scholarly journals Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique

Biosensors ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 524
Author(s):  
Tania García-Maceira ◽  
Fé I. García-Maceira ◽  
José A. González-Reyes ◽  
Luis A. Torres-Sánchez ◽  
Ana Belén Aragón-Gómez ◽  
...  
Keyword(s):  
Mortality Rate ◽  
Recombinant Protein ◽  
Sandwich Elisa ◽  
Covalent Immobilization ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microtiter Plates ◽  
Elisa Technique ◽  
Density Surface ◽  
Capture Antibody

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.

Rapid, sensitive two-site ELISA for detection of cows' milk in goats' or ewes' milk using monoclonal antibodies

1994 ◽  
Vol 61 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Didier Levieux ◽  
Annie Venien
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factors Affecting ◽  
Assay Performance ◽  
Capture Antibody ◽  
Constant Quality ◽  
Species Specific ◽  
Β Lactoglobulin

SummaryA sandwich ELISA (enzyme-linked immunosorbent assay) of the two-site type has been successfully developed for the detection of cows' milk in goats' or ewes' milk. The assay uses two monoclonal antibodies (MAb) raised in mice against cows' β-lactoglobulin (β-lg). These MAb recognize different epitopes of the β-lg, which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. One of the MAb recognizes a species-specific epitope of the bovine β-lg and was adsorbed to a plastic microtitration plate (capture antibody). The second MAb was labelled with peroxidase and used to detect the captured cows' β-lg. Factors affecting assay performance were investigated. The optimized assay is highly specific, reproducible (intra- and inter-assay CV were 8 and 13% respectively) and sensitive: as little as 5 ng β-lg/ml or 1 part cows' milk per 100000 parts goats' or ewes' milk can be detected. The technique is robust, cheap, rapid, reliable and suitable for high sample throughput, semi-automation and screening surveys. The MAb used guarantee the high specificity of the assay and indefinite reagent supply of constant quality once approved by collaborative national or international trials.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

scholarly journals Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

2020 ◽  
Author(s):  
Pinpin Ji ◽  
Jiahong Zhu ◽  
Xiaoxuan Li ◽  
Wenqi Fan ◽  
Qianqian Liu ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Immunosorbent Assay ◽  
Newcastle Disease ◽  
Tertiary Structure ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Permanent Storage ◽  
Capture Antibody ◽  
The Cost

Abstract Background: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents.Results: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. Conclusions: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

scholarly journals Protein G Binding to Enriched Serum Immunoglobulin from Nondomestic Hoofstock Species

2003 ◽  
Vol 15 (3) ◽  
pp. 253-261 ◽  
Author(s):  
Joely A. Kramsky ◽  
Elizabeth J. B. Manning ◽  
Michael T. Collins
Keyword(s):  
Recombinant Protein ◽  
Animal Species ◽  
Positive Control ◽  
Negative Control ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein G ◽  
Animal Populations ◽  
Wide Range ◽  
Binding Curves

Quick and cost-effective serologic assays, such as those based on enzyme-linked immunosorbent assay (ELISA) technology, are useful for screening animal populations for infectious diseases. Recombinant protein G is described as an almost universal ELISA conjugate for the detection of antibodies from a wide range of animal species. However, there is limited data documenting the ability of protein G to bind immunoglobulin (Ig) from many captive and free-ranging nondomestic hoofstock (Order Artiodactyla, e.g., elk, antelope, bison). Protein G binding to Ig from 11 species within this taxonomic order (addax, antelope, bison, bontebok, elk, impala, kudu/nyala, muntjac, oryx, sheep, and white-tailed deer) and 2 control species (bovine and chicken) was assessed. A serum Ig enrichment protocol, using high-performance liquid chromatography (HPLC), was optimized in bovids ( Bos taurus) and then applied to the other study species. Binding assays were performed by adding protein G to microtiter wells coated with titrated dilutions of enriched artiodactyl Ig. Optical densities were measured and binding curves generated. Differences in protein G binding were observed, both within and among species, as well as within taxonomic families. Significant intraspecies binding variation was observed for 7 species tested (antelope, oryx, sheep, muntjac, impala, bontebok, and addax). No statistically significant intraspecies differences in protein G binding were found for Ig from bison, elk, kudu/nyala, white-tailed deer, plus control species (cattle and chicken). Binding of protein G to Ig from impala, muntjac, and elk was statistically different from the positive control (cattle), with muntjac binding curves statistically comparable with the negative control (chicken). For the other 7 species tested, binding curves illustrated the ability of protein G to bind Ig as well as, or better than, the positive control. These findings expand the list of animal species whose Ig is capable of being detected using recombinant protein G, with the caveat that protein G does not bind Ig uniformly in closely related species. It is concluded that recombinant protein G conjugates may serve as useful reagents for serodiagnosis by ELISA in nondomestic hoofstock, although different assay interpretation algorithms and assay protocols may need to be developed on a per species basis for maximum diagnostic effectiveness.

scholarly journals Awareness about ELISA technique among dental students

2020 ◽  
Vol 11 (SPL3) ◽  
pp. 806-810
Author(s):  
Nithyanandham Masilamani ◽  
Dhanraj Ganapathy
Keyword(s):  
College Students ◽  
Indirect Elisa ◽  
Sandwich Elisa ◽  
Educational Programs ◽  
Dental Students ◽  
Competitive Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Sectional ◽  
Direct Elisa ◽  
Elisa Technique

The enzyme-linked immunosorbent assay (ELISA) can be used to recognize proteins, peptides, antibodies as well as hormones. Often known also as an enzyme immunoassay (EIA), ELISA is used as a diagnostic test in the field of biomedicine and science. This study was conducted to determine the understanding of the ELISA technique among dental students. This survey was performed for assessing the awareness about ELISA technique amongst the dental students. This study was a questionnaire oriented, cross-sectional type of survey comprising 100 dental college students in Chennai. A self-designed questionnaire with 10 questions eliciting the knowledge and awareness about applications of ELISA technique among dental college students. Questionnaires were circulated through an online website survey planet. The questions explored the awareness on ELISA technique diagnostic indications, Direct ELISA, Indirect ELISA, Sandwich ELISA, Competitive ELISA and mechanism of ELISA technique. After the responses were received from 100 participants, data were collected and analysed.67% of the respondents were aware of the ELISA technique .52% were aware of direct ELISA technique. 45%% were aware of the indirect ELISA technique. 42% were aware of the sandwich ELISA technique, 38% were aware of the competitive ELISA technique. 35% were aware of the mechanism of the ELISA technique. The awareness about the ELISA technique in diagnostic medical applications was less among dental students. Increased awareness and educational programs should be initiated to spread knowledge about the ELISA technique among all students and clinicians.

scholarly journals Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

2020 ◽  
Author(s):  
Pinpin Ji ◽  
Jiahong Zhu ◽  
Xiaoxuan Li ◽  
Wenqi Fan ◽  
Qianqian Liu ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Immunosorbent Assay ◽  
Newcastle Disease ◽  
Tertiary Structure ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Permanent Storage ◽  
Capture Antibody ◽  
The Cost

Abstract Background Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. Results A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 2 2 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. Conclusions In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

D-Dimer Determination to Exclude Pulmonary Embolism: a Two-Step Approach Using Latex Assay as a Screening Tool

1994 ◽  
Vol 72 (01) ◽  
pp. 089-091 ◽  
Author(s):  
P de Moerloose ◽  
Ph Minazio ◽  
G Reber ◽  
A Perrier ◽  
H Bounameaux
Keyword(s):  
Pulmonary Embolism ◽  
Screening Tool ◽  
Screening Tests ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Elisa Technique ◽  
Diagnostic Step ◽  
D Dimer ◽  
Rule Out ◽  
Latex Test

SummaryD-dimer (DD), when measured by a quantitative enzyme-linked immunosorbent assay (ELISA), is a valuable test to exclude venous thromboembolism (VTE). However, DD ELISA technique is not appropriate for emergency use and the available agglutination latex assays are not sensitive enough to be used as an alternative to rule out the diagnosis of VTE. Latex assays could still be used as screening tests. We tested this hypothesis by comparing DD levels measured by ELISA and latex assays in 334 patients suspected of pulmonary embolism. All but one patient with a positive (DD ≥500 ng/ml) latex assay had DD levels higher than 500 ng/ml with the ELISA assay. Accordingly, ELISA technique could be restricted to patients with a negative result in latex assay. This two-step approach would have spared about 50% of ELISA in our cohort. In conclusion, our data indicate that a latex test can be used as a first diagnostic step to rule out pulmonary embolism provided a negative result is confirmed by ELISA and the performance of the latex assay used has been assessed properly.

scholarly journals Use tumor suppressor genes as biomarkers for diagnosis of non-small cell lung cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chuantao Zhang ◽  
Man Jiang ◽  
Na Zhou ◽  
Helei Hou ◽  
Tianjun Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Mortality Rate ◽  
Tumor Suppressor ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Network Effects ◽  
Small Cell ◽  
The Other ◽  
Small Cell Lung ◽  
High Efficient

AbstractLung cancer is the leading cause of death worldwide. Especially, non-small cell lung cancer (NSCLC) has higher mortality rate than the other cancers. The high mortality rate is partially due to lack of efficient biomarkers for detection, diagnosis and prognosis. To find high efficient biomarkers for clinical diagnosis of NSCLC patients, we used gene differential expression and gene ontology (GO) to define a set of 26 tumor suppressor (TS) genes. The 26 TS genes were down-expressed in tumor samples in cohorts GSE18842, GSE40419, and GSE21933 and at stages 2 and 3 in GSE19804, and 15 TS genes were significantly down-expressed in tumor samples of stage 1. We used S-scores and N-scores defined in correlation networks to evaluate positive and negative influences of these 26 TS genes on expression of other functional genes in the four independent cohorts and found that SASH1, STARD13, CBFA2T3 and RECK were strong TS genes that have strong accordant/discordant effects and network effects globally impacting the other genes in expression and hence can be used as specific biomarkers for diagnosis of NSCLC cancer. Weak TS genes EXT1, PTCH1, KLK10 and APC that are associated with a few genes in function or work in a special pathway were not detected to be differentially expressed and had very small S-scores and N-scores in all collected datasets and can be used as sensitive biomarkers for diagnosis of early cancer. Our findings are well consistent with functions of these TS genes. GSEA analysis found that these 26 TS genes as a gene set had high enrichment scores at stages 1, 2, 3 and all stages.

Sign in / Sign up

Export Citation Format

Share Document