Production of antibodies that inhibit the binding of insulin to its receptor

In this study, we report a procedure for producing antisera that block the binding of ¹²⁵I-insulin to its receptor. After 2 injections with intact IM-9 cultured human lymphocytes, the antisera from 8 of 17 $\text{Balb}\text{C}$ mice inhibited the binding of ¹²⁵I-insulin to its receptor on IM-9 cells by 50% or greater. One antiserum at dilutions of 1:200 and 1:50 inhibited the binding of ¹²⁵I-insulin by 50% and 80%, respectively. Four lines of evidence indicated that the inhibition of ¹²⁵I-insulin binding by this antiserum was due to a specific immunoglobulin directed against the insulin receptor. First, removal of the immunoglobulin fraction of the antiserum resulted in a complete loss of its inhibitory activity. Second, the antiserum inhibited the binding of ¹²⁵I-insulin to its receptor on both human cultured lymphocytes and human placenta particles. Third, the antisera bound solubilized insulin-receptor complexes. Finally, the antiserum did not inhibit the binding of ¹²⁵I-human growth hormone to its receptor on IM-9 lymphocytes. These studies demonstrate therefore, a simple method for producing antibodies that block the binding of ¹²⁵I-insulin to the human insulin receptor.     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

Development of radioimmunoassay for motilin

A highly specific and sensitive radioimmunoassay for motilin has been developed. High titers of motilin antisera were produced in rabbits by repeated immunization with synthetic porcine motilin coupled to bovine serum albumin. Motilin was radioiodinated with Na[¹²⁵I] by the chloramine-T method and was purified using a CM-Sephadex C-25 column eluted with ammonium acetate gradient at pH 5.9. A specific radioactivity of 300 to 400 μCl/μg was usually obtained. Untreated plasma showed significant nonspecific interference in the assay; however, the interference could be minimized by extracting the plasma with methanol. All of the known gastrointestinal hormones were tested and showed no cross-reactivity at 10 ng/ml. Plasma motilin levels in dogs were significantly increased within 3 min, after an intraduodenal bolus injection of 50 ml of 0,1 n HCl. This increase coincided with a marked increase in myoelectrical activities of the duodenum.     

Immunoassay of 5-methyltetrahydrofolate: Use of 125I-labeled Protein A as the tracer molecule for specific antibody

A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. ¹²⁵I-Labeled staphylococcal Protein A (¹²⁵I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of ¹²⁵I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific: 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15–20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits.     

Separation of mitochondrial creatine kinase (MiMi-CK) from cytosolic creatine kinase isoenzymes by Cibachrome-Blue affinity chromatography

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.     

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

A non-chromatographic radioimmunoassay for serum dehydroepiandrosterone using a mixture of antisera

A simplified method for evaluating serum dehydro-epiandrosterone (DHEA) without chromatography has been developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ⁵ antiserum and anti-11-deoxy-17-ketosteroid antiserum, in which cross-reactivity of each antiserum is reduced to a negligible amount. Serum (20 μ1) was extracted with 1 ml of n-hexane. One milliliter of 80% methanol was added to the n-hexane extract which was stirred and centrifuged. The n-hexane layer was discarded, and the methanol layer was evaporated to dryness. The residue was incubated with an antiserum mixture containing DHEA-7α-³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α- ³H. The accuracy, precision, sensitivity and specificity were satisfactory. Good agreement was found between the serum DHEA levels obtained by the present radioimmunoassay and those obtained by radioimmunoassay with paper chromatography, making this method suitable for routine use.     

Analysis of the calcium-dependent interaction of calmodulin with bovine serum albumin

An air-driven ultracentrifuge has been used to investigate the calcium-dependent association between calmodulin and bovine serum albumin. Procedures were described which allowed the interaction to be analyzed to yield the equilibrium constant. At low ionic strength (25 mm Tris-HCl, pH 7.5, pCa 6.68, 9°C) the equilibrium constant for the interaction was estimated to be 2.1 × 10⁴m⁻¹, while at high ionic strength (25 mm Tris-HCl, pH 7.5, 150 mm KCl, pCa 6.68, 9°C) the value was 4.5 × 10³m⁻¹. Under similar conditions, calmodulin was also found to interact with β-lactoglobulin A and gelatin, but no detectable association was observed with ovalbumin.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D₂ with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D₂-methoxamine coupled to bovine serum albumin. A (¹²⁵I)-Histamide prostaglandin D₂-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D₁-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D₂ in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D₂ destruction. The unstability of this prostaglandin is due to the presence of a β-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Interaction of hepatic asialoglycoprotein receptor with asialoorosomucoid and galactolyzed lysosomal α-glucosidase

An asialoglycoprotein receptor was isolated from murine liver and purified more than 1600-fold using 2-fold affinity chromatography on asialoorosomucoid-Sepharose. The purified receptor did not interact with ¹²⁵I-orosomucoid, but bound to ¹²⁵I-asialoorosomucoid. The binding of the receptor to asialoorosomucoid was saturable. The dissociation constant of the receptor-asialoorosomucoid complex was 0.4·10⁻⁹ M. The molecular mass of the receptor, as determined with the use of specific antibodies by the immunoblotting method, was 43 kDa. High concentrations of unlabeled asialoorosomucoid and of n-aminophenyl-β-d-galactosyl derivatives of bovine serum albumin, ovalbumin and acid α-glucosidase from human liver inhibited the binding of the receptor to ¹²⁵I-asialoorosomucoid almost completely. The binding of the receptor to ¹²⁵I-galactolyzed α-glucosidase was pH-dependent, with the pH optimum at 8.0–9.0. It was shown that, as in the case of ¹²⁵I-asialoorosomucoid, the binding of the ¹²⁵I-galactosyl derivative of α-glucosidase occurred in the presence of Ca²⁺ and was inhibited by N-acetylgalactosamine. Glycoproteins containing galactose as a terminal residue inhibited the interaction of the receptor with ¹²⁵I-galactolyzed α-glucosidase. The possibility of directed transport of the galactolyzed α-glucosidase derivative into parenchymous liver cells using receptor-mediated endocytosis is discussed.     

Rat liver phosphofructokinase isozymes

The labile phosphofructokinase activity of rat liver was found to be stabilized and efficiently extracted in 50 mm Tris-HCl, pH 8.0, 50 mm NaF, 10 mm dithiothreitol, and 1.0 mm ATP. By the method of DEAE-cellulose chromatography liver phosphofructokinase activity could be resolved into two isozymes. The major isozyme which was 85% of the total isolated activity was purified to homogeneity. This 15,000-fold purified isozyme had a specific activity of about 90 IU/mg protein with 25–30% recovery of the total activity. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of the sodium dodecyl sulfate-treated isozyme indicated a subunit molecular weight of 65,000. Antiserum to the major isozyme was obtained from rabbits, and immunotitration of the two isozymes indicated that they were immunologically different. Kinetic properties of the two isozymes indicated that the major isozyme was more susceptible to ATP and citrate inhibition as well as relief of ATP and citrate inhibition by fructose-6-P, AMP, and ammonia. With the use of DEAE-cellulose chromatography and antiserum titration of 100,000g supernatant fluids, it was shown that the two hepatic isozymes were always found together in adult, embryonic, and neoplastic liver and in kidney.     

Possible evidence for the existence of a growth hormone fragment in porcine pituitary

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126–180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of ¹²⁵I-labelled fragment B porcine growth hormone required a 10³ M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of ¹²⁵I-labeled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possibly representing a growth hormone fragment.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Studies on adenosine 3′,5′-monophosphate phosphodiesterase of human erythrocyte membranes

In this work we show the existence of cyclic AMP phosphodiesterase (EC 3.1.4.17) in human erythrocyte membranes and have clarified some properties of the enzyme. In human erythrocytes, about 23% of the total cyclic AMP phosphodiesterase activity is in a membrane-bound form. Although it could be solubilized with Triton X-100 in 5 mM Tris-HCl buffer (pH 8.0), it was not solubilized by a low or high concentration of salt. The enzyme seems to be localized in the cytoplasmic surface, since it is detected in sealed inside-out vesicles of human erythrocyte membranes, but not in intact human erythrocytes. The optimum pH was found to lie between 7.4 and 8.0, and Mg²⁺ was found to be necessary for its activity. Ca²⁺ and calmodulin could not stimulate the activity of this enzyme. Theophylline was a strong inhibitor, but cyclic GMP could not inhibit the enzymic hydrolysis of cyclic [³²P]AMP and this membrane-bound enzyme therefore seems to be specific to cyclic AMP.     

The use of homologous and hetebologous 125 I-radioligands in the radioimmunoassay of progesterone

Eight homologous and heterologous¹²⁵ I-radioligand systems for the radioimmunoassay of progesterone were examined. Using an antiserum raised to 11α-hydroxyprogesterone 11-succinyl-bovine serum albumin, standard curves were set up with the homologous radioligands, 11α-hydroxyprogesterone 11-succinyl-[¹²⁵I]-iodotyramine, -[¹²⁵I]-iodohistamine and -[¹²⁵I]-iodotyrosine methyl ester. Heterologous bridge systems were represented by progesterone-11α-oxycarbonyl-[¹²⁵I]-iodotyrosine methyl ester and 11α-hydroxyprogesterone 11-phthalyl-[¹²⁵I]-iodotyrosine methyl ester, and heterologous site systems by progesterone-3-(O-carboxymethyl)oxime-[¹²⁵I]-iodotyramine, progesterone-12-(O-carboxymethyl) oxime-[¹²⁵ I]-iodotyramine, and progesterone-20-(O-carboxymethyl) oxime-[¹²⁵I]-iodohistamine. The preparation of the steroid derivatives and iodination by a two-phase method are described. The curves obtained from the homologous radioligands were relatively insensitive compared with a tritiated system, with the tyrosine methyl ester derivative providing a more sensitive assay than the corresponding tyramine or histamine analogues. The heterologous bridge systems gave more sensitive curves than the homologous tracers whilst the 3- and 12-(O-carboxymethyl) oxime derivatives of progesterone furnished curves as sensitive as the tritiated reference. Progesterone-20-(O-carboxymethyl)oxime-[¹²⁵I]-iodohistamine was not bound by the antibody.     

Specific antisera suitable for solid-phase radioimmunoassay of 11beta-hydroxyandrost-4-ene-3,17-dione

Specific antiserum has been developed for use in measuring 11β-hydroxyandrost-4-ene-3, 17-dione by radioimmunoassay (RIA). Rabbit antiserum was generated by employing the conjugate prepared by coupling 6β,11β-dihydroxyandrost-4-ene-3,17-dione 6-hemisuccinate with bovine serum albumin. The antiserum bound 68% of 50 picograms of 11β-hydroxyandrost-4-ene-3,17-dione-[1,2,6,7-³H] during characterization at a dilution of 1:12,500. Among the numerous steroids tested for cross-reactivity, 5α-androstane-3,17-dione, androst-4-ene-3,17-dione, and 11β-hydroxy-5α-androstane-3, 17-dione showed 2%, 5%, and 30% cross-reactivity respectively. The Rivanol-treated antiserum was coupled to Enzacryl AA, in order to study the feasibility of a solid-phase RIA, and this complex showed 50% binding with the labeled antigen at a dilution of 1:3000. The complex retained high specificity and should prove useful in a simple solid-phase RIA.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

A solid-phase immunoassay for human immunoglobulin E: use of 125I-labeled protein A as the tracer

A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of ¹²⁵I-labeled protein A (¹²⁵I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of ¹²⁵I-PA. The degree of inhibition of ¹²⁵I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays.     

A radioimmunoassay procedure for quantitating parotid hormone

The methodology and characterization of a double antibody radioimmunoassay (RIA) for quantitating parotid hormone (PH) in biological fluids are reported. A specific antiserum against PH was raised in rabbits using PH conjugated to human serum albumin. Its binding capacity and association constant were 22 μg/ml and Ka = 1.01 × 10^?12 M^?1, respectively. The sensitivity of the RIA was 1.5 pg PH when a sequential incubation schedule was used. This RIA makes possible the quantitation of PH in biological fluids and tissue extracts.     

Quantification of creative kinase isoenzymes by a radioimmunoassay

Summary It is not known whether loss of enzyme activity from the circulation is due to denaturation, inactivation or removal of intact enzyme molecules. This is in part due to the lack of an assay to measure enzyme protein concentration since available assays measure only enzyme activity. Radioimmunoassays for plasma enzymes and isoenzymes have not been possible because of oxidation in radioactive labelling by conventional methods and the problem of subunit dissociation. In the present study, antibodies specific to the B and M subunits of creatine kinase isoenzymes were obtained by immunization of rabbits with canine BB and MM creatine kinase. Anitgens (MM and BB) were radioactively labelled with ¹²⁵I by acylation, avoiding the problem of oxidation and subunit stabilized by mercaptoethanol (0.020 m) and Trisbuffer (1.6 m). A radioimmunoassay capable of detecting picogram amounts of CK isoenzymes was developed which measures the concentration of enzyme protein rather than activity. The method was shown to provide a sensitive quantitative method for analysis of plasma CK isoenzymes in dogs after myocardial infarction produced by coronary occlusion. This technique may provide a prototype for the development of radioimmunoassays for other plasma isoenzymes and should help to elucidate the nature of the disappearance of isoenzymes from the circulation.Work from the authors' laboratory was supported in part by the National Institutes of Health Grant HL 17646, SCOR in Ischemic Heart Disease