The use of microtiter plates for the simple and sensitive determination of insulin by an ELISA method

A method of insulin determination using a commercially available ELISA kit was modified for use in microtiter plates. The adapted assay, based on the binding of porcine anti-guinea pig insulin antibodies to microtiter plates and insulin-peroxidase conjugate as displacer, is sensitive between 0.5 and 30 ng/ml. Since it uses only 10-40 microliter of sample material it enables the determination of 5-100 pg of insulin. The rapid (5-6 h), automatable, reproducible and reliable assay makes it possible to determine many samples in a short time.     

Detection of varicella zoster virus DNA in human tissue by standard and nested polymerase chain reaction

Results and conclusion The sensitivity of the PCR assay was determined with cloned VZV DNA. About 200 copies of the target sequence were necessary for detectable amplification by standard PCR and less than 20 copies by nested PCR. Out of 24 human trigeminal ganglia five tested positive for VZV DNA by standard PCR (21%), in eleven cases VZV DNA was detectable using nested PCR (46%). Sequences specific for VZV could be detected in PMBC from children with acute varicella up to six days after the onset of rash by standard (one child) or nested (three children) PCR. This confirms that at the time of haematogenous spread before and during the rash viral DNA can be found within the mononuclear cells. Thus the use of nested primers enhances the sensitivity of the assay and allows the detection of only a few genomic copies of viruses in human tissues.     

The use of microtiter plates for the simple and sensitive determination of insulin by an ELISA method

Summary A method of insulin determination using a commercially available ELISA kit was modified for use in microtiter plates. The adapted assay, based on the binding of procine anti-guinea pig insulin antibodies to microtiter plates and insulin-peroxidase conjugate as displacer, is sensitive between 0.5 and 30 ng/ml. Since it uses only 10–40 l of sample material it enables the determination of 5–100 pg of insulin. The rapid (5–6 h), automatable, reproducible and reliable assay makes it possible to determine many samples in a short time.     

Comparison of different techniques for semiquantitative detection of HBV DNA by PCR

Conclusions Our results indicate that the choice of the probe for ELOSA is of major concern. In our panel we had seven sera which contained about 100 molecules/ml and further five sera which contained less than 1000 molecules/ml. Most of them were detected by the long probe but not by the short probe. When PCR for the S-or PreS-gene was included it was possible to detec all 24 HBV-positive sera (not shown) by ELOSA. The reliable lower quantification limit for the long probe is 250 molecules/ml and for the short probe 2500 molecules/ml. Surprisingly, chemiluminescence did not produce better qualitative or quantitative results. The data suggest that the usage of several replicates allows relative quantification in most cases. One possible drawback we see is the hybridization efficiency. Six of our positive samples showed great differences between the number of target molecules suggested by agarose gel electrophoreses or by hybridization (Southern blot or ELOSA). All of them contained more than 10⁶ molecules/ml. For these cases and for the samples where the short probe and the long probe gave discordant result (2 cases) we think that competitive PCR will be the method of choice, but in most cases ELOSA with the long probe gives reliable results and is highly sensitive.     

Comparison of DNA isolation methods from blood for use in trypanosome specific polymerase chain reaction

Conclusions The three methods tested are convenient for the preparation of samples to be analyzed by PCR for the repetitive satellite DNA sequences of trypanosomes. Despite a slightly reduced sensitivity of detection of free trypanosome DNA, the preparation method based on the isolation of cell nuclei seems to be the most suitable and rapid technique for the routine analysis of a large number of blood samples.     

Quantification ofMycobacterium tuberculosis DNA using PCR and a simple dot blot-based DNA enzyme assay (DEA)

Conclusions Use of PCR for quantification of microorganisms has certain limitations. Methods currently under investigation, such as covalent binding of modified DNA onto the surface of microwells or coupling to magnetic beads with the aid of biotin-avidin, are hampered by problems concerning immobilization of DNA on a solid phase. Moreover, these strategies are laborious, including several washing steps and complicated detection systems (sandwich hybridization).We have developed an alternative method, exploiting the reliability of covalent DNA binding to positively charged nylon membranes enabling easy to handle direct hybridization. The method is adaptable for routine use in clinical laboratories. We have shown results of a quantitative assay to measure bacterial load of MTB. Quantification of MTB may have value in: (i) monitoring patients under anti-myobacterial therapy, and (ii) early in vitro drug susceptibility testing.     

DNA in wheat seeds from European archaeological sites

We have used hybridization analysis to detect ancient DNA in wheat seeds collected from three archaeological sites in Europe and the Middle East. One of these samples, carbonizedT. spelta dated to the first millennium BC, has yielded PCR products after amplification with primers directed at the leader regions of the HMW (high molecular weight) glutenin alleles. Sequences obtained from these products suggest that the DNA present in the Danebury seeds is chemically damaged, as expected for ancient DNA, and also indicate that it should be possible to study the genetic variability of archaeological wheat by ancient DNA analysis. Finally, we describe a PCR-based system that enables tetraploid and hexaploid wheats to be distinguished.     

A simplified assay for cyclic AMP using protein kinase binding

Summary The protein kinase binding assay for cAMP was modified by substitution of adsorption by QAE cellulose for the membrane filtration. This modification obviates the variation of recovery of cAMP with the volume of buffer used to wash the filter. The assay is reproducible and technically simpler than those currently employed.Acknowledgment. This work was partially supported by grants HL-16583 and HL-18827 from the National Heart and Lung Institute.     

Direct evidence favouring the notion that erythropoietin alters iron transport across the isolated intestinal tract of the rat.

A simple and reproducible method, using the isolated not everted intestine of the rat, for the study of iron transport is presented. Erythropoietin (ESF) enhanced significantly the passage of 59Fe across the intestine augmenting its movement at mucosal and serosal layers of the intestinal well.     

Interspecific differences between the DNA restriction profiles of canine adenoviruses

Summary Identification of canine adenovirus-1 (CAV-1) and canine adenovirus-2 (CAV-2) strains was done by electrophoresis of restriction endonuclease-fragmented viral DNA. Results obtained with this sensitive and reproducible technique clearly show that CAV-2 is not a variant of CAV-1 but a distinct virus.This work was supported by grant number IAF-82-927 from the Conseil des Recherches et Services agricoles du Québec.     

Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants

Many virus and transposon DNAs can integrate into the host genome. In this review, techniques, including inverse polymerase chain reaction (IPCR), novel Alu-PCR and vectorette- or splinkerette-PCR are introduced as possible strategies for cloning flanking DNA regions of the integrants. Targeted gene-walking PCR, restriction-site PCR, capture PCR, and panhandle PCR and boomerang DNA amplification are also described. The principles, advantages and limitations of each approach are discussed. Received 9 July 1998; received after revision 2 October 1998; accepted 7 October 1998     

Flow microcalorimetric bioassay of polyene antibiotics: Interaction with growingSaccharomyces cerevisiae

Summary Microcalorimetric investigation of the interaction of polyene antibiotics with mid-exponential cells of a growing culture ofSaccharomyces cerevisiae has been used as the basis of a bioassay procedure. The assay is rapid, sensitive and reproducible. The results are compared to classical assays and potency ranking orders.This work was supported by the Science Research Council. (Studentship for B.Z. Chowdhry). We are grateful to the Central Research Fund of London University for funds to purchase the flow nephelometer used to support this study.     

Rapid isolation of mitochondrial DNA. Mitochondrial DNA fromDrosophila serrata

A simple and rapid method for isolation of high quality mitochondrial DNA (mtDNA) is presented in this report. Using this method, isolation and restriction site maps for 10 enzymes of the mtDNA ofDrosophila serrata were established.     

A simple technique for testing the in vitro response of rabbit lymphocytes to PHA and allogeneic cells.

Lymphocytes of rabbits can be separated from small quantities of heparinized whole blood using a simple density gradient of Ficoll-Ronpacon 1.09. This separation technique yields a pure suspension of viable cells allowing reproducible results from cultures stimulated either with PHA or allogeneic lymphocytes isolated by the same technique.     

Rapid isolation of mitochondrial DNA. Mitochondrial DNA from Drosophila serrata.

A simple and rapid method for isolation of high quality mitochondrial DNA (mtDNA) is presented in this report. Using this method, isolation and restriction site maps for 10 enzymes of the mtDNA of Drosophila serrata were established.     

A simple technique for testing the in vitro response of rabbit lymphocytes to PHA and allogeneic cells

Summary Lymphocytes of rabbits can be separated from small quantities of heparinized whole blood using a simple density gradient of Ficoll-Ronpacon 1.09. This separation technique yields a pure suspension of viable cells allowing reproducible results from cultures stimulated either with PHA or allogeneic lymphocytes isolated by the same technique.Supported by the Swiss Science Foundation No. 3.890-0.77.     

Development of an enzyme linked immunosorbent assay (ELISA) for measurement of vitellogenin concentrations in cockroaches

Summary A competitive enzyme linked immunosorbent assay was developed for the quantification of a large lipoprotein, namely the yolk protein vitellogenin, in the haemolymph of cockroaches (Nauphoeta cinerea). This assay was found to be specific, reproducible and it has a high sensitivity (approximately 10 ng).Acknowledgments. We would like to express our thanks to Prof. H. Fey and Ms H. Pfister, Veterinary Bacteriological Institute, University of Berne, for introducing us into the ELISA methodology, to P. Beyeler for technical assistance and to M. Kaltenrieder for drawing the graphs. We also thank the Sandoz Foundation for a fellowship to M. Dumas and the Swiss National Science Foundation (grant No. 3. 188-0.77) for financial support.     

DNA microarray-based gene expression profiling of estrogenic chemicals

We summarize updated information about DNA microarray-based gene expression profiling by focusing on its application to estrogenic chemicals. First, estrogenic chemicals, including natural/industrial estrogens and phytoestrogens, and the methods for detection and evaluation of estrogenic chemicals were overviewed along with a comprehensive list of estrogenic chemicals of natural or industrial origin. Second, gene expression profiling of chemicals using a focused microarray containing estrogen-responsive genes is summarized. Third, silent estrogens, a new type of estrogenic chemicals characterized by their estrogenic gene expression profiles without growth stimulative or inhibitory effects, have been identified so far exclusively by DNA microarray assay. Lastly, the prospect of a microarray assay is discussed, including issues such as commercialization, future directions of applications and quality control methods.     

Diagnostic value of human cytomegalovirus DNA PCR regarding different clinical specimens

Conclusions In transplant recipients, a positive HCMV PCR in PBLs is a sign of the possible development of HCMV disease which should be confirmed by further diagnostic approaches. However, detection of HCMV DNA in the CSF appears to be a specific marker of pathological processes, exemplified in a case of HCMV encephalitis.     

Heat evolution of cultured human keratinocytes

The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83 +/- 12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134 +/- 35) pW/cell was obtained when the transformed keratinocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.