Polyclonal immunization
A modified GnRH-I peptide sequence (CHWSYGLRPG- NH2, 90% purity, synthesized by Immune Systems Ltd, Torquay, UK) was conjugated to tetanus toxoid (TT, a gift from Dr Mark Lavery, Intervet UK Ltd, Cambridge, UK).24 Male NZB · Balb ⁄ c mice (F1), 6 weeks old were immunized intraperitoneally (i.p.) in study weeks 0, 2, 4 and 6 with 100 lL, 50 lg equivalent of GnRH-I, adsorbed on to an equal volume of Imject Alum (Perbio Science Ltd, Cheshire, UK). Three days after immunization, tail bleeds (100 lL ⁄ mouse) were carried out into capillary tubes (Hawksley and Sons Ltd, West Sussex, UK) and antiserum was obtained following centrifugation at 200 g for 20 min. The specific anti-GnRH-I antibody response was measured by indirect enzyme-linked immuno assays (ELISAs), using a BSA-CHWSYGLRPG-NH2 coated onto 96 well tissue culture plates and 1 : 1000 dilution of sera.25 Mice with the highest titers were boosted i.p. in study week 9 with a double dose of immunogen, made up to 200 lL with phosphate buffered saline (PBS) (0.02 m sodium phosphate buffer, pH 7.4, containing 0.14 m NaCl).
Hybridization and production of Mabs
Hybridomas secreting anti-GnRH-I antibodies were developed by fusion of SP2⁄0 myeloma cells with splenocytes isolated from mice immunized with TTCHWSYGLRPG- NH2 conjugate, using an adapted protocol.26 Approximately 4–6 · 106 immunized spleen cells and 2–3 · 106 mouse myeloma cells were fused using 35% (w ⁄ v) polyethylene glycol (Sigma Aldrich Ltd, Dorset, UK), molecular weight 1500 Da. Invitr o growth of the hybridomas was carried out in complete RPMI-1640 medium (Sigma Aldrich Ltd) containing 10% (v ⁄ v) fetal calf serum (Harlan Sera Laboratory Ltd, Leicestershire, UK), 2% (v ⁄ v) hypoxanthine, aminopterin, thymidine (HAT-50x) or 2% (v ⁄ v) hypoxanthine, thymidine (HT-50x), 1% (v ⁄ v) L-glutamine and 1% (v ⁄ v) penicillin and streptomycin (all supplied by Life Technologies Ltd, Paisley, UK).
Cross reactivity of the Mabs; indirect ELISA
Ninty-six well plates were coated with 0.1 lg ⁄ well equivalent of GnRH-I or GnRH-II or 1 lg ⁄ well equivalent of GnRH-III modified peptides conjugated to bovine serum albumin (BSA) in 100 lL PBS. In order to evaluate the difference between N and C terminal conjugation cross-reactivities, plates were coated with the GnRH-I conjugates, BSA-CHWSYGLRPG- NH2 or HWSYGLRPGC-BSA. The GnRH-II and GnRH-III modified peptide sequences used were BSA-CHWSYGWYPG-NH2 and BSA-CHWSHDWKPG- NH2, respectively, to evaluate cross reactivity of the Mabs to the isoforms. The peptides were synthesized by Immune Systems Ltd and conjugated to BSA (Sigma Aldrich Ltd) using the bifunctional reagent sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1- carboxylate (S-MCC; Perbio Science Ltd).25 Plates were also coated with 3% (w ⁄ v) milk powder (Marvel, Premier Beverage Ltd, Merseyside, UK) containing Tween-20 (0.05%, v ⁄ v) in PBS, to assess the level of non-specific binding.
Assessment of antibody avidity
A competitive enzyme-linked immumnosorbent assay (ELISA)24 was used to assess the binding avidity of the Mabs to the GnRH isoforms. Plates were coated and blocked as for the indirect ELISA using the modified GnRH-I, II and III sequences, conjugated to BSA. Competition was generated by simultaneous incubation of the plate at 37C, for 1 hr, with 50 lL ⁄ well 10- fold serial dilutions of native GnRH-I (Sigma Aldrich Ltd), GnRH-II (Sigma Genosys Ltd, Cambridge, UK) or GnRH-III (Sigma Genosys Ltd) (100 lg, 10 lg, 1 lg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg ⁄ well) in PBS and 50 lL ⁄ well hybridoma cell culture supernatant. The plates were washed and developed as for the indirect ELISA.
Antibody isotyping
Capture ELISAs were carried out to determine the class and subclasses of the polyclonal and monoclonal antibodies using a Sigma immunotypeTM (ISO-2) kit, according to the manufacturer’s instructions (Sigma- Aldrich Ltd).
Examination of GnRH-I tissue distribution
Adult male Sprague–Dawley rats were sacrificed by cervical dislocation. The brain with intact pituitary, liver, spleen, heart and testes were removed and fixed for 24 hr in 10% (v ⁄ v) buffered neutral formalin. The tissues were paraffin-embedded and sagittal sections (5–8 lm) made of the brain and transverse plane cross sections made of the other tissues. These were placed onto silanized slides and rehydrated in PBS.24 Cell smears consisting of peritoneal macrophages were prepared by injecting 10 mL ice cold RPMI-1640 into the peritoneal cavity of a rat using a 21-G needle. The abdominal fluid was withdrawn using a 5-mL sterile pipette and 20 lL smeared onto a silanized slide. In order to obtain spleen and thymus cell suspensions, RPMI-1640 was injected into the spleen (10 mL) and thymus (5 mL) several times. The cell suspensions were collected in a 20-mL universal tube from which smears (20 lL ⁄ slide) were made. Finally, tail blood (100 lL) was collected in heparinized capillary tubes, centrifuged at 200 g for 20 min and the white blood cells resuspended with 200 lL PBS and smeared (20 lL ⁄ slide) onto slides. All the slides were air dried and fixed in Carnoy’s fluids for 10 min, followed by rehydration in descending concentrations of ethanol (100, 96, 80 and 50%) for 3 min in each, followed by PBS for 30 min. Both the tissue and cell smear slides were incubated for 20 min, at room temperature with 0.3% (v ⁄ v) H2O2 in PBS, to remove endogenous peroxidase activity. Immunohistochemistry was achieved by incubating the slides at 37C in a humidified chamber for 30 min, with 7% (v ⁄ v) normal goat serum in PBS, containing 0.05% (v ⁄ v) Tween-20 and then for 2 hr with undiluted 7B101D10 hybridoma cell culture supernatant. Control slides were incubated with either PBS or an unrelated Mab, 1B52F10,27 at 37C in a humidified chamber for 2 hr. After washing 3 times for 5 min in PBS, containing 0.05% (v ⁄ v) Tween-20, the slides were incubated at 37C for 45 min in a humidified chamber, with a 1 : 100 dilution of biotinylated goat antimouse IgG (Sigma Aldrich Ltd). The slides were washed 3 times in PBS, for 5 min and then incubated at 37C for 20 min with a 1 : 50 dilution of extravidin peroxidase (Sigma Aldrich Ltd). Development was carried out at room temperature with 0.1% (w⁄v ) 3¢, 3¢-diaminobenzidine tetrahydrochloride substrate (DAB; Sigma ldrich Ltd) in Tris- HCl buffer, pH 7.6, containing 0.03% (v ⁄v ) H2O2. The reaction was terminated by dipping the slides in distilled water for 5 min, counter-stained for 2 min in Mayer’s Hematoxylin (Sigma Aldrich Ltd) and rinsed in running tap water for 5 min before being mounted in Aqua Immune (Thermo Shandon Inc, PA, USA).
Examination of GnRH-I binding activities
CHWSYGLRPG-NH2 (1 mg), was dissolved in 500 lL PBS and sulfo-NHS-LC-LC-biotin (3 mg, Perbio Science Ltd) was dissolved in 100 lL distilled water. The two solutions were incubated together at room temperature for 30 min, in order to obtain a biotinylated CHWSYGLRPG-NH2. The tissue sections and cell smear slides, were incubated at room temperature for 20 min with 0.3% (v ⁄v ) H2O2 in PBS and blocked at 37C for 45 min in a humidified chamber with 7% (v ⁄ v) normal goat serum in PBS, containing 0.05% (v ⁄ v) Tween-20. They were then incubated for 2 hr with a 1 : 200 dilution of biotinylated CHWSYGLRPG-NH2 in PBS or PBS only for controls. After washing 3 times for 5 min in PBS, the slides were incubated at 37C, for 20 min with a 1 : 50 dilution of extravidin peroxidase conjugate (Sigma Aldrich Ltd) in PBS. The slides were developed, counter-stained and mounted as described before.