scholarly journals Design of 3D Structure Membrane for the Increased Sensitivity in Enzyme Linked Immunosorbent Assay (mELISA)

2019 ◽  
Vol 9 (19) ◽  
pp. 4171 ◽  
Author(s):  
Anna Go ◽  
Young Ju Park ◽  
Min-Ho Lee
Keyword(s):  
Immunosorbent Assay ◽  
Binding Capacity ◽  
Membrane Surface ◽  
3D Structure ◽  
Fold Increase ◽  
Thyroid Stimulating Hormone ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coated Membranes ◽  
Plastic Membrane ◽  
Self Assembled Layer

The Enzyme Linked Immunosorbent Assay (ELISA) technique has been widely used for the identification and quantification of biochemical markers. The typical ELISA requires a number of washing steps to eliminate the unbound proteins which sometimes cause the desorption of protein due to their weak bonding between protein and well plate. In this study, we have developed a meshed type of plastic membrane in order to increase the reliable binding efficiency between proteins and the membrane surface, and to provide easy steps of washing. The use of our developed solid membrane has significantly increased the binding capacity of the biomolecules because this membrane ELISA (mELISA) provides 3D binding surfaces which increases the surface area when compared to the conventional 2D surface well plate. The columns were pretreated to form a self-assembled layer (SAM) on the surface for the stable conjugation of a target antibody. The SAM-coated membranes could be stored for one month without any further deterioration of stability. The measured optical density (O.D.) shows a 1.2-fold increase in IgG antigen (25 μg/mL) from the plastic membrane as compared with the conventional ELISA method. The concentrations of thyroid stimulating hormone were also monitored using the mELISA method and it shows good linearity against the concentrations.

scholarly journals Cystatin Capture Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Human Clonorchiasis and Profile of Captured Antigenic Protein of Clonorchis sinensis

2001 ◽  
Vol 8 (6) ◽  
pp. 1076-1080 ◽  
Author(s):  
Tae Yun Kim ◽  
Shin-Yong Kang ◽  
Sun Hyo Park ◽  
Kom Sukontason ◽  
Kabkaew Sukontason ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Proteinase Inhibitors ◽  
Binding Capacity ◽  
Clonorchis Sinensis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Moderate Degree ◽  
Cysteine Proteinases ◽  
Capture Elisa ◽  
Crude Extracts ◽  
High Degree

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) with crude extracts of adult Clonorchis sinensis has been reported to have a high degree of sensitivity with a moderate degree of specificity for the serodiagnosis of clonorchiasis. The cystatin capture ELISA was investigated for its usefulness for the serodiagnosis of human clonorchiasis. Cystatin bound specifically to cysteine proteinases in crude extracts of adult C. sinensis worms, and its binding capacity was not hindered competitively by the other proteinase inhibitors tested. The cystatin capture ELISA for clonorchiasis showed a higher degree of specificity than the conventional ELISA, which produced some cross-reactivities to sera from patients with cysticercosis, sparganosis, and opisthorchiasis. Immunoglobulin G antibodies to C. sinensis cysteine proteinases were produced in experimental rabbits at week 3, and their levels increased rapidly and remained at a plateau after 8 weeks of infection. Of the proteins from the C. sinensis crude extract captured with cystatin, seven proteins were reactive with the serum from patients with clonorchiasis. The cystatin capture ELISA is indicated to be a sensitive and highly specific immunodiagnostic assay for serodiagnosis of human clonorchiasis.

scholarly journals HUBUNGAN KANDUNGAN IODIUM GARAM RUMAH TANGGA DENGAN STATUS IODIUM WANITA USIA SUBUR DI KABUPATEN WONOGIRI

2020 ◽  
Vol 12 (1) ◽  
pp. 27-38
Author(s):  
Taufiq Hidayat ◽  
Muhamad Arif Musoddaq ◽  
Alfien Susbiantonny ◽  
Prihatin Broto Sukandar
Keyword(s):  
Immunosorbent Assay ◽  
Ammonium Persulfate ◽  
Thyroid Stimulating Hormone ◽  
Enzyme Linked Immunosorbent Assay ◽  
Salt Iodization ◽  
Stimulating Hormone

Latar Belakang. Status iodium merupakan penentu utama gangguan tiroid pada wanita. Wanita usia subur (WUS) merupakan kelompok populasi berisiko tinggi. Gangguan fungsi tiroid pada WUS akan meningkatkan risiko kehamilan dan berdampak negatif terhadap perkembangan anak. Tujuan. Penelitian ini untuk mengetahui hubungan kandungan iodium garam rumah tangga dan status iodium WUS di Kabupaten Wonogiri. Metode. Studi potong lintang dilakukan di Kabupaten Wonogiri. Total 170 responden wanita berusia 15-49 tahun, dilakukan pengukuran terhadap kandungan iodium garam rumah tangga, konsentrasi iodium urine (KIU), dan kadar thyroid stimulating hormone (TSH) serum. Analisis kandungan iodium garam rumah tangga dilakukan dengan metode titrasi iodometrik, KIU dengan metode ammonium persulfate digestion, dan kadar TSH serum dengan metode enzyme-linked immunosorbent assay (ELISA). Hasil. Analisis 170 sampel menunjukkan cakupan garam beriodium rumah tangga yang memadai yaitu 53,5 persen. Median KIU WUS 178,5 μg/L, dengan proporsi nilai KIU < 100 μg/L dan < 50 μg/L masing-masing 17,7 persen dan 7,1 persen. Kandungan iodium garam rumah tangga berhubungan bermakna dengan KIU WUS (p<0,05) dan tidak berhubungan bermakna dengan kadar TSH serum WUS (p>0,05). Kesimpulan. Cakupan garam beriodium tingkat rumah tangga di Kabupaten Wonogiri di bawah sasaran universal salt iodization (USI) (cakupan ≥ 90 persen). Nilai KIU < 100 μg/L dan < 50 μg/L masing-masing kurang dari 50 persen dan 20 persen, menunjukkan asupan iodium memadai. Kandungan iodium garam rumah tangga berpengaruh terhadap tingkat asupan iodium.

scholarly journals Immunoaffinity Column as Cleanup Tool for a Direct Competitive Enzyme-Linked Immunosorbent Assay of Cyclopiazonic Acid in Corn, Peanuts, and Mixed Feed

1998 ◽  
Vol 81 (6) ◽  
pp. 1169-1176 ◽  
Author(s):  
Wanjun Yu ◽  
Joe W Dorner ◽  
Fun S Chu
Keyword(s):  
Immunosorbent Assay ◽  
Binding Capacity ◽  
Cyclopiazonic Acid ◽  
Chromatographic Behavior ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoaffinity Column ◽  
Mixed Feed ◽  
Buffer Solution ◽  
Sample Extract ◽  
Sepharose 4B

Abstract An immunoaffinity column (IAC) for cyclopiazonic acid (CPA) was prepared by coupling a CPA-specific monoclonal antibody to CNBr-activated sepharose 4B. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was used to study the chromatographic behavior of a 0.2 ml_ gel column with a binding capacity of 4 μg CPA/column as well as to evaluate its efficacy as a cleanup tool for analysis of naturally occurring CPA. Sample extract either in buffer solution or in a solution containing up to 35% methanol could be loaded onto the column. After the column is washed with 5 ml_ deionized water and 5 ml_ 50% methanol, CPA could be quantitatively eluted with 2 ml_ 100% methanol. The column could be regenerated at least 10 times by washing with 10 mL equilibrating buffer and then storing in a cold room overnight before reuse. Recoveries of CPA added to corn, peanut, and mixed feed extracts in the range 10-200 ng/g were 88-105,86-100, and 90-110%, respectively. Detection limits were 2.0,4.4, and 4.7 ng/g for corn, mixed feed, and peanuts, respectively. Twenty-two peanut samples naturally contaminated with CPA were subjected to both IAC and solvent partition cleanup followed by dc-ELISA. Although a good correlation between data obtained from lAC-dc-ELISA and from SP-dc-ELISA (r = 0.75, p &lt; 0.0001) was obtained, the slope of the linear regression was low (0.67), indicating loss during solvent partition cleanup. The overall data showed that the combination of IAC and dc- ELISA is an effective method for CPA analysis.

scholarly journals Automation and ISO 17025 Test Validation of a Toxoplasma Gondii Antibodies Enzyme-Linked Immunosorbent Assay

2003 ◽  
Vol 8 (5) ◽  
pp. 42-50
Author(s):  
John T. Y. Wu ◽  
Lester S. Y. Wong ◽  
Eva Y. W. Chow ◽  
Evelyn E. Bowlby
Keyword(s):  
Toxoplasma Gondii ◽  
Immunosorbent Assay ◽  
High Throughput Screening ◽  
Fold Increase ◽  
Threshold Value ◽  
Enzyme Linked Immunosorbent Assay ◽  
Iso 17025 ◽  
Test Configuration ◽  
Porcine Serum ◽  
Panel Testing

The authors automated an enzyme-linked immunosorbent assay to detect porcine serum antibodies to Toxoplasma gondii. Two thousand swine sera can be assayed in two eight-hour shifts using a robotic workstation. The automated-ELISA programming is not complex and the test configuration is flexible. This high-throughput screening (HTS) a-ELISA can achieve a 10-fold increase (100→ 1000 tests) in test capacity over the manual method. The assay has been validated according to the requirements of the ISO/IEC 17025 standard. These include repeatability, reproducibility, and optimal threshold value studies. Other requirements are proficiency panel testing, analyst training, standard operation procedure, and equipment certifications.

α2–plasmin Inhibitor And α2-Macroglobulin-Plasmin Complexes In Plasma. Quantitation By An Enzyme-Linked Immunosorbent Assay

1981 ◽  
Author(s):  
P Harpel
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme System ◽  
Fold Increase ◽  
Fibrinolytic Enzyme ◽  
Enzyme Linked Immunosorbent Assay ◽  
Normal Individuals ◽  
Α2 Macroglobulin ◽  
Low Levels ◽  
Enzyme Complexes ◽  
Plasmin Inhibitor

An enzyme-linked immunosorbent assay has been developed for the quantitation of α2-plasmin inhibitor-plasmin and α2-macroglobulin-plasmin complexes. In this method, the inhibitor-plasmin complex is bound to a surface by an inhibitor specific antibody, then the plasmin bound to the inhibitor is quantified by a second antibody labeled with alkaline phosphatase. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase (UK). The concentration of plasmin added was well below the plasma concentration of α2-plasmin inhibitor or of α2-macroglobulin so that neither inhibitor would be fully saturated with enzyme. Under these conditions, increasing amounts of plasmin generated an increase in both α2-plasmin inhibitor-plasmin and α2-macroglobulin- plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors and the complexes that formed were quantitated by immunoassay. These studies made it possible to quantitate the distribution of plasmin between the two inhibitors in plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with α2-macroglobulin. In contrast, between 19-51% of the plasmin generated in UK-activated plasma was bound to α2-macroglobulin. Thus major changes in the distribution of plasmin were observed depending upon whether plasmin was added to plasma or whether plasminogen was activated endogenously. 23 normal individuals had low levels of α2-plasmin inhibitor-plasmin complexes (4.1±3.5 fmol/ml) whereas six patients with laboratory evidence for DIC demonstrated a 16 to 35-fold increase in the concentration of these complexes. These data indicate that a useful new probe for the study of the fibrinolytic enzyme system has been developed.

scholarly journals Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice

Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 4044 ◽  
Author(s):  
Zhichang Sun ◽  
Xuerou Wang ◽  
Qi Chen ◽  
Yonghuan Yun ◽  
Zongwen Tang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
Solvent Tolerance ◽  
Enzyme Linked Immunosorbent Assay ◽  
One Step

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis

2017 ◽  
Vol 20 (2) ◽  
pp. 355-362
Author(s):  
Ri-E Bu ◽  
Jin-Liang Wang ◽  
Jin-Hua Wu ◽  
Gao-Wa Xilin ◽  
Jin-Long Chen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Immunosorbent Assay ◽  
Streptococcus Agalactiae ◽  
Membrane Surface ◽  
Bovine Mastitis ◽  
Assay Method ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Serum Dilution

Abstract Objective: The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. Methods: The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. Results: The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. Conclusions: The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

scholarly journals A Simpler, More Sensitive Competitive Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Antibody to Malignant Catarrhal Fever Viruses

2001 ◽  
Vol 13 (4) ◽  
pp. 361-364 ◽  
Author(s):  
Hong Li ◽  
Travis C. McGuire ◽  
Uwe U. Müller-Doblies ◽  
Timothy B. Crawford
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Fold Increase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Sensitivity ◽  
Malignant Catarrhal Fever ◽  
Pcr Assay ◽  
Antibody Titers ◽  
Polymerase Chain ◽  
Time Required

An earlier competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed for detection of specific antibody against malignant catarrhal fever (MCF) viruses (MCFV) in ruminants. In this study, the indirect CI-ELISA was improved by conjugating the monoclonal antibody 15-A directly with horseradish peroxidase and by developing a method of producing precoated, dried antigen plates. This new test is referred to as a direct CI-ELISA. The reformatted test yielded a significantly improved sensitivity, and the time required was reduced to about one-sixth of the previous time. Of 37 MCF cases in cattle that were confirmed by histopathology and polymerase chain reaction (PCR) assay, 37 (100%) were positive by the new test, whereas the indirect CI-ELISA detected only 23 (62%). The direct CI-ELISA detected antibody to MCFV in 100% of 48 sheep that had been defined as infected with ovine herpesvirus 2 (OvHV-2) by PCR, whereas the indirect CI-ELISA detected only 41 (85%). Comparison of antibody titers measured by the 2 assays for sera collected from OvHV-2-infected sheep and from cattle, bison, and deer with clinical sheep-associated MCF revealed that the direct CI-ELISA offered a 4-fold increase in analytical sensitivity over the indirect format. The number of seropositive animals detected by the direct CI-ELISA among apparently normal cattle and bison was 2–3 times greater than the number detected by the indirect CI-ELISA, indicating that a significant percentage of normal cattle and bison are subclincally infected with MCFV.

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