for 3-methyl-1H-indole (skatole) which is the main determinant of malodor so that the results point to promising strategies for reducing pig odor emission.

Development of a rapid and accurate method for the determination of key compounds of pig odor.

Sampling of odor substances in the emissions from swine production facilities is still the limiting step for routine odor quantification. Solid sorption techniques based on cartridges were developed for three categories of substances (indoles, volatile fatty acids and methylthiol) and were standardized to a sampling time of 15 min. These cartridges also trap dust particles which transport odor substances. Quantification was performed by RP-HPLC or GC. Reliability criteria revealed excellent values for sensitivity (lower ppb level) and repeatability (R.S.D. < 10%), thus they are comparable to fiber solid-phase micro extraction sorption techniques. Parallel determinations in feces and air revealed high correlations (r = 0.99, P < 0.01), so that microbial processes during digestion determine odor quality and hence provide a possibility to reduce odor via feeding.