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There is no specific treatment for recurrent Henoch-Schönlein purpura nephritis (HSPN) in a transplanted kidney. We herein report a case of a kidney transplant recipient with recurrent HSPN that was successfully treated with steroid pulse therapy and epipharyngeal abrasive therapy (EAT). A 39-year-old Japanese man developed HSPN 4 years ago and had to start hemodialysis after 2 months despite receiving steroid pulse therapy followed by oral prednisolone, plasma exchange therapy, and cyclophosphamide pulse therapy. He had undergone tonsillectomy 3 years earlier in the hopes of achieving a better outcome of a planned kidney transplantation and received a living-donor kidney transplantation from his mother 1 year earlier. Although there were no abnormalities in the renal function or urinalysis 2 months after transplantation, a routine kidney allograft biopsy revealed evidence of mesangial proliferation and cellular crescent formation. Mesangial deposition for IgA and C3 was noted, and he was diagnosed with recurrent HSPN histologically. Since the renal function and urinalysis findings deteriorated 5 months after transplantation, 2 courses of steroid pulse therapy were performed but were ineffective. EAT using 0.5% zinc chloride solution once per day was combined with the third course of steroid pulse therapy, as there were signs of chronic epipharyngitis. His renal function recovered 3 months after daily EAT and has been stable for 1.5 years since transplantation. Daily EAT continued for >3 months might be a suitable strategy for treating recurrent HSPN in cases of kidney transplantation.

A 57-year-old woman with chronic renal failure due to the thrombotic thrombocytopenic purpura complained of palpitation. A 12-lead ECG showed supraventricular tachycardia with a cycle length of 375 ms. During the electrophysiological study, a tachycardia with a cycle length of 375 ms was reproducibly induced and terminated by atrial extrastimulation. The tachycardia exhibited an inverse relationship between the coupling interval of extrastimulus initiating the tachycardia, and the first postpacing return cycle, as well as an increasing pattern of resetting the tachycardia with an atrial extrastimulus. Ventricular burst pacing during tachycardia produced AV dissociation. Intravenous injections of a low dose (4 mg) of adenosine triphosphate (ATP) terminated the tachycardia without a preceding atrio-His bundle block. The tachycardia was diagnosed as an ATP-sensitive micro-reentrant atrial tachycardia. Real-time endocardial activation mapping using an electroanatomical mapping system revealed that the earliest activation site of the tachycardia was located at the midlateral portion of the crista terminalis. The tachycardia was abolished by focal ablation targeting the earliest activation site during tachycardia. This is the first reported case of an ATP-sensitive micro-reentrant atrial tachycardia associated with thrombotic thrombocytopenic purpura.

A 57-year-old woman with chronic renal failure due to the thrombotic thrombocytopenic purpura complained of palpitation. A 12-lead ECG showed supraventricular tachycardia with a cycle length of 375 ms. During the electrophysiological study, a tachycardia with a cycle length of 375 ms was reproducibly induced and terminated by atrial extrastimulation. The tachycardia exhibited an inverse relationship between the coupling interval of extrastimulus initiating the tachycardia, and the first postpacing return cycle, as well as an increasing pattern of resetting the tachycardia with an atrial extrastimulus. Ventricular burst pacing during tachycardia produced AV dissociation. Intravenous injections of a low dose (4 mg) of adenosine triphosphate (ATP) terminated the tachycardia without a preceding atrio-His bundle block. The tachycardia was diagnosed as an ATP-sensitive micro-reentrant atrial tachycardia. Real-time endocardial activation mapping using an electroanatomical mapping system revealed that the earliest activation site of the tachycardia was located at the midlateral portion of the crista terminalis. The tachycardia was abolished by focal ablation targeting the earliest activation site during tachycardia. This is the first reported case of an ATP-sensitive micro-reentrant atrial tachycardia associated with thrombotic thrombocytopenic purpura.

Abstract To develop chymopapain-induced chemonucleolysis as an established treatment, it is necessary to determine the kinetics of chymopapain in blood and urine following intradiscal injection. To investigate the rate of blood metabolism and urinary excretion of chymopapain following intradiscal injection, we developed a high-sensitivity enzyme immunoassay for chymopapain. The sensitivity for this assay was 1?pg/tube (40?amol). After injecting chymopapain into the nucleus pulposus of humans, levels of blood chymopapain were measured by enzyme immunoassay. The level of chymopapain in blood decreased gradually, with a half-life of 2–3 days. The half-life for urinary excretion was a little longer, at 3 days. It was also found that chymopapain in blood was not present as a free molecule but formed a complex that had a molecular weight of about 120??kDa. These findings suggest that most chymopapain would not have activity in blood.

To develop chymopapain-induced chemonucleolysis as an established treatment, it is necessary to determine the kinetics of chymopapain in blood and urine following intradiscal injection. To investigate the rate of blood metabolism and urinary excretion of chymopapain following intradiscal injection, we developed a high-sensitivity enzyme immunoassay for chymopapain. The sensitivity for this assay was lpg/tube (40amol). After injecting chymopapain into the nucleus pulposus of humans, levels of blood chymopapain were measured by enzyme immunoassay. The level of chymopapain in blood decreased gradually, with a half-life of 2-3 days. The half-life for urinary excretion was a little longer, at 3days. It was also found that chymopapain in blood was not present as a free molecule but formed a complex that had a molecular weight of about 120kDa. These findings suggest that most chymopapain would not have activity in blood.

Mutants of Chlorellathat form dark green colonies under heterotrophic conditions were generated from the parental Chlorella regularisS-88 by MNNG mutagenesis. The mutant Y-21 was the highest in chlorophyll and carotenoids contents among these isolates. Y-21 grew a little slower than its parental S-88 under heterotrophic and photoautotrophic conditions. Although chloroplasts were generated in both S-88 and Y-21 during glucose starvation and under illumination, the chlorophyll and carotenoid contents of Y-21 were approximately 1.5–1.8 times those of S-88 under both growth conditions, suggesting that relative size of chloroplast is larger in Y-21 than in S-88. Consistent with these results, photosynthesis genes on chloroplast DNA were more highly up-regulated in Y-21 than in S-88 during glucose starvation and under illumination. These results lead us a model in which chloroplast generation is sugar-repressed and photoinduced in Chlorella. Y-21 appears to be a mutant in which sugar repression of chloroplast generation is weak.

ABSTRACTAntibody IgG to HIV-1 reverse transcriptase (RT) was measured in three different ways. In immunoassay I, a polystyrene bead noncovalently coated with recombinant RT (rRT) was allowed to react with anti-RT IgG and rRT-β-D-galactosidase (GAL). In immunoassay II, a polystyrene bead noncovalently coated with (anti-2, 4-dinitrophenyl group) IgG was allowed to react with 2, 4-dinitrophenyl-bovine serum albumin (BSA)-rRT, anti-RT IgG and rRT-GAL. In immunoassay III, 2, 4-dinitrophenyl-biotinyl-BSA-rRT and (anti-human IgG γ-chain) Fab'-GAL were substituted for the corresponding conjugates in immunoassay II. The immune complex(es) of the three or/and four components formed on the polystyrene bead was quickly (only 2.5 min) eluted with detergents such as Triton X-100, Tween-20 and CHAPS in the absence or presence of ɛN-2, 4-dinitrophenyl-L-lysine and was transferred to a polystyrene bead successively coated with biotinyl-BSA (covalently), streptavidin and biotinyl-(anti-human IgG γ-chain) Fab' (immunoassays I and II) or with biotinyl-BSA (covalently) and streptavidin (immunoassay III ). By immune complex transfer with detergents, the sensitivity to anti-RT IgG was improved 280 to 800-fold in immunoassay I, 1,800 to 2,600-fold in immunoassay II and 100 to 500-fold in immunoassay III over that of immunoassay I without immune complex transfer, that is, a widely used conventional enzyme immunoassay.

•Pyrolysis gas constitutes gaseous hydrocarbons with 54.7–69.1 wt% yield at 650- °C reaction temperature and 1–3.1-kg/h feeding rate.•We simulated self-supplied operation of a 200-kg/h commercial plant.•The simulation approach yielded a waste-management cost reduction of approximately US $177,000/year.

A mild and efficient two-spet procedure for conjugating rabbit Fab' and horseradish peroxidase using maleimide compounds was developed, and the conjugate obtained was evaluated as an analytical tool in enzyme immunoassay. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxyeyclogexylmethyl) maleimide to introduce maleimide groups. Then, the maleimideenzyme was allowed to react with thiol groups of Fab'. MOre than two thirds of Fab'and the enzyme used was converted to conjugates with a minfaml polymerization, and both activities of the enzyme and antibodies were well preserved in the conjugate. The conjugate Obtained by this method was more suitable for enzyme immunossay thatn polymerized conjugates prepared by the periodate method. The conjugation using N-hydroxysuccinimide ester of m-maleimidobenzoic acid provided similarly less polymerized preparations but was less efficient.

Effect of temperature was examined on the sensitivity of sandwich enzyme immunoassay for human chorionic gonadotropin (hCC) with anti-hCG Fab'-horseradish peroxidase conjugates prepared by using three different reagents (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, glutaraldehyde and metaperiodate). The non-specific bindings of the conjugates to anti-hCC IgG-coated polystyrene balls were much lower at 20°C than at 37°C, and the specific bindings were slightly higher at 20°C than at 37°C. The lowest non-specific binding and the highest specific binding were obtained by incubation at 20°C with the maleimide conjugate. As a result, the sensitivity could be more easily improved by incubation at 20°C than at 37°C and the highest sensitivity was obtained with the maleimide conjugate. Similar results were also obtained for other macromolecular antigens such as human ferritin and -fetoprotein.

A highly sensitive sandwich enzyme immunoassay (EIA) for human ferritin was developed using rabbit anti-ferritin IgG-coated polystyrene balls and affinity-purified rabbit anti-ferritin Fab' labelled with -D-galactosidase from Escherichia coli and compared with the corresponding sandwich radioimmuno assay (RIA). The specific and nonspecific binding of labelled anti-ferritin to the polystyrene balls were examined in relation to the amount of labelled anti-ferritin used per tube, and the highest sensitivity of each immunoassay (0.2 amol/tube in EIA and 2.5 amol/tube in RIA) was obtained by using the minimal amount of the corresponding labelled anti-ferritin (0.71 fmol in EIA and 4.5 fmol=4436 cpm in RIA) which gave a reliable calibration curve. The sandwich RIA was less sensitive, largely because the specific radioactivity of 125I-labelled anti-ferritin used was not sufficiently high.

A sensitive sandwich enzyme immunoassay for human inter-leukin-2 (hIL-2) using monoclonal antibody IS described. A monoclonai anti-hIL-2 IgG-coated poiystyrene ball was incubated with hIL-2 and subsequently with affinity-purified rabbit anti-hIL-2 Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. There was no cross-reaction with interleukins 1 and 1bT, epidermal growth factor, insulin and other protein hormones. The detection limit of hIL-2 was 3 pg/tube or 30 ng/1 using 0.1 ml of cuiture supernatant. When peripheral blood mononuclear cells from healthy subjects aged 22-62 yr were cultured in the absence and presence of phytohemagglutinin P for 48 h, hIL-2 levels in the culture supernatants were <0.03-0.10 g/1 and 0.18-3.7 g/1, respectively.

A small scale sandwich enzyme immunoassay for macromolecular antigens using -D-galactosidase from Escherichia coli and horseradish peroxidase as labels is described. An antibody IgG-coated glass ball of 1.0 mm in diameter was incubated with antigen in 5 l and subsequently with affinity-purified Fab'-enzyme conjugate in 5 l. Using B-D-galactosidase, the detection limits of ferritin, IgE, a-fetoprotein and chorionic gonadotropin were 0.001 amol (600 molecules), 0.03 amol, 0.03 amol and 0.07 amol, respectively. Using peroxidase, the detection limits of ferritin, -fetoprotein and chorionic gonadotropin were 0.005 amol, 0.1 amol and 0.2 amol, respectively. These detection limits were 3 to 30-fold less than those obtained by the previous sandwich enzyme imnunoassays using antibody IgG-coated polystyrene balls of 3.2 mn in diameter in 150 l.

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. Insulin bound to anti-insulin antibodies was separated from free insulin by precipitation with polyethylene glycol. Anti-insulin antibodies in the precipitates were dissociated from insulin and inactivated by incubation with 0.1 mol/1 HCl. The amount of insulin dissociated was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab′-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was improved 1,000-fold as compared with that of the enzyme immunoassay previously described, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab′-horseradish peroxidase conjugate.

A Simpler method for the preparation of monomeric affinity-purified Fab'-ß-D-galactosidase conjugate is described. Rabbit (anti-human IgG) serum was subjected to successive processes of pepsin digestion to convert IgG to F(ab')2′ reduction with 2-mercaptoethy on a column of human IgG-Sepharose 4B. The affinity-purified Fab' thus obtained without using gel filtration was reacted with excess of maleimide groups introduced into ß-D-galactosidase from Escherichia coli. The monomeric Fab'-ß-D-galactosidase conjugate formed was separated from unconjugated Fab' by gel filtration and from unconjugated ß-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B. By immunoenzymometric assay technique for human IgG, the monomeric conjugate was compared with a monomeric conjegate prepared by a previously reported complexmethod and non-monomeric conjugate which contained 3.7 Fab' molecules per ß-D-galactosidase molecule. The present monomeric conjugate provided as sensitive a dose-response curve as the previously reported monomeric conjugate and a more sensitive dose-response curve than the non-monomeric conjugate.

The level of human chorionic gonadotropin (hCG)-like substance in normal serum was measured using 50 l of serum specimen by a highly specific and sensitive sandwich enzyme immunoassay for hCG which did not significantly cross-react with hLH. The level was calculated using a standard hCG and expressed in mIU/ml as that of hCG. In normal male (20–59 years of age) and normal, non-pregnant female (20–42 years of age) adults, the level was below 0.6 mIU/ml with mean levels of 0.16 mIU/ml (n=209) and 0.14 mIU/ml (n=36), respectively. In older subjects, however, the level tended to rise. In normal male adults, aged 60–91 years, the level was mostly below 0.8 mIU/ml (mean=0.56 mIU/ml, n=99), but levels of 15 specimens out of 99 were 1.1–5.5 mIU/ml. In normal, non-pregnant female adults, aged 45–88 years, the level was widely distributed between 0.1 and 11.7 mIU/ml (mean=1.6 mIU/ml, n=141). In these older female adults, levels of hCG-like substance were not well correlated to either levels of hLH and hFSH, while levels of hLH and hFSH were well correlated to each other.

An improved method for the conjugation of Fab' to B-D-galactosidase from Escherichia coli was developed. The enzyme with thiol groups was treated with excess of N, N'-o-phenylene-dimaleimide to introduce maleimide groups, and the maleimide-enzyme was reacted with thiol groups in the hinge of Fab' to form Fab'-B-D-galactosidase conjugate. Gel filtration of Fab' was required only once, and the recovery of Fab' in the conjugate was 71–81%, while in the previous method, gel filtration was required twice and the recovery was 31–38 %. Although the enzyme activity was decreased slightly (8–12%) by the dimaleimide treatment, the conjugate was as useful for sandwich enzyme immunoassay as that obtained by the previous method. 1gG also could be conjugated to B-D-galactosidase more efficiently in the present method than in the previous one.

A monomeric Fab'-horseradish peroxidase conjugate was prepared from a small quantity of rabbit affinity-purified anti-human chorionic gonadotropin (hCG) F(ab')2 using normal goat F(ab')2 as a carrier. Rabbit affinity-purified anti-hCG F(ab')2 (about 0.04 mg) was mixed with 1 mg of normal goat F(ab')2, reduced to Fab' and conjugated to peroxidase using N-succinimidyl 6-(N-maleimido)hexanoate. The rabbit Fab'-peroxidase conjugate was separated from normal goat Fab'-peroxidase conjugate by passage through a rabbit (anti-goat IgG) IgG Sepharose 46 column and shown to be comparable, in both the purity and the ability for a sensitive sandwich enzyme immuncassay of hCG, to that prepared from a sufficient amount of affinity purified anti-hCG F (ab')2 without using a carrier.

β-d-galactosidase from Escherichia coliand horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human α-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The non-specific binding of Fab'–peroxidase conjugates to IgG-coated polystyrene balls was less than that of Fab'-β-d-galactosidase conjugates, and the affinity-purified Fab'–peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding β-d-galactosidase conjugates. However, a large quantity of Fab'–peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than the β-d-galactosidase assay. Other advantages and disadvantages of the two enzymes are discussed.

A solid-phase, enzyme-linked immunoassay for the quantitation of circulating immune complexes was developed using C1q. In this assay, the immune complexes in human serum are adhered to C1q-coated polystyrene tubes and then detected by a β-D-galactosidase-conjugated monovalent fragment (Fab′) of rabbit (anti-human IgG, γ chain-specific) IgG. The enzyme activity bound to the tubes was determined fluorometrically using 4-methylumbelliferyl-β-D-galactopyranoside as substrate. The sensitivity of this assay was as little as 100 ng/tube, which corresponded to 6 μg/ml when 16·7 μl of serum was assayed. The specificity of the assay was demonstrated by the following observations: (1) absence of cross-reaction of monomeric IgG; (2) non-detectability of immune complex in most of the sera from normal subjects; (3) parallelism of the standard curve with dilution of reference serum. The precision of the assay was proved by the demonstration of sufficient within-assay coefficients of variation (10·7–14·9%).

By using IgG-coated polystyrene balls and β-d-galactosidase-labelled Fab', sandwich enzyme immunoassays for human ferritin, immunoglobulin E (IgE), and thyroid-stimulating hormone (TSH) were developed, and their sensitivities were shown to be largely limited by the purity, binding efficiency, and amount of β-d-galactosidase-labelled Fab' used. (1) Their sensitivities were enhanced 10 to 50-fold by using affinity-purified Fab' labelled with β-d-galactosidase. (2) Their sensitivities depended upon the efficiency of specific binding of the labelled Fab' to antigens adsorbed on antibody IgG-coated polystyrene balls. (3) Their sensitivities were improved by using the minimal amount of labelled Fab' that gave a reliable calibration curve. Under optimal conditions the sensitivities obtained for ferritin, IgE, and TSH were 0·09 pg (0·2 amol), 0·48 pg (2·4 amol, 0·2 mIU), and 3 · 2 mμU (5 · 7 amol) per tube, respectively.

Levels of human growth hormone (hGH) in urine samples were determined by a highly specific and sensitive sandwich enzyme immunoassay, which had little cross-reaction with human luteinizing hormone, human follicle-stimulating hormone, human thyroid-stimulating hormone and human prolactin. The volume of urine samples used was 0.05 ml. Urine hGH levels in normal subjects, aged less than 2 yr, 2-5 yr, 6-15 yr and 28-35 yr (male) were 78-113, 19-51, 7.8-25 and 1.1-5.2 ng/g of creatinine, respectively. In dwarfism patients aged 6-15 yr, urine hGH levels were < 0.2-6.0 ng/g of creatinine.

In the previous immune complex transfer enzyme immunoassay for anti-HTLV-I IgG, the transfers of polystyrene beads in and out of test tube were handled with tweezers, and the bound -D-galactosidase activity was measured with a fluorometer. The use of tweezers was considered the primary cause of false-positivity by carryover. Furthermore, the testing of many samples using a tweezer as a means of transfer was difficult. In the present immune complex transfer enzyme immunoassay, polystyrene beads were attached to plates through cylindrical bars and were used in microplate wells. Therefore, no tweezers were required. The bound -D-galactosidase activity was measured with a fluororeader. This allowed the elimination of false-positivity due to carryover and made it easier to test many samples with higher sensitivity and reliability.

A highly specific and sensitive sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. Monoclonal anti-hTSH -subunit IgG1-coated polystyrene balls were incubated with serum samples and subsequently with affinity-purified polyclonal anti-hTSH -subunit Fab′-horseradish peroxidase conjugate. There was no cross-reaction with 38 mIU/tube of human follicle-stimulating hormone, 40 mIU/tube of human luteinizing hormone or 10 IU/tube of human chorionic gonadotropin. The detection limit of hTSH was 0.3 nU/tube or 0.006 mU/1 of serum. Therefore, it was possible to measure very low levels of serum hTSH in pregnant women with Graves' disease. Serum hTSH levels in normal subjects and patients with Graves' disease were 2.45 ± 1.49 (SD) mU/1 (range : 0.61–6.09 mU/1) and 0.086 ± 0.060 (SD) mU/1 (range : < 0.006-0.18 mU/l), respectively. However, the present sandwich enzyme immunoassay was expensive, since affinity-purified polyclonal anti-hTSH -subunit Fab′-peroxidase conjugate was prepared from rabbit anti-hTSH serum.

A highly sensitive sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. Monoclonal anti-hTSH -subunit IgG1-coated polystyrene balls were incubated with serum samples and subsequently with polyclonal anti-human chorionic gonadotropin Fab1-horseradish peroxidase conjugate. Neither hTSH nor anti-hTSH serum was required except for a small amount of hTSH as a standard. The detection limit of hTSH was 0.1 nU/tube or 0.005 mU/1 for most serum samples except for those from pregnant women, since there was no cross-reaction with 209 IU/1 of human follicle-stimulating hormone and 439 IU/1 of human luteinizing hormone when 20 1 of serum sample was used. Serum hTSH levels in normal subjects and Graves1 disease were 1.22 ± 0.70 (SD) mU/1 (range: 0.26–2.65 mU/1) and 0.080 ± 0.062 (SD) mU/1 (range: < 0.005–0.17 mU/1), respectively.

A novel and sensitive noncompetitive enzyme immunoassay for angiotensin I as a peptide model is described. Angiotensin I in buffer containing bovine serum albumin or in plasma was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. the biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. the biotinylated angiotensin I eluted was reacted with anti-angiotensin I Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. the detection limit of angiotensin I was 13 fg (10 amol)/tube and 6.5 ng/1 of plasma, which was 10 to 480-fold lower than that previously reported by competitive radioimmunoassay and competitive enzyme immunoassay. and other peptides could also be measured more sensitively by the present method than by competitive radioimmunoassay.

A method to eliminate human chorionic gonadotropin (hCG) in the sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. hTSH in serum containing hCG was reacted with dinitrophenyl monoclonal mouse anti-hTSH -subunit IgG1, and the complex formed between the dinitrophenyl IgG1 and hTSH was trapped onto affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. hCG in the test serum was largely eliminated by washing the polystyrene balls. Subsequently, the complex on the polystyrene balls was reacted with affinity-purified rabbit anti-hCG Fab′-peroxidase conjugate followed by washing. The complex of the dinitrophenyl IgG1, hTSH and the conjugate was eluted with dinitrophenyl-L-lysine from the polystyrene balls, to which hCG had been nonspecifically adsorbed, and was trapped onto clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. Peroxidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls in the absence and presence of hTSH was not significantly affected by the presence of up to 75,000 IU of hCG per liter of serum. As a result, serum hTSH could be sensitively measured with little interference by hCG.

A highly sensitive bioluminescent assay of -D-galactosidase from Escherichia coli is described. D-Galactose was released from 2-nitrophenyl--D-galactopyranoside as a substrate by the catalytic action of -D-galactosidase, and subsequently NADH was formed using galactose dehydrogenase. NADH was measured by a bioluminescent assay using NAD(P)H:FMN oxidoreductase and luciferase from Photobacterium fischeri. The detection limits of -D-galactosidase for 100 and 1,000 min assays were 2 × 10-21 mol and 2 × 10-22 mol, respectively. When the volume of the reaction mixture for -D-galactosidase assay was reduced from 2 to 0.5 1, the detection limits were reduced to half.

An antibody IgG-coated polystyrene ball was incubated with an antigen and then with affinity-purified Fab'-enzyme conjugate in the presence of normal IgG, F(ab')2, Fab' or Fab'-bovine serum albumin conjugate. After washing by incubation at 30&(d)grave;C for 10 min with shaking, the enzyme activity bound to the polystyrene ball was assayed. The non-specific binding of the Fab'-enzyme conjugate to the polystyrene ball considerably decreased in the presence of normal IgG and the other related proteins, while the specific binding decreased only slightly. As a result, the detection limit of hCG, human IgE and human -fetoprotein was improved 3 to 10-fold.

Urine samples of patients with Graves′ and Hashimoto′s diseases were dialysed and subjected to a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG. Anti-thyroglobulin IgG in urine was reacted simultaneously with 2,4-dinitrophenylated thyroglobulin and thyroglobulin--D-galactosidase conjugate. The immune complex formed consisting of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N-2,4-dinitrophenyl-L-lysine and transferred onto polystyrene balls coated with (anti-human IgG -chain) IgG. -D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Anti-thyroglobulin IgG was detected in most of the patients. The detection of anti-thyroglobulin IgG in urine by the immune complex transfer enzyme immunoassay was suggested to be useful as a diagnostic aid for autoimmune thyroid diseases.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in serum is described. Anti-thyroglobulin IgG in serum was reacted simultaneously with 2,4-dinitrophenyl-thyroglobulin and biotinyl-thyroglobulin. The immune complex formed of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG and, after washing, reacted with streptavidin--D-galactosidase conjugate. After washing, the immune complex was eluted from the polystyrene balls with N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with (anti-human IgG -chain) IgG. -D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Biotinylthyroglobulin and streptavidin--D-galactosidase conjugate could be prepared more easily than thyroglobulin--D-galactosidase conjugate used in the previous immunoassay. Inactive -D-galactosidase, used to eliminate interference by anti--D-galactosidase antibodies in the previous immunoassay, was not required. The present immunoassay was 300-fold more sensitive than the conventional enzyme immunoassay, although 10-fold less sensitive than the previous immunoassay. Antithyroglobulin IgG was demonstrated in all patients with autoimmune thyroid diseases and 54% of healthy subjects.

The nonspecific binding of rabbit anti-ferritin Fab′-B-D-galactosidase conjugate to rabbit anti-ferritin IgG-coated polystyrene balls in two-site enzyme immunoassay for ferritin was reduced to various extents by the presence of nonspecific rabbit F(ab′)2, inactive -D-galactosidase and related substances with only small decreases in the specific binding, and the detection limit of ferritin was improved up to 10-fold. The use of nonspecific F(ab′)2, inactive -D-galactosidase and related substances was suggested to be useful for improving the detection limit of other antigens.

A novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens is described. Antigens were reacted with dinitrophenyl monoclonal mouse antibody IgG1 and rabbit antibody Fab′-ß-D-galactosidase conjugates. The complex formed of antigens with dinitrophenyl monoclonal mouse antibody IgG1 and rabbit antibody Fab′-ß-D-galactosidase conjugates was trapped onto affinity-purified rabbit (antidinitrophenyl bovine serum a1bumin) IgG-coated polystyrene balls. After eliminating excess of the conjugates, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. ß-D-Galactosidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed by fluorimetry. Nonspecifically bound ß-D-galactosidase activity considerably decreased with less decrease in specifically bound ß-D-galactosidase activity. As a result, the detection limits of human thyroid-stimulating hormone (0.01 nu, 0.02 amol) and human growth hormone (10 fg, 0.5 amol) by the present enzyme immunoassay were 30-fold lower than those by the conventional enzyme immunoassay, in which antigens were incubated with monoclonal mouse antibody IgG1-coated polystyrene balls and rabbit antibody Fab′-ß-D-galactosidase conjugates.

A novel and sensitive enzyme immunoassay for anti-thyroglobulin antibodies is described. Anti-thyroglobulin antibodies in test serum were reacted simultaneously with dinitrophenyl thyroglobulin and thyroglobulin-horseradish peroxidase conjugate. The complex formed of anti-thyroglobulin antibodies, dinitrophenyl thyroglobulin and thyroglobulin-peroxidase conjugate was trapped onto two affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as substrate. This assay was more sensitive than the conventional enzyme immunoassays, and anti-thyroglobulin antibodies were demonstrated in part of healthy subjects and in all patients with Graves' disease and chronic thyroiditis.

Human growth hormone (hGH) was demonstrated in normal urine by a highly specific and sensitive sandwich enzyme immunoassay. The molecular weight of hGH in normal urine was shown to be 22,000 by gel filtration, and levels of urine hGH were 15–64 ng/1 or 50–83 ng/g of creatinine in normal subjects aged 1.2–5.9 yr.

A micro-scale method was developed for conjugating a small amount of affinity-purified Fab' to enzymes through thiol groups in the hinge of Fab'. 2,4-Dinitrophenyl groups were introduced into rabbit anti-hCG F(ab')2 before affinity-purification, and the 2,4-dinitrophenyl F(ab')2 (0.2–2.0 mg) was affinity-purified by elution from a column of hCG-Sepharose 46 with 0.1 M glycine-HC1 buffer, pH 2.5, containing normal rabbit F(ab')2 (1.0 mg) as a carrier. The affinity-purified 2,4-dinitrophenyl F(ab')2 mixed with normal F(ab')2 was split into Fab' by reduction, reacted with maleimide groups introduced into enzymes and subjected to gel filtration to separate the conjugates from unconjugated components. Finally, the affinity-purified 2,4-dinitrophenyl anti-hCG Fab'-enzyme conjugates were separated from normal Fab'-enzyme conjugates and unconjugated enzyme, if any, by affinity chromatography on a column of (anti-2,4-dinitro-phenyl) IgG-Sepharose 4B. The conjugate preparations obtained by the micro-scale method were satisfactory in purity, antigen-binding activity and usefulness for sandwich enzyme immunoassay.This method is applicable to the conjugation not only with horseradish peroxidase but with other enzymes, while a previously reported method is applicable only to the conjugation with horseradish peroxidase.

A highly sensitive bioluminescent assay of dehydrogenases was performed. NADH was produced by the catalytic action of alcohol and glucose-6-phosphate dehydrogenases and subsequently measured with high sensitivity by a bioluminescent assay using NAD (P) H : FMN oxidoreductase and luciferase from Photobacterium fischeri. The minimal amount of dehydrogenases that could be measured was 0.0055 amol (5.5 × 10--21 mol).

A “simultaneous” enzyme-linked immunoassay for the measurement of thyroglobulin in human serum was established using polyclonal rabbit antibody, in which rabbit anti-human thyroglobulin IgG-coated silicone rubber rod, rabbit anti-human thyroglobulin monovalent fragment (Fab')--D-galactosidase conjugate and serum sample were mixed at the same time. In order to improve the assay performance, an appropriate amount of Fab' --D-galactosidase conjugate was carefully chosen.The present assay was simple, rapid, highly sensitive and excellently reproducible. The sensitivity of the assay was approximately 1.5 amol/tube corresponding to 0.5 ng/ml using 2 1 of serum, which was higher than that in the two-step sandwich enzyme-linked immunoassay previously reported. The coefficients of variation for different levels of serum thyroglobulin determined by the present assay ranged from 3.4-7.2 % and 2.5-6.1 % for within and between run, respectively.

A highly sensitive sandwich enzyme immunoassay for insulin in human serum has been developed using capybara anti-insulin serum. Capybara anti-insulin IgG-coated polystyrene balls were incubated with serum samples in the presence of 0.4 M NaCl and then with capybara anti-insulin Fab'-horseradish peroxidase conjugate.The peroxidase activity bound to the polystyrene balls was correlated to the amount of insulin to be assayed. Serum interference was eliminated by the presence of 0.4 M NaC1, and there was no need to add insulin-free serum to a standard curve. The sensitivity was 4 nU/tube or 0.2 U/ml of serum when 20 1 of serum samples was used. The recovery of insulin added to human serum was 92–97 %. The coefficients of within-assay and between-assay variations were 5.1–7.2 % and 7.6–9.2 %, respectively. The regression equation and correlation coefficient to radioimmunoassay were Y(EIA)=0.91×(RIA)-2.4 and 0.97 (n=76), respectively.

A “simultaneous” enzyme-linked immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum was studied, in which human thyroglobulin-coated silicone rubber rod, ß-D-galactosidase- or horseradish peroxidase-conjugated human thyroglobulin and serum sample were mixed at the same time. In order to obtain a suitable range of the measurable antibody, an appropriate amount of human thyroglobulin-enzyme conjugate was carefully selected. A successful simultaneous assay for antithyroglobulin was obtained using thyroglobulin-ß-D-galactosidase conjugate, in which the minimum amount of detectable antibody was approximately 10 ng/ml using 5 l of serum. This sensitivity was 20-fold higher than that in two-step sandwich enzyme-linked immunoassay reported previously.

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for arginine vasopressin in plasma is described. Plasma (0.3 ml) was diluted 1.3-fold with an appropriate buffer and filtered by centrifugation in a micro-concentrator with polysaccharide membrane to eliminate plasma proteins. Arginine vasopressin in plasma filtrates was biotinylated and trapped onto anti-arginine vasopressin IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated arginine vasopressin was eluted from the polystyrene balls with HCl and was reacted with anti-arginine vasopressin Fab′-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The detection limit of arginine vasopressin was 11 fg (10 amol)/tube. This was 45-fold lower than that by competitive enzyme immunoassay using the same antiserum as used in this study and 9 to 400-fold lower than those previously reported by competitive radioimmunoassays. The assay range of arginine vasopressin in plasma was 0.14–140 ng /l using 100 l of plasma filtrates corresponding to 75 u1 o f plasma. Plasma levels of arginine vasopressin i n 8 healthy subjects aged 25–41 yr with, adlibitum water in take and normal activity approximately 4 h after breakfast were 0.72 ± 0.22 (SD) ng /l (range, 0.42–1.04 ng /l).

-D-Galactosidase from Escherichia coli was measured by bioluminescent assay coupled with enzymatic cycling. D-Galactose released from 2-nitrophenyl--D-galactoside by the catalytic action of -D-galactosidase was incubated with excess of galactose dehydrogenase and NAD+ to produce NADH. After degradation of NAD+ by treatment at alkaline pH, NADH was amplified by enzymatic cycling using alcohol dehydrogenase and malate dehydrogenase and

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-insulin IgG in guinea pig serum is described. Anti-insulin IgG in guinea pig serum was reacted with dinitrophenyl bovine serum albumin-insulin-horseradish peroxidase conjugate, and the complex formed between guinea pig anti-insulin IgG and the conjugate was trapped onto affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls in the presence of bovine serum albumin. After eliminating nonspecific guinea pig IgG in test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-guinea pig IgG)IgG. Peroxidase activity bound to the (anti-guinea pig IgG) IgG-coated polystyrene balls was measured by fluorimetry. The detection limit of anti-insulin IgG in guinea pig serum was lowered 1,000 to 10,000-fold as compared with that by the conventional enzyme immunoassays.

hIL-1a and hIL-1 were demonstrated and measured in culture supernatants of peripheral blood monocytes and mononuclear cells by sensitive and specific enzyme immunoassays. Two components for both hIL-1 and hIL-1 were demonstrated by gel filtration of culture supernatants: one with the same molecular weight as recombinant hIL-1 or hIL-1 and the other with a molecular weight of about 30,000. The production of hIL-1 and hIL-1 increased 1.6 to 38-fold in the presence of Escherichia coli (E. coli) lipopolysaccharide. Levels of hIL-1 and hIL-1 became almost plateau within 24 h in culture supernatants of monocytes but became maximal within 12–18 h and decreased afterwards in culture supernatants of mononuclear cells, suggesting that hIL-1 and hIL-1 were produced by monocytes and consumed or degraded by other cells. In addition, hIL-1 and hIL-1 were detected in sera from patients with various disorders but not in sera from healthy subjects.

A “simultaneous” enzyme-linked immunoassay for the measurement of thyroglobulin in human serum was established using polyclonal rabbit antibody, in which rabbit anti-human thyroglobulin IgG-coated silicone rubber rod, rabbit anti-human thyroglobulin monovalent fragment (Fab')--D-galactosidase conjugate and serum sample were mixed at the same time. In order to improve the assay performance, an appropriate amount of Fab'--D-galactosidase conjugate was carefully chosen.The present assay was simple, rapid, highly sensitive and excellently reproducible. The sensitivity of the assay was approximately 1.5 amol/tube corresponding to 0.5 ng/ml using 2 1 of serum, which was higher than that in the two-step sandwich enzyme-linked immunoassay previously reported. The coefficients of variation for different levels of serum thyroglobulin determined by the present assay ranged from 3.4–7.2% and 2.5–6.1 % for within and between run, respectively.

A highly sensitive sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) was developed. hGH to be assayed was incubated with an anti-hGH IgG-coated polystyrene ball, and then the polystyrene ball after washing was incubated with anti-hGH Fab1 -peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was correlated to the amount of hGH to be assayed. Polystyrene balls were coated by physical adsorption with rabbit anti-hGH IgG which was not affinity-purified. Rabbit anti-hGH Fab1 which had been affinity-purified was coajugated with horseradish peroxidase using N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate.The sensitivity was 0.06 pg of hGH per tube or 6 pg/ml of serum when 0.01 ml of serum samples was used. No significant interference was observed in the presence of 8 ng/tube of human prolactin, 8 U/tube of human thyroid-stimulating hormone, 50 mU/tube of human luteinizing hormone, 10 mU/tube of human follicle-stimulating hormone, or 5 U/tube of human chorionic gonadotropin. The recoveries of hGH added to human sera were 96.2-121 %. The coefficients of within-assay and between-assay variations were 3.6-10.0 % and 5.6-9.8 % respectively. The regression equation and coefficient for correlation to radio-immunoassay (RIA) were Y(EIA)=1.04X(RIA)-0.60 and 0.98 (n=137), respectively.