Development of a Sensitive Enzyme-Linked Immunosorbent Assay for Cattle, Sheep, Rat, and Mouse Luteinizing Hormone1

Abstract

This manuscript reports the development of a rapid, sensitive, and specific-enzyme linked immunosorbent assay (ELISA) suitable for measuring luteinizing hormone (LH) in cattle, sheep, rats, and mice. The LH ELISA used #15 anti-LH serum-coated 96-well plates and peroxidase-labeled bovine luteinizing hormone (bLH). Bovine LH labeled with peroxidase by the periodate method was stored at -15 degrees C for over 20 mo without appreciable loss of activity. With bLH-B5 used as the standard diluted in assay buffer, the LH ELISA had a sensitivity of 79.6 +/- 31 pg/ml, and a 50% displacement point of 359 +/- 69 pg/ml. With rat LH-RP-2 (rLH) used as the standard diluted in assay buffer, the LH ELISA had a sensitivity of 102 pg/ml and a 50% displacement point of 531 pg/ml. The LH ELISA was highly specific for LH from several mammalian species. This LH ELISA was seven times more sensitive for bLH than a radioimmunoassay (RIA) with comparable sample volumes. The LH ELISA was validated for measurement of LH in buffer, tissue culture media, and sera. Depending on the sensitivity desired, the LH ELISA can be conducted in 3 to 48 h, produces no hazardous waste, and can easily be automated. Use of this LH ELISA offers improvements in speed, convenience, economy, sensitivity, and safety over comparable RIA procedures. The LH ELISA was also conducted with other monoclonal and polyclonal antibodies to LH. The LH ELISA can be performed with other LH antisera, provided the antisera has a high affinity and specificity for LH and can be uniformly coated on 96-well plates.

Targeted Loss of Androgen Receptor Signaling in Murine Granulosa Cells of Preantral and Antral Follicles Causes Female Subfertility1

“…Mouse serum luteinizing hormone (mLH) was determined using a species-specific immunofluorometric assay, as previously described [23,48], with modification. The capture antibody used was the anti-LH antibody (5303 SPRN-1; Medix Biochemica), and the detection antibody was the anti-LH antibody (MAb 518B7; supplied by Dr. J Roser, Dept of Animal Science, UC Davis) [49] directly labeled with a Europium chelate using the DE-LFIA Eu-labeling kit (Perkin Elmer) as per suppliers methodology. The mLH detection limit was 0.02 ng/ml, the quantification limit was 0.05 ng/ml, and the within-assay QC was 6.8% at low (0.25 ng/ml), 4.7% at mid (0.49 ng/ml), and 7.4% at high (1.18 ng/ml) range.…”

Detection of Anti-HPV11-L1 Antibodies in Immune Sera from Patients Suffering from Recurrent Respiratory Papillomatosis Using ELISA

“…Moreover, HPV-16 denatured antigens linked to a poly-histidine tag produced in E. coli were previously used for coating in ELISA assays of antisera from HPVinfected women (7). Coating of microplates with antisera is rarely performed, but it has been described in the literature (1,34). To challenge the above-cited studies and to further reduce financial costs, our microplates for sandwich ELISAs were coated first with human antisera, and viral antigen was added after blocking of human antibodies.…”

Development of a Simple Enzyme Immunoassay for the Determination of Ovine Luteinizing Hormone

“…This antibody has been well characterized (Matteri et al, 1987) and shows very high specificity for the LH molecule from diverse mammals. It has been used in development of both RIA and ELISA assays for LH (Matteri et al, 1987;Spearow and Trost, 1987) in a number of species, including sheep. The cross-reactivity of this antibody with ovine FSH and TSH was reported to be 4.7% and 2.12% respectively (Illera et al, 1996).…”

“…Various EIA methods for the quantification of ovine LH have previously been developed (Spearow and Trost, 1987;Maurel, 1991;Dupont et al, 2003;Peclaris et al, 2003;Yildiz et al, 2003). The first three to be developed were based on the coating of plates with an anti-o LH antibody.…”

“…The first three to be developed were based on the coating of plates with an anti-o LH antibody. Spearow and Trost (1987) described a competitive LH EIA based on the coating of plates with the 518b7 monoclonal anti-bovine LH β subunit antibody (Matteri et al, 1987), which has high specificity for the LH molecule of several mammalian species, including sheep. Recently, Peclaris and colleagues (2003) described a competitive EIA in which the LH antigen is used to coat the plates.…”