Anti-rabbit Gamma Globulin Research Articles

Development of Colorimetric Enzyme‐Linked Immunosorbent Assay for Human Chorionic Gonadotropin

The present study demonstrated the development of a solid phase competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of human chorionic gonadotropin (hCG) in serum and urine. Polyclonal antisera raised against the β‐ subunit of peak‐I hCG was used in the assay. The Peak‐IA hCG‐penicillinase was used as tracer. The performance of this antiserum and tracer was compared against hCG‐β antisera of NIH, USA and penicillinase conjugated to hCG‐β obtained from NIH, respectively. Almost parallel standard curves were obtained in both cases, suggesting that these antisera and enzyme label have much potential for developing ELISA system. To the anti‐rabbit gamma globulin (ARGG) coated polystyrene tubes, standard or serum or urine samples (50 µL), 100 µL of hCG‐β antiserum, 100 µL of peak‐I(A) hCG‐penicillinase conjugate and 350 µL of assay buffer were incubated at 37°C for 2 hours. Bound enzyme activity was measured using Penicilline V as substrate. In this new strategy, locally available polystyrene tubes were ground from inside and coated with ARGG. The sensitivity of the assay was 17 mIU/mL in urine and 18 mIU/mL in serum. The intra‐assay and inter‐assay coefficients of variation (CVs) appeared to be within acceptable limits of 10%. The serum and urinary hCG values, obtained by this method, correlated well with those obtained by radioimmunoassay (RIA) r=0.98 (n=100 for serum samples; n=250 for urinary samples).

Development and Validation of an ELISA for Hemoglobin‐A2: A Novel Method for β‐Thalassemia Screening in Developing Countries

Hemoglobin‐A2 (HbA2) measurement in human hemolysates has great significance, since its level can indicate β‐thalassemia carrier status in other-wise healthy individuals. An ELISA for HbA2 using antiserum mono‐specific to the δ chain of HbA2 and affinity purified antirabbit gamma globulins (ARGG) conjugated to horseradish peroxidase (HRP) have been developed. The monospecific antiserum used does not cross react with other hemoglobins. Hemolysates from volunteers are used for measurement of HbA2. In a limited trial for β ‐thalassemia carrier screening (n = 350), the results obtained with the developed ELISA are comparable with those obtained with a micro‐column chromatography method (r ≥ 0.89). The developed ELISA is simple, accurate, precise, inexpensive, and several samples can be processed simultaneously with ease, making this system a suitable candidate for transforming into a user friendly kit.

Identification of CD4-independent HIV receptors on spermatozoa.

Human immunodeficiency virus (HIV) has been demonstrated to bind and enter into the spermatozoa facilitating the transmission into urogenital cells. However, spermatozoa has been reported to be devoid of the conventional CD4 receptors for HIV. This suggests that there exists an alternate modality of HIV entry into spermatozoa using receptors other than CD4. Present communication describes the identification of HIV receptors on the spermatozoa. The sperm proteins were solubilized using Triton X-100 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using cell-free HIV or gp120 envelope glycoprotein as a probe. HIV or gp120 bound protein band was then visualized by using alkaline phosphatase (AP) labeled anti-gp120 antibody as well as by using anti-gp120 antibody and subsequently by AP-labeled anti-rabbit gamma globulin. The results obtained demonstrate for the first time that cell-free HIV and gp120 protein bind specifically to 160 kDa sperm protein that could be the receptor for HIV entry into spermatozoa. A 160 kDa sperm protein could be the CD4-independent HIV receptor for HIV to bind and enter into the spermatozoa. Further characterization of this 160 kDa HIV receptor on sperm will provide an insight in understanding the mechanism and probable mode of intervention or prevention of HIV transmission at the initial stage of infection.

Equine trypsin: purification and development of a radio-immunoassay.

Shock is accompanied by generalised splanchnic hypoperfusion, and splanchnic organs like the pancreas can be damaged, as shown in animal experimental models and in humans, by the presence of high plasma concentrations of trypsin and other pancreatic enzymes. In order to design a radioimmunoassay technique (RIA) for the measurement of equine trypsin-like immunoreactivity (TLI) in biological fluids, trypsin was purified (with purity > or = 96%) from the equine pancreas by extraction in an acid medium, ammonium sulfate precipitations, gel filtration chromatography and, after activation of trypsinogen into trypsin, affinity chromatography. Gel polyacrylamide electrophoresis showed a monomeric enzyme with a molecular weight of 27 kDa. The purified equine trypsin served for the immunisation of rabbits in order to obtain a specific antiserum, and the labelled antigen was prepared by iodination of equine trypsin with 125I. The RIA was based on the binding of the antigen to the antibody followed by the separation of the antigen-antibody complex by immunoprecipitation in the presence of sheep anti-rabbit gammaglobulins and the assay of the radioactivity in the precipitate. The RIA showed good sensitivity, specificity, precision, accuracy and reproducibility. The reference mean value of TLI in the plasma of healthy horses (n = 20) was 30.01+/-6.84 ng/mL (upper confidence limit 50.52 ng/mL; p < 0.01). Three horses with non strangulating intestinal obstruction without shock showed TLI values within normal limits whereas 5 of 7 horses with strangulation obstruction showed TLI levels above the upper confidence limit. Further studies using the RIA and the enzymatic assay should be performed in order to confirm the role of the pancreas in equine intestinal obstruction.

Comparative Studies with Penicillinase, Horseradish Peroxidase, and Alkaline Phosphatase as Enzyme Labels in Developing Enzyme Immunoassay of Cortisol

Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37°C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around −2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2–3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market.

Innervation of cutaneous structures in the mouse hind paw: A confocal microscopy immunohistochemical study

The normal innervation of structures in mouse foot pads was investigated with immunohistochemistry and confocal microscopy. Nerves were visualized by incubating Zamboni fixed, thick, frozen sections with antibodies to protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and protein zero. The antibodies were localized using cyanine 3.18 labeled anti-rabbit gamma globulin. PGP 9.5 immunolocalization showed dense nerve bundles at the base of the foot pad with branches to larger blood vessels, sweat glands and epidermis. Sweat gland tubules were surrounded by numerous sudomotor axons; single fibers accompanied the sweat duct toward the skin's surface. Nerve bundles containing myelinated and unmyelinated axons ran through and around the centrally located sweat gland cluster to end in free nerve endings and Meissner's-like corpuscles at the apex of the foot pad. Other bundles running parallel to the epidermis gave arcuate branches that supplied epidermis on the sides of the pads with a rich nerve network, principally with free nerve endings that often reached the most superficial cell layers of epidermis. Calcitonin gene-related peptide-immunoreactive (-ir) nerves were distributed to dermis and epidermis in lower density than PGP 9.5-ir fibers. Substance P-ir fibers were less numerous; most terminated as free endings in deeper layers of epidermis. Vasoactive intestinal peptide-ir nerves almost exclusively innervated sweat glands, ducts and blood vessels, but not epidermis. The mouse hind paw has potential to serve as a model system for investigations of functional and morphological changes that affect peripheral and autonomic nerves under diverse experimental conditions.

Radioimmunoassay for Hydroxyphosphinyl-3-hydroxybutanoic Acid (SQ 33,600), a Hypocholesterolemia Agent

A specific and sensitive radioimmunoassay (RIA) for the measurement of SQ 33,600 in biological fluids has been developed. The assay utilizes a SQ 33,600 polyclonal antibody, [125I]iodohistamine-SQ 33,600 radiolabel, and standards in serum. Satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved by employing polyethylene glycol-goat anti-rabbit gamma-globulin (PEG-GARG) separant. A quantitative recovery in serum and urine of the exogenous analyte was obtained at all concentrations of SQ 33,600 tested. Intra-assay coefficients of variation (CVs) were 6.19 and 5.57% for the low and high controls, respectively. Interassay CVs were 6.64 and 6.06% for the low and medium controls, respectively. Results from the parallelism studies were acceptable for both serum and urine samples. Comparison of results from samples which were assayed by RIA and high-performance liquid chromatography (HPLC) demonstrated a significant correlation (r = 0.994; HPLC = 1.09 RIA + 57.98; n = 45). The present RIA has been successfully used to assay clinical specimens from pharmacokinetic studies.

A radioimmunoassay for the new antiviral agent 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil.

A specific and sensitive radioimmunoassay (RIA) for the measurement of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in biological fluids has been developed. The assay has a range of 2.5-1,000 ng/ml and 10-1,000 ng/ml for serum and urine, respectively, and has the sensitivity to detect 2.5 and 25 ng/ml of BV-araU in serum and urine, respectively. A satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free ligand was achieved by employing polyethylene glycol-goat anti-rabbit gamma globulin separant. A quantitative recovery of the exogenous analyte was obtained at all concentrations of BV-araU tested. The assay is specific for the parent drug and is not affected by the presence of its metabolite, BV-U (bromovinyl uracil) or serum components (nucleotides, nucleosides, or sugars). Intraassay coefficients of variation were 3.1-4.4% and 2.5-4.2% for serum and urine controls, respectively. Interassay variability was < 8.6% for all serum and urine controls. Linear regression analysis showed that the correlation between RIA and high-pressure liquid chromatography was excellent (r = 0.997). The ascending dosage studies have been analyzed by the BV-araU RIA, and results indicate that the values of area under the serum concentration-time curve increased proportionally with the administered dose of BV-araU up to 80 mg. Cumulative urinary excretion data showed that approximately 50% of unchanged BV-araU was excreted in the urine within 24 h.

Radioimmunoassay for ceronapril, a new angiotensin-converting enzyme inhibitor, and its application to a pharmacokinetic study in healthy male volunteers.

Ceronapril is a member of a new chemical class of angiotensin-converting enzyme inhibitors being developed by The Bristol-Myers Squibb Pharmaceutical Research Institute. A radioimmunoassay (RIA) has been developed for the measurement of ceronapril in biological fluids. The RIA has a range of 0 to 500 ng/ml and has the sensitivity to detect 1.0 ng/ml of ceronapril. Satisfactory zero binding and sensitivity were obtained after a 2-h incubation at room temperature or overnight at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved by employing polyethylene glycol-goat anti-rabbit gamma-globulin separant. A quantitative recovery of the exogenous analyte was obtained at all concentrations of ceronapril tested. Intraassay coefficients of variance (CV's) were 3.9% and 4.6% for the low and medium controls, respectively. A highly significant statistical correlation between RIA and [14C]TLRC was observed for both plasma and urine samples. Clinical samples from the ascending dosage studies have been analyzed by the ceronapril RIA. The maximum concentration and the area under the plasma concentration-time curve did not increase in a dose-proportional manner for doses above 100 mg.

Radioimmunoassay of Corticosterone using &lt;SUP&gt;125&lt;/SUP&gt;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.

Importance of Selection of Separation System in the Development of Enzyme Immunoassay: An Experience with Follicle Stimulating Hormone (FSH) Assay

An antiserum to follicle stimulating hormone (FSH) obtained as a gift from National Institute of Health (NIH), U.S.A. could not be adsorbed on microtitre ELISA plates, although two other FSH antisera raised in authors' laboratory could be adsorbed. A good precision profile for the FSH assay using these three antisera could be achieved with only one separation system viz. solid phase anti rabbit gamma globulin (ARGG), out of the five separation systems tried. The study suggests that a few antisera used for radio-immunoassay (RIA) purposes may not by themselves get adsorbed on plastic plates. However, they could be effectively used for ELISA purposes using solid phase second antibody.

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.

A Radioimmunoassay for SQ 27,519, the Active Phosphinic Acid-Carboxylic Diacid of the Prodrug Fosinopril in Human Serum

Fosinopril (SQ 28,555) is a member of a new chemical class of angiotensin converting enzyme inhibitors being developed by The Squibb Institute for Medical Research. During or following absorption, fosinopril, a prodrug, is hydrolyzed pharmacologically to the active diacid, SQ 27,519. A specific radioimmunoassay (RIA) for the measurement of SQ 27,519 in human serum has been developed. The assay utilizes a specific SQ 27,519 antibody, 125I-iodohistamine-SQ 27,519 radiolabel, and human serum standards. Satisfactory zero binding and assay sensitivity are achieved after a 2-h incubation at room temperature. Separation of the antibody-bound and free radiolabeled antigens is achieved by using polyethylene glycol-goat anti-rabbit gamma globulin separant. Recovery efficiencies ranged from 97.2 to 109.4%. The assay exhibited little or no cross-reactivity with captopril. Cross-reactivities for prodrug (SQ 28,555) and phenolic SQ 27,519 were 5 and 9%, respectively. Intra-assay variability (3.3-5.6%) and interassay variability (7.1-6.6%) were observed. Linear regression analysis indicates that RIA and [14C] thin-layer radiochromatography (TLRC) methods gave a highly significant correlation (RIA = 1.0 [14C]TLRC + 0.17, r = 0.991). Pharmacokinetic profiles of patient sera containing SQ 27,519 obtained by RIA and [14C]TLRC are identical. The RIA has been used routinely in support of the bioavailability and pharmacokinetic studies of fosinopril in humans.

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.

Localization of xanthine dehydrogenase in chicken liver.

The immunohistochemical localization of xanthine dehydrogenase in chicken liver was examined by indirect immunostaining, using anti-chicken xanthine dehydrogenase antisera and peroxidase-labeled anti-rabbit gamma-globulin. The enzyme activity of xanthine dehydrogenase in isolated hepatocytes was assayed by the biochemical method. Immunostaining of hepatic tissue sections showed that the cytoplasm of hepatocytes, Kupffer cells and endothelial cells lining the hepatic sinusoid was intensely stained. In chickens fed with a standard food containing 20% protein, the specific activity of 100, 000xg supernatant fraction of homogenate of isolated hepatocytes was almost the same as that of the whole liver. When the chickens were fed on a protein-rich diet, the enzyme activity in supernatant of homogenized isolated hepatocytes was 2.3 times higher than that in normal liver. Both the immunohistochemical and biochemical data in this study are comparable to each other, and suggest that xanthine dehydrogenase mainly exists in hepatocytes.

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.