Physico-chemical characterization of growth hormone from water buffaloes (Bubalus bubalis)

A purified preparation of growth hormone from pituitaries of water buffaloes (Bubalus bubalis) has been extensively characterized with regard to physico-chemical properties. The molecular size of buffalo GH (buGH) by electrospray ionization mass spectroscopy (ES-MS) was found to be 21394.00+/-8.44Da and its stokes radius was determined as 2.3 nm. Size heterogeneity in buffalo GH was checked both by electrophoresis and molecular sieve chromatography using 125I-labelled buffalo GH. Similar size heterogeneity was found in standard preparations of ovine and bovine growth hormones. Isoelectric focussing and chromatofocussing indicated charge heterogeneity in buffalo GH preparation. Major charge isoforms having pI of 7.2, 7.7 and minor forms in the pI range of 5.7 to 7.0 were found. Lectin chromatography on Concanavalin A matrix showed that less than 1% of buffalo GH was glycosylated. Heterogeneity in NH2-terminal sequence was also observed, with alanine, phenylalanine and methionine as the NH2-terminal residues as checked by dansyl and DABITC methods. Estimation of tryptophan residue indicated that a single tryptophan residue was present. Ellman's method showed presence of two disulfide bridges per mole of buffalo GH. Intrinsic fluorescence spectrum of buffalo GH exhibited lambda emission maximum at 337 nm. UV-CD spectrum showed that almost 48% of the secondary structure of buGH was constituted by alpha-helicity. The T(M) of buGH as determined by differential scanning calorimetric (DSC) studies was found to be 63 degrees C.     

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.     

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.     

Luteinizing hormone in blood plasma of post-partum buffaloes (Bubalus bubalis)

The changes in luteinizing hormone concentration were measured by heterologous double-antibody radioimmunoassay in blood plasma of 28 Murrah buffaloes. The LH concentration fluctuated between 0.22 to 0.78 ng/ml during first 21 days post-partum. The level increased significantly (P < 0.001) at estrus. The basal LH concentration of second and third week post-partum was inversely related to the first post-partum ovulation interval.     

An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.     

An enzyme-linked immunosorbent assay (ELISA) of denatured cartilage link protein

Antiserum (MB007) was raised in rabbits to SDS-denatured cartilage link protein in order to develop an enzyme-linked immunosorbent assay (ELISA) to quantify link protein in cartilage extracts. The antibodies were characterized by using native and denatured link protein, either as the immobilised or the inhibiting antigen in the assay, and shown to bind more effectively to denatured link protein. At low concentrations, neither hyaluronate (0-30 micrograms/ml), proteoglycan (0-50 micrograms/ml) nor hyaluronate-binding region (0-3 micrograms/ml) competitively inhibited the link protein assay. However, at higher concentrations of proteoglycan (50 micrograms/ml-4 mg/ml) and hyaluronate-binding region (3-40 micrograms/ml) inhibition was observed. A more highly purified proteoglycan and a further purified hyaluronate-binding region preparation showed identical behaviour. The inhibition produced by proteoglycan and hyaluronate-binding region occurred at approximately equivalent molar concentrations (assuming Mr of 10(6) and 7 X 10(4), respectively). These results suggest that a significant proportion of these polyclonal antibodies recognize an epitope common to link protein and hyaluronate-binding region. However, the possibility that these effects are due to contamination with covalently bound link protein cannot be excluded. Trypsinated aggregates (0-10 micrograms/ml) produced no inhibition in the link protein ELISA, but as higher concentrations the inhibition was approximately 2000-fold lower than might have been expected from the link protein concentration present. Thus, the accessibility and/or binding of the antibodies to link protein was substantially decreased, illustrating masking of the link protein antigenic sites, as found by A. Ratcliffe and T.E. Hardingham (Biochem. J. 213 (1983) 371-378). These studies indicate that link protein in tissue extracts may be quantified in the concentration range 30-200 ng/ml and in the presence of hyaluronate, proteoglycan and hyaluronate-binding region, provided that both the immobilised and extracted link proteins are denatured.     

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.     

Effect of Bromocryptine on hormones and milk secretion in Murrah buffaloes ( Bubalus bubalis)

An experiment was carried out on 10 advance pregnant Murrah buffaloes to determine the role of hormones in milk secretion around parturition. Experimental animals were administered with a single injection of bromocryptine, @ 100 μg/kg BW, for 5 days before expected calving, whereas control group buffaloes were injected with placebo injections. Blood samples collected before parturition (-5,-4,-3,-2,-1 days), on day of parturition (day-0) and on day 1, 2, 3, 4, 5, 10 and 15 post partum were analyzed for growth hormone (GH), insulin like growth factor-I (IGF-I) and prolactin (PRL) by radioimmunassay methods. Milk samples were collected daily for 5 days and on day 10 and 15 after parturition. Milk fat, protein, lactose, citric acid, non-esterified fatty acids (NEFAs) and somatic cell counts (SCCs) were determined in milk samples. Bromocryptine treatment significantly (P < 0.01) decreased pre partum PRL and increased GH levels (P < 0.01) on day of parturition in experimental buffaloes without influencing plasma IGF-I level. Milk yield was significantly lower (P < 0.01) in experimental than in control group. Further, effect of bromocryptine on milk yield was only for a week. Milk yield increased (P < 0.01) gradually and was similar to control group on day 15 post partum. Bromocryptine treatment significantly increased milk SCC (P < 0.01) and protein content (P < 0.01) but there was no effect of treatment on fat, lactose, citric acid, glucose, milk and plasma NEFA concentration. It was concluded that prepartum suppression of PRL by bromocryptine impairs milk secretion temporarily in ensuing lactation. The significant rise in GH level before parturition and on day of parturition suggests a role of it in milk secretion of buffaloes.     

Optimality assessment in the enzyme-linked immunosorbent assay (ELISA)

An optimality criterion is proposed for evaluating the precision of alternative designs in the enzyme-linked immunosorbent assay. Assay profiles are represented as four-parameter logistic functions with parameter estimation based on either a weighted nonlinear regression or a simple nonlinear regression after a logarithmic transformation. Assay design changes are characterized in terms of their effects on parameters in the four-parameter logistic model. General optimality results are derived for the variance of relative potency estimates in routine assay applications.     

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.     

Induction of parturition in water buffaloes (Bubalus bubalis )

Parturition was induced in ten buffaloes by combining treatments of dexamethasone and vetoestrol, so that they calved about one month before the expected term. Either dexamethasone alone (Group B) or dexamethasone and vetoestrol (Group C) was used. Another five animals served as controls (Group A). Calving was induced in four animals in group B and three animals in group C after two injections of the compounds four days apart. The average time from first injection to calving for these animals was 117.22 hr and 117.66 hr for groups B and C respectively. Induced calves weighed less at birth (P < 0.05) averaging 24.4 and 26.2 kg for groups B and C respectively, than controls (Group A; 30.20 kg). The body weight gain/week among calves was not significantly different (P > 0.05). The service period, number of services/conception and the milk yield of the buffaloes induced to calve was not significantly different (P > 0.05) from their previous records.     

Enumeration of rhizobia by enzyme-linked immunosorbent assay (ELISA)

The use of the enzyme-linked immunosorbent assay (ELISA) to enumerate rhizobia in peat carrier and in soil has been investigated. The ELISA technique takes less time than the conventional plant infection technique often used to enumerate rhizobia present in the presence of other micro-organisms. A minimum of 10₂–10₃ cells are required for a detectable ELISA reaction, limiting the use of this technique when the number of rhizobia is low.     

Measurement of apolipoprotein A-I concentration in nonhuman primate serum by enzyme-linked immunosorbent assay (ELISA)

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for nonhuman primate serum apolipoprotein A-I (apoA-I) is described. The assay is a noncompetitive, sandwich ELISA in which polystyrene microtiter plates were used with purified, monospecific goat anti-monkey apoA-I antibodies adsorbed on the wells. The serum samples were added to the coated wells, incubated, and after washing, antibodies conjugated to horseradish peroxidase were added. After further washing, the bound label was assayed. A heat treatment step, 52 degrees C for 3 hr, was used to maximize the apoA-I immunoreactive sites in diluted serum. Serum samples extracted with chloroform-methanol, delipidated with tetramethylurea, or denatured by heating gave essentially equivalent results. The working range of the apoA-I standards was 0.5 to 5 ng and parallel responses were observed for apoA-I in serum, in isolated HDL, and in buffer as a purified apoprotein. Recovery of apoA-I added to serum was quantitative (106 +/- 3%). The intra- and interassay coefficients of variation were 6.2 and 6.9%, respectively. The enzyme immunoassay yielded values that compared favorably with those obtained by radial immunodiffusion (r = 0.84). ApoA-I concentration in African green monkey serum was highly correlated with the HDL cholesterol concentration (r = 0.86). It is concluded that this ELISA is an accurate and precise method for determination of apoA-I concentrations in primate serum.     

Serodiagnosis of extrapulmonary tuberculosis by enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycobacterium tuberculosis antigenic glycolipids were determined by enzyme-linked immunosorbent assay (ELISA). The 720 sera were collected from adult patients under investigation, suspected with extrapulmonary tuberculosis. The test performance was estimated according to definitive diagnosis in terms of specificity, sensitivity, positive predictive value and negative predictive value. These parameters calculated on 142 sera from patients with extrapulmonary tuberculosis and on 578 sera from patients with different nontuberculosis diseases were 92%, 81.6%, 70.9% and 95.1%, respectively. The specificity decreased to 85% when tuberculosis was associated with cancer or hepatic cirrhosis. In reactivated tuberculosis the sensitivity and the positive predictive value were 86.9% and 83.3%, respectively. Our results showed that ELISA was conclusive for patients with active tuberculosis, before the initiation of the treatment. The sensitivity decreased to 30% in inactive forms. It was demonstrated that ELISA was positive in cases with negative microscopy genitourinary tuberculosis. ELISA could be used as a supporting test in the laboratory diagnosis of active extrapulmonary tuberculosis in adults, disregarding the site involved.     

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of praziquantel concentration in serum

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of praziquantel in the serum was developed. Since praziquantel has no functional group to conjugate with carrier protein, praziquantel was first converted to a compound with an amino group similar to praziquantel. This compound was then conjugated to bovine serum albumin for use as an immunogen, and to horseradish peroxidase, as enzyme-labeled praziquantel, respectively. The conjugate of praziquantel-bovine serum albumin conjugate was used to raise anti-praziquantel antiserum in mice. The direct competitive ELISA was conducted by simultaneously incubating praziquantel and horseradish peroxidase-labeled praziquantel conjugate with anti-praziquantel antiserum over a second antibody and the enzyme activity of the remaining horseradish peroxidase-labeled praziquantel conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0 to 30 ng ml(-1), and the limit of the detection was 0.3 ng ml(-1). The cross reactivities of anti-praziquantel antibody with compounds related to praziquantel were negligible. Using this ELISA, serum levels of praziquantel were easily determined in male Wistar rats up to 8 h following a single intraperitoneal injection at 2 mg kg(-1) of body weight.     

Dot enzyme-linked immunosorbent assay (dot ELISA) for antibodies to Encephalitozoon cuniculi

A dot-ELISA procedure was developed to detect antibodies against Encephalitozoon cuniculi. Sera from 84 rabbits, 22 dogs, 18 squirrel monkeys and 200 mice were tested by dot-ELISA and most also were tested by immunofluorescence. Comparison of the two tests showed that there was excellent agreement of the results (Kappa values greater than or equal to 0.74) in all four species. Dot-ELISA is a simple, quantitative, rapid alternative to immunofluorescence when large numbers of serum samples must be evaluated.