An enzyme-linked immunoassay for the detection of antibodies to endoplasmic reticulum

A simple, sensitive solid-phase assay for the detection of antibodies to endoplasmic reticulum is described. The assay is dependent upon the amount of antigen bound to the solid support and upon the amount of antibody bound to the support via the relevant antigen. The assay can be used to measure both polyclonal and monoclonal antibody to endoplasmic reticulum. It has been used to isolate several monoclonal antibodies which can recognize and precipitate specific proteins of the endoplasmic reticulum. In addition, it has been used to probe the membrane orientation of endoplasmic reticulum antigens.     

Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR

Aim

Create a method for highly sensitive, selective, rapid and easy‐to‐use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously.

Methods and Results

Test‐systems for real‐time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test‐systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 μl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA^® amplifier.

Conclusions

Preloaded 30‐reaction micromatrices having shelf life of 3 and 6 months (for RNA‐ and DNA‐based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg).

Significance and Impact of the Study

The accurate, rapid and user‐friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.     

An immunofluorescence test for detection of serum antibody to rodent coronaviruses

An indirect immunofluorescence test, using cultured cells infected with two strains of mouse hepatitis virus, was developed for detection of antibody to rodent coronaviruses. The immunofluorescence test detected serum antibody to mouse hepatitis virus and rat sialodacryoadenitis virus earlier than the neutralization test. In the case of mouse hepatitis virus, the results of the immunofluorescence test closely paralleled those obtained with a commercially available enzyme-linked immunosorbent assay.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

Development of antibody array for simultaneous detection of foodborne pathogens

Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.     

Use and detection of nonradioactive iodine-labeled antibodies for immunoassay

Traditionally radioactively labeled iodine has been used as a reporter group for the detection of antibodies in immunoassay. We have recently developed a microassay system for the detection of very low concentrations of iodide which eliminates the need for the use of radiolabeled iodine (O'Kennedy, R. et al. 1989, Anal. Biochem. 179, 138-144). The successful application of this assay for the detection of mouse IgG is described. The performance of this system compared with enzyme immunoassay was evaluated.     

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.     

Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.     

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.     

Superhydrophobic surface-based magnetic electrochemical immunoassay for detection of Schistosoma japonicum antibodies

In this paper, a magnetic electrochemical immunoassay that uses a superhydrophobic surface-based analytical platform (SSAP) has been initially developed for detection of Schistosoma japonicum (Sj) antibodies (SjAb). The SSAP is fabricated by modifying the inner surfaces of plastic test tubes with superhydrophobic polycarbonate coatings that show a water contact angle up to 160° and a water rolling angle less than 5°. In a noncompetitive sandwich format, the SjAb immunoassay with magnetic particles is based on sensitive stripping voltammetry analysis coupled with the copper enhanced Au nanoparticle tag amplification. This technique is quantitatively sensitive to SjAb concentrations ranging from 2 ng ml(-1) to 15 μg ml(-1), with a detection limit of ～1.3 ngml(-1). Moreover, the results of assaying several serum specimens prove its feasibility of practical applications. The self-cleaning SSAP can be reused, because no aqueous samples reagents or contaminate the superhydrophobic polycarbonate during the experiments. The comparison study additionally demonstrates that the SSAP-based magnetic electrochemical immunoassays can offer preferable advantages over the existing approaches for SjAb detection, in terms of volumes of samples and reagents, assay time, and detection limit.     

Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes

Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. ? 2011 International Society for Advancement of Cytometry.     

Multiplex real‐time PCR assays for detection of four seedborne spinach pathogens

Aims

To develop multiplex TaqMan real‐time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach.

Methods and Results

Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real‐time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real‐time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed.

Conclusions

The real‐time PCR assays were species‐specific and sensitive. Singular or multiplex real‐time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed.

Significance and Impact of the Study

The freeze‐blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real‐time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.     

Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish

A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.     

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

A multiplex PCR (mPCR) method was designed for the simultaneous detection of 4 major fish pathogens, Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas aeruginosa, and P. putida. Each of the 4 pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were 2 colony-forming units (CFU) of F. psychrophilum, 3 CFU of L. garvieae, 3 CFU of P. aeruginosa, and 5 CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria. High amounts of DNA from 1 bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these were present in lower concentrations in the multiplex reaction. The mPCR assay proved useful for the detection of the bacteria in naturally infected fish. The assay is a sensitive, specific, and reproducible diagnostic tool for the simultaneous detection of 4 pathogenic bacteria that cause disease in fish and offers a potentially useful alternative to the conventional culture-based method.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.