The quantitation of rabies-specific antibodies. III. A comparative evaluation of modified counter immunoelectrophoresis, haemagglutination inhibition and serum neutralization titres of human sera

The rabies-specific antibodies of 73 serum samples from vaccinated humans were determined by the modified counter immunoelectrophoresis (MCIE), and the haemagglutination inhibition test (HAI) by using the conventional serum neutralization test (SN) as a yard-stick. Both MCIE and HAI were found to be sensitive and specific for the estimation of rabies antibodies. In general, the unitages obtained by the MCIE and SN showed statistically insignificant differences (P greater than 0.05) and the correlation coefficient between the two methods was 0.697 (P less than 0.05). Although the unitage of the sera detected by HAI tests was lower by a factor of 0.155 from the unitage of SN tests, there was statistically insignificant differences between the two techniques (P greater than 0.05) with a correlation coefficient of 0.556 (P less than 0.05).     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

The quantitation of rabies-specific antibodies. II. Factors affecting the sensitivity of the haemagglutination inhibition test

Various factors affecting the HAI test for the quantitation of rabies-specific antibodies have been evaluated with a view to obtaining maximum sensitivity and reproducibility in tests using tissue culture antigens prepared in vero cells and concentrated by dialysis. Goose erythrocytes treated with proteolytic enzyme bromelian at a concentration of 0.025% were much more susceptible to HA than those that were untreated or erythrocytes treated with neuraminidase. In addition, other parameters like the use of a phosphate buffered saline (PBS) as a diluent at pH 6.2, incubation at 0-4 degrees C for 1.5-3 h were found to be most critical for achieving maximum HA activity. To remove non-specific inhibitors, serum samples were treated with aerosil, acetone in combination or alone. Of the 73 serum samples tested, removal of non-specific inhibitors by aerosil alone occurred in up to 54.79% of the samples, whereas using acetone-aerosil treatment followed by adsorption with goose erythrocytes, the inhibitors were removed in 98.67% of the samples to a level that was undetectable at the 1:4 starting dilution in the HAI test.     

A rapid and sensitive method for the identification and quantitation of sub-picomolar amounts of neurohypophysial peptides

A simple, rapid and sensitive method, using isocratic reversed-phase HPLC, is described for the concomitant identification and quantitation of low levels of the various neurohypophysial peptides in biological tissues and fluids. The method requires little or no sample preparation and utilises UV peak detection at 215 nm with a serial signal amplification system to achieve a usable maximum sensitivity of less than 200 fmol of peptide.     

A rapid chemiluminescent method for quantitation of human DNA.

A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.     

A rapid method for quantitation of immunofluorescence on human lymphoblastoid cell suspensions.

A quantitative fluoroimmunoassay for antibodies to, and surface antigens of, human lymphoblastoid cells (IM-1) with photon-counting spectrofluorometry is described. IM-1 cell suspensions were reacted with rabbit antiserum to human spleen vesicular membranes, were washed, and then were reacted with an excess amount of fluorochrome-conjugated (fluorescein or rhodamine) goat anti-rabbit immunoglobulin G (IgG). Under appropriate conditions, antibodies to IM-1 cells could be detected with experimental/control fluorescence ratios ranging between 5 and 40. Moreover, detectable levels of antibody-saturated cells approached 5 × 10³ cells per milliliter or a total of 1.7 × 10³ cells per assay. Inhibition of the fluoroimmunoassay was performed with either viable IM-1 cells or IM-1 vesicular membrane preparations and demonstrated a dose-dependent antigen inhibition. Fluorescence of sensitized cells reactive with either fluorescein- or rhodamine-labeled antiglobulins could be quantitatively distinguished in dual-labeled preparations.     

A rapid and sensitive method for the quantitation of montelukast in sheep plasma using liquid chromatography/tandem mass spectrometry

A rapid LC-MS/MS method was developed and partially validated for the quantitation of montelukast in spiked sheep plasma. A total run time of 1.5 min was achieved using a short monolithic column and employing a rapid gradient. Sample preparation involved protein precipitation with twofold acetonitrile by volume during which a deuterated internal standard (montelukast D-6) was incorporated. The MRM transitions for montelukast and the deuterated internal standard were 586/422 and 592/427, respectively. A linear dynamic range of 0.25-500 ng/mL with a correlation coefficient of 0.9999 was achieved. Precision was below 5% at all levels except at the LOQ (0.36 ng/mL) which demonstrated an overall of R.S.D. of 8%. Post-column infusion experiments were performed with precipitated plasma matrix and showed minimal interference with the peaks of interest.     

A rapid and sensitive method for HPLC cholesterol determination in bile.

A relatively little time consuming simple method based on the treatment of bile with cholesterol oxidase and subsequent high performance liquid chromatography measurement of the 3-ketocholesterol produced in order to determine the level of the cholesterol concentration is described. The method avoids bilirubin interferences, has high reproducibility and recovery assays give 100% values. It is highly sensitive and suitable for use in the determination of cholesterol concentrations in bile and other bilirubin containing biological fluids.     

A sensitive method for the quantitation of lysophosphatidylcholine in canine heart

We have developed a procedure for the determination of small amounts of lysophosphatidylcholine in cardiac tissue. Lysophosphatidylcholine from canine heart was separated from the major phospholipids by column chromatography, and then acetylated with labeled acetic anhydride. The acetylated lysophosphatidylcholine was isolated by thin-layer chromatography and the lysophosphatidylcholine content was calculated from the radioactivity associated with the acetylated product. Although the sensitivity of the assay depends on the specific radioactivity of the acetic anhydride used, as low as 0.5 nmol of lysophospholipid in tissue samples can be readily quantitated. The results obtained from the control and ischemic canine cardiac tissues by this assay compares favorably with those obtained by lipid-phosphorus assay. The sensitivity and specificity of the present procedure allows us and other investigators to assay for lysophosphatidylcholine content in very small (10 mg wet weight) tissue samples.     

A rapid and sensitive method for measuring cell adhesion

We have adapted the CyQuant® assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant® assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.     

A rapid and sensitive method for the analysis of cyanophycin

A method has been devised for the quantitative analysis of cyanophycin, based on (1)H nuclear magnetic resonance (NMR) spectroscopy, allowing determination of the nitrogen status of cyanobacteria. Cyanophycin is extracted with minimal washing from small volumes of cells and quantified by integration of the NMR peak attributed to the protons attached to the delta-carbon of arginine. Linear relationships were found between the amount of cyanophycin determined by this method and both known concentrations of cyanophycin solutions and the amount of cyanophycin determined using the standard chemical arginine assay.     

A rapid sensitive collagenase assay.

A rapid, sensitive collagenase assay has been developed using¹⁴C-acetylated collagen as a substrate. Acid-soluble calfskin collagen was labeled with [1-¹⁴C]acetic anhydride at pH 8. The acetylated collagen had a specific activity of 6.25 × 10⁵ dpm/mg protein. Collagen was not denatured as evidenced by its resistance to nonspecific proteolysis and sensitivity to bacterial collagenase. Polyacrylamide gel electrophoresis of the acetylated protein showed that the radioactivity was present in the three bands corresponding to the α, β, and γ components of collagen. The rate of release of ¹⁴C from labeled collagen by Clostridium histolyticum collagenase was proportional to enzyme and substrate concentration.     

A method for rapid quantitation and preparation of antithrombin III-high-affinity heparin fractions

An electrophoretic method for the quantitation and preparation of antithrombin III-high-affinity heparin using agarose beds is described. The method allows the determination of high-affinity heparin fractions in several samples in one single step. The incubation mixture containing heparin and antithrombin III is submitted to agarose gel electrophoresis in 0.06 m barbital buffer, pH 8.6. A sharp separation between free antithrombin III, the complex antithrombin III-heparin, and free heparin occurs under these conditions. Around 30% of heparin molecules present in commerical preparations bind to antithrombin. This bound heparin has an anticoagulant activity of 240 IU. Negligible binding of other sulfated mucopolysaccharides to antithrombin III was observed. The whole procedure takes less than 6 h and can also be used as a semipreparative method for high-affinity heparin.     

Simple, sensitive and rapid LC-MS method for the quantitation of indapamide in human plasma--application to pharmacokinetic studies

A sensitive and specific method using liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) has been developed and validated for the identification and quantification of indapamide in human plasma. A simple liquid-liquid extraction procedure was followed by injection of the extracts on to a C18 column with gradient elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different plasma batches. Linearity was established for the concentration range 0.5-100.0 ng/ml, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (RSD%) was lower than 10%, and accuracy ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.2 ng/ml with 0.2 ml plasma. The proposed method enables the unambiguous identification and quantification of indapamide for pre-clinical and clinical studies.     

A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations.

By using the fluorescence enhancement of ethidium bromide when it binds to RNA, a very rapid, simple and sensitive assay for the concentration of ribosomal RNA in complex mixtures has been devised.