Effects of early experience with low-quality roughage on feed intake, digestibility and metabolism in lambs.

The objective of this study was to compare the feed intake, digestibility and metabolism in lambs fed low-quality roughage with those of lambs fed normal roughage from an early stage of their life. The study consisted of two treatments [low-quality roughage group (LR) and control group (C)] over three time periods (P1, P2 and P3; 2 months each). Four lambs (4 months old) were allocated to each treatment. In P1 and P2, LR was fed sudangrass hay (CP: 5.1% DM; NDF: 70.4% DM), whereas C was fed timothy hay (CP: 8.4% DM; NDF: 60.3% DM). In P3, all lambs were fed sudangrass hay. Although the feed intake was significantly greater (p < 0.05) in C in P1, there were no differences between the groups in P2. The digestibility, serum glucose (GLU), urea nitrogen (SUN) and insulin-like growth factor 1 (IGF-1) did not differ between the groups in P1 and P2. The average nitrogen retention, triiodothyronine (T3) and thyroxine (T4) were significantly higher in C (p < 0.05) during P1 and P2. No interaction was observed between the treatment and periods. In P3, the feed intake was greater in C (p < 0.05), whereas digestibility and nitrogen retention tended to be greater in LR. The body weight did not differ between the treatments. T4 and T3 were numerically lower in LR, while the SUN was greater in LR (p < 0.05). These results suggest that the early experience with low-quality roughage may have improved feed digestibility and nitrogen metabolism in lambs after 4 months of rearing. Furthermore, the experienced lambs became more efficient at utilizing the low-quality roughage. The lower thyroid hormone concentrations observed in LR suggest an adaptive change occurred in experienced lambs that to a lower basal metabolic rate.

A possible role of chorion protease in shell membrane degradation during development of quail embryos.

In the eggs of the quail Coturnix japonica, the limiting membrane demarcates the shell membrane at the interface with the albumen and decreases in width during the hatching process. This study was done to identify agents that affect the width of this limiting membrane. Zymography tests on extracts from extraembryonic tissues, yolk sacs, or chorioallantoic membranes, or all three, showed proteolytic activities during d 4 to 10 of incubation. Localization experiments on these activities, performed on d 5 eggs, indicated that they were located in an avascular chorion. Electron microscopic analysis showed there were secretory cells specifically located in the avascular chorion. After partial purification of d 5 avascular chorion extracts using QA52 and Sephadex G-200 column chromatography, the proteolytic activity of 20 kDa was isolated. The protease showed a high level of activity toward succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide. It had an optimal pH of 9 and digested the limiting membrane. These enzymatic activities were inhibited moderately by EDTA and strongly by leupeptin and aprotinin. It was concluded that it is the 20-kDa protease, showing collagenase-like activity produced by the avascular chorion, that affects the limiting membrane.

The crystal polymorphism of calcium carbonate is determined by the matrix structure in quail eggs.

Two calcified structures, the eggshell and sperm-associated body (SB), are present in the eggs of the Japanese quail, Coturnix japonica. X-ray diffractometry showed that calcium carbonates take the form of calcite in the eggshell and aragonite in the SB. The aim of the present study was to identify the factors that determine the morphology of calcium carbonate crystals. The matrix of EDTA-treated eggshell was a meshwork of vesicles, 200 to 500 nm in diameter, connected by fine fibers or fibrous sheets. The matrix of SB cortex was a radiation of rod-shaped projections approximately 130 nm in width. In vitro crystal formation was achieved by adding dissociated matrix substances to test solutions. When eggshell matrix material was added, formation of calcite crystals, which had many vesicular holes on their surface, was observed. When SB matrix material dissociated by sonication was added, rhombohedral calcite crystals formed at protein concentrations of 100 microg/mL or lower, and elongated and bundled crystals formed at concentrations of 150 microg/mL or higher. When SB matrix material dissociated by pipetting was added, aragonite crystals formed. These observations indicate that the matrix structure is the principal factor in determining the crystal polymorphism of calcium carbonate.

Novel approach on the risk assessment of oxidized fats and oils for perspectives of food safety and quality. I. Oxidized fats and oils induces neurotoxicity relating pica behavior and hypoactivity.

Food poisoning caused by deteriorated fat and oil in instant noodles was first reported in Japan approximately 40 years ago. In these cases, many people developed neurotoxic symptoms such as emesis and discomfort. The degree of oxidation of the fat and oil in the instant noodles that induced food poisoning was at least 100 meq/kg in peroxide value (PV). No general toxicity studies with animals, however, have examined the toxicity of fat and oil oxidized to that extent. In this study, pica behavior, a behavior characterized by eating a nonfood material such as kaolin and that relates to the degree of discomfort in animals, and alterations of locomotor activity of rats eating deteriorated fat and oil were measured. The groups fed fat and oil with at least 138.5 meq/kg PV consumed significantly more kaolin compared to the control group. Furthermore, rats that ate deteriorated fat and oil with at least 107.2 meq/kg PV had significantly decreased locomotor activity compared to control rats. These phenomena suggest that oxidized fat and oil with at least 100 meq/kg PV induce neurotoxicity. The toxicity of oxidized fat and oil has only been addressed using general toxicity tests, but the present results reveal the importance of evaluating toxicity by using other measures.

Monoclonal antibodies as probes to detect conformational changes in the rat cysteine proteinase inhibitor cystatin A.

Five monoclonal antibodies (MAbs), 77, 114, 138, 175 and 187, were established for rat cystatin A. MAbs 77, 114, 138 and 175 were shown to belong to the IgG1 subclass, whereas MAb 187 was an IgM. These MAbs partially suppressed inhibitory activity of rat cystatin A to papain. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs with NH2-terminally truncated forms and fragments of rat cystatin A by an enzyme-linked immunosorbent assay (ELISA), and by reactivity with the inhibitor on immunoblotting. In competitive binding assays the MAbs did not compete with each other, indicating that the epitopes recognized by these MAbs were substantially different. The conformational epitope recognized by the three MAbs 114, 138 and 175 belonged to one group that was highly sensitive to denaturation, but those epitopes were unchanged by NH2-terminal truncation. MAb 187 was able to recognize a linear epitope present in amino acid residues 15-50 in the NH2-terminal region. MAbs 77 and 114 reacted weakly with mouse cystatin A but not at all with human cystatin A, whereas MAb 187 reacted similarly with mouse cystatin A but at about half that level with human. The MAbs produced in this study should be useful tools for detecting conformational changes in the rat cystatin A molecule.

Involvement of thyrotropin-releasing hormone receptor, somatostatin receptor subtype 2 and corticotropin-releasing hormone receptor type 1 in the control of chicken thyrotropin secretion.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

Human papillomavirus in squamous cell carcinoma of the vulva by polymerase chain reaction.

OBJECTIVE: To investigate the prevalence of human papillomavirus (HPV) DNA in squamous cell carcinoma of the vulva by polymerase chain reaction (PCR). METHODS: Archival diagnostic phase biopsies from 74 patients with squamous cell carcinoma of the vulva were investigated for HPV DNA by PCR. We used both consensus primers located in the open reading frame L1 and type-specific primers for HPV 6 (open reading frame E5), HPV 11 (open reading frame L1), HPV 16, HPV 18, and HPV 33 (open reading frame E6). RESULTS: HPV DNA was detected in 27 (36%) of the 74 patients, of whom 19 had HPV 16, nine had HPV 18, one had HPV 33, and one had unclassified HPV DNA. No case of HPV type 6 or 11 was detected. Two squamous cell carcinomas were positive for both HPV 16 and 18, and one was positive for both HPV types 16 and 33. Three squamous cell carcinomas positive for E6 gene using type-specific primers were negative using L1 consensus primers. CONCLUSION: Our PCR methods using both consensus open reading frame L1-derived primers and type-specific open reading frame E6-derived primers of HPV types 16, 18, and 33 seemed to be an appropriate combination for the detection of HPV DNA in archival tissues of vulvar carcinoma. Both HPV types 16 and 18 were associated with squamous cell carcinoma of the vulva, although the prevalence of HPV 16 was considerably lower than in cervical carcinoma. It appears that vulvar and cervical carcinomas are not identical etiologically and that factors other than HPV are important in vulvar carcinogenesis.

Disinfection potential of electrolyzed solutions containing sodium chloride at low concentrations.

Electrolyzed products of sodium chloride solution were examined for their disinfection potential against hepatitis B virus (HBV) and human immunodeficiency virus (HIV) in vitro. Electrolysis of 0.05% NaCl in tap water was carried out for 45 min at room temperature using a 3 A electric current in separate wells installed with positive and negative electrodes. The electrolyzed products were obtained from the positive well. The oxidation reduction potential (ORP), pH and free chlorine content of the product were 1053 mV, pH 2.34 and 4.20 ppm, respectively. The products modified the antigenicity of the surface protein of HBV as well as the infectivity of HIV in time- and concentration-dependent manner. Although the inactivating potential was decreased by the addition of contaminating protein, recycling of the product or continuous addition of fresh product may restore the complete disinfection against bloodborne pathogens.

Clinical study of the effectiveness of a dual amplified immunoassay (IDEIA PCE Chlamydia) for the diagnosis of male urethritis.

A clinical study of patients with male urethritis (n=316) was undertaken to determine the sensitivity potential for a new dual amplified immunoassay (IDEIA PCE Chlamydia). Increased sensitivity (98.8%, 84/85) was obtained for IDEIA PCE Chlamydia compared to a conventional antigen detection test (IDEIA Chlamydia, 81.2%, 69/85) when testing urine samples. In a smaller patient population (n=104) the positivity rate for the first-void urine tested with IDEIA PCE Chlamydia of 30.8% (32/104) was similar to the 27.9% (29/104) obtained from urethral swabs tested with a DNA probe assay (PACE 2). The increased sensitivity of the test was confirmed with a commercial PCR kit (Amplicor) and nested PCR. The IDEIA PCE Chlamydia kit has the sensitivity potential to be a clinically reliable alternative for detecting Chlamydia trachomatis.

Chicken lutropin acts like follitropin in rat ovarian follitropin receptor: an isoelectric focusing study.

This study investigates whether chicken lutropin (LH) specifically binds to rat ovarian follitropin (FSH) receptor and exerts FSH-like bioactivity. Glycoprotein fraction, prepared from the chicken anterior pituitary gland, was fractionated using isoelectric focusing within a pH range of 3.5-11. Analysis of the focused fractions, by a radioreceptor assay (RRA) specific for FSH in rats using rat ovarian homogenate as receptor source, and 125I-labeled rat FSH as radioligand, detected a large component having an isoelectric point of 10.25. This focusing profile obtained by RRA was quite similar to that obtained by a specific radioimmunoassay (RIA) for chicken LH, but clearly different from that obtained by a specific RIA for chicken FSH, indicating this RRA specifically recognizes chicken LH. Chicken LH fraction prepared from the electrofocused material was used for further studies. The chicken LH preparation was three times more potent than rat FSH in the RRA in displacing the radioligand bound to rat ovarian receptor, while chicken LH facilitated an 8-fold less production of estradiol in dispersed rat granulosa cells than rat FSH. These results suggest that chicken LH acts like rat FSH in rat ovarian FSH receptor, but receptor-binding activity is much higher than biological activity.

Evaluation of a sensitive, in vitro bioassay for chicken thyroid stimulating hormone using FRTL-5 cells, a rat thyroid cell line.

An in vitro bioassay for mammalian thyroid stimulating hormone (TSH) based on TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production in FRTL-5 cells, a rat thyroid cell line, was used to measure chicken TSH. The addition of chicken pituitary homogenate equivalent to > or = 25% of a chicken pituitary gland to cultured FRTL-5 cells increased cAMP within these cells in a dose-dependent manner. The glycoprotein fraction derived from the pituitary homogenate was further fractionated by isoelectric focusing within a pH range of 5 to 11. Analysis of the focused fractions by the bioassay detected three major components with isoelectric points of 9.30, 7.12, and 3.82, in addition to several minor ones distributed over a wide range of pH, from alkaline to acidic. The isoelectric focusing profile obtained by the bioassay was clearly different from those obtained by radioimmunoassay for chicken LH and radioreceptor assay for chicken FSH, indicating that fractions contained chicken TSH. The homogenate of the cephalic portion of the chicken anterior pituitary gland was 4.46 times more active than that of the caudal portion in the bioassay, which is consistent with previous findings on localization of TSH in the chicken pituitary. We conclude that the bioassay using FRTL-5 rat thyroid cells is a sensitive, specific, and time-saving method of measuring chicken TSH.

[Bactericidal effect of acidic electrolyzed water--comparison of chemical acidic sodium hydrochloride (NaOCl) solution].

Acidic electrolyzed water is made recently by various kinds of machines and is widely utilized. In this study, we intended to clarify the relationship between the concentration of chloride and pH in the bactericidal effects with acidic electrolyzed water. The effects of weak or strong acidic electrolyzed water were compared with a pseudo-acidic water of pH adjusted by diluted hydrochloric acid and sodium hydroxide, on Staphylococcus aureus, Staphylococcus epidermidis and Pseudomononas aeruginosa. At pH 5.0 approximately 6.0, 3 bacterial strains were killed soon after being exposed to the acidic water containing chloride 50 mg/liter, and the amount of chloride did not change after allowing to stand open for 6 hours. At pH 2.67 approximately 2.80, the bactericidal effects was observed at the concentration of chloride 5 mg/liter, and 80% of chloride remained after allowing to stand for 6 hours. These results indicated that newly made strong acidic water is more effective under a smaller amount of chloride at pH 2.7, and that weak acidic electrolyzed water should be used, if stable bactericidal effect is expected in cleaning the surroundings.

[Detection of human papillomavirus DNA in the normal genitalia of men].

The study of human papillomavirus (HPV) in the genitalia of all normal males is difficult, therefore cases of chronic prostatitis, a common disease treated in urological clinics, were chosen to identify HPV DNA by using Vira Type (Life Technologies Inc.). Smears of glans and sulcus coronalius of 177 subjects, showed a HPV positive rate of 3.4%, while 86 cases of those cases were negative for HPV in urethral smears. The lack of clinical findings suggests that HPV is an asymptomatic infection. In a follow up examination of 5 HPV positive cases some weeks later, smears of glans, sulcus coronalius and urethra were all negative for HPV. Examination by Vira Type showed that HPV disappeared spontaneously in these cases.

[Detection of human papillomavirus DNA in cases of sexually transmitted diseases (STD)].

Human papillomavirus (HPV) has been detected on the genitalia without any macroscopic abnormality and the possibility of latent infection of HPV has been suggested. Using Vira Type (Toure Co.), we have detected 7 genotypes of HPV DNA under a high stringent condition on the genitalia of patients with sexually transmitted diseases (STD), who were suspected of having had many sexual partners. In male cases of STDs other than condyloma acuminatum, the HPV-positive rate of the glans and sulcus coronarius was 4.7% (5/106). In healthy men, the HPV-positive rate was 6.1% (2/33), while in chronic prostatitis cases it was 3.4% (7/205) and in benign prostatic hypertrophy cases HPV was not detected. In female cases of STDs other than condyloma acuminatum, the HPV-positive rate of uterine cervix was 5.1% (3/58). In pregnant women, the HPV-positive rate was 4.6% (9/197). With regard to the HPV-positive rate within different age groups of STD and non STD cases, the rate tended to be higher in young people. After several weeks, follow-up studies were conducted on HPV-positive cases. HPV DNA was detected in one case of 10 males STD cases and two of 10 pregnant women, and the HPV DNA was the same type as at the first examination. However, after 3-4 months, all three of these cases had become negative for HPV DNA.

[Immunosuppressive acidic protein (IAP) and immunosuppressive substance (ISS) in patients with renal cell carcinoma].

To clarify their usefulness as markers for renal cell carcinoma, serum immunosuppressive acidic protein (IAP) and serum immunosuppressive substance (ISS) were evaluated by TIA (turbidometric immunoassay) for IAP and by SRID (single radial immunodiffusion) for ISS. The mean level of IAP and ISS was beyond each upper normal limit (500, 700 micrograms/ml) in every stage, and especially high in the M1 group. The levels of IAP and ISS were significantly correlated with each other. The determination of IAP and ISS levels after treatment showed a good correlation to the clinical course of the disease. The positive rates of IAP and ISS increased as the stages progressed, respectively. When the influences of pretreatment IAP and ISS level on survival period were investigated, the low IAP or ISS level group (less than two times of the upper normal limit) tended to have a better prognosis than the high level group (more than two times of the upper normal limit) in the M1 patients. These findings suggested that IAP and ISS could be used as markers for monitoring a disease and predicting the prognosis in patients with renal cell carcinoma. As for the positive rate in the combination assay for IAP, TPA and ferritin, or ISS, TPA and ferritin, more than 80% of the patients with low stage renal cell carcinoma had at least one positive marker. This suggested that the combination assay of these three markers was clinically valuable as a disease monitor in patients with renal cell carcinoma.

[Studies of human papillomavirus (HPV) in urological tumors].

Urological tumors were examined for the presence of human papillomavirus (HPV) DNA by using Southern blot hybridization. In 20 male patients with condyloma acuminatum, HPV type 6 was found at 85% (17/20), HPV type 11 at 95% (19/20), HPV type 16 at 5% (1/20) and HPV type 18 at 0% (0/20). In 2 female patients with condyloma acuminatum, HPV types 6, 11, 16 and 18 were found at 100% (2/2), 100% (2/2), 50% (1/2) and 0% (0/2), respectively. All 6 of the patients who were positive for HPV type 6, were also positive for HPV type 11. Two patients were positive for HPV types 6, 11 and 16, the last of which was frequently found in penile cancer and uterine cervical cancer. In 6 patients with penile cancer, two patients were positive for HPV type 16 and negative for HPV types 6, 11 and 18. The remaining 4 patients were negative for all these HPV types. One patient who was positive for HPV type 16 had penile cancer after three previous episodes of penile condyloma acuminatum. From this information, a malignant change in the condyloma acuminatum was assumed to indicate the possible association of HPV type 16 with the process of malignant degeneration. HPV types, 6, 11, 16 and 18 were not detected in a female patient with vulvar cancer. Although HPV was thought to participate in the development of urological tumors except for external genital tumors, all patients examined, consisting of 2 with benign prostatic hypertrophy, 5 with prostatic cancer and 24 with bladder cancer, were negative for HPV types 6, 11, 16 and 18. Eight patients with bladder cancer were negative for HPV type 33.

[Rapid detection of Mycoplasma pneumoniae-specific IgM].

The rapid diagnosis of Mycoplasma pneumoniae infection is important in carrying out chemotherapy in appropriate manner. It is also essential to detect the specific immunoglobulin M (IgM) in order to diagnose infectious diseases. The ImmunoCard Mycoplasma kit (TFB. Inc./Meridian Diagnostics, Inc.) is a 10-min-card-based enzyme-linked immunosorbent assay (ELISA) of IgM antibodies to M. pneumoniae. The ImmunoCard was compared with high density particle agglutination (HDPA) and cold hemagglutinin (CHA). The ImmunoCard test had 98.3% sensitivity, 51.4% specificity, and 72.5% agreement with HDPA (>or =320), but it had 94.3% sensitivity, 87.5% specificity, and 92.2% agreement with clinical diagnosis. Our results indicate that the ImmunoCard Mycoplasma IgM assay is a rapid, simple and valuable procedure which can be analyze small numbers of specimens using a less complicated technique and with no other equipment required. This means that the ImmunoCard is a cost-effective, energy saving and rapid procedure for the detection of M. pneumoniae-specific IgM.

Ontogenic expression profiles of thyroid-specific genes in embryonic and hatching chicks.

The last trimester of the embryonic life of chickens is marked by a steady increase in circulating thyroxine (T(4)) levels, reaching a maximum around hatching. We have measured thyroidal mRNA expression levels of several genes involved in the biosynthesis of T(4), namely sodium/iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptor (TSHR), and thyroid transcription factor 1 (TTF-1), during this period. Subsequently, we measured the expression of these genes in more detail during the entire hatching process and compared the gene expression profiles with concomitant changes in intrathyroidal and circulating thyroid hormone levels. We found that NIS and TPO mRNA expression increased significantly in the perinatal period, whereas Tg mRNA expression rose gradually throughout the last week of embryogenesis but was stable during hatching. TSHR and TTF-1 mRNA levels did not change significantly during the last week of embryonic development and hatching. Our results suggest that the elevated plasma T(4) levels observed in the developmental period studied are caused by an increased synthesis and secretion of T(4) by the thyroid gland. Augmented expression of Tg may play an important role in the increasing T(4) production during the last week of embryonic development, whereas increased NIS and TPO expression around hatching allows the thyrocytes to boost T(4) synthesis even further.

Melanopsin expression in dopamine-melatonin neurons of the premammillary nucleus of the hypothalamus and seasonal reproduction in birds.

Melanopsin (OPN4) is a photoreceptive molecule regulating circadian systems in mammals. Previous studies from our laboratory have shown that co-localized dopamine-melatonin (DA-MEL) neurons in the hypothalamic premammillary nucleus (PMM) are putatively photosensitive and exhibit circadian rhythms in DAergic and MELergic activities. This study investigates turkey OPN4x (tOPN4x) mRNA distribution in the hypothalamus and brainstem, and characterizes its expression in PMM DA-MEL neurons, using in situ hybridization (ISH), immunocytochemistry (ICC), double-label ISH/ICC, and real time-PCR. The mRNA encoding tOPN4x was found in anatomically discrete areas in or near the hypothalamus and the brainstem, including nucleus preopticus medialis (POM), nucleus septalis lateralis (SL), PMM and the pineal gland. Double ICC, using tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis)-and OPN4x antibodies, confirmed the existence of OPN4x protein in DA-MEL neurons. Also, tOPN4x mRNA expression was verified with double ISH/ICC using tOPN4x mRNA and TH immunoreactivity. PMM and pineal gland tOPN4x mRNA expression levels were diurnally high during the night and low during the day. A light pulse provided to short day photosensitive hens during the photosensitive phase at night significantly down-regulated tOPN4x expression. The expression level of tOPN4x mRNA in PMM DA-MEL neurons of photorefractory hens was significantly lower as compared with that of short or long day photosensitive hens. The results implicate tOPN4x in hypothalamic PMM DA-MEL neurons as an important component of the photoreceptive system regulating reproductive activity in temperate zone birds.

Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations.

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.