Enzyme-linked Oligosorbent Assay Research Articles

Characterization of an Aptamer Targeting Neu5Gc, as an Endogenous Pathogenic Factor Derived from Red Meat.

N-glycolylneuraminic acid (Neu5Gc), a sialic acid predominantly found in the non-neurohumoral fluids of hind-mouthed animals, is incapable of synthesizing Neu5Gc due to a deletion in the CMAH exon of the gene encoding human CMP-Neu5Gc hydroxylase. But consumption of animal-derived foods that contain Neu5Gc, such as red meat, can instigate an immune response in humans, as Neu5Gc is recognized as a foreign substance by the human immune system. This recognition leads to the production of anti-Neu5Gc antibodies, subsequently resulting in chronic inflammation. When Neu5Gc is consumed excessively or frequently, it may contribute to the development of heart disease and cancer. This makes Neu5Gc, an endogenous pathogenic factor derived from red meat, a new hot topic in red meat safety research. In this study, aptamers obtained by the magnetic bead SELEX technique were subjected to homology and secondary structure prediction analysis as well as affinity determination. The result indicated that the aptamer 2B.N2A9 exhibited a robust binding affinity, with an affinity constant (Ka) of 1.87 × 108 L/mol. This aptamer demonstrated optimal binding specificity within a pH range of 5.4 to 7.4. Molecular docking analysis further revealed that aptamer 2B.N2A9 formed stable binding interactions with the target Neu5Gc at specific sites, namely G-14, C-15, G-13, G-58, G-60, and C-59. An Enzyme-Linked Oligonucleotide Sorbent Assay (ELOSA) methodology was established to detect the endogenous pathogenic factor Neu5Gc present in red meat. This method demonstrated a limit of detection (LOD) of 0.71 ng/mL, along with an average recovery rate of 92.23%. The aptamer obtained in this study exhibited favorable binding properties to Neu5Gc. The assay was relatively convenient and demonstrated good sensitivity. Further investigation into the distribution of Neu5Gc in various red meats is of public health significance and scientific potential. A practical detection method should be provided to guide red meat diets and ensure the nutrition and safety of meat products.

꽃치자나무 추출물의 HIV-1 효소 억제 활성과 QSAR에 의한 활성인자 예측

꽃치자나무 추출물의 HIV-1 reverse transcriptase, protease 및 alpha-glucosidase에 대한 억제활성실험을 실시하였다. ELOSA 방법으로 실험한 역전사효소 억제활성 실험에서는 꽃치자나무 잎 MeOH 추출물 <TEX>$100{\mu}g/ml$</TEX> 농도에서 13.4%의 약한 억제활성이 관찰되었고, 줄기의 메탄올추출물에서는 32.5%의 높은 HIV-1 protease 억제활성과 줄기의 메탄올 추출물에서는 26.1%의 alpha-glucosidase 억제활성이 관찰되었다. 그리고 HIV-1 복제 억제활성은 MT-4 세포에 대한 HIV-1 유도 세포변성억제를 광학현미경으로 관찰하여 살펴본 결과 잎과 줄기의 모든 추출물에서 HIV-1 바이러스 증식억제에 억제활성이 관찰되지 않았다. In silico 실험결과 주요성분인 crocetin이 81.8%의 높은 역전사 효소활성이 예측되었으며, genipin이 55.39%, geniposidic acid aglycone이 64.5% 역전사효소 활성이 예측되었다. 주로 aglycone의 활성이 높게 예측되었으며 배당체의 경우 활성이 현저하게 저하되는 것으로 나타났다. In this study, we conducted the anti-HIV-1 enzymes assay in vitro and its active components were predicted by QSAR in silico for the elucidation of action mechanism on anti-HIV of natural resources. The extracts of Gardenia jasminoides var. radicans Makino were tested for their inhibitory effects on the reverse transcriptase (RT), protease and <TEX>${\alpha}$</TEX>-glucosidase. In the enzyme inhibition assay, the methanol extracts of Gardenia jasminoides var. radicans Makino stem showed a strong activity of 32.5% on the enzyme activity to cleave an oligopeptide, resembling one of the cleavage sites in the viral polyprotein which can only be processed by HIV-1 protease. Moreover the methanol extracts of stem exhibited alpha-glucosidase inhibitory activities of 26.1%. The methanol extracts (<TEX>$100{\mu}g/ml$</TEX>) of stem showed a weak activity of 13.4% on anti-HIV-1 RT using Enzyme Linked Oligonucleotide Sorbent Assay (ELOSA) method. However, all extracts of leaf and stem didn't exhibit the HIV-1-induced cytopathic effects with IC (inhibitory concentration) of <TEX>$100{\mu}g/ml$</TEX> in HIV-1-infected human T-cell line. From these results, it is suggested that Gardenia jasminoides var. radicans Makino extracts may possibly be involved in the inhibition of reverse transcriptase, protease and alpha-glucosidase but can't vitally concerned with the viral replication in vitro.

DNA probe based colorimetric method for detection of rifampicin resistance of Mycobacterium tuberculosis

Rifampicin resistance of Mycobacterium tuberculosis is due to the occurrence of point mutations of the rpoB gene and the site of mutations vary geographically. Commercialized molecular based methods are not able to comprehensively detect rifampicin resistance as they target a limited number of gene mutations which are thought to be common. The aim of the study was to establish a low cost DNA probe based colorimetric method that can be customized for detection of rifampicin resistance of M. tuberculosis. Thus, enzyme-linked oligosorbent assay (ELOSA) was developed for the detection of polymerase chain reaction (PCR) amplified fragments of rpoB gene of M. tuberculosis DNA on microtiter plates. Forty two M. tuberculosis isolates (rifampicin resistant and susceptible isolates identified by agar proportion method) were used for developing and validating the assay. The point mutations of resistant isolates had been previously determined by DNA sequencing. Two fragments of rpoB gene were labeled with digoxigenin by PCR. The amplified products were hybridized with selected allele specific probes for three mutations and its wild types (six probes) which were captured onto streptavidin coated microtiter plates and detected by color development. Both sensitivity and specificity of all probes were ≥96% and there was excellent discrimination (area under the curve (AUC)>0.9) between rifampicin susceptible cases and resistant cases. The probe-based colorimetric assay (PCR–ELOSA) developed in this study showed good agreement with reference mutations that were confirmed by DNA sequencing. In conclusion, PCR–ELOSA is a reliable and economical assay that can be customized for detection of rifampicin resistance.

Development of Innovative and Versatile Polythiol Probes for Use on ELOSA or Electrochemical Biosensors: Application in Hepatitis C Virus Genotyping

The aim of this study was to develop versatile diagnostic tools based on the use of innovative polythiolated probes for the detection of multiple viruses. This approach is compatible with optical enzyme-linked oligosorbent assay (ELOSA) or electrochemical (biosensors) detection methods. The application targeted here concerns the rapid genotyping of Hepatitis C virus (HCV). HCV genotyping is one of the predictive parameters currently used to define the antiviral treatment strategy and is based on the sequencing of the viral NS5b region. Generic and specific NS5b amplicons were produced by real-time polymease chain reaction (RT-PCR) on HCV(+) human plasma. Original NS5b probes were designed for genotypes 1a/1b, 2a/2b/2c, 3a, and 4a/4d. Robust polythiolated probes were anchored with good efficacy on maleimide-activated microplates (MAM) and gold electrodes. Their grafting on MAM greatly increased the sensitivity of the ELOSA test which was able to detect HCV amplicons with good sensitivity (10 nM) and specificity. Moreover, the direct and real-time electrochemical detection by differential pulse voltammetry enabled a detection limit of 10 fM to be reached with good reproducibility. These innovative polythiolated probes have allowed us to envisage developing flexible, highly sensitive, and easy-to-handle platforms dedicated to the rapid screening and genotyping of a wide range of viral agents.

능소화 잎 및 줄기 추출물의 Human Immunodeficiency Virus Type I 억제활성

능소화 추출물의 HIV-1에 대한 항바이러스 효과를 복제 관련 효소에 대한 억제실험과 바이러스 복제억제 실험을 통하여 살펴보았다. 역전사효소 억제활성을 ELOSA 방법으로 실험한 결과 능소화 줄기의 물 추출물이 100 <TEX>${\mu}g$</TEX>/ml 농도에서 각각 37.9%의 활성을 나타내었고, 능소화 잎과 줄기의 메탄올 추출물에서 33.6% 및 31.5%의 HIV-1 protease 억제활성 나타 내었다. 그리고 HIV-1 복제 억제활성은 MT-4 세포에 대한 HIV-1 유도 세포변성억제를 광학현미경으로 관찰하여 살펴본 결과 줄기의 물 추출물이 100 <TEX>${\mu}g$</TEX>/ml 농도에서 HIV-1 바이러스 증식을 완전히 억제하였다. For the elucidation of action mechanism on anti-HIV of natural resources, the extracts of <TEX>$Campsis$</TEX> <TEX>$grandiflora$</TEX> were tested for their inhibitory effects on HIV-1 replication and its essential enzymes as the reverse transcriptase (RT), protease and <TEX>${\alpha}$</TEX>-glucosidase. In the assay of HIV-1-infected human T-cell line, water extracts of stem inhibited the HIV-1-induced cytopathic effects with IC (inhibitory concentration) of 100 <TEX>${\mu}g$</TEX>/ml. Moreover water extracts (100 <TEX>${\mu}g$</TEX>/ml) of stem showed strong activity of 37.9% on anti-HIV-1 RT using Enzyme Linked Oligonucleotide Sorbent Assay (ELOSA) method. In the HIV-1 protease inhibition assay, methanol extracts of stem and leaf extract showed 33.6% and 31.5% inhibition of the enzyme activity to cleave an oligopeptide resembling one of the cleavage sites in the viral polyprotein which can only be processed by HIV-1 protease, but did not exhibited glucosidase inhibitory activities. From these results, it is suggested that the inhibition of the viral replication <TEX>$in$</TEX> <TEX>$vitro$</TEX> is due to the inhibition of reverse transcriptase by water extracts of stem of <TEX>$Campsis$</TEX> <TEX>$grandiflora$</TEX>.

Development and evaluation of a sensitive enzyme-linked oligonucleotide-sorbent assay for detection of polymerase chain reaction-amplified hepatitis C virus of genotypes 1–6

A high-throughput polymerase chain reaction (PCR)-based enzyme-linked oligonucleotide-sorbent assay (ELOSA) was developed for use in the diagnostic testing of serum from patients who may be infected with different hepatitis C virus (HCV) genotypes. Twelve genotype-specific 5′-aminated DNA-coated probes were designed based on the variable 5′-untranslated region sequences of the HCV genotypes 1–6. Using 100 clinical serum samples, the performance of the PCR-ELOSA method was compared with Roche's COBAS Amplicor HCV Monitor V2.0 assay and the VERSANT HCV genotype assay (LiPA), and the overall agreement was 99% at the level of HCV genotypes with a detection range of 2.0 × 10 2 to 1.0 × 10 7 IU/ml for PCR-ELOSA. The PCR-ELOSA was more comprehensive as demonstrated by the fact that ∼20% of the samples with different subtypes could be discriminated by this method but not by LiPA. In addition, the PCR-ELOSA system showed high accuracy (CV ≤ 6.36%) and even higher reproducibility (CV ≤ 5.55%). Thus, this novel PCR-ELOSA system provides a sensitive and versatile alternative to current HCV detection assays.

Association between the level of circulating bioactive tumor necrosis factor α and the tumor necrosis factor α gene polymorphism at −308 in patients with rheumatoid arthritis treated with a tumor necrosis factor α inhibitor

The tumor necrosis factor alpha (TNFalpha) -308A polymorphism has been associated with high production of TNFalpha and poor response to anti-TNFalpha therapy, but these associations remain controversial. The aim of this study was to explore the association between circulating TNFalpha bioactivity, the TNFalpha -308 polymorphism, and the clinical response to infliximab in patients with rheumatoid arthritis (RA). One hundred ninety-eight patients with RA were treated with infliximab and methotrexate. Responses at 6 months according to the American College of Rheumatology (ACR) preliminary criteria for improvement in RA were recorded. Genotyping for the TNFalpha -308 polymorphism was performed by enzyme-linked oligosorbent assay. Circulating TNFalpha bioactivity was evaluated in 50 patients with RA by assessing the production of interleukin-6 (IL-6) in synoviocytes induced by a small amount of TNFalpha plus plasma. IL-6 production in 48-hour supernatants and the levels of TNFalpha protein and IL-6 were measured by enzyme-linked immunosorbent assay. The TNFalpha -308 polymorphism was not associated with the ACR response to infliximab. The level of circulating TNFalpha bioactivity was higher in patients with the TNFalpha -308 A/A or A/G genotype than that in patients with the G/G genotype (median 50.0 ng/ml [interquartile range (IQR) 31.5-62.0] versus 33.0 ng/ml [IQR 16.5-47.5]; P < 0.02). However, no difference was observed for the TNFalpha protein level according to genotype (median 0.62 pg/ml [IQR 0.00-8.85] for G/G versus 3.35 pg/ml [IQR 1.55-4.63] for A/A or A/G; P not significant). The level of circulating TNFalpha bioactivity was higher in good responders (> or =50% improvement) than in poor responders (< or =20% improvement) (median 45.0 ng/ml [IQR 21.0-59.0] versus 28.0 ng/ml [IQR 14.0-39.0]; P = 0.05). However, the level of TNFalpha protein was similar in both groups. The level of functional circulating TNFalpha is partially genetically determined and is predictive of the clinical response to infliximab. Nonresponders to anti-TNFalpha therapy are likely to have a disease that is not primarily driven by TNFalpha.

Carbon nanotube-based labels for highly sensitive colorimetric and aggregation-basedvisual detection of nucleic acids

A novel carbon nanotube (CNT) derived label capable of dramatic signal amplification ofnucleic acid detection and direct visual detection of target hybridization has beendeveloped. Highly sensitive colorimetric detection of human acute lymphocytic leukemia(ALL) related oncogene sequences amplified by the novel CNT-based label wasdemonstrated. Atomic force microscope (AFM) images confirmed that a monolayer ofhorseradish peroxidase and detection probe molecules was immobilized along thecarboxylated CNT carrier. The resulting CNT labels significantly enhanced the nucleic acidassay sensitivity by at least 1000 times compared to that of conventional labels usedin enzyme-linked oligosorbent assay (ELOSA). An excellent detection limit of1 × 10−12 M(60 × 10−18 molin 60 µl) and a four-order wide dynamic range of target concentration were achieved.Hybridizations using these labels were coupled to a concentration-dependent formation ofvisible dark aggregates. Targets can thus be detected simply with visual inspection,eliminating the need for expensive and sophisticated detection systems. The approachholds promise for ultrasensitive and low cost visual inspection and colorimetricnucleic acid detection in point-of-care and early disease diagnostic application.

Force Measurements Between Emulsion Droplets-ssDNA Conjugates: A New Tool for Medical Diagnostics

We describe here a new system involving direct force measurements between biomolecules that could be used in biomedical diagnostics. The method consists in the use of magnetic emulsion droplets bearing immobilized single stranded DNA fragments (ssDNA, Deoxyribo Nucleic Acid). The immobilized ssDNA fragments are able to recognize complementary DNA molecules via specific hydrogen binding (hybridization process). The ssDNA used in this study are 32 bases oligonucleotides functionalized at their 5' extremity with biotin and then immobilized onto the magnetic nanodroplets via interactions with streptavidin previously chemically grafted onto the nanomagnetic support. The aim of this work is to evaluate the possible detection of captured nucleic acid targets via single force measurements as an alternative to classical ELOSA (Enzyme Linked Oligo Sorbent Assay). The obtained results are discussed mainly in terms of electrostatic interactions.

Lack of Association of IL-10 Promoter Gene Variants with Type 1 Diabetes in a French Population

Although genetic predisposition to type 1 diabetes shows a strong association with human leukocyte antigen (HLA) class II alleles, additional genes may influence the immune process and the progression of beta cell loss. Preliminary reports suggested that IL-10 gene polymorphisms contribute to susceptibility to type 1 diabetes. We analyzed the frequencies of three main variants of the promoter region of the IL-10 gene at the positions −1082, −819, and −592 in a cohort of 358 type 1 diabetic patients representing the same regional population pattern and 519 controls from the same region using an enzyme-linked oligonucleotide sorbent assay. We did not find any statistical association in the entire cohort or after stratification for high-risk HLA-DQ alleles. However, the IL-10 −1082 polymorphism was significantly associated with GAD and IA-2 antibodies at clinical onset. Such polymorphism is known to be associated with the reduction of secreted IL-10 which may support the concept of accelerated Th-1 T-cell reactivity. In conclusion, IL-10 promoter gene variants may contribute, but to a minor extent, to disease susceptibility in juvenile type 1 diabetes and should not be included in the routine genetic screening of high-risk individuals.

Water-Soluble Dendrigrafts Bearing Saccharidic Moieties: Elaboration and Application to Enzyme Linked OligoSorbent Assay (ELOSA) Diagnostic Tests

The synthesis of a series of water-soluble galactopyranose-functionalized polystyrene-polyvinyl ether dendrigrafts and their characterization (in solution and thin solid deposits) have been achieved. The presence of external galactopyranose groups on dendritic polymers has been exploited to prepare dendrigraft-oligonucleotide conjugates using a simple one-step coupling procedure with amino-ended oligonucleotides (ODNs). Several parameters such as the peripherical density of hydrophilic branches, the polymerization degree of polystyrene or poly(hydroxyethyl vinyl ether) blocks, and the number of galactopyranose groups were tuned. A capture test with short labeled complementary ODNs (25 bases) confirmed the presence of covalently bound ODNs on various kinds of dendrigrafts. The ability of the dendritic polymers to enhance the sensitivity of enzyme-linked oligosorbent assay (ELOSA) diagnostic tests (detection of hepatitis B virus, DNA target of 2400 bases) was then evaluated, especially the influence of the macromolecular architecture and the impact of the structural parameters. The dendrigraft-ODN conjugate with the lower saccharide external density was found to lead to a very significant amplification of the fluorescence signal, corresponding to a limit of sensitivity of 10(9) DNA copies per milliliter (instead of 10(11) DNA copies per milliliter without using dendrigrafts). Conversely, the dendrigrafts exhibiting a very high number of branches and galactopyranose groups at their periphery were not able to induce a better sensitivity due to steric hindrance generated by the peripheral congestion on these polymers.

Monitoring system for the biocontrol agent Pichia anomala strain K using quantitative competitive PCR‐ELOSA

A quantitative‐competitive PCR (QC‐PCR) method with enzyme‐linked oligosorbent assay (ELOSA) was developed to monitor Pichia anomala strain K, a biocontrol agent against postharvest diseases on apples. The procedure involved: (i) extraction of strain K DNA; (ii) coamplification of a constant amount of the extracted DNA (containing a strain K DNA marker of 262 base pairs (bp) specifically amplified with SCAR primers K1 and K2) with an internal standard (IS) titration; and (iii) differential hybridization with two specific biotinylated probes either for the target or for the IS sequence on microplate. The IS sequence differed from the target by only a short internal region of 35 bp providing the differential detection between both sequences. Both target and IS sequences proved to be competitive in PCR as well as in sandwich hybridization. Two copies of the target sequence were detected in the strain K genome by means of enzymatic restriction and Southern blot analysis. Varying amounts of strain K cell suspension were quantified in the phosphate buffer used for recovering cells from the apple surface. An accurate estimate of the strain K population was obtained from 103 to 106 yeast cells per apple. The threshold of the method was found to be at 1000 colony‐forming units per apple, which was around 100 times more sensitive than the plating method for monitoring strain K.

Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA.

Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.

THE USE OF (OLIGONUCLEOTIDE-MALEIC ANHYDRIDE COPOLYMER) CONJUGATES IN NUCLEIC ACID DIAGNOSTIC ASSAYS: EFFECT OF THE NUMBER OF OLIGONUCLEOTIDES PER POLYMER ON TEST SENSITIVITY IMPROVEMENT

(Polymer-oligonucleotide) conjugates were obtained via direct ON synthesis from the poly(ethylene-alt-maleic anhydride: PEMA) grafted onto glass bead surfaces. The effect of the ON number per polymer chain on binding was evaluated through Tm experiments. Results were correlated with ELOSA (Enzyme Linked Oligosorbent Assay) tests run with conjugates in the capture step.

Oligonucleotide-polymer conjugates: effect of the method of synthesis on their structure and performance in diagnostic assays.

Oligonucleotide-polymer conjugates have been described to improve the sensitivity of an enzyme-linked oligosorbent assay diagnostic test. To understand the influence of their structure and conformation in solution on the efficiency of the test during the capture step, two different ways of synthesizing these conjugates were compared. The first consisted of coupling 5' amino modified oligonucleotides to poly(maleic anhydride-alt-methylvinyl ether) and poly(maleic anhydride-alt-ethylene). The second resulted from direct synthesis of oligonucleotides from poly(maleic anhydride-alt-ethylene) previously grafted onto a controlled pore glass support. The different conjugates were analyzed by size-exclusion chromatography and viscometry. The former method for conjugate synthesis produced aggregates, which was not the case for the latter. These conjugates were then used in the capture phase of a hybridization assay using a HBV DNA target, on a bioMérieux VIDAS instrument. Different parameters were studied, such as the purity of the conjugate solution and the number of oligonucleotides per polymer chain. The amount of conjugate coated on the solid-phase receptacle surface at the time of the capture phase was evaluated by radioactive labeling. Finally, it was demonstrated that conjugates produced an amplification factor of 50 versus the capture oligonucleotide probe used as the reference. The detection limit reached 10(8) copies/mL.

Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA)

Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide. Routine laboratory diagnosis of PE is done by amplification of L. intracellularis -specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide. We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L. intracellularis 328-bp PCR amplified products. The ELOSA involved determination of optical density value at 450 nm (OD450) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex. A positive ELOSA cut-off value of ≥0·375 was established using the mean OD450of negative control specimens plus three times the standard deviation. Using this value, the detection limit of PCR amplified L. intracellularis -specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6·1 ng, between 0·8 and 3·0 ng, and 0·8 ng of DNA, respectively. Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L. intracellularis -specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy. The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective Íinterpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis.

Detection of bovine immunodeficiency-like virus infection in experimentally infected calves.

Detection of BIV virus infection by serological means, PCR and virus isolation in experimentally infected calves is described. Viral sequences were specifically detected by PCR in peripheral blood mononuclear cells (PBMCs), with primer systems located in the gag, pol and tat regions of the viral genome. An enzyme-linked oligosorbent assay (ELOSA) in microtiter plates is described, for the detection of PCR products, the sensitivity of which was shown to be comparable to that of membrane hybridization detection. Serological response of the animals against the BIV p26 protein was shown, using a recombinant fusion protein ((His)6p26) expressed in E. coli and purified by metal affinity chromatography, in ELISA and Western blot studies. The presence of infectious virus was demonstrated by its rescue, by virus isolation in cell cultures, from PBMCs during a one year follow-up.

An RT‐PCR Test for the Detection of Tomato Spotted Wilt Tospovirus Incorporating Immunocapture and Colorimetric Estimation

AbstractA reverse transcription‐polymerase chain reaction (RT‐PCR)‐based assay for the detection of tomato spotted wilt virus (TSWV) has been modified and improved by the use of immunocapture of virus by solid phase adsorbed antibodies and colorimetric detection of nested PCR products, into which digoxigenin has been incorporated, by an ELOSA (enzyme‐linked oligonucleotidesorbent assay)‐like assay. The use of the colorimetric assay for the detection of tospoviruses by using ‘universal’ primers in the RT‐PCR stage is also described.

Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay

Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild-type nef region with a mimic competitive nef gene template carrying mutations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per micrograms of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per microgram of DNA. QC- and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC- and IC-PCR-ELOSA were identical for 77% of patient samples. The copy level ranged between 443 +/- 156 and 21,453 +/- 13,511 copies per 10(5) CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC- and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.

Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1

An enzyme-linked oligosorbent assay (ELOSA) was developed for the detection on microtiter plates of polymerase chain reaction (PCR)-amplified human immunodeficiency virus type 1 (HIV-1) DNA. The denatured PCR product was hybridized with a passively adsorbed oligonucleotide capture probe and a horseradish peroxidase-labeled oligonucleotide detection probe. The sensitivity and specificity of the PCR-ELOSA technique depended to some extent on the nucleotide sequences of the oligonucleotide primer and probe quartet used in the amplification and detection. We evaluated five oligonucleotide quartets located in the gag, pol, vpr, env, and nef regions of HIV-1. DNAs from 39 HIV-1-seropositive individuals and 27 healthy HIV-1-seronegative controls were amplified by the PCR procedure, and the products were detected by ELOSA. Ten copies of HIV-1 DNA against a background of 1 microgram of human DNA were specifically detected by PCR-ELOSA. Specificities and sensitivities were, respectively, 100 and 95% for the gag system, 100 and 97% for the pol system, 100 and 85% for the vpr system, 96 and 95% for the env system, and 100 and 95% for the nef system. The simplicity of ELOSA makes it suitable for automation and applicable to genetic testing and detection of viral and bacterial DNAs or RNAs in most routine laboratories.