Quantitative Radioimmunoassay Research Articles

Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using 125I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodies in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women.

The Use of Functional and Quantitative Assays to Study Glycoprotein Ib in Platelets Stored Under Various In Vitro Conditions

There is much evidence to suggest that platelet membrane glycoprotein Ib is involved in the haemostatic function of platelets and it has been suggested that loss of this glycoprotein may occur during in vitro storage of platelet concentrates. In this study two quantitative radioimmunoassays were developed to measure the content of glycoprotein Ib in platelets stored under a range of conditions used in blood banks. One assay involved the use of iodinated Maclura pomifera lectin and the other the binding of a monoclonal antibody (AN51) specific for glycoprotein Ib. The results showed that there was no significant reduction in the glycoprotein Ib content of platelets under the storage conditions used. These results suggest that any loss of haemostatic effectiveness which occurs on in vitro storage of platelet concentrates is not attributable to a selective loss of glycoprotein Ib from the platelet surface.

Assessment of Anti-Streptokinase Antibody Levels in Human Sera Using a Microradioimmunoassay Procedure

A high capacity, quantitative radioimmunoassay procedure has been developed to measure IgG antibodies to streptokinase (SK) in human serum. Results showed that the use of low concentrations of 125I-SK (100 ng/ml) and ambient temperature incubation conditions minimised degradation of the target labelled antigen and afforded antibody binding values which could be confidently related to the native intact SK protein. SK-complexed to plasminogen was also shown to retain the equivalent antigenic activity of native SK. Comparison of the absolute anti-SK levels with streptokinase resistance titres demonstrated that the two measurements of antibody correlated statistically but afforded quantitatively poor agreements. A survey of IgG levels to SK in sera from 93 normal healthy volunteers showed that all individuals displayed readily measurable anti-SK antibodies, with some 80% having IgG concentration capable of binding in excess of 1 microgram SK per ml serum.

A proenzyme from chicken plasma similar to human plasma prekallikrein.

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.

Seasonal variation of antibody levels among pigeon fanciers.

IgG antibody against avian antigens was measured by quantitative radioimmunoassay in serum samples obtained regularly from twenty pigeon fanciers over a 1-year period. A seasonal variation was seen in nine antibody-positive subjects; eight of whom had symptoms of pigeon breeder's disease (PBD), and a clear peak of antibody production occurred during late summer, corresponding with the period of maximum avian contact in the sporting season. All subjects with insignificant specific IgG levels were asymptomatic and displayed minimal changes throughout the year despite a similar exposure pattern to antigen for all individuals. Raised total IgG was a feature of six symptomatic subjects, two of whom had raised total IgA. Three of these six subjects had maximum hypergamma-globulinaemia coinciding with peak specific-antibody levels, but in general the total immunoglobulin levels tended to remain high throughout the year with only marginal fluctuations. The total immunoglobulin levels in the other individuals were within normal limits and displayed no remarkable changes during the year. The subjects with pigeon breeder's disease had a more active immune responsiveness to avian contact, and the association of the highest levels with periods of maximal contact with antigen may have an important bearing on the dynamic nature of this condition.

Development and applications of a solid-phase radioimmunoassay for the PO protein of peripheral myelin.

This is the first report of a quantitative radioimmunoassay for PO. The assay uses antigen-coated plastic microwells, with antibody binding detected by 125I-labeled protein A. Either peripheral myelin proteins or purified PO may be used as the antigen. Optimal extraction of tissue samples for PO immunoassay requires careful attention to the sodium dodecyl sulfate-to-protein ratio. Sodium dodecyl sulfate interference with antibody binding can be minimized by adding an excess of nonionic detergent and carrier protein to the incubation buffer. This method allows the detection of 0.8 ng of PO (20 ng/ml). Results from this assay showed little or no immunoreactivity in extracts of brain, centra myelin, liver, purified myelin basic proteins, cultured, purified secondary Schwann cells, or membrane preparations from these cells. PO was clearly detectable in Schwann cell cultures from 3- to 4-day-old rats at 12-18 h after dissociation (4% of the level in adult sciatic nerve) and in extracts of one-day-old rat sciatic nerve (2% of the level in adult nerve). Myelin basic protein radioimmunoassays showed that the ratio of PO to myelin basic protein is essentially constant in extracts of sciatic nerve from ne-day-old, four-day-old, and young adult rats. Another result was that PO levels are reduced in the trembler mouse sciatic nerve.

A quantitative radioimmunological screening method for specific gene products

A rapid, sensitive, and quantitative radioimmunoassay for specific translation products was developed using Escherichia coli cells grown in 96-well microtiter plates. A simple and inexpensive apparatus that facilitates the simultaneous transfer of all 96 detergent-lysed cultures to nitrocellulose within 30 s is described. Following this quantitative transfer, selected proteins are screened using specific antisera and 125I-Protein A. The technique works with either cytoplasmic or membrane proteins. As little as a two- to threefold increase of a gene product over normal levels can easily be detected.

Local application of capsaicin to one sciatic nerve of the adult rat induces a marked depletion in the peptide content of the lumbar dorsal horn

In an earlier study we have shown that local application of capsaicin directly to one sciatic nerve induces a decrease of substance P and cholecystokinin octapeptide (CCK8)-like peptide from the dorsal spinal cord using immunocytochemical analysis. 1 Here the effect of locally applied capsaicin on seven peptides known to be present in the L4 segment was assessed by radioimmunoassay and immunocytochemistry. The peptides investigated were substance P, somatostatin and CCK8-like peptide (which are present in small diameter primary afferent fibres), neurotensin, enkephalin (which are intrinsic to the spinal cord), neurophysin (of supraspinal origin) and bombesin (whose origin is unknown). Fourteen days after a single application of 49 mM solution of capsaicin a significant depletion of substance P and somatostatin was detected. These results were confirmed by parallel immunocytochemical analysis which localised the dramatic decreases of substance P and somatostatin to lamina 1 and lamina 2. In addition a depletion of CCK8-like immunoreactivity was observed by immunocytochemistry in this area, but quantitative radioimmunoassay of CCK8-like peptide did not detect this depletion. The capsaicin-induced changes were dose-dependent and reversible. Small decreases were noted with concentrations of capsaicin as low as 0.1 mM. The changes were apparent from day 9 onwards, maximal depletion seen by day 14. By 200 days post-operatively, a recovery to normal peptide levels in the ipsilateral dorsal horn was observed. In addition, a significant depletion of cutaneous substance P was noted in the area of the skin innervated by the capsaicin-treated nerve. These changes were accompanied by a significant increase in noxious thermal response (hind paw immersion test, T = 49°C, ipsilateral leg 9.11 ± 1.3 seconds, contralateral leg: 5.1 ± 1.3 seconds, P = < 0.005). The peptides neurotensin, enkephalin, neurophysin and bombesin were not affected by capsaicin treatment. These findings suggest that local application of capsaicin induces an indiscriminate depletion of peptide-containing primary sensory afferent fibres which is dose-dependent, long-lasting, but reversible.

Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.

A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of [3H]methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme.

Nature of the antigenic complex recognized by T lymphocytes. IX. Direct immunochemical demonstration of nominal antigen on the macrophage cell surface.

As an initial approach to macrophage (M phi) antigen handling, guinea pig M phi pulsed with the radiolabeled terpolymer L-glutamic acid, L-lysine, L-tyrosine (GLT) were modified with the cell surface-specific probe 2,4,6-trinitrobenzenesulfonic acid either immediately after pulsing (fresh) or 24 h after pulsing (aged); nonidet P-40 extracts were prepared from these cells and analysis of these extracts with anti-trinitrophenyl antisera was performed in a quantitative radioimmunoassay. Such experiments indicated that fresh GLT-pulsed M phi contained a pool of intracellular GLT as well as a distinct pool of cell surface GLT. In contrast, aged GLT-pulsed M phi lacked intracellular GLT but expressed GLT on the cell surface. In addition, a pool of cell surface GLT was also detected on Ia-M phi and on M phi from guinea pigs which were nonresponders to GLT. Thus, this study provides a direct demonstration of the presence of exogenous antigen on the M phi cell surface. However, the cell surface expression of GLT does not appear to be an exclusive property expressed by functional Ia+ antigen-presenting M phi.

Comparison of enzyme-linked immunosorbent assay and radioimmunoassay for prostate-specific acid phosphatase in prostatic disease.

We compared results by an enzyme-linked immunosorbent assay (ELISA) with those by a standard radioimmunoassay (RIA) for detection and quantitation of prostate-specific acid phosphatase (EC 3.1.3.2) in serum. Control subjects, patients with benign prostatic hyperplasia, and patients in all four clinical stages of prostatic adenocarcinoma were tested. The upper limit of normal (95% of the population) by the ELISA was 2.0 micrograms/L, and by the RIA was 2.2 micrograms/L. In prostatic adenocarcinoma stage I (not detectable by digital rectal examination), ELISA was slightly more sensitive than RIA, but sensitivity was still relatively low (20%). As tumor mass increased (stages II through IV), the frequency of increased concentrations of prostatic acid phosphatase in serum also increased. We confirmed this increase in circulating enzyme in some cases of benign prostatic hyperplasia and suggest that this finding is related to either acinar cytolysis or an increase in acini size and number. Although prostate-specific acid phosphatase is not a cancer-specific enzyme, we conclude that its measurement may be of considerable value in monitoring prostatic disease.

A general approach to standardization of the solid-phase radioimmunoassay for quantitation of class-specific antibodies

The feasibility of using an anti-human immunoglobulin/human immunoglobulin/[ 125I]anti-human immunoglobulin ‘sandwich’ in a solid-phase radioimmunoassay to produce a standard curve which could be used to quantitate antigen-specific antibody of a particular immunoglobulin class was investigated. The amount of secondary antibody (SAb) bound was determined as a function of whether the primary antibody (PAb) was bound to its specific solid-phase antigen or by a solid-phase anti-human immunoglobulin. No significant difference between the two values was observed. Quantitation of anti-tetanus toxoid antibody by this method was in good agreement with quantitative precipitin tests. Comparison of SAb binding as a function of the way the PAb is bound was extended to class-specific PAb by use of murine monoclonal antibodies to meningococcal antigens. In most cases somewhat greater binding of SAb occurred when PAb was bound to antigen, but in several cases where low avidity antibody and/or poor quality antigens were used, greater SAb binding occurred when PAb was bound by anti-mouse immunoglobulin. The results indicate that this approach may be useful as a general method for standardizing the SPRIA and other solid-phase immunoassays such as the ELISA to measure class-specific antibody.

Radioimmunoassay for detection of anti-oligodendrocyte antibodies

A solid phase radioimmunoassay (RIA) for detection and quantitation of rabbit anti-oligodendrocyte antibody has been developed using bovine oligodendroglia preparation. The assay is simple, rapid, reproducible and economical. It is approximately 150× as sensitive as immunofluorescence. Specificity has been established by using different bovine tissue antigens as substrate and absorption studies. This assays represents a potentially powerful tool for the detection and quantitation of anti-oligodendroglial antibodies and oligodendroglial antigens in serum and CSF of man and experimental animals.

Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site.

Calmodulin is a small, acidic, calcium-binding protein that exhibits multiple in vitro biochemical activities. Although calmodulin has no known enzymatic activity, it stimulates several enzyme activities in calcium-dependent manner. Because of its ubiquitous distribution and highly conserved structure, it has been difficult to elicit anti-calmodulin sera of useful titer. We describe here a reproducible and rapid method for producing anti-calmodulin sera. This method requires the injection of performic acid-oxidized calmodulin, but the antisera react equally well with unoxidized calmodulin. A response was elicited in 11 out of 11 rabbits using three variations of this method. Antisera titers were high enough to enable development of a quantitative radioimmunoassay using dilutions of whole sera, immunoglobulin fractions, or immunoglobulin fractions purified on calmodulin-Sepharose conjugates. For the majority of the antisera, the immunoreactive site is contained in a unique region of the calmodulin molecule. Based on the quantitative reactivity of overlapping tryptic and cyanogen bromide peptides, we propose that a major immunoreactive site is fund within an 18-residue region in the COOH-terminal domain of calmodulin.

A solid phase, direct competition, radioimmunoassay for quantitation of secretory IgA in human serum

A solid phase radioimmunoassay is described for measurement of secretory IgA (sIgA) in human serum. The assay is based on direct competition between sIgA in serum and purified labelled milk sIgA for anti-secretory-component antibodies coated on disposable plastic cups. The assay is relatively rapid, reproducible and of adequate sensitivity. A wide range of sIgA concentrations (5–120 μg/ml) can be tested with the same dilution of human serum. The arithmetic mean ± S.D. of the serum sIgA levels of 120 random blood donors (both sexes) was 10.9 ± 4.6 μg/ml. Compared to previously published methods and results of quantitation of sIgA in human sera, the present assay is an improvement which should yield interesting data in human hepatic physiopathology.

Physical, chemical, and immunochemical studies of apolipoprotein A-I from pigeon plasma high density lipoproteins

Pigeon plasma high density lipoproteins (HDL) were isolated by ultracentrifugation between the densities of 1.063 and 1.21 g/ml. Gel filtration of delipidated HDL in 5 M guanidine-HCl on Sephadex G-150 yielded a major fraction which eluted at the same position as human apolipoprotein A-I isolated from HDL. In SDS-gel electrophoresis, the isolated apolipoprotein co-migrated with human apolipoprotein A-I with a molecular weight of approx. 28000. The amino acid composition was similar to the apolipoprotein A-I isolated from human and hen plasma. The isolated apolipoprotein from pigeon plasma had therefore been designated as apolipoprotein A-I. As judged by circular dichroism (CD), the apolipoprotein A-I displayed a maximum mean residue ellipticity of approx. −28 000 at 222 nm while at concentrations greater than 0.2 mg/ml. Calculations of α-helicial content gave values of 85%. Lowering the concentration of apolipoprotein A-I was found to concomitantly decrease the ellipticity (absolute value) suggesting that there was some conformational change when the apolipoprotein A-I concentration varied. The isolated pigeon apolipoprotein A-I was found bound to the phospholipid (dimyristoyl phosphatidylcholine) and there was no significant conformational change upon lipid binding as judged by CD. Under the same experimental conditions, human apolipoprotein A-I exhibited a drastic conformational change by increasing its helicity in the presence of phospholipid. The helical content of human apolipoprotein A-I was increased from 48 to 85%. This finding suggests that the apolipoprotein may not necessarily increase its helical content during lipid binding. Moreover, immunochemical studies showed that rabbit antiserum prepared against pigeon apolipoprotein A-I could partially react with human apolipoprotein A-I as determined by quantitative radioimmunoassay.

On the mode of incorporation of human transplantation antigens into lipid vesicles.

Highly purified HLA (A and B) antigens in octylglucoside were quantitatively incorporated into egg lecithin vesicles, and it was demonstrated that vesicles of different densities could be prepared by varying the ratio of lipid to protein. The HLA antigens were bound to the vesicles by the hydrophobic stretch of amino acids normally integrated into the plasma membrane, and most if not all HLA antigens in the lipid vesicles occurred in the right-side-out orientation, as demonstrated by pronase digestion and by quantitative radioimmunoassay determinations on intact HLA antigen-containing vesicles. Both the lipid and the HLA antigens aggregated at octylglucoside concentrations below the critical micelle concentration. The protein aggregates appeared stable and could be incorporated into preformed lipid vesicles. It is suggested that vesicle formation preceeds the incorporation of protein into the lipid vesicles.

A simple method for preparation of C3c fragment from trypsinized serum-reacted zymosan.

A simple method for preparing relatively pure C3c fragment from serum-reacted zymosan is described. Although hyperimmunization studies indicate contamination of the "C3c" product by C3d and immunoglobulin antigenic materials, these are present in such small amounts that they do not impair the effectiveness of the product as a reagent for absorbing anti-C3c reactivity from polyspecific antisera. Furthermore, two of the three hyperimmune sera contained such weak anti-C3d that they could serve as monospecific anti-C3c antiglobulin reagents if used at moderate dilution. Neutralization of accompanying anti-IgG and weak anti-IgM is easily accomplished with readily available purified immunoglobulins. Preliminary studies indicate that the C3c product should be adaptable to quantitative radioimmunoassay of anti-C3c antibody concentrations and equilibrium constants.

Antibody potential of human peripheral blood lymphocytes differentially expressing surface membrane IgM.

The appearance of circulating pokeweed mitogen-reactive B lymphocyte subpopulations responsible for the synthesis of IgM and IgG anti-tetanus toxoid antibody after in vivo booster immunization has been analyzed for differential expression of surface membrane IgM. B cells were rosetted with human mu-chain specific antiglobulin-coupled ox red blood cells and separated by density centrifugation into mu+ and mu- populations. After 8 days in vitro culture with irradiated T cells and pokeweed mitogen, antibody synthesis was assessed by quantitative radioimmunoassay. Mu+ cells synthesized both IgG and IgM anti-tetanus toxoid antibodies, whereas mu- cells predominantly synthesized IgG anti-tetanus toxoid antibodies. Limiting dilution analysis revealed a greater number of IgG anti-tetanus toxoid precursors in the mu+ population at all times after immunization compared with mu- cells. However, the activity, as measured by the amount of antibody produced per IgG anti-tetanus toxoid antibody precursor, was 2 to 4 times greater in the mu- population than that in the mu+ population. These results indicate that functionally distinct human circulating B lymphocytes can be identified by their differential expression of surface membrane IgM.

IgE-based radioimmunoassays for the quantitation of allergens.

IgE-based radioimmunoassays for the quantitation of allergens.