Highly sensitive second antibody format enzymeimmunoassay for determination of 13,14-dihydro-15-keto-PGF2alpha in blood plasma of mithun (Bos frontalis)

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in blood plasma of mithun (Bos frontalis; bovine) on microtitreplates using second antibody coating technique and PGFM-horseradish peroxidase as a label has been developed. The wells of the microtitreplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20microl plasma. The PGFM standard curve, with doses ranging from 0.1 to 50pg/well was linear. The sensitivity of the assay was 5pg/ml. PGFM standard curve in buffer showed parallelism with serially diluted mithun plasma containing high endogenous PGFM. Plasma PGFM concentrations estimated by using the developed EIA and commercially available PGFM EIA kit in the same samples were significantly correlated (r=0.98) and showed linearity. Intra- and inter-assay coefficients of variation were below 7%. Recovery of known concentrations of added PGFM in charcoal stripped plasma was linear (r=0.99). The developed EIA was further validated biologically by estimating PGFM in cyclic cows for the entire estrous cycle and in peri-parturient cows beginning day 7 prior to calving till day 30 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of PGFM directly in bovine plasma.     

Standardization and validation of a simple,sensitive, second antibody format enzyme immunoassay for growth hormone determination in mithun (Bos frontalis) plasma

To facilitate research into the action of growth hormone (GH) in mithun (Bos frontalis), we standardized and validated a simple and highly sensitive enzyme immunoassay (EIA) for GH determination in mithun blood plasma on microtiter plates using biotin‐streptavidin amplification system and the second antibody coating technique. Biotin was coupled to GH and used to bridge between streptavidin‐peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 25 µl mithun plasma. The GH standards ranging from 0.25 ng/well/25 µl to 128 ng/well/25 µl were prepared in charcoal‐treated plasma collected from an aged (>10 years) senile mithun. The sensitivity of EIA procedure was 1.0 ng/ml plasma; the 50% relative binding sensitivity was seen at 36 ng/ml plasma. Plasma volumes for the EIA, namely 12.5 and 25 µl, did not influence the shape of standard curve even though a drop in the optical density (OD)₄₅₀ observed with higher plasma volumes was due to higher inherent GH content in mithun plasma collected from an aged (>10 years) senile mithun. For the biological validation of assay, two mithuns were administered with synthetic bovine GH‐releasing factor (GRF; 10 µg/100 kg body weight; intravenous) and another two were administered sterile normal saline (controls). Jugular blood samples were collected at ?60, ?45, ?30, ?15, ?10, ?5, 0, 5, 10, 15, 30 min and thereafter at 15‐min intervals beginning 1 hour before GRF injection up to 8‐hr post treatment, and samples were assayed for plasma GH. In two animals, a peak of GH was recorded at 15 min of GRF administration, which confirms the biological validation of the EIA. Plasma GH estimated in blood samples collected for 6 consecutive weeks from two different age groups of mithun (Group I, age 0–3 months; Group II, age 3–4 yr) showed higher (P < 0.0001) mean plasma GH in younger than in adult mithuns and consequently higher growth rates in the younger group. A parallelism test conducted between a buffer standard curve of bovine GH and GH measured from serial dilution of mithun plasma containing high concentration of endogenous GH showed good parallelism with a standard curve. In conclusion, the EIA developed for GH determination in mithun blood plasma is sufficiently reliable, economic, and sensitive enough to estimate mithun GH in all physiologic variations. Zoo Biol 0:1–11, 2005. © 2005 Wiley‐Liss, Inc.     

Development of a biotin-streptavidin amplified enzyme immunoassay for oxytocin and its application during milk ejection and the reproductive cycle in the mithun (Bos frontalis)

Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductive cyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocin is, therefore, important for studying the control of its secretion and its role in reproduction of the mithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique was therefore developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIA was conducted directly in 200 microl of unknown mithun plasma. Standards prepared in hormone-free plasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA (50, 100, and 200 microl) did not influence the shape of standard curve, even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare endogenous mithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with the bovine standard curve. For biological validation of the assay, plasma oxytocin was measured in the blood samples collected before, during, and after milking in three mithun cows and in six non-lactating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly after udder stimulation was observed. A high level of oxytocin was maintained during milking, falling sharply thereafter. The mean plasma oxytocin concentration was different on different days of the estrous cycle (P < 0.001). Two peaks of oxytocin were recorded, one at day 6 and another at day 18 of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma oxytocin levels in mithuns. Apart from being non-radioactive, the EIA procedure described here also utilizes a highly stable biotinalyted hormone which has a shelf life of several years, unlike the short shelf life of iodinated tracer used in RIA procedures.     

Development and validation of a highly sensitive economic enzymeimmunoassay for prolactin determination in blood plasma of mithun (bos frontalis) and its application during milk let down and cyclicity

The objective of the present study was to develop and validate highly sensitive and economic enzymeimmunoassay (EIA) for prolactin determination in mithun blood plasma on microtitreplates using the biotin-streptavidin amplification system and second antibody coating technique and to apply this procedure during milk let down and cyclicity in mithuns (Bos frontalis), a semi-wild ruminant. Biotin was coupled to prolactin and used to bridge between streptavidin peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 50 microl mithun plasma. The sensitivity of the EIA procedure was 0.1 ng/ml plasma. Plasma volumes viz., 12.5, 25 and 50 microl did not influence much the shape of standard curve though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma prolactin with bovine prolactin standards used in the assay. It showed good parallelism with the bovine standard curve. Plasma prolactin was estimated in six cyclic mithun cows during an estrous cycle. Mean plasma prolactin concentrations around the day of estrus were recorded to be higher than any other day of the cycle. Prolactin profiles were also obtained in three mithuns before, during and after milking. A sharp release of prolactin shortly after udder stimulation was observed. High levels of prolactin were maintained during milking, falling sharply thereafter. In conclusion, the EIA developed for prolactin determination in mithun blood plasma is sufficiently reliable, economic and sensitive enough to estimate prolactin in all physiological variation in mithun.     

Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17β

A sensitive test system has been developed for estimation ofestradiol-17β (E₂) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E₂ is estimated by displacement of biocytinyl-E₂, that was produced by ligation of estradiol-17β, d-glucuronic acid and biocytin. Bound biocytinyl-E₂ is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to ≈120fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E₂ were found in bovine plasma: male or female calves <2.7pg/ml, cycling cow 0.5–7 pg/ml, cow during last month of pregnancy 9–310 pg/ml, mature bull 5–30 pg/ml. However, up to 1110 pg E₂/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sentitive estimation ofestradiol-17β in plasma from all type of cattle and for control of improper use of E₂ after commitment of a threshhold level.     

Application of sensitive enzymeimmunoassays for oxytocin and prolactin determination in blood plasma of yaks (Poephagus grunniens L.) during milk let down and cyclicity

Highly sensitive and specific enzymeimmunoassays for oxytocin and prolactin determination in yak plasma using the biotin-streptavidin amplification system and the second antibody coating technique were validated and applied for determining their profiles during milk let down and cyclicity in yaks. Oxytocin EIA was conducted taking duplicate 200 microl of unknown plasma samples and standards per well. The lowest detection limit was 0.2 pg/well, which corresponded to 1pg/ml plasma. Prolactin EIA was carried out directly in 50 microl of yak plasma. The sensitivity of EIA procedure was 5 pg/well prolactin, which corresponded to 0.1 ng/ml plasma. Mean plasma prolactin concentrations although high at estrus were not statistically different (P > 0.05) from the hormone concentrations on other days. Mean plasma prolactin concentrations during non-breeding season were significantly higher (P < 0.001) than that recorded in breeding season. Oxytocin and prolactin profiles were also obtained in two yaks before, during and after milking. A sharp release of oxytocin and prolactin shortly after udder stimulation was observed. High levels of oxytocin and prolactin were maintained during milking, falling sharply thereafter.     

Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma.

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin-peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 microl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 microl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 microl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl-FSH label (in contrast to the short half life of iodinated FSH in RIA).     

Development of a sensitive enzymeimmunoassay for oxytocin determination in bovine plasma

A highly sensitive and specific second antibody enzymeimmunoassay (EIA) on microtiterplates for oxytocin determination in bovine plasma using the biotin–streptavidin amplification system was developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in the competitive assay. The assay was carried out directly in 200 μl of bovine plasma. Oxytocin standards prepared in hormone-free plasma were used. The sensitivity of the assay was 0.25 pg/well which corresponded to 1.25 pg/ml plasma; the 50% relative binding was seen at 2.8 pg/well. Plasma volumes for the assay ranging from 50 to 200 μl did not influence the shape of the oxytocin standard curve; however a distinct drop in the OD₄₅₀ was observed with higher plasma volumes. The oxytocin antiserum used in the assay showed no significant cross-reaction with other octapeptides tested. The assay was compared with a radioimmunoassay (RIA) procedure employing prior solvent extraction of plasma samples. The oxytocin concentrations assayed by EIA and RIA in plasma samples obtained from four cows before, during and after milking were highly correlated and very similar (r=0.97). Hence the assay developed offers an attractive alternative to the RIA since no prior laborious plasma extraction is needed. Further, the assay has the distinct advantage of being non-radioactive in nature.     

Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei.

A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted.     

Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei.

A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted.     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

A bovine-specific FSH enzyme immunoassay and its application to study the role of FSH in ovarian follicle development during the postnatal period

The primary aim of this study was to develop a FSH enzyme immunoassay (EIA) for the bovine species. The newly developed EIA was validated for FSH determination in bovine plasma by comparison with an existing bovine FSH radioimmunoassay. The EIA detected bovine FSH with a high sensitivity (0.1 ng/ml). Cross-reactivity of the EIA was 0.01% with bovine LH, 51% with ovine FSH, <0.1% with porcine FSH and <0.01% with equine FSH. Using this EIA on different time series of plasma in cows, we have confirmed the presence of a FSH pre-ovulatory peak at estrus, of periodic FSH fluctuations accompanying the waves of terminal follicular development, and of FSH pulses, mainly asynchronous with LH ones, in the peri-ovulatory phase of the cycle. In a second objective, the EIA was used to assess the role of FSH in regulating the development of ovarian follicles up to the small antral stage in young calves. To answer this question, six calves were submitted to weekly blood sampling during their first 3 months of life, and FSH changes were studied concomitantly to those of anti-Müllerian hormone (AMH), a well-established endocrine marker of the ovarian population of small antral follicles in cows. In the ovaries of 3-month calves, the population of 3 to 5 mm follicles contained the highest intra-follicular AMH amounts, and the number of 3 to 5 mm follicles on ovaries was closely correlated with AMH concentrations in the plasma of calves at this age (r_s = 0.94). Before 3 months of age, only two out of six calves showed a clear postnatal FSH peak in plasma, and no correlation was found between plasma FSH and AMH concentrations. These results indicate that female calves undergo different patterns of FSH secretion and that postnatal activation of follicular growth up to the small antral stage appears independent and not directly related to circulating FSH levels.     

Development and validation of a simple, sensitive enzyme immunoassay (EIA) for quantification of prolactin in buffalo plasma

A simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for prolactin quantification in buffalo plasma (on a microtitreplate) using the biotin-streptavidin-peroxidase amplification and immobilized antiserum in a competitive assay. Prolactin standards (range: 5-5000 pg/(well 50 microL)) were prepared in hormone-free plasma collected from minimal stress non-lactating buffalo heifers in temperate weather. The sensitivity of the EIA procedure was 5 pg/(well 50 microL) (corresponds to 0.1 ng/mL plasma); the 50% relative binding sensitivity occurred at 160 ng/(well 50 microL). Plasma volumes for the EIA, viz. 12.5, 25, and 50 microL, did not influence the shape of standard curve. A parallelism test was carried out to compare the endogenous buffalo plasma prolactin with bovine prolactin standard. To validate the assay biologically, 11 Murrah buffaloes were given a third-generation antiprolactin (Norprolac; 10 mg/animal, i.m.). Blood samples were collected 1 d prior to the start of Norprolac administration and continued up to seventh day in an Ovsynch treatment program. In all animals, there were abrupt declines in prolactin concentrations following Norprolac treatments, which confirmed the biological validation of the EIA. After development and validation of EIA procedure, the concentration of plasma prolactin was determined efficiently in samples collected during both summer and winter samples.     

Radioimmunoassays for free and conjugated trienbolone and for trienbolone acetate in bovine tissue and plasma samples.

A specific, sensitive, precise and accurate radioimmunoassay has been developed for the quantitation of the synthetic anabolic steroid trienbolone acetate (TBA) and its major metabolites, free and conjugated trienbolone (TBOH) in bovine tissues and plasma. With the extraction procedure described unspecific interference with the antigen-antibody reaction could be ruled out. The assay can significantly detect amounts of more than 40 pg TBOH and 70 pg TBA. 0.1 - 2.0 g tissue and 0.1 - 1 ml plasma are sufficient for 1 determination. Residues (range 0.1 -2.0 ng/g) were still present in calves 69 days after implantation of "Revalor" (20 mg estradiol-17beta and 140 mg TBA) with the highest concentrations found in liver and TBA could only be quantitated in fat.     

A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate.

Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing. We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N'-dimethylcasein. The substrate 5-(biotinamido)pentylamine is covalently incorporated into N,N'-dimethylcasein by transglutaminase in a calcium-dependent reaction. The biotinylated product is detected by streptavidin-alkaline phosphatase and quantitated by measuring the absorbance at 405 nm following the addition of p-nitrophenyl phosphate. The assay is sensitive, specific, and linear at plasma factor XIIIa concentrations between 0.08 and 1.25 micrograms/ml and at purified guinea pig liver transglutaminase concentrations between 0.05 and 0.8 microgram/ml. The intra-assay coefficient of variation is less than 8%. The solid-phase assay was used to quantitate the transglutaminase activity in Escherichia coli extracts expressing recombinant factor XIII A-chains and to analyze factor XIIIa inhibitors. This method will facilitate the analysis of structure-function relationships of the transglutaminases using recombinant DNA methods. Furthermore, screening of natural and synthetic factor XIIIa inhibitors will be expedited by this solid-phase microtiter plate assay.     

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.     

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).     

A simple, rapid and large capacity ELISA for biologically active native and recombinant human IFN gamma

A rapid and sensitive enzyme-immunoassay for native and recombinant human interferon gamma is described. The test is performed in one step at room temperature and is based on the sandwich principle. The IFN gamma preparation is distributed with horse radish peroxidase-labeled monoclonal antibody to IFN gamma in microtiter plates previously coated with a second mab against IFN gamma. The amount of the IFN gamma mab sandwich fixed in the microtiter plate wells is proportional to the color developed after the addition of peroxidase-specific substrate. The two mab's used in the test neutralize IFN gamma and are directed against the same epitope. For this reason they can only detect the biologically active dimeric form of IFN gamma. The IFN gamma-ELISA works in phosphate buffer as well as in tissue culture medium or human serum. As the assay is routinely performed in 2 hours, the limit of detection is 3 U/ml of IFN gamma (0.3 ng/ml). If the assay is performed in 16 hours, the limit of detection decreases to 0.5 U/ml IFN gamma (0.06 ng/ml). The conditions to preserve the activity of IFN gamma preparation as standard are discussed.     

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.