A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Comparative methods of detecting HbsAg in chronic glomerulonephritis patients in Nigeria

Abstract A comparative evaluation of the enzyme-linked immunosorbent assay (Elisa), passive haemagglutination (PHA) and counterimmunoelectrophoresis (CIE) methods were carried out. Approx. 6% of control samples were positive with the Elisa assay, while the values for Behring Institute (BI) passive haemagglutination, Wellcome Research Laboratory (WRL) passive haemagglutination and CIE were 2.7%, 2.2% and 3.3%, respectively. The above data contrast with values of 21.3%, 10.6, 10.3 and 12.3 obtained in sera from chronic glomerulonephritis patients.
Our observation suggests that HbsAg may be associated with chronic glomerulonephritis.     

Use of phage display for detecting single-nucleotide differences in genes]

Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E. coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles. The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads. DNA heteroduplexes have been obtained from biotin-labeled PCR product. During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides. The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides). Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes. Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.     

Use of highly sensitive radioautography for determining the optimal conditions for obtaining hybridomas

To detect the hybrid cells forming as a result of fusion of the mouse myeloma cells with the immunic mouse splenocytes, a radioautographic method was used which involved the application of 3H-hypoxanthine as a labelled precursor of nucleic acids and of high-sensitive UK emulsion for accelerated preparations of autographs. The optimal conditions of hybridization providing for the maximum yield of hybrid cells and hybrid clones in our experiments were: use of polyethylene glycol ("L?ba", mol. weight 4000) in 50% (w/v) concentration, of NS-O or X-653 myeloma cells in the ratio of parental cells 1:5 (myeloma cell: splenocyte).     

Use of non-radioactive biotin-labelled probes for detecting hepatitis A virus]

The biotin-labeled DNA probes were constructed on the basis of the hybrid bacteriophage M13nip 9 single-stranded DNA containing the fragments of the hepatitis A viral cDNA. The probes were biotin treated by chemical modification of the DNA by the peraminating reagent or photochemically. The labeled DNA probes were used in molecular hybridization experiments with the nuclear acids fixed on the nitrocellulose filters. The biotin treated DNA was determined by the avidin-gold colloid conjugate with the subsequent physical silver amplification or by the streptavidin-alkaline phosphatase conjugate. The sensitivity of both probes was identical and permitted the determination of 5 x 10(-11)-5 x 10(-12) g of the control DNA and 10(-9) g of the hepatitis A virus. The developed test systems were used for detection of the viral RNA in blood from patients.     

Limitations of highly sensitive enzymatic presence-absence tests for detection of waterborne coliforms and Escherichia coli.

This study presents evidence for the unfeasibility of enzymatic presence-absence tests to detect one total coliform or one Escherichia coli organism in 100 ml of drinking water within a working day. The results of field trials with prototype chemiluminometric procedures indicated that the sensitivity-boosting measures that are essential to achieve the required speed compromise the specificity of the tests.     

Phagocytosis of chloramine B sensitive and resistant staphylococci isolated from healthy persons and patients

Phagocytic reaction with respect to antibiotic and chloramine B sensitive and resistant staphylococci isolated from healthy persons and patients, air and stock of medical institutions was studied on albino mice. It was shown that the staphylococcal isolates included strains simultaneously sensitive to antibiotics and chloramine, sensitive to antibiotics and resistant to chloramine, resistant to antibiotics and sensitive to chloramine and simultaneously resistant to antibiotics and chloramine. Activity, intensity and completeness of phagocytosis by leucocytes from mouse abdominal cavity exudates with respect to the staphylococcal strains sensitive to antibiotics and resistant to chloramine, resistant to antibiotics and sensitive to chloramine and simultaneously resistant to antibiotics and chloramine were lower than the values of the phagocytic reaction with respect to the isolates simultaneously sensitive to antibiotics and chloramine. This suggested that not only antibiotic resistance of microbes but also their resistance to disinfectants could be referred to complicating factors of hospital infections.     

Statistical tests for detecting gene conversion

Statistical tests for detecting gene conversion are described for a sample of homologous DNA sequences. The tests are based on imbalances in the distribution of segments on which some pair of sequences agrees. The methods automatically control for variable mutation rates along the genome and do not depend on a priori choices of potentially monophyletic subsets of the sample. The tests show strong evidence for multiple intragenic conversion events at two loci in Escherichia coli. The gnd locus in E. coli shows a highly significant excess of maximal segments of length 70-200 bp, which suggests conversion events of that size. The data also indicate that the rate of these short conversion events might be of the order of neutral mutation rate. There is also evidence for correlated mutation in adjacent codon positions. The same tests applied to a locus in an RNA virus were negative.      

A highly selective and sensitive boronic acid-based sensor for detecting Pd2+ ion under mild conditions

Herein, a boronic acid-based sensor was reported selectively to recognize Pd²⁺ ion. The fluorescence intensity increased 36-fold after sensor binding with 2.47 × 10⁻⁵ M of Pd²⁺ ion. It was carried out in the 99% aqueous solution for binding tests, indicating sensor having good water solubility. In addition, it is discernible that Pd²⁺ ion turned on the blue fluorescence of sensor under a UV–lamp (365 nm), while other ions (Ag⁺, Al³⁺, Ba²⁺, Ca²⁺, Cr²⁺, Cd²⁺, Co²⁺, Cs²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Li⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺ and Zn²⁺) did not show the similar change. Furthermore, sensor has a low limit of detection (38 nM) and high selectivity, which exhibits the potential for the development of Pd²⁺ recognition in practical environments.     

Comparative evaluation of the commercial Sevatest ELISA immunoenzyme set and other serological tests for detecting Toxoplasma gondii antibodies

This work analyzes the results of 4 serologic tests used for the diagnosis of toxoplasmosis: the complement fixation (CFT), indirect immunofluorescence (IIF), passive hemagglutination (PHAT) tests, and the enzyme-linked immunosorbent assay (ELISA). The last-mentioned one was made with the use of the commercial kits Sevatest ELISA IgG/Toxo Micro I. The results of ELISA were in good correlation with those yielded by the traditional tests: 70% coincidence with CFT, 80% with IIF, 84% with PHAT; besides, ELISA has shown a higher sensitivity in the screening of sera.     

Highly sensitive electroimmunodiffusion method of detecting antigens on cellulose acetate film]

A highly sensitive electro-immunodiffusion test suggested by the authors for antigen detection on cellulose-acetate films consists of three stages: antigen concentration in a discontinuous buffer system on cellulose-acetate films; antigen detection on the same films by immunodiffusion using standard test system; to detect the precipitation bands the washed films are stained with protein dyes in case the reaction takes place in the zone of vision, or subject to further treatment by means of "plating" the precipitates with antiglobulin antibody or by radioautography. The method permits one to reveal the nanogram levels of alfa-fetoprotein and it may be applied for detection of antigens with different molecular weights and electrophoretic mobility.     

Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins

A rapid and sensitive method for determining protein concentrations using fluorescamine has been characterized for use in the analysis of intact lipoproteins. It was shown that there is no interference with the assay due to the presence of lipid-associated turbidity or primary amine content. The assay was shown to be sensitive to as little as 0.3 microgram of lipoprotein and to yield similar results when compared to the Lowry method.     

Characteristics of individual lymphocyte populations in the blood of virtually healthy persons]

The number of individual lymphocyte populations in the blood of apparently healthy persons was determined by spontaneous rosette-formation with SRBC (T cells) and mouse red blood cells (B cells). In the majority of persons examined the percentage of T-lymphocytes constituted 47.3 +/- 1,6, of B--16.8 +/- 1, and of "zero" cells--33.5 +/- 3.4. There was less T- and B-lymphocytes in the blood of elderly persons (over 50 years of age) than in the young ones. It was also shown that the T-lymphocyte population forming "active" rosettes could be assessed by the number of SRBC sorbed on their surface.     

Risk factors for ANA positivity in healthy persons

Introduction  

The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.     

A fast and highly sensitive method for detecting freemartinism in bovine twins using immunomagnetic beads and Y-specific PCR primers

A method for detecting male cells in the blood of the female calf in bovine heterosexual twin pregnancies has been established. Nucleated cells were isolated from full blood by immunomagnetic separation, lysed by boiling and then subjected to polymerase chain reaction (PCR) amplification with Y chromosome specific primers. Diagnosis was achieved within one day. The method was successfully used on blood samples that had been stored at +4°C for more than one month. Dilution of male blood in female blood showed that XY cells were detectable down to a concentration of 0.1%. This method should be amenable to automatization and can be adapted to any PCR-based genetic test.