A sensitive two-site enzyme immunoassay for human epidermal growth factor (urogastrone)

A sensitive enzyme immunoassay (EIA) was developed for human epidermal growth factor (hEGF) or urogastrone, which was isolated from human urine. Our EIA system is based on the sandwiching of an antigen between anti-hEGF IgG coated on a polystyrene tube and anti-hEGF antibody Fab'-linked beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). This method has the advantages that the procedures are simple and rapid and that the antibody Fab'-beta-D-galactosidase complex is more stable than radioisotope-labeled IgG. Purified hEGF is detectable at as low as 100 pg/ml, which is very sensitive compared to the radioimmuno-assays or radioreceptor assays already reported. Using this new EIA system, hEGF levels in human urine were examined. The values for normal males and females were 48.4 and 83.5 ng/mg creatinine, respectively, which shows that females excrete 1.7 times more hEGF than males.     

Enzyme immunoassay of gastrin releasing peptide (GRP)-like immunoreactivity in milk

A sensitive and specific enzyme immunoassay (EIA) for gastrin releasing peptide (GRP)-like immunoreactivity was developed using enzyme-labeled antigen. The synthetic carboxy-terminal fragment of human GRP(12-27) was conjugated with beta-D-galactosidase for EIA. The minimum amount of GRP-like immunoreactivity detectable by this method was 0.24 femtomol/well (6 picomol/liter). The level of GRP-like immunoreactive substance in bovine foremilk was about 150 nanomol/liter, the level of which was more than hundredfold higher than that in normal milk or calf serum.     

Radioimmunoassay of secretin in plasma.

A sensitive, specific and reproducible radioimmunoassay for secretin is described. Antibodies were readily produced against low microgram quantities of synthetic secretin. The secretin antibodies did not cross-react with the structurally similar G.I.P., V.I.P., or glucagon. Synthetic secretin was iodinated using Chloramine "T" and purified by a two-state procedure incorporating gel filtration and radient elution from a cation exchange column. Plasma samples were found to produce variable interference in the assay necessitating the incorporation of secretin-free "blands" for each patient's plasma. Production of secretin-free plasma was by incubation of plasma samples at 37 degrees C for 96 hours. The sensitivity of the assay was 12.5-25 pg/ml. Normal fasting secretin levels were 21 +/- S.E. 7 pg/ml. A mean rise in plasma secretin to 220 pg/ml was observed after intraduodenal acidification.     

Radioimmunoassay for secretin using Nalpha-tyrosylsecretin and [Tyr1]-secretin.

Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.     

Development of a direct microplate enzyme immunoassay for the determination of pregnanediol-3 alpha-glucuronide in urine

A sensitive direct enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was developed. The assay system involves the use of an antiserum against pregnanediol-3 alpha-glucuronide and an enzyme-labelled antigen chemically prepared by linking beta-D-galactosidase to 20 alpha-hydroxy-5 beta-pregnane 3(O-carboxymethyl)oxime. Free from antibody-bound antigen was separated by a solid-phase double antibody method, using a microplate coupled with goat anti-rabbit gamma-globulin. This solid-phase enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was validated in terms of specificity, accuracy and sensitivity. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid phase enzyme immunoassay and conventional radioimmunoassay methods agreed well (n = 30, r = 0.922). This assay system has an advantage over radioimmunoassay, because it does not require the use of radioisotopes. The procedure of this method is very simple, since it does not require purification steps of the biological samples.     

Comparison of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique

beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.     

Peripheral distribution of nervous system-specific S-100 protein in rat

S-100 protein, a nervous system-specific protein, was determined in a soluble extract of various rat tissues with a sensitive enzyme immunoassay method, which consisted of a solid-phase with immobilized anti-S-100 antibody and the antibody labeled with beta-D-galactosidase from Escherichia coli. The minimum detectable amount of S-100 protein was 3 pg/assay. Central nervous tissues (cerebrum, cerebellum, and brain stem) contained 1.4 to 2.8 micrograms S-100 protein/mg protein, whereas most of the peripheral tissues contained less than 0.05 microgram/ml of the specific protein. However, the level of S-100 protein was high in adipose tissue (0.5--1.1 micrograms/mg) and in trachea (about 0.5 microgram/mg), which involves cartilage. The S-100 protein levels in several tissues were significantly higher in female rats than in males at ages of 5 to 6 weeks.     

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.     

Sex-Dependent and Sex-Independent Distribution of the β-Subunit of Nerve Growth Factor in the Central Nervous and Peripheral Tissues of Mice

Levels of the beta-subunit of nerve growth factor (beta-NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich-type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. beta-NGF-specific antibody isolated on a column of Sepharose CL-4B coupled with purified beta-NGF reacted only with beta-NGF. The assay for beta-NGF was performed by incubation of F(ab')2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab' fragments labeled with beta-D-galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of beta-NGF in almost all organs of mice. The amount of beta-NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3-5 mg/g of tissue). In serum, some of the 2-month-old males, but none of the females, exhibited a fairly high level of beta-NGF (greater than 100 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitive immunoassay for rat parvalbumin: tissue distribution and developmental changes

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.     

Highly Sensitive Immunoassay for Rat Brain-Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods

Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. Beta-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and Beta-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angiotensin I IgG in serum were 10-30 ng/liter (0.2-0.6 pg/assay). These methods were 330 to 1,000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.     

Tissue distribution and developmental profiles of immunoreactive alpha B crystallin in the rat determined with a sensitive immunoassay system

In order to determine the quantitative distribution of alpha B crystallin (alpha B) in non-lenticular tissues, we have established a sensitive immunoassay system for specific measurement of alpha B. Antisera were raised in rabbits by injecting alpha B purified from bovine lenses, or C-terminal decapeptide (KPAVTAAPKK) of alpha B (alpha Bpep). The antibodies to alpha B and alpha Bpep were purified by the use of alpha B-coupled Sepharose column. The F(ab')2 fragments of antibody IgG to alpha B were immobilized on polystyrene balls and the Fab' fragments of antibody IgG to alpha Bpep were labeled with beta-D-galactosidase from Escherichia coli. The sandwich-type enzyme immunoassay consisted of the above two antibodies was sensitive, and the minimum detection limit of the assay was 10 pg alpha B without any crossreactivity with alpha A. By using the assay method, it is revealed that the alpha B was distributed in most of the tissues examined. Among the non-lenticular tissues, alpha B was present at high levels in the heart and striated muscles, especially in the soleus muscle, and kidney. High levels of alpha B in the muscle tissues were also seen in various animals. Developmental increases of alpha B in rat muscle tissues and kidney were observed from 16 days of gestational age to 1 or 5 weeks of postnatal age. In contrast, the alpha B in the brain kept a low level during the same period. After 5 weeks of age, alpha B concentrations in the brain increased sharply, reaching the adult levels at 9 weeks of age. Immunohistochemical staining with anti-alpha Bpep revealed that alpha B was positive not only in glial cells, in the central nervous tissues, but also in some neurons of spinal cord, brainstem, hippocampus, and olfactory bulb. Spermatocytes in the testis were also immunopositive for alpha B.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

Enzyme-immunoassay of thromboxane B2 at the picogram level

A highly sensitive and reproducible enzyme-immunoassay for the measurement of thromboxane B2 was developed. Thromboxane B2 (TxB2) was coupled with beta-D-galactosidase by mixed anhydride reaction. Thromboxane B2-antiserum was generated in rabbits and used at a final dilution of 1:480,000. The separation of immunocomplex from the free form of TxB2 was accomplished by the double antibody method. The second antibody was sheep anti rabbit IgG. The precipitated enzyme activity was measured fluorometrically with 4-methyl-umbelliferyl-beta-D-galactoside as substrate. This method allowed to measure TxB2 in the range of 0.002-5 picomole per tube. The cross-reactivity of the anti-thromboxane B2-antiserum with 2,3-dinor thromboxane B2 was about 20%, but it was less than 0.2% for the other prostanoids tested. TxB2 extracted from human urine was measured by enzyme-immunoassay (y) and radioimmunoassay (x) which has been found closely correlated to values obtained by gas chromatography-mass spectrometry. Regression analysis of the data comparing enzyme-immunoassay and radioimmunoassay gave the equation y = 0.996 x + 0.470, correlation coefficient r = 0.9947. Inter-assay coefficient of variation was 3.1%. The assay was further simplified by coating the second antibody on glass beads. The regression equation between this solid-phase enzyme immunoassay (y) and radioimmunoassay (x) was y = 0.9860 X 1.927, r = 0.9895, and enzyme immunoassay (y) was y = 0.9749 X -0.94808, r = 0.9887. Thus, the enzyme-immunoassay shows specificity and sensitivity comparable to radioimmunoassay making use of radioactive tracer unnecessary.     

Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain

Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column. Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined. The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.     

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes.

A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicillinase-labeled testosterone-11 beta-carboxymethyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.