Double-antibody Assay Research Articles

Maternal Serum Hormone Concentrations in Long-Evans Rats

Abstract The purpose of this study was to determine the effect of pregnancy on serum concentrations of estradiol, progesterone and prolactin in Long-Evans rats. Female Long-Evans rats, both pregnant and non-pregnant, were used. Four groups (n = 10 rats in each group) were studied: Non-pregnant rats; pregnant rats at 7, 14, and 20 gestational days. Maternal blood was collected transcardially, and serum estradiol, progesterone, and prolactin were measured by radioimmunoassay. Commercial kits from Diagnostic Products Corporation for estradiol and progesterone, and an established in-house double antibody assay for prolactin, were utilized. Statistical analysis was performed using ANOVA, followed by Newman-Keuls post hoc test when applicable. Estradiol concentrations were increased throughout gestation from 16.2 ± 6.2 pg/mL (mean ± SD) in the non-pregnant rat to 23.2 ± 8.8, 25.0 ± 7.7, and 30.3 ± 29.6 pg/mL on gestational days 7, 14, and 20, respectively. Progesterone showed the same trend, increasing from 7.2...

Allergenic materials in the house dust of allergy clinic patients.

Environmental agents including animal, fungal, tree, and weed antigens are known to cause allergic rhinitis and asthma. The following study was performed to measure the antigen concentration of several of these in house dust of children seen in an allergy clinic. Comparisons are made between household allergen levels of children seen for asthma and children seen for other reasons. Dust samples were solicited from patients in a pediatric allergy specialty clinic and other individuals associated with the clinic. Persons submitting dust were asked to complete a questionnaire describing their house. Samples were extracted, centrifuged, and filtered for sterility. Samples were stored in 50% glycerol at -20 degrees C. Specific antigens for Alternaria, Cladosporium, Aspergillus, Candida, Dermatophagoides farinae, cat, dog, oak, fescue, ragweed, plantain, and cockroach were measured using inhibition assays developed with whole antigen extract. Allergens Der p1, Der f 1, Alt a 1, and Alt a 70 kD were measured using double monoclonal antibody assays. Significant concentrations of whole antigen from cat, dog, oak, Alternaria, and Cladosporium were detected. Between 0.1 and 18 microg of Der f1 and Der p1 per gram of dust were also measured. Alt a 1 and Alt a 70 kD levels varied between 3.0 and 1000 U/g of dust. Significant positive correlations were observed in levels of dust mite and Alternaria allergen for patients with an evaluation of asthma. We found measurable levels of fungal antigens (Alternaria, Cladosporium), mite antigens, and animal antigens (dog and cat) in the majority of dust samples in this self-selected set of allergy clinic patients. Specific allergens Alt a 1, Alt a 70kD, and Der p 1 were significantly higher in the homes of asthmatic patients when compared with patients seen for reasons other than asthma. These studies support the hypothesis that fungal allergen exposure is an important component in the pathogenesis of the clinical condition known as asthma.

125I Radioimmunoassay for the Dual Detection of Amphetamine and Methamphetamine

A radioimmunoassay that exhibits a nearly equivalent response to D-amphetamine and D-methamphetamine in urine over the assay range of 0 to 1000 ng/mL while displaying low cross-reactivity to L-amphetamine and L-methamphetamine (4.6% and 2.4%, respectively) has been developed. In addition, methylenedioxy-amphetamine (MDA) and methylenedioxymethamphetamine (MDMA) were detectable in the assay with cross-reactivity levels of > 100% and 77% respectively. Little cross-reactivity was observed with the commonly encountered over-the-counter (OTC) drugs and this cross-reactivity was further reduced by the addition of sodium periodate into the reaction mixture to oxidize the beta-hydroxylamines. The double (second) antibody assay uses 125I-radiolabeled derivatives of both D-amphetamine and D-methamphetamine as tracers in combination with two highly specific sheep antisera directed against D-amphetamine and D-methamphetamine. The assay exhibits a dose-response of approximately 90,000 dpm from 0 to 1000 ng/mL of D-amphetamine or D-methamphetamine with a minimum detectable dose for either drug of approximately 25 ng/mL. With a cut-off level of 500 ng/mL, the assay gave a positive result for 100% of the 111 clinical samples containing GC/MS confirmed (at or above the NIDA GC/MS cut-off values) levels of amphetamine and/or methamphetamine. Eighty-eight samples that screened negative in a clinical laboratory were all negative in the assay. Nineteen samples which were incorrectly identified as positive by other commercially available amphetamine assays were negative in this RIA.

Circadian Rhythms of Melatonin Release from Chicken Pineal In Vitro: Modified Melatonin Radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.

Astrovirus and Adenovirus Associated with Diarrhea in Children in Day Care Settings

The relative importance of astrovirus and adenoviruses as etiologic agents of diarrhea among children in day care was examined. Stool specimens from this prospective study were screened for both astrovirus and adenovirus hexon with two new indirect double-antibody assays and for enteric adenoviruses with an EIA specific for serotypes 40 and 41. Astrovirus was detected in a significantly greater percentage of children with diarrhea (4%, 21/524) than of those without (less than 1%, 1/138) (P less than .05); however, no difference between such such children with adenovirus infections was found (8%, 43/565, and 8%, 10/129, respectively). Overall, 30% (13/43) of all adenovirus hexon-positive specimens were enteric serotypes, and by extrapolation, enteric adenoviruses were identified in an equal percentage of children (2%) with and without diarrhea. This study documents the presence of astrovirus and enteric adenoviruses among children in day care in the United States, associates astrovirus with diarrhea in this setting, and suggests that viral agents may be the most common enteric pathogens among children with diarrhea in day care.

Radioimmunoassay of human eosinophil cationic protein (ECP) by an improved method. Establishment of normal levels in serum and turnover in vivo

A radioimmunoassay was developed allowing measurement of the cytotoxic cationic ECP. The assay, which has a total incubation time of 3.5 hr, is a double antibody assay with radiolabelled ECP, covering the concentration range of 2-200 micrograms/l. Performance data show a detection limit of less than 2 micrograms/l and a cross-reactivity with eosinophil protein X (EPX/EDN) of less than 0.06%. The coefficient of variation (%) within the measuring range was, within assay 4.8-10.4, and total 6.6-12.0. The assay is useful for measurement in various body fluids including serum, nasal secretions and bronchoalveolar lavage fluid, and dilution of samples prior to analysis was generally not required. Sera from 100 apparently healthy individuals revealed a geometric mean of 6.0 micrograms ECP/l and a range (95%) of 2.3-15.9 micrograms/l. The elimination rate of ECP, t1/2, in vivo was estimated to be 65 min when ECP was measured in serum. Comparisons between this assay and a method previously described showed that the new method is superior with regard to precision and assay procedure.

Analysis of the Humoral Immune Response to Treponema pallidum in the Different Stages of Untreated Human Syphilis

It has been shown that using treponema-specific and lipoidal assays for the demonstration of antibodies to Treponema pallidum subsp. pallidum in pooled sera of syphilitic patients, each stage of the untreated infection can be characterized by a typical immune response as far as specific immunoglobulin M (IgM) and G (IgG) antibody reactivity is concerned. In primary syphilis, IgM antibodies predominate over those of the IgG class. Moreover, IgM antibody reactivity with decreased titres can be detected in the later stages of untreated treponema) infection. Furthermore, it has been shown by using the double anti-body radioimmunoprecipitation assay with detergent-solubilized [ 35S] methionine-labelled Treponema pallidum as antigen followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography, that IgM and IgG antibodies in all stages of infection are directed against eight major antigenic proteins of Treponema pallidum with a Mr of 15500, 33000, 34500, 37000, 41000, 44500, 47000 and 71000.

Solving the problem of antibody interference in commercial "sandwich"-type immunoassays of carcinoembryonic antigen.

We evaluated the effect of human anti-murine antibodies (HAMA) on apparent concentrations of carcinoembryonic antigen (CEA) as measured in serum with commercial enzyme immunoassay (EIA) kits manufactured by Abbot ("two-step" double monoclonal antibody assay), Roche, and Hybritech (room-temperature protocol). In sera from patients given parenteral murine monoclonal antibody for experimental diagnostic and immunotherapy studies, HAMA titers were determined with Immunomedics' "ImmuSTRIP HAMA-EIA" kit reagents. "True" CEA titers were established by using the ImmuCEA/MA-EIA and heat-extraction to destroy HAMA before assay for CEA. The concordance of the ImmuCEA/MA assay with the Abbott and Roche CEA EIAs was established with sera from normal individuals and from patients who had not received parenteral injections of murine monoclonal antibody. At high (100 mg/L) concentrations of HAMA, false-positive results were observed with all three kits. The Hybritech and Roche assays were more sensitive to interference by HAMA than was the Abbott CEA-EIA, false-positive results being observed at HAMA concentrations between 1 and 10 mg/L. Similar sensitivity of the three kits to interference by primate anti-MAb sera was demonstrated. Use of primate anti-MAb sera to create controls with HAMA activity and of analyte is recommended to evaluate MAb assays for potential HAMA interference and for use to devise methods to eliminate HAMA interference.

Increased plasma levels of the terminal complement complex in patients with evidence of complement activation.

The terminal C5b-9 complex of human complement has recently been described and quantified in normal human plasma by an enzyme-linked immunosorbent assay (ELISA). We collected EDTA plasma samples from 20 patients clinically suspected to have complement activation. The terminal complement complex (TCC) and C3d split products were measured. The TCC was increased in 8 patients, and 6 of these also had increased C3d values, whereas 4 patients had increased C3d and normal TCC values. Two different double-antibody assays were used to detect terminal pathway activation: the combination of anti-C6 and anti-C9 detecting only the whole complex, and the combination of anti-C6 and anti-C5 detecting intermediate complexes as well. There was a close correlation between the observations in these two assays, suggesting that in general the whole cascade including C9 is involved when the terminal pathway of complement is activated in vivo. Quantification of TCC in plasma is an important supplement to already established methods for the evaluation of complement activation in vivo.

Heterologous enzyme immunoassay for puromycin aminonucleoside using β- D-galactosidase as a label

A heterologous enzyme immunoassay (EIA) was developed to quantify puromycin aminononucleoside (PA). This double antibody assay was based on the use of anti-puromycin (PU) antibody and used β- D-galactosidase-labeled PA conjugate prepared via N-( m-maleimidobenzoyloxy)succinimide. The standard curve of the assay ranged from 1 ng to 30 ng, and the lower limit of detection was 22.7 nM (1 ng/tube). The EIA was found to be approximately 20 times more sensitive than the homologous EIA for PA with anti-PA antibody and PA-β- D-galactosidase conjugate. The heterologous EIA was free from interference by any purine or pyrimidine analogs and drug levels were easily determined in rat tissue following i.v. administration at a dose of 15 mg/kg. The sensitivity and specificity of the EIA should provide a valuable new tool for use in pharmecokinetic and toxicity studies of PA.

Isolation of sheep spleen ferritin

Ferritin was isolated from the spleen of sheep and found to contain an iron/protein content of 21 per cent. Electrophoretic behaviour on 6 per cent polyacrylamide gel revealed three protein bands of Rf values of 5.7, 19.3 and 76.1. Sheep ferritin has a molecular weight of about 475,000 daltons and is high in glutamic acid, leucine and aspartic acid and low in methionine and cysteine. Sheep ferritin is not crystallisable by 10 per cent cadmium sulphate and has an ultraviolet-visible spectrum similar to that of bovine ferritin. Anti (sheep) ferritin antibodies raised in rabbits showed a 57.1 per cent binding at a serum dilution of 1:800 and 30 per cent binding at a 1:12,800 dilution, in a titre determination using double antibody assay procedure.

Radioimmunoassays for protein A of Staphylococcus aureus

Radioimmunoassays have been developed that can detect nanogram amounts of protein A (SpA), a product generated by Staphylococcus aureus that binds selectively to the Fc region of IgG from most mammalian species. Competition assays for fluid phase SpA utilize antibodies produced in chickens, 125I-labeled SpA as the tracer molecule, and either F(ab′) 2 fragments of rabbit IgG anti-chicken IgG or 40% ammonium sulfate as the precipitating agent to separate antigen-antibody complexes from free antigen. The double antibody assay could be carried out in serum from species that form only soluble complexes with SpA (e.g., rabbit), that react poorly with SpA (e.g., rat), or under appropriate conditions in serum from species (e.g., dog) that show high reactivity with SpA and form precipitating complexes. Chicken antibodies prepared by affinity chromatography on SpA-Sepharose and labeled with 125I were used in a direct binding assay for SpA present either on the cell wall of Cowan strain I or Wood 46 bacteria, in insoluble complexes prepared from SpA and whole serum or purified IgG, or in C1q binding complexes that were formed by passage of serum from normal or tumor bearing humans or dogs over SpA-collodion charcoal. Since both types of assays could detect SpA even in the presence of serum or IgG, they offer advantages over other techniques in which the SpA-Fc interaction may interfere.

Boosterable IgE antibody response in mice without the use of adjuvant.

A mouse model is presented giving boosterable IgE antibody responses preceding the IgG antibody responses without using adjuvants. Groups of CBA mice were injected subcutaneously with minute doses of penicilloyl bovine gammaglobulin, 3.8-94 ng daily for one or two 10-day periods. Some groups of animals were simultaneously given Bordetella pertussis bacteria intraperitoneally. The IgE antibody responses were recorded with passive cutaneous anaphylaxis (PCA) in rats as well with RAST. IgG antibody responses were determined with a double antibody assay. An overall good agreement between the antibody activity in the PCA and RAST was seen, both tests showing boosterable IgE antibody responses. Some discrepancies between the two tests are discussed.

A Double Antibody Assay for the Direct Determination of [N-(4-Amino)Phenyl] Aminophenyl Iminodiacetic Acid and its Derivatives in Serum

A double antibody assay for the determination [N-(4-amino)phenyl] aminophenyl iminodiacetic acid (I) and a number of its derivatives in serum has been developed. The same antiserum and tracer were used for the determination of the serum levels of derivatives of (I). The minimum detectable concentration of I that was detectable was 6 ng/ml. The precision error was less than 1% and the accuracy as indicated by the recovery ratios was satisfactory for all derivatives of I.

Preparation and characterization of antisera to the myelin-associated glycoprotein.

Rabbits were immunized with the myelin-associated glycoprotein (MAG) that had been purified from isolated rat brain myelin by selective extraction with lithium diiodosalicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. Antibodies to MAG were detected qualitatively by immunodiffusion and quantitatively by a double antibody assay utilizing [3H]fucose-labeled MAG as antigen. The antisera were capable of precipitating between 300 and 500 micrograms of MAG/ml of serum under the conditions of the assay. Preincubation of the anti-MAG serum with other glycoproteins or glycolipids did not inhibit the precipitation of labeled MAG. Similarly, preincubation of the antiserum with LIS-phenol extracts of non-neural tissues did not inhibit the immune precipitation of MAG. The specificity of the antiserum was also indicated by the selective double antibody precipitation of MAG from solubilized whole myelin that contained a heterogeneous mixture of [3H]fucose-labeled glycoproteins. The antibodies to MAG were not effectively absorbed by whole brain homogenate or purified myelin, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification. Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant.

A cell surface glycoprotein virus inhibitor that is not interferon

The possible relationship between a newly isolated glycoprotein virus inhibitor and interferon was assessed. Comparisons of the cell surface glycopeptide, obtained from mouse cerebral cortex, and interferon included antiviral activity, radioimmune assays, and the ability of antibodies raised against the brain cell surface glycoprotein (BCSG) and against mouse L cell interferon to precipitate the biological activity. BCSG was able to inhibit virus replication but only in a transient fashion. Although anti-BCSG precipitated a major portion of the radiolabelled inhibitor in a double antibody assay, anti-mouse interferon did not. Over 90% of the inhibitory activity was removed with anti-BCSG and Staphylococcus protein A while anti-mouse interferon removed little, or none, of the activity under similar reaction conditions. Other properties of the BCSG that distinguish it from interferon are presented.

Simple hanging drop (20 μl) double antibody radioimmunoassay of human IgM, IgG, IgA, IgD and IgE

A simple hanging drop radioimmunoassay has been developed for different classes of human immunoglobulin from serum or culture supernant samples. The imune precipitate was formed in inverted Terasaki plates (20 μl per well) using 4 aliquots (5 μl) as follows: (A) a standard or unknown sample; (B) rabbit antisera to the appropriate immunoglobulin class; (C) 125I-labelled Human immunoglobin of the appropriate class; (D) a developing goat-anti rabbit serum. The immune precipitate in the hanging drop was harvested and washed without centrifugation with a simple Terasaki plate harvester. The method gives a great saving both in time and in sample and reagent volumes in comparison with an established double antibody assay in tubes (4 × 100 μl aliquots).

Measurement of IgG antibodies against timothy pollen allergens. A high capacity microtiter assay procedure.

This study describes a microtiter double antibody assay for the measurement of specific IgG, which is believed to be identical with blocking antibodies. The precipitates were harvested on glass fiber filters, and the activity from iodinated antigens fixed in the precipitates was used as a measure of the level of the serum blocking antibodies.

C-terminal parathyrin (parathyroid hormone) radioimmunoassay in serum with commercially available reagents.

We describe a sequential double-antibody assay for measuring C-terminal parathyrin in serum with commercially available reagents. Intact bovine hormone, used as a working standard, is iodinated by conventional Chloramine T procedures. The antibody affinity is characteristic of high affinity binding (1.4 to 1.6 x 10(10) L/mol of intact parathyrin). The antibody also cross reacts with a C-terminal parathyrin fragment (amino acids 53-84) but not with a synthetic N-terminal parathyrin fragment (amino acids 1-34). The assay thus also measures both a C-terminal fragment of parathyrin and the intact hormone. The detection limit (250 ng/L; 500 int. units/L) is below the reference interval for healthy adults (430-1860 ng/L; 860-3720 int. units/L). Several commonly recognized problems with iodinated parathyrin are eliminated and accuracy and precision of the procedures for standard preparation and calibration are improved. Overall CV (between-run imprecision) is 10-17%. Analytical recovery is 80-90%, and C-terminal parathyrin measured in fresh sera and in sera stored for seven days at 30 degrees C is equivalent.

Respiratory allergy to ragweed: Correlation of bronchial responses to allergen with bronchial responses to histamine and circulating immunoglobulin E

Flow-volume loops were recorded before and after quantitative inhalation of increasing concentrations of histamine (H) and ragweed (R) in 26 subjects allergic to ragweed. A threshold response (TR) was defined by a change in forced expiratory volume in 1 sec (FEV 1 ) greater than -10% and the presence of wheezing. In 20 subjects with reproducible TRR and TRH, 14 with asthma and hay fever and six with hay fever only, we measured: (1) total IgE with a solid-phase (paper radioimmunosorbent test [PRIST]) and a double-antibody assay; and (2) specific IgE with four tests using as solid phase paper disks (commerical or laboratory prepared radioallergosorbent test [RAST]), microcellulose, and sepharose. The double-antibody assay and PRIST produced highly correlated results (r = 0.94), but total IgE was not related to TRR. Although the four tests for specific IgE gave highly correlated results, they did not correlate with TRR or TRH. There was a linear correlation between TRH and TRR (r = 0.71). When compared with the corresponding slope reported in sensitized guinea pigs, TRR/TRH was shifted toward lower antigen doses. In summary: (1) in opposition to total or specific IgE, histamine airway responses may be important in the bronchial localization of allergic reactions to ragweed; and (2) the airways seem to be more sensitive to allergen in spontaneous asthma in humans than in anaphylactic “asthma” in guinea pigs.