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PubMed1985

Hidden autoantibodies against common serum proteins in murine systemic lupus erythematosus. Detection by in vitro plaque-forming cell assay.

The autoantibodies found in human and murine systemic lupus erythematosus (SLE) are generally directed against cells or components of cells such as nuclear antigens. This predilection may be due to the unusual immunogenicity of certain autoantigens, or to unusual patterns of antibody crossreactivity. Alternatively, the observed spectrum of reactivities may reflect the in vivo absorption of those autoantibodies directed against soluble antigens. To test whether hitherto undetected autoantibodies against serum proteins might exist in murine SLE, we developed assays that were independent of the possibility of absorption of autoantibodies by serum autoantigens; large numbers of plaque-forming cells (PFC) directed against mouse albumin and mouse transferrin were easily detected in the spleens of MRL/Mp-lpr/lpr, BXSB, and NZB mice. The secreted antibodies were relatively specific for the mouse proteins, since only limited cross-reactivity was seen with albumin and transferrins of other species in inhibition experiments. The production of these hidden antibodies could not be the result of diffuse polyclonal B cell activation, since the PFC to mouse transferrins and albumin were not always accompanied by comparable numbers of PFC against related albumins and transferrins. The results indicate that autoantibody production in murine lupus is a generalized phenomenon, not limited to the production of autoantibodies to nuclear or other cell-bound antibodies. However, the relative specificity of the autoantibodies for self-antigens indicates that diffuse polyclonal B cell activation cannot be the mechanism responsible, and argues that a selective mechanism, probably driven by antigen, accounts for production of autoantibodies in SLE.

Animals↗

PubMed2006

Assays to determine allelic usage of gene expression in the placenta.

Mammalian placentas express a large number of so-called imprinted genes. Imprinting refers to mono-allelic or biased expression based on which parent contributed the allele. Many of these imprinted loci encode factors involved in growth and cell-cycle regulation, as well as maternal behavior. In general, paternally expressed genes tend to enhance growth, whereas maternally expressed genes inhibit growth. Methods are described for developing assays to test the allelic usage of a gene. The approaches described are best utilized within a system where multiple strains are available, and it is possible to perform reciprocal crosses. Only polymerase chain reaction-based methods are examined in any detail.

Alleles↗

PubMed1985

Sensitive quantification of isoforms of canine MM creatine kinase with an immunoblot procedure suitable for large numbers of samples.

To quantify individual isoforms of canine myocardial MM creatine kinase (CK) we developed an immunoblot procedure suitable for analysis of large numbers of samples. Isoforms in plasma were separated by agarose electrophoresis, immobilized on nitrocellulose, detected with anti-MM followed by a labeled second antibody, and quantified by well counting. The amount of non-CK protein was reduced with sequential ethanol precipitation. Reproducibility of the assay developed was high with a standard deviation of 5.9% of mean values for each isoform. Variation of results after serial dilutions of samples constituted with 33.3% of each of the three isoforms was modest with a standard deviation of 3.5%. The method developed provided sensitive, discrete resolution of individual MM CK isoforms under nondenaturing conditions and should prove useful for improved, objective definition of the time of onset of irreversible injury to myocardial tissue rendered ischemic and hence improved stratification of patients studied in clinical trials concerned with protection and salvage of myocardium.

Animals↗

PubMed1995

The E-screen assay: a comparison of different MCF7 cell stocks.

MCF7 human breast cancer cells have been studied extensively as a model for hormonal effects on breast cancer cell growth and specific protein synthesis. Because the proliferative effect of natural estrogen is considered the hallmark of estrogen action, it was proposed that this property be used to determine whether a substance is an estrogen. The E-screen assay, developed for this purpose, is based on the ability of MCF7 cells to proliferate in the presence of estrogens. The aim of our study was to characterize the response of four MCF7 cell stocks (BUS, ATCC, BB, and BB104) and determine which of them performed best in the E-screen test. The four stocks assayed were distinguishable by their biological behavior. In the absence of estrogen, MCF7 BUS cells stopped proliferating and accumulated in the G0/G1 phase of the cell cycle; estrogen receptors increased, progesterone receptors decreased, and small amounts of pS2 protein were secreted. Of all the MCF7 stocks tested, MCF7 BUS cells showed the highest proliferative response to estradiol-17 beta: cell yields increased up to sixfold over those of nontreated cells in a 144-hr period. The differences between estrogen-supplemented and nonsupplemented MCF7 BUS cells were due mostly to G0/G1 proliferative arrest mediated by charcoal dextran-stripped serum. MCF7 BUS cell stocks and others showing a similar proliferative pattern should be chosen for use in the E-screen test, or whenever a proliferative effect of estrogen is to be demonstrated.

Benzhydryl Compounds↗

PubMed1982

Zona pellucida antibodies in human sera.

A quantitative indirect immunofluorescent assay developed for the measurement of antibodies produced in rabbits against porcine zonae pellucidae was modified and used to detect autoantibodies specific to human zonae pellucidae. An undetermined number of human serum constituents which appear to vary quantitatively between individuals had the capacity to bind non-specifically the porcine zonae used as antigen in this assay. Binding was reduced or eliminated by altering the chemical composition of the assay system until the only positive results were for sera from infertile, sub-fertile and menopausal women. These positive sera were adsorbed with washed porcine blood cells, spleen cells, and liver cells. Each tissue completely removed the positive binding to porcine zonae. The positive sera were also tested against human oocytes with and without porcine tissue adsorption, and no positive fluorescence was detected. It is therefore unlikely that the antibodies in human sera which bind to porcine zonae pellucidae could be a causal factor in infertility in women.

Animals↗

PubMed1997

Monoclonal antibodies and an indirect ELISA for detection of psychrotrophic bacteria in refrigerated milk.

Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an indirect ELISA format for the detection of Pseudomonas spp. and related psychrotrophic bacteria in refrigerated milk. The immunorecognition of monoclonal antibodies adsorbed to bacteria bound to the wells of a microtiter plate was performed with rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase. Subsequent enzymic conversion of the substrate resulted in distinct absorbance differences when assaying milk samples containing psychrotrophic bacteria in the range 10(5) to 10(9) CFU ml(-1) . The detection threshold for the ELISA assay developed in this work is 10(5) CFU ml(-1).

Animals↗

PubMed2005

Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR.

A pentaplex reverse-transcription polymerase chain reaction (Pentaplex RT-PCR) in a single tube was developed for the simultaneous detection of the pome fruit viruses: Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). This is the first report of the simultaneous detection of all four viruses and host mRNA as an internal specific control. Pentaplex RT-PCR was applied successfully throughout the year, using different plant organs (leaves or dormant buds). The sensitivity of detection by monoplex- and pentaplex RT-PCR assays was comparable. Different combinations of mixed infections of viruses were identified in samples of infected apple and pear trees from different geographical regions. The pentaplex RT-PCR assay developed was sensitive, simple, rapid, and reliable for simultaneous detection of the four viruses in extracts of leaves or dormant buds.

Enzyme-Linked Immunosorbent Assay↗

PubMed2006

Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus.

The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.

Animals↗

PubMed2006

Tracking problems and possible solutions in the quantitative determination of small molecule drugs and metabolites in biological fluids using liquid chromatography-mass spectrometry.

During the last decade, quantification of low molecular weight molecules using liquid chromatography-tandem mass spectrometry in biological fluids has become a common procedure in many preclinical and clinical laboratories. This overview highlights a number of issues involving "small molecule drugs", bioanalytical liquid chromatography-tandem mass spectrometry, which are frequently encountered during assay development. In addition, possible solutions to these issues are proposed with examples in some of the case studies. Topics such as chromatographic peak shape, carry-over, cross-talk, standard curve non-linearity, internal standard selection, matrix effect, and metabolite interference are presented. Since plasma is one of the most widely adopted biological fluid in drug discovery and development, the focus of this discussion will be limited to plasma analysis. This article is not intended to be a comprehensive overview and readers are encouraged to refer to the citations herein.

Biological Assay↗

PubMed1984

Diagnostic implications of tear protein profiles.

Concentrations of lysozyme, lactoferrin, ceruloplasmin, IgA, and IgG have been measured in tears by the ELISA (enzyme-linked immunosorbent assay) technique. Tears were collected on weighed filter paper discs, after which they were eluted into buffer and transported frozen to a remote laboratory for assay. Patients with sicca, questionably dry eyes and ocular pemphigoid were sampled, as were 54 normal volunteers. Tear protein profiles were established which were unique for each condition and clearly differed from the normal controls. The assay developed is considered suitable for other proteins such as IgE, and could also be used for monitoring the effects of drugs on the lacrimal gland.

Adult↗

PubMed2001

Detection of horses infected naturally with equine infectious anemia virus by nested polymerase chain reaction.

A nested polymerase chain reaction (PCR) amplifying a region of the gag gene of equine infectious anemia virus (EIAV) was developed for the rapid and direct detection of proviral DNA from the peripheral blood of naturally infected horses and was compared with the Coggins test. DNA prepared from white blood cells of 122 field horses from 15 stables with reported cases of EIAV and one seronegative stable were analysed. Amplifications of expected size fragments were obtained by nested PCR for 88 horses using two different sets of primers targeting the gag region. The specificity of the amplified products was confirmed by hybridization using a digoxigenin-labeled probe. Gag-nested PCR-restriction fragment length polymorphism analysis distinguished two different subtypes of gag gene, A and B. Subtype A was found to be the most prevalent among the infected horses that were tested. The PCR-gag amplified sequence of subtype A shared 84.6% nucleotide and 93% deduced amino acid sequence identities with the prototype Wyoming strain whereas subtype B sequence was almost 100% identical to the prototype. Sequence analysis of gag subtype A suggests the presence of a novel EIAV variant among infected horses in Canada. The nested PCR assay developed in the present study detected more EIAV positive animals and was found as specific as the agar gel immunodiffusion (Coggins) assay and offers great potential a diagnostic test for the detection of EIAV infections in field horses.

Animals↗

PubMed2005

Fertility evaluation of frozen/thawed semen.

In vitro semen analyses have been used for more than half a century to estimate the fertilizing potential of a semen sample. Unfortunately, none of the assays developed provide results that consistently correlate well with fertility. The reasons for this lack of consistency, due in part to the complexity of the spermatozoon itself, the collection of fertility data, and factors beyond control of the semen analyses themselves, are discussed. Different spermatozoal attributes that are necessary for a spermatozoon to fertilize an oocyte are presented and assays used to evaluate each attribute described. Although laboratory assay results do not correlate well with semen fertility, the importance of conducting laboratory assays on every semen sample used for artificial insemination or to attempt to determine causes for infertility, is discussed.

Animals↗

PubMed1987

Radial diffusion as a simple and rapid method for screening superoxide dismutase activity.

Superoxide dismutases are of great interest due to their increasing medical applications in therapy and diagnosis of some diseases. The radial diffusion assay was evaluated for its usefulness as a simple, cheap and accurate assay for screening superoxide dismutase activity. In this assay O2- radicals were generated from the interaction of reduced riboflavin with molecular oxygen upon exposure of agar gel containing riboflavin and N,N,N,N-tetramethylethylene diamine (TEMED) to light. If nitrotetrazolium dye is also present, it will be reduced to the blue insoluble formazan, whilst if superoxide dismutase is present it will prevent this blueing. The developed assay was found to give reproducible estimates of pure samples of superoxide dismutase with a lower limit of measurements of about 10 mg. It can be adapted to measure the levels of superoxide dismutase in various crude biological samples.

Animals↗

PubMed1993

Antisera specific for carcinogen-DNA adducts and carcinogen-modified DNA: applications for detection of xenobiotics in biological samples.

The development of immunoassays and immunoaffinity chromatography methods for determination of carcinogen-DNA adducts and carcinogen-modified DNA samples rests upon eliciting and characterizing polyclonal and monoclonal antisera against these haptens. The use of such antisera has widespread application in investigating chronic carcinogen administration in animal models and in monitoring human tissues for evidence of carcinogen exposure. Radioimmunoassays and enzyme-linked immunosorbent assays developed with carcinogen-DNA adduct antisera are exceedingly sensitive, measuring 1 adduct in 10(8) nucleotides. Not only can DNA damage be quantified directly by immunoassay, but the antisera have also been used to isolate DNA adducts of a particular chemical class by immunoaffinity chromatography before application of more chemically-specific end-points. Both of these methodological approaches have made seminal contributions to the newly-emerging field of molecular epidemiology. This chapter will focus on methods for preparing immunogens, the establishment of immunoassays, characterization of antisera and specific problems encountered with biological samples in addition, the use of immunoaffinity chromatography for preparative concentration of DNA adducts of a particular class will be included.

Animals↗

PubMed1997

Regulation of follistatin production by rat granulosa cells in vitro.

The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function. The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard. Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive. There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC. Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages. It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.

Activins↗

PubMed1996

Sensitive and rapid bioassay for analysis of P-glycoprotein-inhibiting activity of chemosensitizers in patient serum.

Clinical studies of agents capable of reversing P-glycoprotein (Pgp)-mediated multidrug resistance have attracted much attention in recent years. One question of interest in such studies is whether the concentrations achieved by chemosensitizers are sufficient to inhibit Pgp function. The goal of the present study was to develop a reliable ex vivo bioassay for analysis of the Pgp-inhibiting activity of chemosensitizer-containing patient serum. The fluorescent Pgp substrates daunorubicin (DNR) and rhodamine 123 (R123) were used as probes for Pgp function. The 8226/DOX6 human myeloma cell line, which expresses Pgp at levels that can be detected in clinical cancers, was used as a model system. The index chemosensitizers tested were dexverapamil (DVPM) and cyclosporin A, with particular focus on DVPM. Using flow cytometry, chemosensitizer effects on 1-h drug accumulation and on drug retention at 30 min were evaluated. In the studies using pooled human serum spiked in vitro with graded chemosensitizer concentrations, the order of assay sensitivity was R123 retention >>> R123 accumulation > DNR retention equal to DNR accumulation. Keeping serum spiked with DVPM for several hours at room temperature or 4 degreesC or for several months at -80 degreesC had no effect on Pgp-blocking activity. Sixteen blood samples from patients with metastatic breast cancer receiving DVPM to overcome epirubicin resistance were analyzed for Pgp-inhibiting activity and for levels of DVPM and nor-DVPM, the major metabolite of verapamil. Each patient sample was found capable of increasing R123 retention in the 8226/DOX6 cells, with activity factors of 3- to 8-fold and good agreement between DVPM blood levels and bioassay activity (r = 0.7168; two-sided P = 0.0018). The R123 retention assay developed and validated in this study seems to be a sensitive, reproducible, and easy-to-use method for analysis of Pgp-inhibiting activity of chemosensitizer-containing human serum. The assay seems capable of estimating DVPM blood levels and could prove to be a valuable tool for monitoring chemosensitizer treatment in cancer patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Construction and expression of recombinant streptolysin-o and preevaluation of its use in immunoassays.

Commercially available immunoassays for assessment of anti-streptolysin-O antibodies use native streptolysin-O obtained by a complex process. We prepared a biologically active recombinant streptolysin-O with higher yield and a simpler purification process. An enzyme-linked immunosorbent assay developed with this recombinant showed good correlation with a commercial test, suggesting that it could be suitable for immunoassays.

Antibodies, Bacterial↗

PubMed1981

Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay developed for the demonstration of respiratory syncytial (RS) virus immunoglobulin G antibodies was used for the detection of RS virus in specimens of nasopharyngeal secretions (NPS) obtained from children with acute respiratory disease. Samples of NPS were incubated with a fixed amount of standard serum (human serum antibodies to RS virus) before being added to the enzyme-linked immunosorbent assay test plate. A decrease in the optical density value determined for this standard serum was seen with majority of NPS specimens from which RS virus had been isolated in tissue culture. The reliability and the specificity of this inhibiton test were supported by experiments with purified RS virus and by tests with NPS specimens containing other respiratory viruses.

Antibodies, Viral↗