Evaluation of Passive Hemagglutination, Solid-Phase Radioimmunoassay, and Immunoelectroosmophoresis for the Detection of Hepatitis B Antigen

Sensitivity and specificity of passive hemagglutination (RCA), solid phase radioimmunoassay (RIA), and immunoelectroosmophoresis (IEOP) were compared under experimental and clinical conditions. In dilution experiments with sera containing hepatitis B antigen (HB Ag) of known subtypes, the sensitivity for an ad subtype serum was RIA (1), RCA (1/2), IEOP (1/256) and for an ay subtype serum RCA (1), RIA (1/8), IEOP (1/128). An evaluation of the National Institutes of Health, Division of Biologics Standards test panel number 2 demonstrated HB Ag in 34 of 60 samples by RIA, in 33 by RCA, and in 25 by IEOP. HB Ag was detected in 57.5% of 200 outpatients with a tentative diagnosis of hepatitis by RIA, in 54% by RCA, and in 42.5% by IEOP. In 1,661 volunteer blood donors, 13 (0.78%) were "positive" for HB Ag by RIA, 11 (0.66%) by RCA, and 3 (0.18%) by IEOP. However, absorption experiments indicated that at least six of the above RIA positive and five of the RCA positive sera exhibited nonspecific positive reactions.     

Measurement of human FSH by radioreceptor assay

We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15 x 10(-10) M. The standard curve was obtained with 0.2-25.6 ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the non-specific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intra-assay and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.     

Interactions of Standard Antibody with Australia Antigens in Au Ag-Ab Radioimmunoassay

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.     

Comparative evaluation of methods for detecting HBsAg in sera

The results of the analysis of 1209 serum samples, made with a view to detecting those containing HBsAg, are presented. This analysis was made by the radioimmunoassay (RIA) on a polyethylene film, by the standard RIA technique with the use of a diagnostic kit obtained from Abbott Laboratories (USA) and by the passive hemagglutination (PHA) test. The RIA film technique was found to have the sensitivity of about 2 ng/ml HBsAg, which is similar to the sensitivity of the kit from Abbott Laboratories and exceeds the sensitivity of the PHA test approximately 50-fold. The percentage of detected HBsAg-positive sera, yielded by analysis with the use of the RIA film technique and the standard RIA technique, is the same. The RIA technique make it possible to detect more positive sera than the PHA test by about 2.5%.     

Endogenous thyrotropin-releasing hormone in the anterior pituitary: sites of activity as identified by immunocytochemical staining.

Thyrotropin-releasing hormone (TRH) immunoreactivity was localized in the rat anterior pituitary with rabbit anti-TRH sera and the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. Stain was present in secretory granules of cells possessing morphological characteristics of thyrotropes, gonadotropes and lactotropes. Antibody absorption studies with anti-TRH sera absorbed with TRH, 3 diastereoisomeric analogues of TRH, gonadotropin-releasing hormone (GnRH), bovine serum albumin, thyrotropin, prolactin, adrenocorticotropin, luteinizing hormone, follicle stimulating hormone were performed to determine the specificity of the staining reaction. Only absorption with TRH resulted in a significant reduction in staining intensity. In vitro experiments were then begun with hemipituitaries to ascertain if intrapituitary TRH might originate by sequestration of exogenous, plasma membrane bound TRH or by de novo synthesis. The results suggest that anterior pituitary TRH is of endogenous origin.     

Reagentless amperometric immunosensor for human chorionic gonadotrophin based on direct electrochemistry of horseradish peroxidase

A novel amperometric immunosensor for determination of human serum chorionic gonadotrophin (HCG) was constructed by immobilization of HCG with titania sol-gel on a glassy carbon electrode and the direct electrochemistry of horseradish peroxidase (HRP) labeled to HCG antibody (HRP-anti-HCG). The morphologies of the HCG membrane were characterized to be chemically clean, porous and homogeneous. HRP-anti-HCG was functionally conjugated with the immobilized HCG after incubation in phosphate buffer (PBS) containing HRP-anti-HCG. A direct electron transfer of HRP with a rate constant of 1.35+/-0.40 s(-1) was observed at the HRP-anti-HCG-HCG/titania sol-gel membrane modified electrode in 0.1 M PBS pH 7.0. With a competitive mechanism the differential pulse voltammetric peak current of the immobilized HRP decreased linearly with an increasing HCG concentration from 2.5 to 12.5 mIU/ml in the incubation solution. The HCG immunosensor showed a detection limit of 1.4 mIU/ml, a good accuracy and acceptable precision and reproducibility with an intra-assay CV of 4.7% at 5.0 mIU/ml and an inter-assay precision of 8.1% obtained at 10 mIU/ml. The biosensor displayed a good stability in a storage period of 30 days.     

Different immunofluorescent titres of Candida albicans blastospores and pseudohyphae

Indirect immunofluorescent staining ofCandida albicans pseudohyphae and blastospores by serial dilutions of sera from patients with different forms of candidiasis showed that the mycelial form fluoresced more intensely at higher serum dilutions than the blastospores.     

Analysis of an estrogen binding macromolecule in human pancreas by radioimmunoassay

A radioimmunoassay for quantitation of the human pancreatic estrogen binding protein (hEBP) was developed using polyclonal rabbit hEBP antiserum and iodinated purified hEBP. Parallel dose-response curves were obtained when serial dilutions of human serum and of cytosols obtained from human pancreas, prostate and colon were analyzed simultaneously with serial dilutions of purified hEBP standard. Very high levels of hEBP (500-1000 mg/kg wet weight) were found in normal pancreas. High as well as medium levels were found in pancreatic carcinoma tissue and medium values (0.1-1 mg/kg wet weight) in prostate, colon and ovarian tissue. Other tissues and serum from healthy volunteers showed low values, usually below 0.1 mg/kg. When serial dilutions of rat pancreatic cytosol were analyzed in the hEBP assay, [125I]hEBP was displaced by the rat preparation, but the dose-response curves were not parallel to the standard curves, indicating similarity but non-identity between the estrogen binding proteins in human and in rat pancreas.     

Development of an avian calcitonin radioimmunoassay using synthetic chicken calcitonin as immunogen

1. A sequential double antibody radioimmunoassay (RIA) has been developed using synthetic chicken calcitonin (CT) as antigen, tracer and standard. 2. The immunoassay has a minimum detection limit of 0.5 ng and effective dose (ED50) of 7 ng. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of CT standard. Extracts of chicken and turkey ultimobranchial glands caused parallel displacement of tracer similar to synthetic CT. 3. Primary antisera (anti-chicken CT) was raised in guinea pigs immunized with RIBI: animals treated with Freund's complete adjuvant failed to respond. 4. Chicken CT was determined to have a half-life of 60 sec in the turkey hen. Development of a homologous RIA for avian CT will allow studies to elucidate the role of this hormone in birds.     

Levels of human chorionic gonadotropin in breast cyst fluid

The results of dosage of human chorionic gonadotropin (HCG) in 75 breast cyst fluids taken from 61 patients suffering from breast gross cystic disease (BGCD), 13 of which had multiple mono and/or bilateral cysts are discussed. The corresponding sera were also examined. Assays were carried out using the ELISA method. For comparison, HCG levels were also determined in 21 breast cyst fluids using RIA method. In 66.7% of the breast cyst fluids examined the hormone levels were higher than normal serum values. Corresponding sera showed HCG to be within the range of normal levels. In 5 patients the multiple and/or bilateral cysts showed widely differing hormone levels. The importance of these observations is discussed.     

A single serum dilution method for the quantitation of neutralizing antibodies to varicella-zoster virus

A one-point serum dilution method for determination of neutralizing antibody in human sera to varicella-zoster (V-Z) virus instead of the serial serum dilution method was investigated. Focus counting was performed under a microscope on day 5 to 6 after inoculation of V-Z virus into 6-well plastic trays in which human embryonic lung cells were grown. A table was constructed to estimate the ND50 titers by the per cent reduction of the focus count from the control at only one dilution of test sera. The estimated ND50 values agreed well with those determined by the serial serum dilution method. Test sera showed a slight nonspecific reactivity at low serum dilutions, but reliable results could usually be obtained at a serum dilution of 1:8 or more. This method, which saves materials and labor, was applied to the quantification of neutralizing antibody against V-Z virus in human sera with satisfactory accuracy and reproducibility.     

A quantitative solid-phase enzymeimmunoassay for 13,14-dihydro-15-keto- prostaglandin F2 alpha in plasma.

Enzymeimmunoassays (EIA) can be viable alternatives to radioimmunoassays (RIA). Indeed, from an environmental perspective, EIA are preferable to RIA. Therefore, the purpose of this project was to develop a quantitative EIA for 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in bovine plasma. Acetylcholine esterase bound covalently to PGFM, rabbit anti-PGFM, mouse monoclonal anti-rabbit IgG, and PGFM were the principle reagents used for the EIA. Validation experiments indicated that: 1) PGFM standard curves, with doses ranging from 391 to 200,000 fg per microtiter well, were linear; 2) assay sensitivity averaged 391 fg per well; 3) for satisfactory results, PGFM had to be extracted from plasma; 4) content of PGFM in ethyl ether extracts of aliquots from serial dilutions of whole plasma with unknown amounts of PGFM and charcoal-stripped plasma supplemented with known amounts of PGFM did not deviate from parallelism with PGFM standard curves in buffer; 5) correlation between EIA and RIA measurements of PGFM in the same plasma samples was .95; 6) the regression of EIA data on RIA data was linear (Y = .93 x + 83.9; r2 = .91); 7) intra- and interassay coefficients of variation were 3.3 and 10.6%, respectively. The EIA developed in this project is a valid and reliable method for quantitating PGFM in extracts of bovine plasma.     

一种快速鉴定猪肺炎支原体免疫显性蛋白抗原的ELISA方法的建立

旨在建立一种快速鉴定猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)血清体液免疫显性蛋白抗原的方法。通过构建p GEX-6P-1-mhp366重组表达质粒并转入大肠杆菌BL21(DE3),将GST-Mhp366重组蛋白进行原核表达。将GST-Mhp366重组菌和GST工程菌裂解液加入谷胱甘肽包被板进行抗原包被,分别与17份Mhp阳性血清和13份Mhp阴性血清反应,通过对抗原包被量、封闭液、血清和二抗稀释度的优化,确定间接ELISA的反应条件。最终确定GST-Mhp366重组菌和GST工程菌裂解液原液为抗原的最佳包被量,PBS+10%FBS+2.5%脱脂奶粉为最佳封闭液,分别将血清按照1∶500稀释、酶标二抗按照1∶40 000稀释作为最佳工作浓度,从而建立了一种基于间接ELISA的快速鉴定Mhp血清体液免疫显性蛋白抗原的方法,同时通过已知的血清体液免疫显性蛋白Mhp156和Mhp364对所建立的方法进行了验证。该方法的建立能够用于在基因组水平高通量筛选Mhp血清体液免疫显性蛋白抗原,并为Mhp初乳体液免疫和黏膜免疫显性蛋白抗原鉴定方法的建立奠定基础。     

Enzyme-linked immunosorbent assay for detection of serum antibody to CAR bacillus

An enzyme-linked immunosorbent assay (ELISA) for detection of CAR bacillus antibody in rat sera was developed by Ganaway et al., in 1985 although the ELISA method was not described in detail. We investigated antigen preparation and test procedures of the ELISA using two strains of CAR bacillus which we isolated from a mouse (CB-M) and a rat (CB-R). Allantoic fluids containing 2.4 X 10(8)/ml of CB-M and 2.0 X 10(8)/ml of CB-R were washed with sterile phosphate buffered saline (PBS), resuspended in a 1/5 volume of sterile carbonate buffer (pH 9.8) and sonicated. Then 1/40 and 1/80 dilutions of CB-M and CB-R lysates in PBS, respectively, were used for antigen solutions of ELISA. Briefly, antibodies in sera are reacted with antigens coated on the surface of microtiter plates. The amount of horse radish peroxidase labeled protein-A or anti-rat IgG bound to the antigen-antibody complexes is measured on the spectro photometer at wave length of 492 nm. A total of 180 mouse and 205 rat sera were tested against both antigens. The optical density (OD) values of 140 mouse and 161 rat sera obtained from SPF mice and rats free from CAR bacillus infection were on the average 0.005 and 0.019, respectively. On the other hand, OD values of the sera collected from CB-M or CB-R infected animals ranged from 0.20 to 1.52. According to these results, the cut-off OD value for positive reaction was set at 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioreceptor assay for cynomolgus monkey serum luteinizing hormone

Serum luteinizing hormone (LH) of cynomolgus monkeys was measured by radioreceptor assay (RRA). The assay system consisted of 100 microliter of standard or unknown samples, 100 microliter of a receptor preparation and 100 microliter of 125I-LH (LER-960). Dispersed interstitial cells of mature rat testes were suspended in Tris-HCl buffer containing MgSO4 (5 mM), bovine serum albumin (0.1%) and sucrose (0.3 M) and used as the receptor preparation. 125I-LH was dissolved in the same buffer. The incubation was carried out at 37 degrees C for 2 to 3 hr. The nonspecific inhibitory effect of sera in the radioreceptor assay system was compensated for by using LH-free diluent prepared by heat treatment of pooled sera. Validation of the assay demonstrated good reliability in terms of accuracy, precision and sensitivity (equivalent to 3.1 micrograms LER-907/ml of serum). Recovery experiments were performed by adding known amounts of cynomolgus pituitary LH to the normal serum and mean recovery and the standard deviation of the mean was 86 +/- 13%. The intra- and inter-assay coefficients of variation were 7 and 9%, respectively. By using this RRA system, cyclic changes in serum LH during the menstrual cycle were determined in three adult female cynomolgus monkeys. These results indicate that this RRA system is useful in measuring serum LH in female cynomolgus monkeys.     

In vitro oncospheral agglutination given by immune sera from mice infected, and rabbits injected, with eggs of Hymenolepis nana.

Oncospheral agglutination given by sera immunized with Hymenolepis nana eggs is described as a new way of assessing H. nana infection. All sera of mice which possessed acquired protective immunity against reinfection by H. nana eggs had the potency to induce oncospheral agglutination in vitro. Only oncospheres, which had been hatched, agglutinated, no agglutination occurred in sera from uninfected mice. Oncospheral agglutination was carried out by mixing 0-1 ml of serial two-fold dilutions of serum and 0-1 ml of Hanks' balanced salt solution containing about 600 hatched oncospheres. Titre of agglutinins was indicated as a reciprocal of the final dilution capable of giving agglutination clusters made of three or more oncospheres. Agglutinins developed within 14 days after a primary infection with 500 shell-free eggs. There was no rapid increase of agglutinins within 4 days following a secondary infection. The titre increase coincided with the increase in dosages of eggs. Agglutinins were thought to be immunoglobulins, because the potency of the serum to agglutinate oncospheres was extinguished after absorption of globulins with rabbit anti-mouse globulin serum. Agglutinins were produced in rabbits by intravenous injections of shell-free eggs. The titres of the rabbit sera were much higher than those of mouse sera.     

A unique property of fetal bovine serum: High levels of protein-glutathione mixed disulfides

Summary Fetal bovine serum has been reported to delay or inhibit “spontaneous” neoplastic transformation in vitro as compared with all other sera tested. The present results indicate that fetal bovine serum is also unique in containing high levels of protein-glutathione mixed disulfides (3 to 7 μg glutathione as mixed disulfide per ml serum). The level of mixed disulfide appears to vary in accordance with the period of gestation of the fetal calves used to prepare the serum, decreasing below detectable levels (less than 0.2 μg per ml) with nearterm fetal calves. Calf, adult bovine, fetal horse, and swine sera did not contain detectable levels of this type of mixed disulfide. This work was supported by a grant from the National Institutes of Health (CA 08348).     

Determination of tamoxifen and metabolites in serum by capillary electrophoresis using a nonaqueous buffer system

Tamoxifen (TAM), an antiestrogen, is widely used to treat hormone-dependent breast cancer in post-menopausal women. TAM may be used as a chemopreventive agent in women of child-bearing age; however, few data exist describing potential TAM-induced fetal toxicity. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, this work describes an analytical technique utilizing capillary electrophoresis (CE) for the detection of circulating levels of TAM, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in maternal rodent serum. Greater than 90% of ³H-labeled TAM was extractable from serum using 98:2 hexane–isoamyl alcohol. Optimum separation of TAM, DMT, and 4-HT was obtained on a 57 cm×50 μm capillary using a nonaqueous buffer system of 1:1 methanol–acetonitrile containing 50 mM ammonium acetate and 1% acetic acid. 4-Dimethylaminopyridine was used as internal standard. Temperature and voltage were optimized at 40°C and 15 kV, respectively. The limit of detection of TAM by UV detection at 214 nm was approximately 800 amol. TAM and DMT were confirmed in serum of female rats 4 h following a single oral dose of 120 mg/kg. Transplacental exposure of TAM to fetal tissue will be evaluated using this technique.     

Coating efficiency of a 1 -acid glycoprotein in competitive enzyme immunosorbent assays with antigen immobilized on the solid phase

Coating efficiency of rat and human serum a 1 -acid glycoprotein (a 1 -AGP) was investigated for competitive enzyme immunosorbent assays with antigen immobilized on the solid phase by using different pHs and buffers. Blocking materials and pH of coating buffer had a marked influence on the amount of a 1 -AGP that binds to plate. Usually, carbonate buffer is used at pH 9.6 or 9.0, but phosphate buffered saline (PBS) at pH 7.2 can be used for an effective coating. At pH 7.2, coating of a 1 -AGP in Tris buffered saline was five - tenfold as effective as in PBS and phosphate buffer. Blocking of uncoated surface with casein was ten - twenty times as effective as with fetal bovine serum albumin for coating of a 1 -AGP.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.