Attempts to vaccinate ewes and their lambs against natural infection with Haemonchus contortus in a tropical environment

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50 μg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3 week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1 month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50 μg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.     

Effects of active immunization of ram lambs and bull calves against synthetic luteinizing hormone releasing hormone

Ram lambs and bull calves were immunized against LH-RH by injections given in weeks 0, 6, 12 and 28 (ram lambs, week 0 = 16 to 20 weeks of age) and weeks 0, 6, 12 and 18 (bull calves, week 0 = approximately 4 weeks of age). The testis size of LH-RH-immunized animals was significantly less than that of controls from week 13 onwards in ram lambs and from week 15 onwards in bull calves. When ram lambs were sampled in week 17 and bull calves in week 20, mean plasma gonadotrophin and testosterone concentrations were consistently lower in LH-RH-immunized animals than in controls. Single intravenous injection of synthetic LH-RH or an analogue of LH-RH in week 27 failed to induce LH or testosterone responses in LH-RH-immunized ram lambs. Motile semen samples could not be obtained from any of the LH-RH-immunized ram lambs in weeks 24, 25 and 26 or from 7 of 10 in week 72, but samples of moderate motility were obtained in week 72 from three rams in which LH-RH antibody titres had fallen. No attempt was made to obtain semen from bull calves. After castration there was no increase in plasma LH in LH-RH-immunized rams and only a small increase in LH-RH-immunized bull calves. Mean testis weight was significantly lower in LH-RH-immunized animals than in controls of both species. Thus the normal development of the reproductive system in ram lambs and bull calves was blocked by active immunization against LH-RH. Some evidence was obtained for natural reversal of the effects with time and falling antibody titres. These findings demonstrate the potential of LH-RH immunization as an alternative to castration.     

Protective immunity in grouper (Epinephelus coioides) following exposure to or injection with Cryptocaryon irritans

The protective immunity of grouper (Epinephelus coioides) against Cryptocaryon irritans was determined after immunisation by surface exposure or intraperitoneal injection. Specific antibody titres of immunised fish serum and skin culture supernatant were determined by enzyme-linked immunosorbent assay and immobilisation assays. Specific antibody can be detected in some immunised fish at Week 1 and in all immunised fish at Week 2, and the peaks were between Weeks 4-6. Specific antibody was still evident in the serum and skin of immunised fish at Week 8, and provided good protection against challenge with C. irritans. These findings indicated that humoral and skin mucosal immunity play important roles in fish against C. irritans infection.     

Seroprotection against serogroup C meningococcal disease in adolescents in the United Kingdom: observational study

Objective To determine the persistence of bactericidal antibody titres following immunisation with serogroup C meningococcal glycoconjugate vaccine at age 6-15 years in order to examine changes in persistence of antibodies with age.Design Observational study.Setting Secondary and tertiary educational institutions in the United Kingdom.Participants Healthy adolescents aged 11-20 years previously immunised between 6 and 15 years of age with one of the three serogroup C meningococcal vaccines.Intervention Serum obtained by venepuncture.Main outcome measures Percentage of participants with (rabbit complement) serum bactericidal antibody titres of at least 1:8; geometric mean titres of serogroup C meningococcal serum bactericidal antibody.Results Five years after immunisation, 84.1% (95% confidence interval 81.6% to 86.3%) of 987 participants had a bactericidal antibody titre of at least 1:8. Geometric mean titres of bactericidal antibody were significantly lower in 11-13 year olds (147, 95% confidence interval 115 to 188) than in 14-16 year olds (300, 237 to 380) and 17-20 year olds (360, 252 to 515) (P<0.0001 for both comparisons). Within these age bands, no significant difference in geometric mean titres of bactericidal antibody between recipients of the different serogroup C meningococcal vaccines was seen. More than 70% of participants had received a vaccine from one manufacturer; in this cohort, geometric mean titres were higher in those immunised at aged 10 years or above than in those immunised before the age of 10.Conclusions Higher concentrations of bactericidal antibody are seen five years after immunisation with serogroup C meningococcal vaccine at age 10 years or above than in younger age groups, possibly owing to immunological maturation. This provides support for adolescent immunisation programmes to generate sustained protection against serogroup C meningococcal disease not only for the vaccine recipients but also, through the maintenance of herd immunity, for younger children.     

Immunogenic and adjuvant properties of Haemophilus ducreyi lipooligosaccharides

Haemophilus ducreyi, the chancroid-causing bacterium, produces lipooligosaccharides (HdLOS) that comprise 5–11 partially sialylated monosaccharides. Subcutaneous immunisation of mice with 5 μg of HdLOS purified from H. ducreyi strains 4438 and 7470 induced high levels of anti-HdLOS IgG. The antibody responses displayed T-cell-independent features, and were dependent upon Toll-like receptor 4/MyD88 signalling pathways as demonstrated using knockout mice. The immunogenicity of HdLOS was found to require the intact lipid A moiety. The specificity studies of the anti-HdLOS antibodies, as revealed by absorption studies, antibody detection in ELISA, and immune thin-layer chromatography, indicated that the majority of the anti-LOS antibodies were specific for the inner core of the HdLOS. Antibodies to HdLOS failed to inhibit LOS induction of TNF-α release from human mononuclear cells. The adjuvanticity of HdLOS7470 was assessed in BALB/c mice that were immunised with bovine serum albumin (BSA) with or without the addition of HdLOS. The addition of 5 μg HdLOS resulted in a 10-fold increase in the total anti-BSA IgG antibody level as estimated by ELISA. The highest increase was noted for IgG2b, which contrasted with the predominantly IgG1 subclass response to immunisation with BSA alone, indicating an immunomodulatory activity of the HdLOS.     

The effect of the stage of the oestrous cycle and exogenous sex steroids on the transport of starch grains through the genital tract of the ewe, a preliminary study

The starch grain test was studied in two ewes to determine the effect of the stage of the oestrous cycle on their transport through the Fallopian tubes. There was very little difference when the test was performed at oestrus or day 10 of the oestrous cycle. Bilateral ovariectomy had very little effect on the transport of starch grains. When progesterone in oil was administered by intramuscular injection to a bilaterally ovariectomised ewe and an intact ewe there was a marked increase in the number of grains which were recovered. Oestradiol benzoate in oil delayed the recovery of grains and reduced their numbers.     

Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund’s Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.     

Whooping cough after stopping pertussis immunisation.

An epidemic of whooping cough occurred in a rural practice in Shetland, containing 144 children under 16. Before July 1974 all children were immunised against pertussis, but after that date immunisation was stopped. Of the 134 children studied, 93 had been immunised. Sixty-five of the children developed whooping cough. The incidence of infection was similar in those who had and had not been immunised. The incidence was also similar in those born before and after July 1974. There was no evidence to support the routine use of pertussis immunisation in rural Shetland.     

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Model multiple antigenic and homopolymeric peptides from non-repetitive sequences of malaria merozoite proteins elicit biologically irrelevant antibodies

Three model peptides containing B-epitopes from conserved, non-repetitive regions of the merozoite surface antigens, MSA2 and MSA1, and the erythrocyte binding protein EBP of Plasmodium falciparum were synthesised. The peptides incorporated GPG spacers and C residues at the N and C termini, and were polymerised by oxidation to form cystine bridges. Multiple copies of essentially the same peptide sequences were also synthesised on a branching lysyl matrix to form a tetrameric multiple antigen peptide. Rabbits were immunised with the polymerised and multiple antigen peptides, in alum followed by Freund’s adjuvant, and the antibody responses examined by IFA and ELISA. Reproducible antibody responses were obtained against the MSA1 and EBP but not MSA2 peptides. IgG antibody levels detected by ELISA after three injections of antigen in alum, increased significantly after further immunisation in Freund’s adjuvant. IgG levels were largely maintained for at least 23 weeks after the final immunisation. IgM antibodies, generally detectable only after immunisation in Freund’s adjuvant, were absent 23 weeks later. Antibody titres against the native protein on fixed parasites, assayed by IFA, were three to five orders of magnitude lower than the corresponding ELISA titres against the peptides. Antibody-dependent inhibition of P. falciparum growth in vitro could not be demonstrated with the immune rabbit sera. The MSA1 and EBP peptides elicited cross-reactive antibodies. The results suggest that the selected non-repetitive sequences are conformationally constrained in the native proteins and only a small proportion of the anti-peptide antibodies bind to the native proteins. The significance of the findings for the development of peptide vaccines and the use of peptides in immunoassays is discussed.     

Luteinizing hormone (LH) release after single injections of a synthetic LH-releasing hormone (LH-RH) in the ewe at three different reproductive stages and comparison with natural LH release at oestrus

The possibility was investigated of using single i.v. injections of a synthetic luteinizing hormone-releasing hormone (LH-RH) to manipulate the reproductive pattern of the ewe.Single i.v. injections of 150 μg synthetic LH-RH were given on Day 12 of the oestrous cycle, during seasonal anoestrus and on Day 16 $\text{post}$-$\text{partum}$ in ewes which lambed during the breeding season. Blood samples were obtained at 5-, 10- or 15-minute intervals for 1 hour before and for 3 hours after treatment. Plasma LH concentrations were measured using a specific double antibody radioimmunoassay, the development of which is described. Laparotomy was performed on each animal 2–3 days after treatment.The treatment induced LH peaks in all animals and ovulation in the majority. There was no significant difference between the groups in the LH response. The LH release was, however, much less than that found in untreated ewes sampled every 15 minutes for 18 hours during oestrus.     

The effects of injecting [D-Ser(But)6] Des Gly-NH2(10) luteinizing hormone releasing hormone ethylamide in early, mid and late seasonal anoestrus on gonadotrophin secretion and luteal function in the ewe

The ability of the luteinizing hormone releasing hormone (LH-RH) analogue [D-Ser(Bu(t))(6)] Des-Gly-NH(2)(10) LH-RH ethylamide to stimulate the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and to induce ovulation and luteal function in seasonally anoestrous ewes was investigated by injecting the analogue at three stages of the anoestrus (day 118, day 182 and day 235 of the year). After injection on day 118, eight of nine ewes ovulated and all of the former secreted progesterone during the subsequent 20 days. After injection on day 182, six of the nine ewes ovulated, of which none showed luteal function. Only two of the nine ewes were not already secreting progesterone on day 235. Both of these responded to the analogue by secreting normal luteal levels of progesterone. The mean LH peak heights in response to injection at the three stages showed no significant differences from one another. The mean FSH peak heightafter injection on day 182 was significantly lower than the mean FSH peak height associated with the other two challenges (P < 0.05). On day 116 of the following year, 20 ewes were treated with the analogue as before. The high progesterone levels confirmed the results of the day 118 challenge in the previous year. However, none of the ewes conceived when inseminated artificially 24 and 36 hours after analogue treatment.     

Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes

The object of our experiments was to characterize the response of plasma follicle stimulating hormone (FSH) within minutes of an i.v. injection of high or low doses of gonadotrophin releasing hormone (GnRH), especially in relation to contemporary changes in luteinizing hormone (LH) concentrations. In the deep anoestrous period (June), three intact ewes and two ovariectomized ewes were injected with 1 mug synthetic GnRH followed 2 h later by a second identical injection. A week later, the same regimen was repeated with the same sheep but with 50 mug GnRH after an interval of 5 h 20 min. Blood samples were collected every 15 sec for 15 min after each injection (early release), then at longer intervals (main release) till the next treatment, followed by sampling for a further 6-h period after the second treatment. FSH was released as soon as the second minute after GnRH injection in all ewes. The mean pituitary FSH response, during this early release, in intact and ovariectomized ewes was similar after either 1 or 50 mug GnRH. However, the main release was less pronounced in the ovariectomized sheep and was not stimulated after the second treatment in all sheep. Three other ewes were injected with 40 mug GnRH and sampled every 15 sec for seven, 6-min periods during the period of release to compare FSH and LH secretion. The profiles reflected a similarity in sensitivity and responsiveness to GnRH, especially soon after GnRH injection. Increases in both hormones were formed by several grouped associated spikes. It is suggested that a readily releasable pool of FSH exists in the ewe. There are probably differences in the mechanisms of synthesis and/or release between pituitary FSH and LH.     

The binding of ethyl carbamate to liver DNA in gonadectomised mice

Castration, with or without subsequent hormone administration resulted in different levels of binding of a metabolite of ethyl carbamate to mouse liver DNA. In ovariectomised and ovariectomy plus testosterone-treated females, the binding persisted for longer than in intact and ovariectomy plus estradiol-treated females, although the maximum levels of binding were similar in all four groups. In males, orchidectomy dramatically lowered the level of binding compared with that in intact males. Twelve hours after dosing, the levels of binding were 276 and 9.8 nanomoles g. DNA^?1 respectively in the two groups. Estradiol treatment did not restore the level of binding whereas testosterone treatment did.     

Clinical trial with inactivated hepatitis A vaccine and recommendations for its use.

OBJECTIVE--To compare the reactogenicity and immunogenicity of an inactivated hepatitis A vaccine in two different immunisation schedules. DESIGN--Randomised trial. SETTING--One London teaching hospital. SUBJECTS--104 healthy adult volunteers (71 men, 33 women aged 19-60). INTERVENTIONS--Hepatitis A vaccine to group 1 (54 volunteers) at 0, 1, and 2 months and to group 2 (50) at 0, 1, and 6 months. MAIN OUTCOME MEASURES--Symptoms at and after each dose; liver function, hepatitis A virus specific serum immune response; and responses in saliva and parotid fluid in immunised volunteers and subjects with natural immunity. RESULTS--The vaccine was well tolerated; 97% (96/99) and 100% of those immunised developed serum antibody after one and two doses of vaccine respectively. Geometric mean titres increased progressively after each dose and were significantly higher in men but not women in group 2 after the third dose (ratio between geometric mean titres 0.265, 95% confidence interval 0.18 to 0.39; p less than 0.001). At one year this group-sex interaction was absent; geometric mean titres for both sexes were significantly higher in group 2 (ratio 0.330, 0.227 to 0.478; p less than 0.0001). Antibody responses were not significantly different between the groups at two years. Compared with naturally infected subjects immunised volunteers developed poor or undetectable virus specific IgG and IgA responses in saliva and parotid fluid. CONCLUSIONS--The vaccine was safe and highly immunogenic, and the differences in the immune responses in saliva and parotid fluid are unlikely to affect its efficacy.     

Evaluation of test immunisation in the assessment of antibody deficiency syndromes.

Antibody responses after immunisation with pneumococcal polysaccharide did not correlate with the severity and frequency of infections in 22 patients with severe hypogammaglobulinaemia, when these were measured by a Farr radioimmunoassay. Five "healthy" patients with severe hypogammaglobulinaemia not only failed to make antipneumococcal polysaccharide antibody, when measured by radioimmunoassay, but also had very low or unrecordable antibody responses to Escherichia coli and failed to produce antibody when immunised with tetanus toxoid. Some of these subjects, however, did make small amounts of IgM antipneumococcal polysaccharide antibody when this was measured by an enzyme linked immunosorbent assay, while others retained some ability to produce IgM or IgA or both in their saliva. These findings show that the measurement of serum antibody responses after immunisation, with the possible exception of IgM antibodies to polysaccharides, is unlikely to be helpful in assessing the requirement for gammaglobulin replacement therapy in patients with hypogammaglobulinaemia.     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

Effects of an opioid antagonist on pulsatile luteinizing hormone secretion in the ewe vary with changes in steroid negative feedback

In ewes during the breeding season, estradiol (E) and progesterone (P) synergistically regulate pulsatile luteinizing hormone (LH) secretion. E primarily inhibits LH pulse amplitude and P inhibits LH pulse frequency. To determine if endogenous opioid peptides (EOP) mediate these negative feedback effects, we administered the long-acting opioid antagonist WIN 44,441-3 (WIN) to intact ewes during the luteal and follicular phases of the estrous cycle and to ovariectomized ewes treated with no steroids, E, P, or E plus P. Steroid levels were maintained at levels seen during the estrous cycle by Silastic implants placed shortly after surgery. WIN increased LH pulse frequency, but not amplitude, in luteal phase ewes. In contrast, during the follicular phase, LH pulse amplitude was increased by WIN and pulse frequency was unchanged. Neither LH pulse frequency nor pulse amplitude was affected by WIN in long-term ovariectomized ewes untreated with steroids. In contrast, WIN slightly increased LH pulse frequency in short-term ovariectomized ewes. WIN also increased LH pulse frequency in ovariectomized ewes treated with P or E plus P. WIN did not affect pulse frequency but did increase LH pulse amplitude in E-treated ewes. These results support the hypothesis that EOP participate in the negative feedback effects of E and P on pulsatile LH secretion during the breeding season and that the inhibitory effects of EOP may persist for some time after ovariectomy.     

Pertussis: should we immunise neurologically disabled and developmentally delayed children?

A total of 400 children with neurological disorders were studied to ascertain whether they had been immunised against pertussis, the reasons for non-immunisation, and the "validity" of these reasons, as judged by interpretation of the recommendations of the Department of Health and Social Security. The results for this group were compared with those for a group of 400 aged matched controls. The study group had a significantly lower rate of immunisation than controls (p less than 0.01); rates for both groups fell sharply after 1975. A total of 192 study patients and 186 controls were not immunised. Those children with cerebral palsy had the lowest rate of immunisation (19%) and the highest number of valid reasons for non-immunisation (63%). Paediatricians apparently advised against immunisation in 61 (32%) of the index group but in only four (2%) of the controls. The risk of serious neurological handicap after pertussis immunisation is small and there is little evidence to support the view that underlying neurological disease predisposes a child to increased risk. The advice currently given by paediatricians may need to be reconsidered.     

The production and characterisation of antisera to 17-hydroxyprogesterone

Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7 alpha-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7 alpha-CETE-KLH) or with 17-OHP-3-(O-carboxymethyl)oxime-KLH (17-OHP-3-CMO-KLH). The resultant antisera were assessed using [3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 X 10(9) 1/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7 alpha-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10-20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO-KLH were further assessed using [125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 X 10(10) 1/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.