Immunodot Assay Research Articles

Use of the dot-immunogold assay for the rapid diagnosis of acute enteric infections

A simple method based on an immunodot assay using colloidal gold labels is proposed for the rapid diagnosis of a range of acute enteric infections. Owing to its rapidity, high sensitivity, and specificity, the method can be recommended for routine use in the laboratory diagnosis of enteric infections.

Prevalence of antibodies against hepatitis C virus in the adult German population.

The prevalence of anti-HCV in Germany has been determined for blood donors and certain risk groups, but the burden of disease in the general population remains unknown. The aim of this study was to determine the prevalence of anti-HCV in a study group representing the normal adult German population. A total of 5312 individuals aged 18-70 years were randomly selected from small, middle-sized and big cities in five different German states. Sera were tested for anti-HCV by enzyme immunoassay and immuno dot assay, as well as for anti-HBc and, in the case of a positive result, for anti-HBs and HBsAg. Serological typing was performed in anti-HCV-positive persons. Thirty-nine individuals were anti-HCV positive; indeterminate results (with antibodies against the viral core protein only) were obtained in 20. There was a tendency to higher prevalence rates with increasing age as well as to a higher prevalence in women. Serological typing revealed the presence of genotype 1 in the vast majority of participants (82%); only a minority showed genotype 3 (7.2%) or other genotypes (7.2%). Markers of HBV were seen in 43.6% of the anti-HCV positive individuals, with nearly one third (29.4%) of the double-infected showing anti-HBc as the only marker for HBV. According to our data, an anti-HCV prevalence of 0.63% (95% confidence interval, CI, 0.42-0.84%) can be assumed in the general adult German population, with higher values in older people and women. Nearly half of the anti-HCV positive individuals also show markers of hepatitis B virus.

Detection of Zooplankton Prey in Squid Paralarvae with Immunoassay

Sustainable management of economically important squid requires monitoring of changes in their abundance, which are related inter alia, to their success in the food chain. the highest mortality is expected in the paralarval stages, which are prone to starvation. Causes of starvation may be linked to the lack of suitable prey. A multiple detection system was developed for the simultaneous identification of five putative zooplankton prey in the guts of paralarval Chokka squid, Loligo vulgaris reynaudii, by employing polyclonal rabbit antisera in conjunction with solid phase immunoassays. Specificities of antisera were validated by ELISA screening against different zooplankton taxa. Cross-reactions observed with ELISA were minimized through manipulation of antibody and antigen concentrations resulting in more specific detection of target prey antigens when used in an immunodot assay. Application of this optimised immunoassay detected multiple predation in paralarval squid samples collected from diverse areas in the Agulhas Bank ecosystem on the south coast of South Africa.

Role of serology in the diagnosis of Lyme disease.

Numerous concerns regarding the potential for misdiagnosis of Lyme disease using commercial assays have been voiced by the US Food and Drug Administration (FDA). We attempted to clarify the clinical value of serologic testing for Lyme disease using the results of commonly marketed assays for detecting antibody to Borrelia burgdorferi, the organism that causes Lyme disease. We reviewed published studies on B burgdorferi test performance published through 1998, package insert labeling from FDA-cleared test kits for B burgdorferi, and Lyme Disease Survey Set LY-A from the College of American Pathologists. We assessed the sensitivity and specificity of commercial serologic tests (enzyme-linked immunosorbent assay [ELISA], immunofluorescence antibody [IFA], and immunodot) for diagnosis of Lyme disease. To reduce this risk of misdiagnosis, it is important that clinicians understand the performance characteristics and limitations of these tests. These tests, in common use in clinical or commercial laboratories, should be used only to support a clinical diagnosis of Lyme disease, not as the primary basis for making diagnostic or treatment decisions. Serologic testing is not useful early in the course of Lyme disease because of the low sensitivity of tests in early disease. Serologic testing may be more useful in later disease, at which time sensitivity and specificity of the test are improved. Positive or equivocal results on an ELISA, IFA, or immunodot assay requires supplemental testing with a Western blot assay. A negative result on the Western blot or ELISA indicates that there is no serologic evidence of infection by B burgdorferi at the time the sample was drawn.

Lipoprotein release by bacteria: potential factor in bacterial pathogenesis.

Lipoprotein (LP) is a major component of the outer membrane of bacteria in the family Enterobacteriaceae. LP induces proinflammatory cytokine production in macrophages and lethal shock in LPS-responsive and -nonresponsive mice. In this study, the release of LP from growing bacteria was investigated by immuno-dot blot analysis. An immuno-dot blot assay that could detect LP at levels as low as 100 ng/ml was developed. By using this assay, significant levels of LP were detected in culture supernatants of growing Escherichia coli cells. During mid-logarithmic growth, approximately 1 to 1.5 microgram of LP per ml was detected in culture supernatants from E. coli. In contrast, these culture supernatants contained 5 to 6 microgram/ml of lipopolysaccharide (LPS). LP release was not unique to E. coli. Salmonella typhimurium, Yersinia enterocolitica, and two pathogenic E. coli strains also released LP during in vitro growth. Treatment of bacteria with the antibiotic ceftazidime significantly enhanced LP release. Culture supernatants from 5-h cultures of E. coli were shown to induce in vitro production of interleukin-6 (IL-6) by macrophages obtained from LPS-nonresponsive C3H/HeJ mice. In contrast, culture supernatants from an E. coli LP-deletion mutant were significantly less efficient at inducing IL-6 production in C3H/HeJ macrophages. These results suggest, for the first time, that LP is released from growing bacteria and that this released LP may play an important role in the induction of cytokine production and pathologic changes associated with gram-negative bacterial infections.

Mapping of linear antigenic determinants on glycoprotein C of herpes simplex virus type 1 and type 2 recognized by human serum immunoglobulin G antibodies.

Using membrane-based dekapeptides, the reactivity of human serum antibodies with linear antigenic determinants of herpes simplex virus (HSV) type 1 and type 2 glycoprotein C (gC-1, gC-2) was studied by pep scan and immunodot assay. The entire coding sequences of gC-1 and gC-2 were screened for the presence of linear epitopes by pep scan. Peptides recognized in an HSV-1 type-specific manner were mainly identified within the N-terminal third and at the C-terminus of gC-1, whereas most type-common antibodies were directed against colinear peptides within the central parts of gC-1 and gC-2. The type-specific reaction of human sera with gC-2 peptides in pep scan was poor. Eight peptides identified as immunoreactive by pep scan were further tested in immunodot assay for their reactivity with a human serum panel. None of the eight HSV-negative sera gave positive results by immunodot assay. Positive reactions with gC peptides were found to be strongly age-dependent, i.e., the rate of positive reactions was significantly higher in HSV-positive adults than in HSV-positive children. Antibody reactivity with two type-common gC peptides was demonstrated in 17 out of 28 HSV-positive sera. A putative type-specific gC-2 peptide employed in immunodot assay was inconsistently recognized by human sera. Twenty HSV-positive sera reacted with at least 1 of 5 type-specific gC-1 peptides. Nine sera showing no reactivity with glycoprotein G of HSV-1 (gG-1) by immunobloting recognized type-specific gC-1 peptides in immunodot assay. Thus, gC-1 peptides might allow the detection of HSV-1-specific antibodies in individuals showing no reactivity with commonly employed HSV-1-specific diagnostic antigenes, i.e., purified or recombinant gG-1.

An epidemiologic study of herpes simplex virus type 1 and 2 infection in Japan based on type-specific serological assays.

A seroepidemiologic study of herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was performed on Japanese adults. Serum samples collected between 1985-9 from a total of 536 healthy adults, female prostitutes, males with sexually transmitted diseases (STD), homosexual men, and pregnant women were studied by immunodot assays using HSV type-specific antigens, glycoproteins G (gG1 and gG2). HSV-1 infections correlated mostly with age and was widely prevalent among subjects < 40 years. HSV-2 prevalence varied greatly among subgroups defined by sexual activity and was associated with risk behaviours for prostitution, infection with STD, and homosexual activity. HSV-2 seroprevalence was highest among prostitutes (80%), lowest among pregnant women (7%), and intermediate in STD patients (23%) and homosexuals (24%). Because HSV-1 infection during childhood has been decreasing, primary genital HSV-2 infection, with its higher frequency of clinical manifestations, will become a greater burden to the public health in Japan.

Detection and direct typing of herpes simplex virus in perianal ulcers of patients with AIDS by PCR.

The presence of herpes simplex virus type 1 (HSV-1) and HSV-2 in perianal ulcerations of 41 AIDS patients was assessed by virus culture and a type-specific PCR-based assay. HSV was isolated from the lesion site in 24 of 41 (58.5%) patients, and HSV DNA was detected by PCR in all 24 (100%) of these specimens. Additionally, PCR was used to detect HSV DNA in 12 of 17 (70.5%) HSV culture-negative samples. Thus, HSV genomic sequences could be demonstrated in 36 of 41 (87.8%) perianal ulcers in this series. Full agreement in HSV typing by either immunodot assay or PCR was seen in 24 samples that were positive by both virus culture and PCR. HSV-2 was demonstrated in 35 of 36 (97.2%) HSV-positive samples.

IgE /Anti-IgE Immune Complexes in Sera from Patients with Crohn’s Disease Do Not Contain Food-Specific IgE

Background: An association of Crohn’s disease (CD) with food allergy has been discussed, but the role of food-specific IgE has not been clarified yet. Since CD is combined with immune complex formation, we examined in the present study whether anti-IgE autoantibodies in such complexes might hinder the determination of specific IgE. Methods: In order to elucidate the role of food-specific IgE in CD, we tested sera from CD patients (n = 107), healthy controls (n = 65) and allergics subjects (n = 7) for their IgE binding to food antigens (yeast, corn, celeriac, wheat) by an immunodot assay. After determining levels of IgE/IgG anti-IgE immune complexes, we purified them from serum pools of patients with CD, allergic subjects and healthy controls by affinity absorption using a monoclonal anti-IgE antibody. These purified immune complexes were treated by low pH (pH = 4) in order to dissolve them and to increase the detectability of food-specific IgE by RAST and CAP assay. Results: In CD sera no food-specific IgE could be detected, but levels of immune complexes of IgE and IgG anti-IgE autoantibodies were statistically significantly increased compared to healthy controls. pH treatment of purified IgE/IgG anti-IgE immune complexes resulted in a significant increase in specific IgE to yeast, corn, wheat and celeriac detected by RAST, however, only in the serum sample purified from allergic subjects. After pH treatment of CD immune complexes, specific IgE levels remained still very low. Conclusion: Thus, even if IgE seems to represent an autoantigen in CD, it is unlike to specifically participate in the pathophysiology of the putative food adverse reactions.

Detection of herpes simplex virus type-specific antibodies by an enzyme-linked immunosorbent assay based on glycoprotein G.

In order to develop a simple and quantitative method to detect herpes simplex virus (HSV) type-specific antibodies, the usefulness of an enzyme-linked immunosorbent assay (ELISA) using HSV glycoprotein G (gG) captured on a plate by monoclonal antibodies as antigen was studied. The gG1- and gG2-specific IgG antibody activities were measured by the ELISA for 54 sera which had been collected from culture-proven genital herpes patients and pre-characterized by an immunodot assay using purified gG antigens. Thirty control sera without antibodies against the HSV whole antigens were also included. In comparison with the immunodot assay as standard, the sensitivities of the ELISA were 88.9% (32/36) for HSV-1 antibody and 89.2% (33/37) for HSV-2 antibody and the specificities were both 100%. Sera taken within a few months after primary infection tended to give false negative results. The HSV type-specific ELISA based on easy-to-prepare gG antigens might be useful to help improve the serological assessment of HSV infections.

Prevalence of herpes simplex virus type 1- and 2- specific antibodies among the acute, recurrent, and provoked types of female genital herpes.

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P < 0.005), and 55% for the recurrent type (P < 0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.

An immuno-dot blot assay for detection of thermostable protease from Pseudomonas sp. AFT-36 of dairy origin.

A dot-ELISA technique for the detection of Pseudomonas protease was developed using IgG of anti-Pseudomonas AFT-36 protease as capture antibody. The detection limit of protease in buffer or milk was 1.01 ng ml-1. The procedure was performed at room temperature, took about 2.5 h and was economical. Protease AFT-36 is immunologically related to five out of seven Pseudomonas spp. The results suggest that the assay could be used to detect proteases in dairy products.

Lipogenic Enzymes and Surfactant Proteins in Fetal Sheep Lung Have Different Ontogenic Patterns. 272

Functional maturation of fetal sheep lung is enhanced by glucocorticoids(GC), but ontogeny and hormonal regulation of enzymes involved in surfactant production have not been studied. Activities of two lipogenic enzymes (fatty acid synthetase, FAS; cholinephosphate cytidylyltransferase, CYT) increase during development and are induced by GC in fetal lung of 2 species (rat, human) in vivo and/or in vitro. We studied these enzymes and surfactant proteins A and B (SPA, SPB) in lungs (n =15) from sheep fetuses of ≈60d gestation to ≈10d postnatal. FAS protein content (western blot), FAS activity (14Cmalate incorp. into fatty acid), CYT activity(14CCDPcholine formed), and SPA and SPB contents (immunodot assay) were determined. FAS specific activity was maximal at ≈120d (0.36±0.05 nmol/min/mg prot), dropping by 75% at term (≈148d, p<0.01); western analysis confirmed a similar decline in FAS protein content. CYT activity in both soluble and particulate fractions was highest at ≈90d(0.36±0.03 and 0.19±0.04 nmol/min/mg prot., respectively), decreasing by 50% at term (p<0.05). Tissue protein/DNA ratio was constant at all ages. Tissue SPA and SPB were undetectable at 60d or 90d, present at 125d gestation, and increased 510 fold by term (p<0.05). To assess shortterm GC effect, fetuses were treated with betamethasone (0.5 mg/kg) via maternal or intraamniotic injection 48h prior to delivery at 125d gestation. Lung compliance increased with treatment (≈50%, p<0.05), but FAS and CYT activities remained constant. We conclude that FAS and CYT enzymes are not developmentally correlated with other markers of surfactant system maturation(increased DPPC, SPA, SPB) in fetal sheep lung, nor responsive to short GC exposure. We hypothesize that either de novo lipogenic activity in fetal sheep lung is adequate by early third trimester or that circulationderived fatty acid is the primary source for surfactant lipid synthesis in the fetal sheep.

The Ecology of Agricultural Pests: Biochemical Approaches.

Introduction. Biochemical systematics: principles and perspectives for pest management. Combined use of biochemical, immunological and molecular assays for infection, species identification and and resistance detection in field populations of Anopheles (Diptera: Culicidae). Studies of interactions between alfalfa weevil strains, Wolbachia endosymbionts and parasitoids. Biochemical approaches to the study of ecological genetics: the role of selection and gene flow in the evolution of insecticide resistance. A molecular and ecological investigation of the large arionid slugs of north west Europe: the potential for new pests. Systematics of brown planthopper and related species using nuclear and mitochondrial DNA. Application of novel molecular markers (DNA) in agricultural entomology. Location of resistance to Brevicoryne brassicae in wild brassica species. The use of DNA markers in population genetics and ecological studies of the desert locust Schistocerca gregaria (Orthoptera: Acrididae). The use of DNA analysis and the polymerase chain reaction in the study of introduced pests in New Zealand. Serological analysis of arthropod predation: past, present and future. Serological diagnosis of parasitism: a monoclonal antibody-based immunodot assay for Microplitis (Hymenoptera: Braconidae). Polyclonal, monoclonal and engineered antibodies to investigate the role of predation in slug population dynamics. The potential of combinatorial antibody libraries in pest/predator relationship studies. Serological analysis of predators of Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) eggs if sorghum-pigeonpea intercropping at ICRISAT, India: a preliminary field study. Using gut content immunoassays to evaluate predaceous biological control agents: a case study. An environmental risk assessment for release of an exotic microsporidium for European corn borer control in North America. Progress in quantifying predation using antibody techniques. Electrophoretic approaches to predator-prey interactions. Genetics of esterases in laboratory and feral cotton boll weevils, Anthonomus grandis Bohemia. Practical applications for techniques to determine the nutritional state and age of field caught tsetse flies.

A Receptor-Based Immunoassay to DetectStaphylococcusEnterotoxin B in Biological Fluids

A rapid, simple, and inexpensive sandwich enzyme-linked receptor based immunodot assay was developed for the detection of staphylococcal enterotoxin B (SEB) in human fluids by using purified glycosphingolipid digalactosylceramide (diGalCer) receptor for SEB. Three micrograms of diGalCer was immobilized on a polyvinyledene difluoride membrane and the membrane was subsequently incubated with primary and secondary alkaline-phosphatase-labeled antibodies. A positive reaction was discerned as a blue spot. As little as 1 ng/ml of SEB could be detected in the assay. SEB did not bind to structurally related glycosphingolipids, such as glucosylceramide, galactosylceramide, and lactosylceramide in this assay. Of five monoclonal anti-SEB antibodies and commercial anti-SEB antiserum tested, the latter was the most sensitive in our assay. The specificity of SEB assay was assessed by comparison with structurally related toxins, for example, staphylococcal enterotoxin A, and toxic shock syndrome toxin 1 (TSST-1). TSST-1 was not detected in the assay. This was because these toxins were not recognized by the anti-SEB antibody and did not bind to diGalCer. In conclusion, we believe that this assay may be widely applicable because it is highly specific for SEB, it does not require special equipment, and the results can be obtained within few hours with the naked eye. Since the receptor for SEB has a long shelf life, it can be easily stored and used for a long time.

Sero- and genotyping of some marine aquatic birnavirus isolates from Norway

Twelve isolates of aquatic birnaviruses (9 field isolates from marine fish and shellfish and the Ab/A3, Sp/A2 and NI strains) were serotyped using an immunodot assay. The assay with 17 monoclonal antibodies revealed some serological variation among the Norwegian isolates, but the N1 strain and the Sp/A, type strain reacted identically. A genotyping assay based upon restriction fragment analysis of a polymerase chain reaction-amplified fragment from the VP2 coding region was run parallel to the serotyping. All the 9 serogroup A serotypes, except He/A, and Can. 3/A8, were assayed in addition to the Norwegian isolates mentioned above. This method differentiated among all serotypes tested, but only small differences were observed among the Norwegian isolates. The NI and Sp/A2 strains reacted identically. Our results indicate homogeneity between Norwegian birnavirus isolates and support earlier studies that concluded that the NI strain belongs to the Sp/A, serotype.

Plasma membrane calcium ATPase in synaptic terminals of chick Edinger-Westphal neurons

The plasma membrane calcium ATPase pump (PMCA) is one of two major mechanisms known to be involved in extruding calcium from cells. The monoclonal antibody 5F10 was used to examine the distribution of PMCA in chick Edinger-Westphal neurons, a population of cholinergic preganglionic neurons whose cells bodies reside in the Edinger-Westphal nucleus in the brainstem and whose axons form synaptic terminals on parasympathetic neurons in the ciliary ganglion. Definitive PMCA immunoreactivity was undetectable in Edinger-Westphal cell bodies in the brainstem. In contrast, immunoreactivity for PMCA was robust in ciliary ganglia and resembled patterns of immunoreactivity for the synaptic vesicle antigen SV-2, suggesting that PMCA is expressed in Edinger-Westphal synaptic terminals. Moreover, PMCA immunoreactivity co-localized with immunoreactivity for enkephalin and substance P, two neuropeptides known to be expressed in Edinger-Westphal synaptic terminals. Fine structure studies revealed that PMCA immunoreactivity is associated with synaptic vesicles rather than the plasma membrane in Edinger-Westphal terminals. In immunodot assays, synaptic vesicles purified fromTorpedo electric organ are also immunoreactive for PMCA as well as SV-2.Torpedo vesicles are negative for the sarcoplasmic/endoplasmic reticulum ATPase, suggesting that the observed PMCA immunoreactivity is not associated with smooth endoplasmic reticulum. Immunoblot analysis confirmed that 51710 recognizes a protein with the correct molecular mass for PMCA in tissue homogenates of chick cerebellum, chick ciliary ganglia, andTorpedo synaptic vesicles. These findings describe a previously unrecognized location for PMCA in the membranes of cholinergic synaptic vesicles. Relevance to previous data and possible functions are discussed.

Cross-reactivities of polyclonal antibodies against lebetase, fibrinolytic enzyme of levantine viper ( Vipera lebetina) venom

Antibodies to two fractions of fibrinolytic enzyme in Vipera lebetina venom were produced by immunizing mice with chromatographically purified lebetase probes. The antibodies were isolated from mice blood by protein A affinity chromatography. The antibodies to both fractions reacted only with the fibrinolytic enzyme in V. lebetina venom as demonstrated by western immunoblotting. Immunodot assays, ELISA and western immunoblotting revealed that 11 snake venoms including species of Viperidae and Crotalidae but not Elapidae cross-react with lebetase antibodies to varying degrees. The molecular weights of cross-reacting components were detected and compared with the earlier published data.

Immunochemical detection of methamphetamine using polymer-protected ultrafine platinum particles

Ultrafine platinum particles protected by poly (methyl acrylate-co- N-vinyl-2-pyrrolidone) were applied to the immunological detection of methamphetamine (MA). The polymer-protected ultrafine particles could chemically bind anti-methamphetamine monoclonal antibody to their surfaces. The antibody-fixed particles behaved like an antibody in the immunoreaction. The antibody-bound particles competitively reacted against the immobilized and the free antigens in the ELISA system. MA could be detected down to a concentration of ca. 10 ng/ml. In the immuno-dot assay, 10–100 ng/ml of MA was detected within 30 min using the ultrafine particles.

Ontogeny of the opioid growth factor, [Met5]-enkephalin, and its binding activity in the rat retina.

The endogenous opioid peptide [Met5]-enkephalin is a tonically active opioid growth factor (OGF) with an inhibitory action on DNA synthesis in the developing rat retina. In this study, the ontogeny of the spatial and temporal expression of OGF and its binding activity was examined. OGF-like immunoreactivity was detected in the retina at gestation day (E) 20, but not at E18, and was localized to ganglion cell and neuroblast layers; immunochemical reaction was no longer seen in the retina by postnatal day 6. Native OGF was further identified and characterized by high-performance liquid chromatography (HPLC) studies and immunodot assays, which revealed that [Met5]-enkephalin was present in the neonatal, but not adult, rat retina. OGF binding activity was detected as early as E18 using [125I]-[Met5]-enkephalin and in vitro receptor autoradiography. Little OGF binding activity was noted for prenatal retinas, but appreciable activity was observed from birth to postnatal day 4; no OGF binding could be detected after postnatal day 5 or in the adult. These results reveal the transient appearance of the OGF, [Met5]-enkephalin, and its receptor binding activity in the developing mammalian retina, and show that their ontogeny coincides with the timetable of DNA synthesis of retinal neuroblasts.