scholarly journals Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Yilian Wang ◽  
Vishwesh Shah ◽  
Angela Lu ◽  
Ella Pachler ◽  
Brian Cheng ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Enzymatic Reactions ◽  
Immunosorbent Assays ◽  
Hydrogel Particle

Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical...

scholarly journals Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

2021 ◽  
Author(s):  
Yilian Wang ◽  
Vishwesh Shah ◽  
Angela Lu ◽  
Ella Pachler ◽  
Brian Cheng ◽  
...  
Keyword(s):  
Dynamic Range ◽  
High Sensitivity ◽  
Microfluidic Devices ◽  
Imaging Systems ◽  
Enzymatic Reactions ◽  
Hydrogel Particles ◽  
Hydrogel Surface ◽  
Hydrogel Particle ◽  
Particle Solution ◽  
Digital Counting

Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical labs. We present a lab-on-a-particle solution to digital counting of thousands of single enzymatic reactions. Hydrogel particles are used to bind enzymes and template the formation of droplets that compartmentalize reactions with simple pipetting steps. These hydrogel particles can be made at a high throughput, stored, and used during the assay to create ~500,000 compartments within 2 minutes. These particles can also be dried and rehydrated with sample, amplifying the sensitivity of the assay by driving affinity interactions on the hydrogel surface. We demonstrate digital counting of β-galactosidase enzyme at a femtomolar detection limit with a dynamic range of 3 orders of magnitude using standard benchtop equipment and experiment techniques. This approach can faciliate the development of digital ELISAs with reduced need for specialized microfluidic devices, instruments, or imaging systems.

scholarly journals Performance of Five Serological Assays for Diagnosis of Rhodococcus equi Pneumonia in Foals

2003 ◽  
Vol 10 (2) ◽  
pp. 241-245 ◽  
Author(s):  
Steeve Giguère ◽  
Jorge Hernandez ◽  
Jack Gaskin ◽  
John F. Prescott ◽  
Shinji Takai ◽  
...  
Keyword(s):  
Breeding Season ◽  
Diagnostic Performance ◽  
High Sensitivity ◽  
Rhodococcus Equi ◽  
Agar Gel ◽  
Cutoff Value ◽  
Serological Assay ◽  
Immunodiffusion Test ◽  
Immunosorbent Assays ◽  
Selection Of

ABSTRACT The performance of four enzyme-linked immunosorbent assays (ELISAs) (ELISA-6939, ELISA-33701, ELISA-VapA, and ELISA-California) and an agar gel immunodiffusion test for diagnosis of Rhodococcus equi pneumonia in foals was evaluated. Antibody concentrations of foals with culture-confirmed R. equi pneumonia (n = 41) were compared to those of age-matched pasturemates that remained clinically healthy during the entire breeding season (n = 24). For each serological assay evaluated, selection of a low cutoff resulted in high sensitivity but low specificity. Increasing the cutoff value resulted in better specificity but to the detriment of sensitivity. The best diagnostic performance was achieved with ELISA-California at a cutoff of 40%, resulting in a sensitivity of 59% and a specificity of 88%. We conclude that current serological assays do not differentiate between diseased and clinically healthy foals.

scholarly journals Comparative Evaluation of Nine Different Enzyme-Linked Immunosorbent Assays for Determination of Antibodies againstTreponema pallidum in Patients with Primary Syphilis

2000 ◽  
Vol 38 (3) ◽  
pp. 1279-1282 ◽  
Author(s):  
Bruno L. Schmidt ◽  
Marzieh Edjlalipour ◽  
Anton Luger
Keyword(s):  
Venereal Disease ◽  
Treponema Pallidum ◽  
High Sensitivity ◽  
Immunoglobulin M ◽  
Routine Testing ◽  
Primary Syphilis ◽  
Immunosorbent Assays ◽  
Late Stages ◽  
Higher Sensitivity

Nine different enzyme-linked immunosorbent assays (ELISAs) with a sonicate or recombinant proteins of Treponema pallidum as antigen have been evaluated comparatively by testing 52 highly selected sera from patients with primary syphilis, all negative in the microhemagglutination test for T. pallidum (MHA-TP). Eight tests exhibited greater sensitivity (48.5 to 76.9%) than the commonly used Venereal Disease Research Laboratory test (44.2%). Higher sensitivity could be related to (i) the volume and dilution of the serum, (ii) the design of the assay (capture and competitive tests showed higher sensitivity than sandwich-based assays), and (iii) the ability to detected specific immunoglobulin M antibodies. The specificity of the ICE Syphilis and the Enzygnost Syphilis tests was 99.5 and 99.8%, respectively, as determined by routine testing of 2,053 unselected sera in comparison with the MHA-TP test. ELISAs tested offered high sensitivity in patients with primary syphilis; however, recommendations to use these tests as screening assays do need further data on specificity and reactivity in late stages of the disease.

scholarly journals Sensitive one-step isothermal detection of pathogen-derived RNAs

2020 ◽  
Author(s):  
Chang Ha Woo ◽  
Sungho Jang ◽  
Giyoung Shin ◽  
Gyoo Yeol Jung ◽  
Jeong Wook Lee
Keyword(s):  
Diagnostic Method ◽  
High Sensitivity ◽  
T7 Rna Polymerase ◽  
Molecular Diagnostic ◽  
Enzymatic Reactions ◽  
Rna Aptamer ◽  
Rna Detection ◽  
Highly Sensitive ◽  
One Step ◽  
Sensitivity Specificity

AbstractThe recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times.

scholarly journals Comparison of Two Enzyme-Linked Immunosorbent Assays for Diagnosis of Mycobacterium Avium Subsp. Paratuberculosis

2005 ◽  
Vol 17 (5) ◽  
pp. 463-466 ◽  
Author(s):  
S. L. B. McKenna ◽  
D. C. Sockett ◽  
G. P. Keefe ◽  
J. McClure ◽  
J. A. VanLeeuwen ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
High Sensitivity ◽  
Kappa Statistic ◽  
Mycobacterium Avium Subsp ◽  
Control Programs ◽  
Test Characteristics ◽  
Unknown Status ◽  
Immunosorbent Assays ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Low Sensitivity

Enzyme-linked immunosorbent assays (ELISAs) are used in Johne's disease (JD) control programs as a first screening for presence of the disease in a herd. A high sensitivity of the ELISA is therefore important, yet the commonly used ELISAs have relatively low sensitivity. The inclusion of an absorption phase, although improving specificity, potentially decreases sensitivity. Sera and feces of 383 adult dairy cows in 8 herds were used to compare the test characteristics of an absorbed and a nonabsorbed indirect ELISA for the detection of JD. The absorbed ELISA is based on a protoplasmic antigen, whereas the nonabsorbed uses a lipoarabinomannan-based antigen. The potential advantage of the nonabsorbed ELISA is that it may be less specific and more sensitive. Two herds certified free of JD were used to compare the specificity of the ELISAs. The other herds used to compare sensitivity were either infected with Mycobacterium avium subsp. paratuberculosis or had unknown status. Using fecal culture as a gold standard, the diagnostic specificity for the absorbed and nonabsorbed ELISAs were 98.4% and 87.9%, respectively. The diagnostic sensitivity was 72.4% and 65.5% for the absorbed and the nonabsorbed ELISA, respectively. Furthermore, a comparison using a fecal DNA probe as the comparison standard resulted in both ELISAs having a sensitivity of 61.9%. Agreement between the 2 ELISAs was moderate, with a kappa statistic of 0.58. The nonabsorbed ELISA did not have a higher sensitivity and had a lower specificity than the absorbed ELISA. Therefore, in this population, there was no advantage gained with using the nonabsorbed ELISA.

scholarly journals Enzyme-Linked Immunosorbent Assays with High Sensitivity for Antigen-Specific and Total Murine IgE: A Useful Tool for the Study of Allergies in Mouse Models

2009 ◽  
Vol 58 (2) ◽  
pp. 225-235 ◽  
Author(s):  
Toshiro Takai ◽  
Yuri Ochiai ◽  
Saori Ichikawa ◽  
Emi Sato ◽  
Takasuke Ogawa ◽  
...  
Keyword(s):  
Mouse Models ◽  
High Sensitivity ◽  
Immunosorbent Assays

scholarly journals Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows

2010 ◽  
Vol 17 (4) ◽  
pp. 588-595 ◽  
Author(s):  
Ana C. A. M. Pajuaba ◽  
Deise A. O. Silva ◽  
José R. Mineo
Keyword(s):  
Brucella Abortus ◽  
High Performance ◽  
Protein A ◽  
High Sensitivity ◽  
Affinity Maturation ◽  
Serum Samples ◽  
Additional Tool ◽  
Igg Avidity ◽  
Immunosorbent Assays ◽  
Peroxidase Conjugate

ABSTRACT This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle.

scholarly journals Characterization of Plasmodium vivax Heat Shock Protein 70 and Evaluation of Its Value for Serodiagnosis of Tertian Malaria

2007 ◽  
Vol 14 (3) ◽  
pp. 320-322 ◽  
Author(s):  
Byoung-Kuk Na ◽  
Jae-Won Park ◽  
Hyeong-Woo Lee ◽  
Klin Lin ◽  
Seon-Hee Kim ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Plasmodium Vivax ◽  
Immunoglobulin G ◽  
Heat Shock Protein 70 ◽  
High Sensitivity ◽  
Igg Subclasses ◽  
Immunosorbent Assays

ABSTRACT We have characterized Plasmodium vivax heat shock protein 70 (PvHSP70) and evaluated serodiagnostic applicability of recombinant PvHSP70 (rPvHSP70). In enzyme-linked immunosorbent assays and immunoblot analyses, rPvHSP70 showed high sensitivity (88.8%; 203/228 cases). P. falciparum-infected sera revealed positive reactions (78.8%). The predominant immunoglobulin G (IgG) subclasses were segregated with IgG1 and IgG3.

scholarly journals Comprehensive Molecular Serology of HumanChlamydia trachomatisInfections by Peptide Enzyme-Linked Immunosorbent Assays

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
K. Shamsur Rahman ◽  
Toni Darville ◽  
Ali N. Russell ◽  
Catherine M. O'Connell ◽  
Harold C. Wiesenfeld ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
B Cell ◽  
Cell Epitope ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Superior Performance ◽  
Content Type ◽  
Peptide Antigens ◽  
B Cell Epitope ◽  
Immunosorbent Assays

ABSTRACTSensitive species-specific detection of anti-Chlamydia trachomatisantibodies is compromised by cross-reactivity of theC. trachomatisantigens used in standard microimmunofluorescence (MIF) testing and enzyme-linked immunosorbent assays (ELISAs). Previously, we discovered 48 strongly reactiveC. trachomatis-specific B cell epitope peptides from 21 immunodominant proteins. Here we comprehensively evaluated the 11 top-rankedC. trachomatis-specific peptide antigens from 8 proteins for use inC. trachomatisserology. Sera from 125 women with nucleic acid amplification test (NAAT)-confirmed activeC. trachomatisinfection and from 49 healthy women with a low risk ofC. trachomatisinfection were used as anti-C. trachomatisantibody-positive and -negative sera. Results obtained for detection of IgG1, IgG3, and IgA1 antibodies against the 11C. trachomatispeptide antigens were compared to results from 4 commercial anti-C. trachomatisIgG ELISAs. Using composite reference standards (CRS) of all assays for anti-C. trachomatisantibody status, commercial ELISAs detected antibodies in antibody-positive women with sensitivities of 51.5% to 64.8%. In contrast, a combination of the results of all 11 peptides detected IgG (IgG1 and IgG3) antibodies with 91.8% sensitivity, and a labor-saving combination of the 5 optimal peptides still detected antibodies in antibody-positive women with 86.5% sensitivity (all at 98% specificity). The superior performance of the combined peptide ELISAs was confirmed by area under the receiver operating characteristic curve (ROC-AUC), likelihood ratio, and predictive value analyses. The higher sensitivity of the peptide assays results from using multiple B cell epitopes of severalC. trachomatisimmunodominant proteins, including OmpA, compared to exclusively using the OmpA antigens used in commercial ELISAs. Thus, ELISAs with combined use of synthetic peptide antigens forC. trachomatisantibody detection have the advantage of simultaneous high sensitivity and high specificity.IMPORTANCEFor detection of anti-Chlamydia trachomatisantibodies by serological assays, use of classical whole-organism chlamydial antigens results in high cross-reactivity. These antigens bind mainly antibodies against the major outer membrane protein (OmpA) and bind antibodies against other immunodominant non-OmpA proteins to a lesser extent, resulting in poor assay sensitivity. The specificity ofC. trachomatisserology is also compromised by the high prevalence of cross-reactive anti-C. pneumoniaeantibodies in human populations. We previously identified 48 highly specificC. trachomatisB cell epitope peptide antigens of 21 immunodominant proteins. This study validated peptide antigen-based novel ELISAs that provide highly specific and sensitive detection of anti-C. trachomatisantibodies. Compared to four commercial ELISAs that achieved only poor sensitivities (51.5% to 64.8%), the combined signals of 5 to 11 peptides provided high sensitivity (86.5% to 91.8%) at the same 98% specificity. Thus, by using multiple peptide antigens of immunodominant proteins, we created simple ELISAs with specificity and sensitivity superior to standardC. trachomatisserodiagnosis.

Radio Measurements of Coronal Magnetic Fields

1994 ◽  
Vol 144 ◽  
pp. 21-28 ◽  
Author(s):  
G. B. Gelfreikh
Keyword(s):  
Magnetic Field ◽  
Magnetic Fields ◽  
Solar Corona ◽  
Active Regions ◽  
High Sensitivity ◽  
Coronal Magnetic Field ◽  
Transverse Magnetic ◽  
Radio Sources ◽  
Radio Waves ◽  
Solar Active Regions

AbstractA review of methods of measuring magnetic fields in the solar corona using spectral-polarization observations at microwaves with high spatial resolution is presented. The methods are based on the theory of thermal bremsstrahlung, thermal cyclotron emission, propagation of radio waves in quasi-transverse magnetic field and Faraday rotation of the plane of polarization. The most explicit program of measurements of magnetic fields in the atmosphere of solar active regions has been carried out using radio observations performed on the large reflector radio telescope of the Russian Academy of Sciences — RATAN-600. This proved possible due to good wavelength coverage, multichannel spectrographs observations and high sensitivity to polarization of the instrument. Besides direct measurements of the strength of the magnetic fields in some cases the peculiar parameters of radio sources, such as very steep spectra and high brightness temperatures provide some information on a very complicated local structure of the coronal magnetic field. Of special interest are the results found from combined RATAN-600 and large antennas of aperture synthesis (VLA and WSRT), the latter giving more detailed information on twodimensional structure of radio sources. The bulk of the data obtained allows us to investigate themagnetospheresof the solar active regions as the space in the solar corona where the structures and physical processes are controlled both by the photospheric/underphotospheric currents and surrounding “quiet” corona.

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