Goat Anti-rabbit Gamma Globulin Research Articles

Radioimmunoassay of Corticosterone using <SUP>125</SUP>I-Labeled Radio-ligand

A radioimmunoassay (RIA) of corticosterone was developed and characterized using 125I labeled radioligand (ICN Biomedicals, U.S.A.) and a single ether extraction procedure. The intra- and interassay coefficients of variation was 7.2% and 14.6% and assay sensitivity was 16.4±3.5 pg per tube. Utilizing this assay, the antiserum for corticosterone (GDN377) made small samples (0.1-1.0μl) use possible. The ease of this technique allows handling of large numbers of samples as compared with RIA using 3H-corticosterone.The specific steps in the procedure of assay were following: 125I-corticosterone was diluted with 0.05M phosphate buffered saline (PBS) containing 1 % bovine serum albumin (BSA). Antiserum (GDN377) was diluted with 0.05M PBS containing 0.05mM ethylenediamine tetraacetic acid, 2Na salt, dihydrate (EDTA) and 0.4% normal rabbit serum (NRS) to 1 : 15000-1 : 20000 dilution. Goat anti-rabbit gamma globulin serum (ARGG) was diluted with 0.05M PBS containing 3.5% polyethylene glycol 6000 (PEG) to 1:150 dilution. Plasma or serum (0.1-1μl) and standard were diluted to 400μl with distilled water in 13 × 100mm culture tubes and extracted once with 2ml of diethyl ether. The samples were allowed to settle for 5 min to ensure separation of ether and aqueous phases. The tubes were then placed in dry ice-ethanol bath to freeze the aqueous phase. The ether phase was then decanted into 12 × 75mm culture tube and dried under reduced pressure with gentle mixing at 50C. One hundred μl of PBS containing 1% BSA, antiserum and labelled ligand (50-100Bq/100μ) were added to each tube and the extract was dissolved by agitation and incubated at 4C for 24 h. Then, 100μl of ARGG was added to each tube. After further incubation for 24 h, the antibody bound hormone was separated from the free hormone by centrifugation at 4C 1700g for 30 min. The supernatant was carefully decanted and the precipitate was counted in automatic gammacounter. Plasma or serum concentrations of corticosterone were calculated using a standard curve.

Radioimmunoassay for salmon pancreatic somatostatin-25

A specific and sensitive radioimmunoassay (RIA) for the measurement of plasma levels of somatostatin-25 (SS-25) in salmon was developed using antisera raised against coho salmon ( Oncorhynchus kisutch) SS-25. Somatostatin-25 was iodinated by the chloramine-T method and repurified on Sephadex G-25. The RIA was performed using a double antibody (goat anti-rabbit gammaglobulin as second antibody) method under disequilibrium conditions. Plasma from several salmonids (coho, chinook, rainbow trout, brook trout, arctic char, lake trout, and whitefish) as well as plasma from some nonsalmonids (sucker, bluegill) crossreacted with the antisera; serial dilutions of plasma from rainbow trout, brook trout, chinook salmon, and coho salmon were parallel to the SS-25 standard curve. Plasma from catfish showed negligible cross-reactivity. None of the mammalian somatostatins (somatostatin-14, somatostatin-28), U II, or other pancreatic hormones (insulin, glucagon) tested showed significant cross-reactivity with the antibody in the assay system. The lowest detectable level of SS-25 was 5 pg/tube; especially reproducible results were obtained in the range of 0.15–1.20 ng/ml, which appears to be the normal range of SS-25 circulating in the plasma of salmonids. Intra- and interassay coefficients of variation were 5.7 and 12.6%, respectively. Injection of glucose into chinook salmon resulted in an elevation of plasma SS-25 titers within 30 min and was coincident with hyperglycemia.

Polyclonal Antibodies against Follitropin (FSH) Receptor Interfere with Hormone Binding, but Mimic the Effects of FSH*

We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a approximately 60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 micrograms/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action.

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

A sensitive direct enzyme immunoassay for urine pregnanediol-3α-glucuronide was developed. The assay system involves the use of an antiserum against pregnanediol-3α-glucuronide and an enzyme-labelled antigen chemically prepared by linking gb- d-galactosidase to 20α-hydroxy-5β-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-3α-glucuronide was validated in terms of specificity, accuracy and sensitivity. When urine samples were assayed for pregnanediol-3α-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.

Localization and Orientation of Subunit Delta of Spinach Chloroplast ATP-Synthase within the CF0CF1 Complex. 1. Distinction of Shielded and Exposed Surfaces of Delta on the Thylakoid Membrane

A new polyclonal antiserum against spinach CF1 subunit delta was produced in rabbits. It decorates only one band at 21 kDa in Western immunoblots of thylakoid proteins and does not react in ELISA with delta-free four subunit CF1(-delta); therefore it is regarded monospecific. The polypeptide used as immunogen had been purified by HPLC. Earlier antisera against CF1 delta cross-react with CF1 subunit beta. The new antiserum 306 contains different antibodies; some can be absorbed with thylakoids, i.e. by delta within the assembled CF0CF1 complex on the membrane, others still react in ELISA with isolated CF1. The former antibodies agglutinate thylakoids and inhibit PMS cyclic photophosphorylation. Therefore we conclude that part of the surface of CF1 subunit delta is exposed within the quaternary structure of the ATP-synthase complex of photosynthetically active thylakoids, but part of the surface of delta is shielded. Trypsination of isolated delta occurs at several sites, but this protease does not attack delta in situ, nor does aminopeptidase. Staphylococcus aureus protease V8 digests CF1 delta after isolation at residues Asp53, Glu61, Glu95 and Glu106, but has no access to these residues of delta in situ. Thus CF1 subunit delta seems to be shielded within the CF0CF1 complex to a large degree. Direct agglutination of thylakoids by anti delta serum 306 was weak and could be improved tenfold by a Coombs serum (goat anti rabbit gammaglobulin), whereas an anti beta serum agglutinated directly. From this we conclude that delta is not accessible at the top of the enzyme; the exposed part is extending below the large subunits alpha and beta and oriented towards the membrane.

Enzyme immunosorbent assay of prolactin with penicillinase as label.

This competitive, rapid, and sensitive enzyme immunosorbent assay for measuring human prolactin in plasma involves penicillinase (EC 3.5.2.6) as label. Microtiter plate wells coated with goat anti-rabbit gamma globulin are filled with antibody to prolactin and plasma sample or reference prolactin and incubated with penicillinase-labeled prolactin for 1 h at 37 degrees C. The enzyme activity of the bound complex is then measured. The assay is as sensitive as radioimmunoassay, the detection limit being 2.5 ng of prolactin per milliliter of plasma. The plasma prolactin values obtained by enzyme immunoassay correlated well with those determined by radioimmunoassay: r = 0.98, slope = 1.00, intercept = 1.11 ng/mL (n = 53).

Enzyme immunoassay of cortisol in human plasma using penicillinase as label

An enzyme immunoassay for cortisol in human plasma using an antiserum raised against cortisol-3- O-carboxy-methyloxime bovine serum albumin and cortisol-21-hemisuccinate conjugated to penicillinase as tracer is described. Although employing immunoassay plates for separation of antigen-antibody complex from the free components was less time consuming, the slope and sensitivity of the standard curve were improved by the addition of goat anti-rabbit gamma globulin for precipitating the complex. There was good correlation between radioimmunoassay and enzyme immunoassay results obtained for cortisol levels present in normal human plasma.

Use of an enzyme linked immunosorbent assay (ELISA) for quantification of proteins on the surface of materials

This study demonstrates the usefulness of an enzyme linked immunosorbent assay (ELISA) for detection and quantification of protein on the surface of materials. Bovine serum albumin (BSA) and bovine gamma globulin (BGG) were the proteins used. Titanium and stainless steel were the materials tested. The proteins were detected with the use of rabbit antiserum specific for BSA and for BGG. This reaction was quantitated by the use of horseradish peroxidase conjugated goat anti-rabbit gamma globulin. The technique is described in detail. The technique was demonstrated to be suitable for quantitation of protein from 0.01 mg/mL to 0.1 mg/mL on the surface of 3 mm X 10 mm materials. The technique was also demonstrated to be suitable for determining the surface area of solid materials. It is a simple technique and suitable for most biomaterials laboratories.

Radioimmunoassay of somatomedin-C in filter paper discs containing dried blood.

We describe a simple radioimmunoassay (RIA) for estimating concentrations of somatomedin-C (Sm-C) in dried blood on filter paper. A single 3.2-mm blood spot specimen on filter paper is eluted overnight into buffer containing antibody and 125I-labeled Sm-C. The following day, bound and free hormones are separated by addition of goat anti-rabbit gamma globulin in 60 g/L polyethylene glycol solution. The correlation between values obtained for such blood-spot discs and the corresponding wet plasma is highly significant (r = 0.90, p less than 0.001). The relative concentrations (arbitrary units) of Sm-C as determined for specimens on filter paper from mothers and infants, and for cord bloods, are similar to those reported by others using acidified serum.

Ontogeny and localization of the alpha-, beta-, and delta- crystallin antigens during lens development in mallard embryos.

The ontogeny of alpha-, beta-, and delta-crystallin antigens in the developing lens of mallard (Anas platyrhynchos) was investigated by indirect immunofluorescence using specific antibodies to mallard alpha-, beta-, and delta- crystallins raised in rabbits. The IgG fraction from each antiserum was isolated by affinity chromatography usign Protein A Sepharose CL-4B. Fluoresceinee (FITC) or rhodamine (TRITC) conjugated goat anti rabbit (GAR) gamma-globulin was used as the secondary antibody. The results show that alpha-, beta-, and delta-crystallins appear simultaneously in the developing mallard lens and are detectable from 66 hr (stage 15/16). This situation is different from the chick where delta- is known to appear first followed by beta-, and alpha-. This could be due to species variation. In the epithelium, however, as in the chick, delta- emerges first followed by beta-, and alpha- but rather late when compared with the chick and this seems probably due to a difference in the incubation time between the two species. Results from immunofluorescence and Tris-SDS gel electrophoresis demonstrate that alpha A subunit of the alpha-crystallin appears much earlier than the alpha B subunit. We also found that the newly discovered epsilon-crystallin (18) appears long after the three major crystallin classes.

Thrombin-induced platelet membrane glycoprotein IIb and IIIa complex formation. An electron microscope study.

The topographic relationships of platelet membrane glycoprotein IIb and glycoprotein IIIa have been studied in stimulated and unstimulated human platelets using immunoelectron microscopy. An indirect approach with ferritin-conjugated goat anti-rabbit gamma-globulin was used to localize the rabbit antibody to glycoprotein IIIa. The second ultrastructural label was keyhole limpet hemocyanin conjugated directly to antibody to glycoprotein IIb. Using the double labels, it was demonstrated that glycoprotein IIb and glycoprotein IIIa were distributed randomly in the unstimulated platelet membrane. After platelet stimulation with thrombin, large clusters of glycoprotein IIb-glycoprotein IIIa complexes were formed. No complex formation between glycoprotein Ib and glycoprotein IIb was observed in control experiments. These observations suggest that thrombin stimulation initiates the specific glycoprotein IIb-glycoprotein IIIa macromolecular complex formation on the platelet surface, which may act as the active fibrinogen-binding site required for normal platelet aggregation.

ELISA assay for measurement of human hemoglobin A and hemoglobin F.

An enzyme-linked immunosorbent assay (ELISA) for the measurement of nanogram quantities of hemoglobin A and hemoglobin F is described. The assay employs hemoglobin bound to polystyrene test tubes. The antigen is quantitated by the addition of rabbit antibody specific for the hemoglobin type, followed by a second antibody of goat antirabbit gamma globulin coupled to alkaline phosphatase. Quantitation of the reaction is achieved by incubating the coated tubes with p-nitrophenyl phosphate. The binding characteristics, the linearity, the ability of the assay to discriminate between hemoglobin A and hemoglobin F, the ability to discriminate between native and denatured gamma globin, and the comparison of this assay with a previously described immunoradiometric (IRMA) assay are discussed.

Serum Concentration of Prednisolone in Children with Nephrotic Syndrome

[Purpose] Recently prednisolone has been used clinically for the therapy in children with nephrotic syndrome. But little information was available about the study of the absorption and excretion of prednisolone in the nephrotic syndrome. So we invented new easy radioimmunoassay (RIA) for prednisolone, and measured the serum concentration of prednisolone in children with nephrotic syndrome. [Methods] Three rabbits were injected with prednisolone 21-BSA hapten and Freund's adjuvant. The rabbit serum(anti-prednisolone antibody) and goat anti-rabbit gamma globulin was used for RIA by double antibody technique. By heating unknown serum in 70°C for 30 minutes, the native prednisolone binding protein was denatured, and it was possible to analyze prednisolone in unextracted and nondiluted serum. Twenty children with idiopathic nephrotic syndrome were administered orally prednisolone several times. The quantity of prednisolone was equal in the each patient. The serum concentration of prednisolone was measured serially. [Results and Discussion] This RIA was capable of detecting 50 picograms of prednisolone in 50 μ1 of undiluted serum. The peak concentration of prednisolone in patients had a tendency to increase gradually with the decrease of the urine protein. The tendency had little correlation with the change of the total protein in serum.

Development and validation of a choriogonadotropin beta-subunit radioimmunoassay for serum choriogonadotropin using commercially available components

A sensitive, specific, accurate and precise radioimmunoassay procedure developed for the beta-subunit of serum choriogonadotropin (hCGbeta) is described. 1. The assay employs an anti-hCGbeta (rabbit) serum generated against hCGbeta, highly purified intact choriogonadotropin (hCG) as standard, and [125I]hCG as the radioactive ligand. The antibody-bound hCG was separated from the free hormone by the addition of goat anti-rabbit gamma-globulin. 2. The detection limit of the assay was approximately 75 microIU hCG which corresponds to a serum concentration of approximately 0.75 mIU/mL. 3. Cross reactivity studies performed with human luteinizing hormone (hLH) and human thyroid-stimulating hormone (hTSH) indicated minimal interferences from these structurally similar glycoproteins. Parallel dose-response curves were demonstrated between dilutions of sera with elevated hCG concentrations and the standard reference preparation. A non-specific binding of less than 2.5% of the total [125I]hCG was routinely observed. 4. The analytical recovery of hCG added to human sera varied from 94 to 110%, with a mean recovery of 101%. 5. The inter-assay variation was determined by assaying (n=30) 3 different quality control pools. The following data were obtained: X1=4.6 +/- 0.5 mIU/mL (CV=10.9%); X2=8.1 +/- 0.9 mIU/mL (CV=11.1%); and X3=36.8 +/- 2.8 mIU/mL (CV=7.6%). 6. The clinical data collected from subjects with trophoblastic disease agreed with previously published studies. All of the reagents are available commercially.

Immunoassay of the adenosine deaminase complexing proteins of human tissues and body fluids.

A sensitive immunoassay for the adenosine deaminase binding protein (complexing protein) of human kidney has been developed. Impetus for the development of the assay was provided by the observations that (a) antibody to complexing protein does not react with the catalytically active adenosine deaminase monomer, and (b) binding of antibody to complexing protein does not affect the binding or catalytic activity of the enzyme monomer. Preformed immune precipitate prepared from rabbit anti-kidney complexing protein serum and goat anti-rabbit gamma-globulin serum is used to selectively insolubilize complexing protein. Quantitation is accomplished by measuring the intrinsic adenosine deaminating activity or adenosine deaminase binding capacity of the protein held in the immune precipitate. As little as 1 ng of kidney complexing protein can be accurately quantitated with the assay. The assay was used to demonstrate that complexing proteins from liver, lung, spleen, fibroblasts, plasma, and urine react with antibody to kidney complexing protein. The shared capacity to bind adenosine deaminase coupled with their antigenic similarity suggests that the complexing proteins of a number of human tissues and body fluids may be products of the same gene.

An acid transporting enzyme in human gastric mucosa.

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.

Localization of the site of adenylylation of glutamine synthetase by electron microscopy of an enzyme-antibody complex.

Antibodies to the nucleosidel,N(6)-ethenoadenosine have been used to localize the site of adenylylation of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of Escherichia coli. Antibodies were induced in rabbits by injection of a bovine albumin-ethenoadenosine conjugate. The resulting antisera strongly bound ethenoadenosine, its 5'-nucleotide, or protein conjugates of the nucleoside; little or no crossreaction was seen to adenosine, AMP, or the protein carrier. Ethenoadenylylated glutamine synthetase was prepared by modification of the enzyme by the E. coli adenylyltransferase, using etheno-ATP as a substrate. The ethenoadenylylated glutamine synthetase was precipitated by antibodies to ethenoadenosine in conjunction with goat anti-rabbit gamma globulin. Electron micrographs of reaction mixtures of ethenoadenylylated glutamine synthetase and anti-ethenoadenosine showed individual enzyme molecules complexed with one or more antibodies and pairs of enzyme molecules crosslinked by a single antibody. The approximate site of adenylylation was located from the apparent area of contact between enzyme and antibody. We conclude that the adenylylation sites are on the periphery of the bilayered hexagonal disc, offset by 15 +/- 10 degrees from the 2-fold axis of symmetry through a vertex of the hexagon and 20 +/- 10 A from the plane between the layers of the disc.

Localization of an adenosine deaminase-binding protein in human kidney.

A protein which specifically complexes with adenosine deaminase (complexing protein) has been localized in human kidney. Thin sections of renal tissue were treated with rabbit anti-complexing protein serum followed by fluorescein-labeled goat anti-rabbit gamma globulin serum. Complexing protein was detected in the proximal tubules of four normal kidneys. The glomeruli in sections from one of the four normal kidneys were also positive. Lung, liver, spleen, cardiac muscle, skeletal muscle, and kidney medulla were negative by this technique. The glomeruli as well as the proximal renal tubules of two patients with combined immunodeficiency disease-adenosine deaminase deficiency and three of seven patients with kidney disease contained easily detectable levels of complexing protein.

Human Alpha-Fetoprotein in the Epidermal Cells of Bowen's Disease

Human alpha-fetoprotein (HAFP) was demonstrated in clusters of atypical cells of an extensive Bowen's disease involving the pubic area. Frozen sections were studied by indirect immunofluorescence using monospecific rabbit anti-HAFP antiserum or purified rabbit anti-HAFP antibody followed by staining with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit gamma-globulin and by a direct technique using FITC-labeled rabbit anti-HAFP Fab2 fragments. Sections of fetal liver served as positive controls and sections of adult human skin and liver were used as negative controls. Preabsorption of the anti-HAFP antibodies with purified HAFP blocked the fluorescence. This finding adds Bowen's disease to the list of premalignant disorders which possess HAFP as an oncofetal marker of their malignant potential. HAFP might possibly enhance malignant behavior through interference with cellular immunosurveillance mechanisms.

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(o-carboxymethyl)oximino-[ 125l]-iodo-histamine as radioligand

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11α-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11α-BSA) serum, progesterone-3-(O-carboxymethyl)oximino-[ 125I]-iodohistamine (progesterone-3-[ 125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11α-BSA serum, the overall assay specificity of the progesterone-3-[ 125I] ria is similar to that of the [ 3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[ 125I] RIA appear comparable to those of [ 3H]-progesterone assays which employ similar anti-progesterone-11α-BSA sera. The progesterone-3-[ 125I] double-antibody RIA, however, is more convenient and less expensive than the [ 3H]-progesterone RIA method.