Progesterone determination in Iberian red deer by time-resolved fluorometry: an alternative method to RIA

The validation for Iberian red deer of a commercially available Time-resolved fluoroimmunoassay (TR-FIA) designed for analysis of progesterone in human beings was carried out. Intra-assay coefficients of variation ranged from 3.6% to 7.4%, while inter-assay coefficients of variation varied from 5.2% to 15.5%. Accuracy, evaluated by comparing results yielded by TR-FIA with those obtained from a validated radioimmunoassay (RIA) in the measurement of 14 samples, provided a high regression coefficient (R(2)= 0.93). Different progesterone concentrations added to pool plasma showed percentages of recovery that ranged between 102.6% and 82.48%. The limit of detection was 0.102 nmol/L. The results obtained indicate that the present method is suitable for the measurement of progesterone in female Iberian red deer.     

抗—HBs国际单位RIA一点法定量测定与计算方法的探讨

在Radio-immuno Assay(RIA)试验测抗－HBs国际单位的简易定量与计算中,采用RIA一点法测定,以分析表达式mIU/ml＝SC（mIU/ml)[exp(0.69315S-NC/SC-NC-1]进行结果计算。此式可以常用对数式表示为mIU/ml=SC(mIU/ml)[lg^-1(0.30103S-NC/SC-NC-1],当（S－NC）在0．3－1．1界线内时,其计算结果的相对误差小于6％,是目前误差最小的简易计算方法。同时还推算求出Holliger公式的阳性对照的最适含量为124．26mIU／ml,Richardson公式的阳性对照为125mIU/ml,中国药品生物制品检定所公式的阳性对照为92．5mIU/ml。     

Improved method for the assay of phenylglycosidase activity with a 4-aminoantipyrine reagent

An improved method for the assay of nmole quantities of phenol with a 4-aminoantipyrine reagent is reported and the use of the method for phenylglycosidase assay is shown. Tissue homogenate, serum, and different buffers did not interfere with the assay, thus making protein precipitation unnecessary.     

In vitro production of feline IgG: quantification by an enzyme-linked immunosorbent assay (ELISA)

We report here on the development of a sensitive and convenient enzyme-linked immunosorbent assay (ELISA) for feline IgG by using commercially available reagents and optimizing their concentrations. The reagents employed include goat anti-cat IgG antibody and alkaline phosphatase-conjugated goat anti-cat IgG antibody. The assay described is sensitive, reproducible, and highly specific for feline IgG. The assay was applied for the measurement of feline IgG synthesized and secreted in vitro by peripheral blood mononuclear cells cultured with or without a polyclonal B-cell activator. The amounts of secreted IgG in the supernatants measured by an ELISA correlated well with the numbers of IgG-secreting cells which were induced upon stimulation with pokeweed mitogen and determined by a reverse hemolytic plaque assay.     

Determination of tularemia antibodies by an immunoenzyme method on a solid-phase carrier (ELISA)

ELISA "sandwich" techniques have been developed and the optimum assay conditions for detecting specific antibodies in human serum samples have been determined. The possibility of using these techniques for the determination of the level of antibodies to tularemia antigens in the sera of persons immunized with live tularemia vaccine has been shown. Statistically significant differences in the level of antibodies to tularemia antigen in the sera of immunized and nonimmunized persons have been established. The comparative study of five serological methods - ELISA, the agglutination test, the passive hemagglutination test, the immunofluorescence test and the defined antigen substrate sera ( DASS ) techniques - has revealed the advantage of ELISA, whose sensitivity has proved to be considerably higher than that of all other methods used in our work.     

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.     

Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan-based real-time PCR assay has been developed.
Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene ( aerA ) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty-one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria , Aer. caviae and Aer. hydrophila . Only Aer. hydrophila strains tested positive by PCR assay.
Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species.
Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.     

Quantification of two cytochrome P-450 isoenzymes by an enzyme-linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) using monoclonal and polyclonal antibodies has been developed to quantify individual cytochrome P-450 isoenzymes in microsomal preparations, namely UT-A and PB-B. This very sensitive method can be used for the rapid processing of large quantities of determinations and requires only limited amounts of antibodies.     

Direct radioimmunoassay (RIA) of salivary testosterone: correlation with free and total serum testosterone

Simple and sensitive direct RIA for determination of salivary testosterone was developed by using RSL NOSOLVEX TM (125 1) kit produced by Radioassay System Laboratories (Carson, California). In addition, a relationship between salivary and serum free and total testosterone concentrations was studied in randomly selected 45 healthy subjects, 5 females on oral contraceptive pills and 28 hypertensive patients on various treatment regimens. The lowest weight of testosterone detectable by our modified method was equivalent to 1 pg/ml of saliva, taking into account analytical variability. Intra- and interassay coefficients of variation were 5.09 +/- 2.7% and 8.2 +/- 5.9% respectively. Statistically significant correlations were found between salivary and serum free testosterone (r = 0.97) and salivary and serum total testosterone concentrations (r = 0.70-0.87). The exception to this was a group of hypertensive females in which no correlation (r = 0.14) between salivary and total serum testosterone was found. It is also of interest that, while salivary testosterone was significantly increased in subjects taking oral contraceptives and most of the hypertensive patients the total serum testosterone concentration was in normal range. Our findings suggest that determination of salivary testosterone is a reliable method to detect changes in the concentration of available biologically active hormone in the circulation.     

Nitrate reductase: an improved assay method for phytoplankton

A new assay for measuring the activity of nitrate reductasein phytoplankton, based upon the permeability of cells treatedwith toluene to substrates and products, is described. The methodis simple and, since the reaction is carried out directly ona glass fiber filter, can be easily performed in the field oron shipboard. In comparison with previous methods, this techniquegave higher absolute amounts of NO^–₂ formed per unit tuneand higher enzymatic activities per sample volume when testedwith axenic algal cultures and with natural phytoplankton populationsfrom Lake Kinneret, the River Jordan and the Eastern Mediterranean.     

Glucosyceramide synthase of mouse kidney: further characterization with an improved assay method

The synthesis of glucosylceramide from ceramide and UDP-[3H]glucose by mouse kidney homogenates is very sensitive to the concentration of tissue. This was shown to be due to the presence of a UDP-glc pyrophosphatase, which could be blocked by adding NAD to the medium. A new solvent partitioning system is described, containing t-butyl methyl ether, isopropyl alcohol, and aqueous sodium sulfate, which separates the original substrate (UDP-[3H]glc) from the enzyme product, [3H]cerebroside. A particular advantage of the solvent system is that only a single partitioning step is needed, without backwashes, and the enzyme product appears in the upper phase, making transfer to a counting vial more reliable. A novel incubation device, a thermostatically controlled ultrasonic bath, is used to produce highly uniform enzyme reaction rates. Ca2+, as well as Mg2+ and Mn2+, was found to be a good stimulator of the glucosyltransferase. The enzyme activity in kidney of 22-day old mice, approximately 240 pmol/h/mg tissue, is significantly greater than previously demonstrated. The enzyme was stable in intact kidneys stored at -70 degrees C but unstable at 4 degrees C. The enzyme, when acting on endogenous ceramides, showed no demonstrable glucosylation of the C24 family of ceramides although this family is the predominant one in kidney.     

Fractionation of cortisol antisera by immunoadsorption chromatography: characterisation and use in an enzyme-linked immunosorbent assay (ELISA)

The use of affinity chromatography in the presence of 20% acetonitrile combined with a decreasing pH gradient has allowed the fractionation of two cortisol antisera into components of varying affinity. The high affinity fractions generate considerably improved ELISA standard curves compared to the intact sera but do not grossly alter the specificity of the antisera. Accordingly, the high affinity fraction of the least cross-reactive antiserum was used for a plasma cortisol ELISA. The cortisol ELISA, although returning slightly higher values than RIA, was comparable in the ability to distinguish dexamethasone suppression and cortisol response to synacthen and should thus prove of value in the assay of plasma cortisol.     

In vitro rabies vaccine potency appraisal by ELISA: advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody

The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.     

Detection of Renibacterium salmoninarum by the enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and identification of Renibacterium salmoninarum. The immune γ-globulin used in the assay was absorbed with two species of cross-reacting bacteria to make a specific test system. R. salmoninarum could be detected in clinically-diseased fish within 30 minutes of preparing a kidney sample, and thus because of its ease of use, the ELISA could be employed as a rapid field test for bacterial kidney disease (BKD), although isolation of R. salmoninarum was more sensitive than the ELISA for detecting individual carrier fish.     

Lectin affinity bioassay: an assay method for glycoprotein enzyme

A simple and sensitive chromogenic microtitre plate assay for glycoprotein enzymes is described, using melanoma tissue plasminogen activator (t-PA) as a model enzyme. The assay is based on the binding of t-PA to immobilised lectin and quantitating the bound enzyme with plasminogen, fibrinogen fragments and chromogenic substrate S-2251 on an ELISA plate reader. Seven different lectins were examined for the binding of t-PA, and of these, concanavalin A was chosen for subsequent studies. The specificity of this binding can be inhibited dose-dependently in the presence of D-mannose and methyl alpha-D-mannoside, but not by D-glucose and D-lactose. The lower limit of the sensitivity of this assay is about 0.5 IU/ml. Comparison of the dose-response curves indicates that the sensitivity of this assay method is very similar to that of bioimmunoassay using anti-t-PA IgG to capture the antigen. The applicability of this method to other glycoprotein enzymes was also evaluated using alkaline phosphatase from bovine mucosa. The specificity of this method was related to the choice of substrate and this was shown by analysis of a mixture of t-PA and alkaline phosphatase. It is suggested that this assay can be adapted for the analysis of complex glycoprotein mixtures with the appropriate choice of lectin and substrate.