A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma.

The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH ß subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH ß subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species.

Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma.

Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level was falsely elevated due to interference from HAs. The interfering antibodies were polyspecific immunoglobulins, with fairly broad species-specificity, which affected the eGH immunoassay by bridging the mouse IgG capture antibody and the rabbit IgG conjugate. This produced artificial sandwiches which led to overestimation of the eGH plasma concentration. Spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma of several species significantly reduced but did not totally eliminate the HAs interference. Immunoglobulins and whole plasma differed in their ability to block the interference, suggesting that HAs may recognize other proteins beside immunoglobulins in animal sera. To investigate whether HAs have any implications in equine clinical practice, we decided to seek information on the incidence of HAs interference in normal animals. We collected single plasma samples from another 114 horses and we found that 5 of these had plasma HAs. Therefore, in total 6 out of the 127 horses examined (4.7%) had plasma HAs generating falsely elevated eGH measures. In conclusion, this study provides the first evidence of HAs in horse plasma interfering with an immunoassay and indicates that veterinary surgeons and diagnostic laboratory staff should be aware of this potential for interference in tests on horse plasma using monoclonal or polyclonal antibody reagents.

Identification of commercial fish species belonging to the orders pleuronectiformes and gadiformes: library of isoelectric focusing patterns.

Isoelectric focusing in polyacrylamide gel was used to establish an identification archive of fish species belonging to the orders Pleuronectiformes, or flat fish, and Gadiformes, or gadoid fish. The 2 orders include species of different commercial value and interest that are frequently requested in European fish markets, but are susceptible to substitution either because they are morphologically similar or because they arrive on the markets already filleted or sliced. The sarcoplasmic protein profiles are species-specific and reproducible. The use of densitometry and image analysis coupled with a simple computer program overcomes the subjective evaluation of the patterns, making it possible to identify species correctly.

Amino acid modifications in canine, equine and porcine pituitary growth hormones, identified by peptide-mass mapping.

Modified amino acid residues in porcine, canine and equine growth hormones purified from pituitary glands were characterised by tryptic mapping and high-performance liquid chromatography with on-line coupled electrospray ionisation mass spectrometry (HPLC-ESI-MS) detection. Hormones from all three species showed the same changes. Conversion of Asp128 to iso-Asp128 was a component of native hormones, while deamidation of Asn12 and Asn98 to Asp and iso-Asp, oxidation of Met4, and cyclisation to the pyroglutamyl derivative of Gln139, probably occurred in vitro, during isolation, storage or hydrolysis. Porcine and canine hormones had indistinguishable protein fingerprints, confirming the assumption, based on their cDNA sequences, that their mature primary structures are identical.

Monoclonal antibody against human growth hormone receptor.

GHR shows a high degree of homology with the prolactin receptor and with the other receptors that belong to the hemopoietic receptor superfamily. This paper describes a monoclonal antibody (MAb) (2B4B6) specific for both the extracellular domain of human GHR and human growth hormone (GH) binding protein. Mice were immunized against a seven-aminoacid peptide sequence screened by FASTA (sequence similarity search served by Genome-Net) from the European Bioinformatics Institute to exclude the existence of human membrane proteins with significant sequence homology. MAbs were screened against the peptide sequence and 2B4B6 was selected for its capability to recognize the full-length hGHBP. As evaluated by both enzyme-linked immunoadsorbent assay (ELISA) and FACS analysis, this MAb seems to recognize and bind to a hGHR positive cell line, IM-9, as well as a murine cell line, BaF3 (8/6), transfected with a chimeric construct, hGHR/hG-CSFR and expressing hGHR on the cell membrane. Studies investigating the biological effects of this MAb showed that anti-hGHR mediated inhibition of cell proliferation was not due to competition with GH binding but rather to prevention of receptor dimerization. Because of its specificity, this MAb may be usefully applied in situations in which GHR and receptors with a high degree of homology, such as PRLR (prolactin receptor), are expressed simultaneously, as occurs in the immune system.

Plasma levels of GH and PRL and concentrations in the fluids of bovine ovarian cysts and follicles.

Prolactin and GH have been detected within the ovary, and it has become increasingly evident that they have a role as intrafollicular regulatory factors. The aim of the present work was to gain an insight into the elements influencing intraovarian GH and PRL in bovine species and to see whether cystic degeneration was accompanied by abnormal bovine GH (bGH) and PRL (bPRL) plasma patterns. We followed the relationships between plasma and ovarian fluid bGH and bPRL concentrations over an entire year in Friesian cows whose ovaries showed distinct types of structures. To assess the presence of bGH and bPRL within ovarian cells, we assayed selected ovarian structures by immunohistochemistry. The results demonstrated that: 1) plasma and ovarian fluid hormonal concentrations were independent, and their ratio was independent of the ovarian structure classes, subclasses and period of the year; 2) in the majority of the cows the concentration of bGH in ovarian fluid was no more than 80% of the level in plasma, whereas in about half the animals bPRL concentrations were higher in the ovary than in peripheral plasma; 3) mean bPRL concentrations in ovarian fluids were significantly higher in summer than in winter; 4) immunoreactive bGH and bPRL were present within granulosa and luteal cells. Thus, it is suggested that in the cow bGH and bPRL levels in the ovary might be regulated in some way independently of the pituitary.

Growth hormone but not prolactin concentrations in the fluid of bovine ovarian cysts are related to the cystic stage of luteinization.

Regulation of follicular growth and ovulation as well as steroid production by the ovary depends principally on gonadotropins. However nonsteroid systemic hormones and autocrine and paracrine factors contribute to the regulation of ovarian function. The objectives of the present work were 1) to asses the presence of growth hormone (GH) and prolactin (PRL) in fluid drawn from normal bovine ovarian follicles, cysts or cystic corpora lutea; 2) to relate the stage of luteinization of the cyst with the GH and PRL concentrations in fluids; and 3) to asses the feasibility of providing a defined nonsteroid hormone marker to distinguish between normal and pathological ovarian structures. Cysts were classified according to histological and morphological appearance as follicular or luteal. Concentrations of GH, PRL, estrogens (E2), progesterone (P4) and testosterone (T) were measured in follicular and cystic fluids. On the basis of the E2 to P4 ratio, ovarian formation classes were further divided into two subclasses (E2 dominant and P4 dominant). The results provide evidence of 1) the presence of immunoreactive GH and PRL in all the follicular and cystic fluids assayed, 2) an increasing concentration of GH correlated to the stage of luteinization of the cyst and a direct correlation between GH and P4 concentrations, 3) a significant variability of intraovarian fluid PRL concentration not related to the histological class of the cyst nor to the concentrations of steroid hormones examined, and 4) the possibility of distinguishing 6 different ovarian formation classes by merely measuring GH, P4, E2 and T concentrations in fluids. These data contribute to a better understanding of the endocrine milieu of bovine ovarian cystic degeneration.

Monoclonal antibodies against recombinant human growth hormone as probes to study immune function.

Monoclonal antibodies were raised against human recombinant growth hormone (rhGH) and those that did not cross-react with other human recombinant proteins like prolactin (PRL), interleukin 2 (IL-2), insulin, or bovine pituitary growth hormone were selected. The selected hybridoma supernatants were studied for their ability to influence T lymphocyte proliferation when induced either by a mitogen, such as phytohemagglutinin (PHA), or by alloantigen. All supernatants inhibited proliferation. Three MAbs were then purified by several passages on antimouse IgG (or IgM)-agarose columns, and characterized. These MAbs recognized three different epitopes, as revealed by competition study, although their inhibitory effect on PHA-induced T cell proliferation was quite similar. The data demonstrate that the MAbs were not cytolytic, that they did not interfere with the PHA binding to T cell membranes, and, as revealed by FACS analysis, did not bind to the membrane. Finally, these MAbs immunoprecipitated a 44-kDa molecule from PHA-activated T cell-concentrated supernatants. These data indicate that the MAbs recognized a soluble factor that plays a central role in T cell proliferation and that is probably the immune growth hormone.

Inverse control of growth hormone and prolactin secretion in clonidine-stimulated dairy cattle.

Clonidine is a specific alpha-2-adrenoreceptor agonist that stimulates growth hormone (GH) release in animals and humans. This drug was used to study the GH and prolactin (PRL) secretory response in dairy cows and heifers. An i.v. infusion of 10 micrograms/kg body weight induced GH release to a peak concentration after 30-60 min, while 2 micrograms/kg had no effect on GH secretory patterns. Plasma PRL decreased significantly (P < 0.01) starting 15-60 min after both doses of clonidine, this effect lasting up to 6 h. Clonidine significantly lowered plasma insulin (P < 0.01) and raised plasma glucose (P < 0.01). The changes in plasma GH, PRL, insulin and glucose differed significantly between doses, the 10 micrograms/kg dose being more effective (P < 0.01). The results of our investigation in dairy cattle provide evidence of (i) an increase in GH release after 10 micrograms/kg clonidine; (ii) a concomitant decrease in PRL secretion, hence GH and PRL secretion in cattle appear inversely controlled; (iii) a significant difference between the effects of the 2 and 10 micrograms/kg doses and (iv) no relationship between the changes in plasma GH and PRL after clonidine and plasma hormone levels before treatment.

Matrix-assisted laser desorption mass spectrometry for the detection of recombinant bovine growth hormone in sustained-release form.

We employed matrix-assisted laser desorption mass spectrometry (LD-MS) to detect recombinant bovine growth hormone (r-bGH) in sustained-release preparations. After preliminary extraction in phosphate buffer, LD-MS provided a precise determination of the molecular mass (M(r)) of the r-bGH contained in 38 sustained-release preparations. The hormone was characterised using enzyme immunoassay, immunoblotting and amino acid sequencing. Rapid detection is essential for analysing large numbers of samples, and for monitoring the use of r-bGH in zootechnical productions and its administration as a "high-tech" drug for therapeutic purposes.

Monoclonal antibodies can reveal immunoreactivity differences between pituitary and recombinant bovine growth hormone.

Monoclonal antibodies (MAbs) to pituitary bovine growth hormone (bGH) were used to assay the immunoreactivity of a recombinant form of bGH. The recombinant hormone used differed from the pituitary principally in the presence of a short amino acid sequence starting with methionine added to the N-terminal end of the molecule. Monoclonal antibody 1D2 recognized the recombinant hormone with greater affinity than the pituitary hormone, whereas MAb 5G1 bound the recombinant molecule with a lower strength than the pituitary. The other MAbs showed different behavior depending on the type of immunoassay used. Results indicate that the recombinant bGH molecule has been altered in its immunological structure, and suggest a possible interaction of the added N-terminal fragment with the three-dimensional structure of the hormone.

Circadian and circannual plasma secretory patterns of growth hormone and prolactin in Fresian heifers: hormonal profiles and signal analysis.

The circadian and circannual secretory patterns of growth hormone (GH) and prolactin (PRL) in Fresian heifers were analysed to identify the significant pulses, the shape and the periodic components of the pulsatile signal that may be relevant to intercellular communication. The possible influence of temperature and photoperiod over these hormonal secretions has also been investigated. In the bovine GH (bGH) secretory patterns the basal levels and number, magnitude and amplitude of significant secretory peaks seem to be independent and not affected by seasonal changes. All the bGH circadian patterns show two periodic components with distinct frequencies. The bovine PRL (bPRL) circadian secretion shows basal levels, number and magnitude of the secretory peaks greater in summer than in winter; the periodic components are only one in winter, but two in summer. There is a significant increase of plasma bPRL in the May-July period and a correlation of the circannual hormone profile with both temperature and daylength.

Binding characteristics of monoclonal antibodies raised against bovine growth hormone.

Monoclonal antibodies have been raised against pituitary bovine growth hormone using the hybridoma procedure. The binding characteristics of the seven selected monoclonal antibodies toward the antigen molecule in its native, chemically or enzymatically treated form have been studied. The reactivities of the monoclonal antibodies with growth hormones from other species and bovine prolactin have also been investigated. The epitopes recognized by four of the produced monoclonal antibodies are conformational, whereas two other monoclonal antibodies bind to sequential determinants. Three antibodies define immunological sites located between residues 6-124 of the bovine growth hormone molecule, and one of this antibody shows higher affinity to human than bovine growth hormone. The immunoreactivity of one monoclonal antibody is enhanced by the previous binding of the antigen to polyclonal antibodies, probably because of a localized conformational change of the bovine growth hormone molecule. This antibody also shows cross-reactivity with all the homologous hormones tested, indicating to recognize a highly conserved antigenic determinant.

Purification to homogeneity of bovine prolactin by high-performance ion-exchange chromatography.

Homogeneous bovine prolactin (bPRL) has been obtained using a procedure based on high-performance anion-exchange chromatography. The procedure enables up to 6 mg of 99.4% pure bPRL to be obtained per hour, with a recovery of 32.4%. The purity of the protein was checked by N-terminal sequencing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The highly purified bPRL obtained with this method is suitable for complete structural and immunochemical studies.

An enzyme-linked immunosorbent assay for the determination of bovine prolactin in plasma.

Highly purified pituitary bovine prolactin (bPRL) has been used in a sensitive non-competitive enzyme-linked immunosorbent assay of prolactin (PRL) concentrations in plasma. In this assay affinity purified polyclonal antibodies were immobilized to the solid phase in order to capture the antigen, and were also used biotinylated as the detector antibody. The method was found to be reproducible (3% variability between calibration curves) and has been optimized for measuring bPRL concentration in plasma samples, giving an intra-assay coefficient of variation of about 5% and an interassay coefficient of variation of about 9%. The sensitivity of the assay was found to be as low as 0.1 ng/ml of bPRL.

A biotin-avidin sandwich enzyme-linked immunosorbent assay of growth hormone in bovine plasma.

Purified pituitary bovine growth hormone (bGH) has been used to develop a homologous sandwich enzyme-linked immunosorbent assay in which affinity-purified antibodies are immobilized on microtiter plates. Bovine GH bound to specific antibody is then revealed with a second anti-bGH antibody labeled with biotin and peroxidase-conjugated avidin. The method requires only 48 h, including the coating step, and has a sensitivity as low as 0.25 ng/ml of bGH. Statistical analyses (test of parallelism, cross-reactivity among bGH and GH of various species and bovine prolactin, recovery test, within- and between-assay variation, comparison with radioimmunoassay) confirm the high specificity and reproducibility of the method.

Thy 1.2 antigen inducing capacity of a calf thymus acid hydrolysate and its fractions.

Splenic null cells of normal mice have been shown to undergo phenotypic induction of Thy 1.2 antigen by various thymic extracts. This experimental model has been employed in characterizing the Thy 1.2 inducing ability of an acid lysate from calf thymus and its various fractions. Incubation of splenocytes from C3H/He mice with unfractionated Thymomodulin induces a dose-response percent increase in the Thy 1.2 antigen bearing cells (9.28 +/- 2.6%, p less than 0.05). Comparable increase appears in fractions 5B (9.46 +/- 0.55%, p less than 0.001) and 5C (8.09 +/- 3%, p less than 0.005), obtained by ultrafiltration and containing peptides of m.w. 600-10,000 d and less than 600 d, respectively. The activity is confined to the acid fraction (6.61 +/- 0.54%, p less than 0.001) isolated by isoelectrofocusing. Control lysate from pig duodenal mucosa was devoid of Thy 1.2 inducing activity. The present findings indicate that Thymomodulin possesses Thy 1.2 inducing capacity on murine null cells and that such activity is maintained and enhanced after various fractionation procedures.