Endocrine Abstracts

JOINT1258

Introduction: To examine the effect of blocking Luteinizing hormone (LH) in several endocrine disorders, a mouse monoclonal antibody against the β-subunit of LH was developed. Here, the in vitro and in vivo validation of the LHβ-specific antibody is presented.

Materials and Methods: The LHβ-specific antibody was generated by immunizing mice using a recombinant protein fragment corresponding to amino acids S21-L141aa of mouse LH. For in vitro validation of the LHβ-antibody we used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). For in vivo validation of the blocking capacity of the LHβ-antibody, male mice were treated with vehicle, GnRH agonist (Leuprorelin, 1.5 mg/kg), or co-treated with GnRH agonist + LHβ antibody (0.2 mg). The GnRH agonist were injection on day 2 and 4, whereas the LHβ antibody was injected on day 1-4 or 1+3. LH levels were measured on day 2, 4 and 5 with ELISA and testosterone levels were measured on day 2 and 5 with liquid chromatography– tandem mass spectrometry (LC-MS/MS). To examine whether the LHβ antibody was also efficient in blocking high LH levels, ovariectomized (OVX) mice were treated with vehicle or LHβ antibody on day 1-4 (0.2 mg). Blood samples were taken on day 2,4, and 5.

Results: The LHβ-antibody bound the LH peptide fragment (S21-L141aa) in an ELISA where a goat anti-mouse IgG was used to detect the bound complexes. Additionally, the antibody against the LHβ-subunit specifically stained the gonadotropic cells in the mouse pituitary. To prove the blocking capacity in vivo, i.e., whether the LHβ-subunit antibody can block LH-signaling we utilized the fact that a GnRH agonist in male mice initially induces high LH levels and subsequently high testosterone levels. Treatment with LHβ antibody completely reduced LH in all groups at all timepoints. At day 5, LH and testosterone was induced with the GnRH agonist alone compared with vehicle (2.3 vs 0.2 ng/ml and 64 vs. 7 nmol/l, respectively). Compared with the GnRH agonist alone co-treatment with the LHβ antibody on day 1-4 completely abolished LH and testosterone levels on day 5 (2.3 vs 0.02 ng/ml and 64 vs 1.3 nmol/l, respectively). The LHβ antibody was also efficient in blocking the high LH levels in OVX mice (vehicle=17.3 vs. LHβ antibody= 0.1 ng/ml).

Conclusion: A specific LH-blocking antibody was successfully generated, and its blocking capacity was demonstrated in vivo. In subsequent studies the possible non-gonadal effects of LH will be investigated using this LH-blocking antibody.