Heterologous Enzyme-linked Immunosorbent Assay Research Articles

Development of a sensitivity-improved immunoassay for the determination of carbaryl in food samples

With the aim of developing a highly sensitive immunoassay for carbaryl, a hapten which had high similarity to carbaryl was synthesised using a safer and more practical approach. After it was conjugated to horseradish peroxidase, direct competitive heterologous enzyme-linked immunosorbent assay (CD-ELISA) was optimised and characterised. The assay performance conditions were investigated in details. Enhanced chemiluminescence ELISA (ECL-ELISA) was also used in preliminary studies. The assay obtained an IC(50) value (the concentration causing 50% inhibition) of 2 microg kg(-1), which was 12-fold more sensitive than previous results of homologous CD-ELISA. In ECL-ELISA, the IC(50) was further decreased 10-fold to 0.2 microg kg(-1). The CD-ELISA developed was applicable for broad conditions, and could be applied on various food samples with a more convenient pre-treatment. Average recoveries were in a range of 88.3-101.7%. The results correlated well with those obtained using high-performance liquid chromatography (HPLC) analysis (R(2) = 0.989). The ELISA developed was a great improvement in the determination of carbaryl, showing that the immunoassay developed was a simple, rapid and efficient method that was reliable for the detection of carbaryl and suitable for rapid quantitative or qualitative determination in food samples.

Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay

The hapten of Flumequine (FLU) with four carbon atoms spacer arm (FLUABA) was synthesized and coupled to bovine serum albumin (BSA) for immunogen by the activated ester method. BALB/C mice were immunized with the artificial immunogen and the splenocytes of immunized mice were fused with SP2/0 cells to obtain the monoclonal antibody (McAb). A hybridoma cell line (named DB6-E7) secreting anti-FLU McAbs was obtained by limited dilution method and screened by heterologous enzyme-linked immunosorbent assay (ELISA). The results showed that the subtype of the obtained McAb was IgG1, and the affinity was about 8.19 × 108 M−1. The haptens of FLU, FLUABA, and FLUACA with different space arms were linked to ovalbumin (OVA) for heterologous or homologous coating antigens. The results of indirect ELISA and indirect competitive ELISA indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. Using FLU-OVA as coating antigen, the ELISA showed an IC50 value of 26.33 μg L−1, a limit of detection (LOD) of 4.0 μg L−1, and the workable range of 8–114 μg L−1 (IC20–IC80). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity (< 0.1%) was detected between the obtained McAbs and the several major quinolones compounds or other structural analogs. The developed ELISA can satisfy the detection criteria of FLU in animal food-products.

Development of a sensitive monoclonal antibody-based ELISA for the detection of clenbuterol in animal tissues

A specific monoclonal antibody (mAb) against clenbuterol, with cross-reactivities less than 0.01% for all test compounds except salbutamol (6.4%), was produced with hybridoma technology. The mAb originated from immunogen of clenbuterol–human serum albumin was combined with coating antigen of salbutamol–ovalbumin to develop a heterologous enzyme-linked immunosorbent assay (ELISA). This assay shows very high sensitivity with IC50 of 0.3 ng/ml and LOD of 0.1 ng/ml when it was run in 0.01 M PBS (pH 7.5). Clenbuterol was spiked in chicken and pork samples and after a simple extraction procedure the extracts at appropriate dilution were analysed by ELISA. Satisfactory results were obtained by both intra-assay, with average recoveries of 81–102% and coefficient variations (CVs) of 3–12%, and inter-assay, with average recoveries of 77–95% and CVs of 5–13%. The survey results of ELISA and HPLC for some real world tissue samples were consistent. It suggests that the mAb-based ELISA will be a feasible quantitative/screening method for clenbuterol residue in animal tissues.

Development of a sensitive competitive indirect ELISA for parathion residue in agricultural and environmental samples

A sensitive competitive indirect enzyme-linked immunosorbent assay (ELISA) for the insecticide parathion was developed. The optimal immunogen was 5-(ethoxy(4-nitrophenoxy)phosphorothioylamino)pentanoic acid. In addition, five competitors were applied for development of a heterologous competitive indirect ELISA. Then several physicochemical factors (organic solvent, ionic strength and pH) that influence assay performance were studied and optimized. The IC 50 and IC 10 of the optimized ELISA were 0.95 and 0.15 ng/mL, respectively, which meant almost 86-fold and 6-fold improvement in the assay sensitivity in comparison with the homologous assay (81.74 ng/mL) and the non-optimized heterologous assay (5.60 ng/mL). Finally, the assay was applied to the analysis of parathion in spiked agricultural and environmental samples without extraction or cleanup. The average recoveries of parathion added to water, soil, cucumber, rice and corn were between 78.57% and 107.67%. The limit of detection (LOD) for water and soil samples was 5 ng/mL, and the LOD for cucumber, rice and corn samples was 10 ng/mL.

Simultaneous Determination of Florfenicol and Its Metabolite Florfenicol Amine in Swine Muscle Tissue by a Heterologous Enzyme-Linked Immunosorbent Assay

A sensitive and heterologous enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) in swine muscle tissue was developed. FFA was conjugated to bovine serum albumin by a formaldehyde coupling method as an immunogen to immunize rabbits. FFA, thiamphenicol glycinate, and modified FF were conjugated to ovalbumin as coating antigens. The effect of different types of hapten heterology on the sensitivity and specificity of the ELISA was evaluated. Using FF glutaric anhydride ester as a coating hapten and antibody raised against modified FFA, an ELISA was developed that showed an IC50 value of 0.48 ng/mL. The antibody showed a cross-reactivity of 100% with FFA, 97% with FF, 6% with thiamphenicol, and a negligible value with chloramphenicol. From fortified swine muscle samples at levels of 4-320 ng/g, the average recoveries of FF and FFA ranged from 58.2 to 96.8%, with coefficients of variation less than 14%. Analysis of incurred samples by the ELISA gave similar results to those by a previously developed liquid chromatographic method. The ELISA could be used as a rapid method for the simultaneous determination of FF and FFA in swine muscle tissue.

Phage-borne Peptidomimetics Accelerate the Development of Polyclonal Antibody-based Heterologous Immunoassays for the Detection of Pesticide Metabolites

Competitive immunoassays for the detection of small analytes, such as pesticides and their metabolites, use haptens that compete with the target compounds for binding to the antibody. This competing hapten can be either the same as the immunizing hapten (homologous assay) or structurally modified mimics of the immunizing hapten (heterologous assay). Polyclonal antibody-based heterologous immunoassays have shown superior sensitivities to homologous ones, butthe synthesis of heterologous haptens may be time-consuming, requiring expertise in synthetic chemistry. In this work we demonstrate that phage display peptide libraries can be used as a source of phage-borne peptidomimetics to facilitate the development of sensitive heterologous assays. Different strategies for the isolation of these peptides were explored using two metabolites of pyrethroid insecticides. The sensitivities of the best competitive phage heterologous enzyme-linked immunosorbent assays were 13 fold and 100 fold better than the homologous assay, for the glycine conjugate of trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzoic acid, respectively. The phage particles were highly versatile as tracer reagents, allowing the use of enzymatic, chemiluminescent, or immuno-polymerase chain reaction detection. The data presented here shows a new systematic procedure that enables the fast generation of several competing haptens for the rapid development of sensitive heterologous immunoassays.

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC 50 values ranged from 0.3 ng mL −1 to 5.5 ng mL −1 by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL −1 to 1.7 ng mL −1 by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1–2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserum G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using D1). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (G1) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1).

Preparation of Anti-Tetracycline Antibodies and Development of an Indirect Heterologous Competitive Enzyme-Linked Immunosorbent Assay to Detect Residues of Tetracycline in Milk

Tetracycline (TC) is a broad-spectrum antibiotic used increasingly in animal husbandry to treat diseases or to promote growth as feed additives. To avoid using labor-intensive instrumental methods to detect residues of TC in food and food products, a simple and convenient indirect heterologous competitive enzyme-linked immunosorbent assay (ELISA) method for TC was developed using polyclonal antibody prepared in this study. Three new immunogens, TC-o-tolidine-bovine serum albumin (BSA), TC- 4-aminobenzoic acid-cationized BSA (cBSA), and TC-1,1'-carbonyldiimidazole-cBSA, were synthesized in this research to develop anti-TC antibodies. All antibodies raised in rabbits and coating antigens synthesized were screened and characterized using homologous and heterologous ELISA formats to select the best combination. An optimized ELISA gave an IC50 value of 3.92 mug/mL toward TC in PBS buffer. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally closely related compounds of chlortetracycline (112%) and oxytetracycline (<2%). The recovery rates from the TC-fortified raw milk samples were in the range of 74-116%, while the intra- and interassay coefficients of variation were <14.5 and <25.0, respectively.

A direct antigen heterologous enzyme immunoassay for measuring progesterone in serum without using displacer

An antigen heterologous enzyme-linked immunosorbent assay (ELISA) for directly measuring progesterone in serum is described. Six combinations of antigens and enzyme conjugates were tested; the enzyme conjugate 17-αOH-progesterone-3-O-carboxymethyloxime-alkalinephosphatase (17-αOH-P-3-CMO-ALP) and the immunogen progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) were found to be best. Fifty microliters of standard or serum sample and 100 μL of the 17-αOH-P-3-CMO-ALP enzyme conjugate were added to the antibody coated wells, and incubated for 1 h at 37 °C. Bound enzyme activity was measured by using p-nitrophenyl phosphate as substrate. The sensitivity of the assay was 0.11 ng/mL, and intra- and inter-assay CVs ranged from 5.1% to 9.6%. The analytical recoveries were 97–105%. The serum progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.97 ( n = 44). Moreover, in this ELISA no displacing agent was used or special means was required to displace progesterone from corticosteroid binding globulin (CBG). Serum progesterone concentrations of subjects, with histories of recurrent spontaneous abortions were also measured, and correlated well with clinical history.

Calcitonin-like immunoreactivity in serum and tissues of the bonnethead shark, Sphyrna tiburo.

Calcitonin is a 32-amino acid peptide hormone that is best known for its actions in maintaining skeletal integrity and calcium homeostasis in mammals. Calcitonin also appears to function in regulating certain aspects of animal reproduction, but the nature of this role remains unclear, particularly in nonmammalian vertebrates. The present study investigated the relationship between calcitonin and reproduction in the bonnethead shark (Sphyrna tiburo), a well-studied member of the oldest living vertebrate group (i.e. elasmobranchs) known to possess a calcitonin-producing organ. Serum calcitonin concentrations were measured in 28 reproductively mature female S. tiburo using a heterologous enzyme-linked immunosorbent assay (ELISA) system. Sites of calcitonin immunoreactivity were detected in tissues of mature female and embryonic S. tiburo using immunocytochemistry. Significant increases in serum calcitonin concentrations of mature female S. tiburo occurred during early stages of gestation, a period characterized by yolk-dependency of developing embryos. Immunoreactive calcitonin was detected in the duodenum and pancreas of embryonic S. tiburo sampled during the same period. The results from this study suggest that calcitonin obtained from endogenous and/or maternal sources may function in regulating yolk digestion in embryonic S. tiburo. Therefore, the association between calcitonin and reproduction in elasmobranchs may reflect an important role for this hormone in embryonic development.

Estrogenicity of domestic and industrial effluents in Sweden

Estrogenicity in Swedish wastewaters was surveyed. Estrogenicity was examined using a recombinant yeast cell test and analyses of the yolk protein precursor in the blood of caged juvenile rainbow trout at sites close to wastewater outlets or in continuous flow tanks of undiluted wastewater. Estrogenic effects corresponding to &amp;lt;0.1 to 15 ng estradiol equivalents were found in municipal effluents. In 9 of 12 industrial effluents, estrogenic effects were below the detection limit of the screen test. None of the six effluents from the pulp and paper industry, from a steel works, and from two chemical industries had a detectable estrogenic effect. Two assays were used for analysis of plasma vitellogenin, a heterologous enzyme-linked immunosorbent assay and a homologous radio immunoassay. Exposure to undiluted municipal wastewater resulted in increased levels of vitellogenin in the plasma of juvenile trout. Using the more sensitive RIA test, increased levels of plasma vitellogenin were detected in cage exposure of rainbow trout in municipal effluent receiving waters. Textile industry effluents mixed with domestic wastewater were estrogenic probably due to contributions from the domestic effluent. In some effluents, toxicity may have masked estrogenic effects.

Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Herbicide Propanil

Although the extensively used postemergence herbicide propanil itself is of low acute toxicity in mammals, it raises environmental concerns due to its effect on aquatic organisms and other adverse impacts. Therefore, in order to obtain a rapid analytical method for this pesticide, an indirect enzyme-linked immunosorbent assay (ELISA) has been developed. Antibodies obtained against a conjugate of 3,4-dichloroaniline coupled to succinylated proteins were tested in hapten-homologous and heterologous indirect ELISA formats using various N -(dichlorophenyl)-succinamic acid derivatives conjugated to carrier proteins as coating antigens. Titers in ELISA were found to be significantly affected by the type and quantity of coating antigen. One of the optimized systems using N -(2,4-dichlorophenyl)-succinamic acid and N -(3,5-dichlorophenyl)-succinamic acid conjugated to ovalbumin allowed serum dilution of 1 : 10,000 and IC 50 values of 2.2 and 2.7 ng/mL for propanil, respectively. The limit of detection (LOD) of the immunoassays is 0.2 ng/mL. Other optimized ELISA systems based on different dichloroaniline-based coating antigens also offered similar sensitivities. The ELISA systems appeared to tolerate methanol and ethanol upto 5% concentration. For confirmatory purposes, the ELISA protocol was compared with a highly sensitive gas chromatographic method coupled with mass spectrometric detection (GC-MS). Spiked propanil content was detected both by ELISA and GC-MS in methanolic rice extract. Detection sensitivities of the two analytical systems appeared to closely correlate with each other in the range of 10-90 ng/mL (0.02-0.18 µg/g), indicating the utility of the immunoanalytical method in detecting propanil content in rice, the main commodity propanil is being applied on.

Assignment of Neisseria meningitidis serogroup A and C class-specific anticapsular antibody concentrations to the new standard reference serum CDC1992.

A new standard meningococcal reference serum designated CDC1992 was prepared to replace meningococcal reference sera ECG and PB-2, which are not available in sufficient quantities for continued use as primary reference sera. CDC1992 was prepared from 14 healthy adult volunteers who underwent plasmapheresis 4 to 12 weeks postvaccination with a single dose of a Neisseria meningitidis quadrivalent polysaccharide vaccine. Total and/or class-specific meningococcal serogroup A and C anticapsular antibody concentrations (in micrograms per milliliter) were assigned to CDC1992 by using homologous and heterologous enzyme-linked immunosorbent assay (ELISA) formats. The reference serum ECG was used as a reference standard to assign total anticapsular antibody concentrations to CDC1992 by a homologous ELISA format. A heterologous ELISA format, with the Haemophilus influenzae type b standard reference serum FDA 1983, was used to assign total and class-specific antibody concentrations to CDC1992. Alkaline phosphatase-labeled mouse anti-human monoclonal antibody conjugates were used as secondary antibodies in both ELISA formats. The total, immunoglobulin G (IgG), IgA, and IgM antibody concentrations, assigned to CDC1992 for serogroup A were 135.8, 91.8, 20.1, and 23.9 micrograms/ml, respectively, and those for serogroup C were 32.0, 24.1, 5.9, and 2.0 micrograms/ml, respectively. Meningococcal serogroup A and C antibody concentrations were in good agreement when homologous and heterologous ELISA format results were compared. Total and class-specific serogroup A and C antibody concentrations were determined in six adult quality control serum samples from the Centers for Disease Control and Prevention by using the homologous ELISA and our assigned antibody concentrations for CDC1992. Antibody concentrations in reference sera ECG and PB-2 were measured in order to provide a historical link to previous studies. The general acceptance of CDC1992 as the standard reference serum and the assigned antibody concentrations will allow investigators to compare antibody levels in serum to those in a single reference preparation.

An enzyme immunoassay for the environmental monitoring of the herbicide bromacil

Competitive enzyme-linked immunosorbent assays (ELISAs) were devised for the environmental monitoring of the herbicide bromacil. The polyclonal antibodies used in this work were raised against two haptens. The bromacil molecule was derivatized at the N-1- and 6-methyl-positions to obtain these haptens with carboxyalkyl [(CH 2 ) n CO 2 H] spacer arms. The antibodies have been examined in several immunoassay formats. Two additional haptens were also synthesized and used for the preparation of coating antigens and enzyme tracers. Some of the heterologous indirect ELISAs in a coating antigen format showed promising sensitivities and, with only a few exceptions, slight cross-reactivities with a series of bromacil metabolites and related compounds

Enzyme-linked immunosorbent assay reactivity of torovirus-like particles in fecal specimens from humans with diarrhea.

Toroviruses are recognized enteric pathogens of cattle and horses; in humans, similar pleomorphic particles have been described, but doubt has been raised concerning their identity as viruses. We screened fecal samples from humans with diarrhea for the presence of torovirus-like particles (TVLPs) by electron microscopy and subsequently used an enzyme-linked immunosorbent assay (ELISA) with bovine torovirus reference reagents to test for the presence of torovirus antigens. To add another selection criterion to this heterologous ELISA, we enriched the TVLPs from the stool specimens by using sucrose density gradients before testing. The results of ELISA and EM correlated significantly, the ELISA having a sensitivity of 68% and a specificity of 86% (chi-square, P < 0.0001). In the gradient, peaks of ELISA reactivity were found at a buoyant density of 1.16 g/ml and were parallel to those found when using bovine torovirus. Furthermore, in 50% of the ELISA-positive gradients, a hemagglutinin for human group O erythrocytes comigrated with the peaks of ELISA reactivity. We were unable to isolate human TVLPs in human colonic tumor or rectal tumor cells. We cloned and sequenced amplification products obtained by low-stringency polymerase chain reaction amplification using consensus primers mapping to the 3' end of the genome of animal toroviruses, but found no significant homologies with animals torovirus sequences. Rabbits were inoculated with material from the gradient peak fractions of human stool specimens, and their sera were assayed for immunologic comparison with bovine torovirus as a reference. A two-way antigenic cross-reactivity was seen between human TVLP and bovine torovirus reagents when tested by ELISA. The rabbit antisera to human TVLP detected a higher number of electron microscopy-positive stool specimens than did the rabbit antisera to bovine torovirus. The application of these assays and reagents should help to elucidate the roles of TVLPs and toroviruses in diarrheal disease in humans.

Development of an enzyme-linked immunosorbent assay for the .beta.-exotoxin of Bacillus thuringiensis

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify the amount of β-erotorin from Bacillus thuringiensis in solution and to evaluate the ability of the antibodies to distinguish among various natural and synthetic compounds related to the β-exotoxin. The β-exotoxin was coupled to several proteins via glutaraldehyde, diazotization, and periodate procedures. The antibodies used in the assay were obtained from antisera raised against β-exotoxin linked to keyhole limpet hemocyanin, and the ELISA coating antigens consisted of β-exotoxin-bovine serum albumin conjugates. Both homologous and heterologous ELISA systems were examined

Determination of follicular fluid estradiol levels by enzyme-linked immunosorbent assay

A direct competitive heterologous enzyme-linked immunosorbent assay (ELISA) was developed and validated to determine follicular fluid estradiol levels of different antral follicular sizes (small, medium, and large) obtained from the ovaries of heifers in the follicular phase of the estrous cycle. Polyclonal antiestradiol antibodies were raised in New Zealand White rabbits using 6-keto-1,3,5(10)-estratriene-3,17β-diol 6-carboxymethyloxime: bovine serum albumin as immunogen and characterized by the usual methods. Horseradish peroxidase conjugated to 1,3,5(10)-estratriene-3,17β-hemisuccinate was used as a label. The concentration ranged used for the standard curve was 0 to 1 ng per well. The low detection limit of the technique was 0.3 pg per well with a sensitivity of 50% binding of 93.62 pg per well. Intraassay and interassay CV (%) were <5.3% and <7.0%, respectively ( n = 10). The recovery rate of the known estradiol concentrations added to follicular fluid averaged 96.1 ± 1.3%. Compared with a radioimmunoassay (RIA), the values of ELISA were highly correlated (r = 0.99, P < 0.005). This assay was used to quantify follicular fluid estradiol concentrations without previous extraction in three antral follicular sizes: small (<5 mm), medium (5.1 to 10 mm), and large (10.1 to 20 mm). The mean ± SE follicular fluid estradiol concentrations were 77 ± 5.2 ng/ ml ( n = 490) in small follicles, 111 ± 19 ng/ ml ( n = 65) in medium follicles, and 496 ± 144 mg/ ml ( n = 45) in large follicles. These values are similar to those previously reported for the same species, age, follicular size, and stage of cycle. We concluded that this technique is useful for determining follicular fluid estradiol concentrations in antral follicles of heifers. Furthermore, its application to other species is now being investigated.

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes

A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicillinase-labeled testosterone-11β-carboxy methyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C 19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation. (Steroids 57:288–294, 1992)

Experimental Infection of Turkeys with Mycoplasma gallisepticum of Low Virulence, Transmissibility, and Immunogenicity

Three-week-old turkeys were inoculated intranasally with approximately 10(6) colony-forming units (CFU) of putative variant Mycoplasma gallisepticum (MG) strains M876, M35, or the virulent S6 reference strain. Uninoculated turkeys in each group served as contact sentinels. The hemagglutination-inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. MG was isolated from 100% and 92% of S6- and M876-inoculated turkeys, respectively, on day 7 PI. However, culture-positive rates among M876-inoculated turkeys declined more rapidly, transmission to contact sentinels took longer and occurred at lower rates, and serologic responses measured by HI and ELISA were lower than in S6-infected turkeys. Testing sera from inoculated turkeys for antibodies to MG in homologous and heterologous ELISA systems indicated that strain M876 was significantly (P less than 0.05) less immunogenic than S6 (days 62 and 95 PI), and that the homologous ELISA was more sensitive (P less than 0.005). MG strain M35 failed to infect turkeys in three attempts, even though the inocula used were viable on culture media.

A rapid and sensitive ELISA for rainbow trout maturational gonadotropin (tGtH II): Validation on biological samples; in vivo and in vitro responses to GnRH

A rapid and sensitive heterologous enzyme-linked immunosorbent assay (ELISA) was developed to measure rainbow trout maturational gonadotropin. Purified salmon maturational gonadotropin (sGtH II) was used as reference hormone. Optimization of the procedure was performed by using an anti-βsGtH serum. Two procedures were developed: an equilibrium assay (which did not involve a preincubation step) which lasted for 8 hr and a nonequilibrium assay (which involved a preincubation step) which lasted for 26 hr. The nonequilibrium assay gave the best sensitivity (70 pg/ml sample). GtH II measurements on in vivo and in vitro samples from GnRH analogs or sGnRH experiments showed that the ELISA procedure could be used over a wide range of concentrations. The method was validated by comparing GtH II concentrations measured by both RIA and ELISA.