Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay

1997 ◽  
Vol 78 (04) ◽  
pp. 1262-1267 ◽  
Author(s):  
Claudia C Folman ◽  
Albert E G K von dem Borne ◽  
Irma H J A M Rensink ◽  
Winald Gerritsen ◽  
C Ellen van der Schoot ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Platelet Transfusion ◽  
Large Scale ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reliable Tool

SummaryIn this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

scholarly journals Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

2006 ◽  
Vol 13 (3) ◽  
pp. 420-422 ◽  
Author(s):  
S. E. Burastero ◽  
C. Paolucci ◽  
D. Breda ◽  
G. Monasterolo ◽  
R. E. Rossi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Positive Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Positive Results

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

Generation of Human Monoclonal Anti-idiotypic Antibodies with Specificity to the Murine Monoclonal Anti-CA 125 Antibody B43.13

1996 ◽  
Vol 11 (4) ◽  
pp. 211-215
Author(s):  
J.B. Oltrogge ◽  
B. Donnerstag ◽  
R.P. Baum ◽  
A.A. Noujaim ◽  
L. Träger
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Peripheral Blood Lymphocytes ◽  
Cross Reactivity ◽  
Tumor Burden ◽  
Ca 125 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Monoclonal Antibodies ◽  
Ebv Transformation

Two human monoclonal antibodies, HID-7E7 and ROB-6F2, were produced by EBV transformation of peripheral blood lymphocytes (PBL). PBL were obtained from a patient with ovarian cancer who had been exposed several times to a Tc-99m labeled murine monoclonal anti-CA 125 antibody (B43.13, Biomira, Edmonton) for immunoscintigraphy. The HID-7E7 and ROB-6F2 producing B-cells were cloned with a limiting dilution technique and have shown stable immunoglobulin secretion within a period of three years. The human monoclonal antibodies HID-7E7 and ROB-6F2 are of the IgG isotype, and bind with significant affinity to the murine monoclonal antibody B43.13, which was used for immunoscintigraphy. Binding affinity of ROB-6F2 to other murine antibodies could not be detected. Cross reactivity of HID-7E7 to a murine anti-CEA monoclonal antibody was observed. In order to verify the anti-idiotypic character of the generated human antibodies, the ability of HID-7E7 and ROB-6F2, respectively, to inhibit the formation of the CA125/B43.13 complex is demonstrated via an enzyme-linked immunosorbent assay. These human anti-idiotypic antibodies are possible candidates for immunotherapy of ovarian cancer in patients with a small tumor burden following surgery and/or chemotherapy.

A Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Determination of Homoharringtonine

Planta Medica ◽  
2018 ◽  
Vol 84 (14) ◽  
pp. 1038-1044 ◽  
Author(s):  
Benyakan Pongkitwitoon ◽  
Seiichi Sakamoto ◽  
Rika Nagamitsu ◽  
Waraporn Putalun ◽  
Hiroyuki Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Myeloid Leukemia ◽  
High Performance ◽  
Sodium Periodate ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Study Support ◽  
Mediated Oxidation

AbstractHomoharringtonine (HHT), also known as omacetaxine, is a natural compound found in the genus Cephalotaxus and is a promising pharmaceutical drug used for the treatment of chronic or accelerated phase chronic myeloid leukemia. As a tool for the quantitative determination of HHT, a specific monoclonal antibody against HHT (MAb 6A1) was generated by conjugates prepared via sodium periodate-mediated oxidation. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) using MAb 6A1 was found to be highly specific and sensitive with a limit of detection for HHT of 48.8 ng/mL. Validation assays to evaluate precision and accuracy of the method were conducted by the use of intra- and inter-assay analysis, recovery test, and comparison analysis between the amounts of HHT determined by ELISA and high-performance liquid chromatography. These results revealed that the established icELISA using MAb 6A1 is specific, sensitive, and reliable enough to be applied to the qualitative analysis for HHT. Furthermore, the results of this study support the usefulness of sodium periodate as a reagent for the conjugation between Cephalotaxus alkaloids and proteins for producing specific antibodies.

Detection of Abrin in Food Using Enzyme-Linked Immunosorbent Assay and Electrochemiluminescence Technologies

2008 ◽  
Vol 71 (9) ◽  
pp. 1868-1874 ◽  
Author(s):  
ERIC A. E. GARBER ◽  
JENNIFER L. WALKER ◽  
THOMAS W. O'BRIEN
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Dairy Products ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ribosome Inactivating Protein ◽  
Limits Of Detection ◽  
Abrus Precatorius ◽  
A Priority

Abrin is a toxic ribosome-inactivating protein present in beans of Abrus precatorius, also known as rosary peas. The possibility that abrin could be used to adulterate food has made the development of assays for the detection of abrin a priority. Rabbit-derived polyclonal antibodies and mouse monoclonal antibodies were prepared against a mixture of abrin isozymes. The specificity and cross-reactivity of the antibodies were evaluated against a challenge library of 40 grains, nuts, legumes, and foods. An enzyme-linked immunosorbent assay (ELISA) and an electrochemiluminescence (ECL)–based assay were assembled and optimized. Polyclonal (capture) and polyclonal (detection) ELISAs, polyclonal and monoclonal ELISAs, and polyclonal and monoclonal ECL assays had limits of detection (LODs) of 0.1 to 0.5 ng/ml for abrin in buffer. The LOD for abrin dissolved into juices, dairy products, soda, chocolate drink, and condiments and analyzed with the ECL assay ranged from 0.1 to 0.5 ng/ml in the analytical sample. In contrast, the LODs for the ELISAs ranged from 0.5 to 10 ng/ml in the analytical sample.

scholarly journals Development of a New Monoclonal Antibody against Brevetoxins in Oyster Samples Based on the Indirect Competitive Enzyme-Linked Immunosorbent Assay

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2398
Author(s):  
Xiya Zhang ◽  
Mingyue Ding ◽  
Chensi Zhang ◽  
Yexuan Mao ◽  
Youyi Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Broad Spectrum ◽  
Limit Of Detection ◽  
Keyhole Limpet Hemocyanin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Neurotoxic Effects ◽  
Rapid Monitoring ◽  
Ic50 Values ◽  
Ester Method

The consumption of shellfish contaminated with brevetoxins, a family of ladder-frame polyether toxins formed during blooms of the marine dinoflagellate Karenia brevis, can cause neurotoxic poisoning, leading to gastroenteritis and neurotoxic effects. To rapidly monitor brevetoxin levels in oysters, we generated a broad-spectrum antibody against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3) and developed a rapid indirect competitive enzyme-linked immunosorbent assay (icELISA). PbTx-2 was reacted with carboxymethoxylamine hemihydrochloride (CMO) to generate a PbTx-2-CMO hapten and reacted with succinic anhydride (HS) to generate the PbTx-2-HS hapten. These haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen reagents, respectively, using the active ester method. After immunization and cell fusion, a broad-spectrum monoclonal antibody (mAb) termed mAb 1D3 was prepared. The 50% inhibitory concentration (IC50) values of the icELISA for PbTx-2, PbTx-1, and PbTx-3 were 60.71, 52.61, and 51.83 μg/kg, respectively. Based on the broad-spectrum mAb 1D3, an icELISA was developed to determine brevetoxin levels. Using this approach, the limit of detection (LOD) for brevetoxin was 124.22 μg/kg and recoveries ranged between 89.08% and 115.00%, with a coefficient of variation below 4.25% in oyster samples. These results suggest that our icELISA is a useful tool for the rapid monitoring of brevetoxins in oyster samples.

scholarly journals Single-Laboratory Validation of the Biosense Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA) for Determination of Domoic Acid Toxins in Shellfish

2007 ◽  
Vol 90 (4) ◽  
pp. 1000-1010 ◽  
Author(s):  
Hans Kleivdal ◽  
Sven-Inge Kristiansen ◽  
Mona V Nilsen ◽  
Lyn Briggs
Keyword(s):  
Immunosorbent Assay ◽  
Domoic Acid ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Method Comparison ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Shellfish Poisoning ◽  
Relative Standard

Abstract Method validation was conducted for an enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins, known to give amnesic shellfish poisoning (ASP) symptoms, in shellfish. The calibration curve range of the assay is approximately 10260 pg/mL, with a dynamic working range for DA toxins in shellfish from 0.01 to at least 250 mg/kg. The ASP ELISA showed no significant cross-reactivity to structural analogs, and proved to be robust to deliberate alterations of the optimal running conditions. The shellfish matrix effects observed with mussels, oysters, and scallops were eliminated by diluting shellfish extracts 1:200 prior to analysis, leading to a limit of detection at 0.003 mg/kg. Thirteen blank shellfish homogenates were spiked with certified mussel material containing DA to levels in the range of 0.125 mg DA/kg, and analyzed in quadruplicate on 3 different days. The relative standard deviation (RSD) under intra-assay repeatability conditions ranged from 6.5 to 13.1%, and under interassay repeatability conditions the RSD ranged from 5.7 to 13.4%, with a mean value of 9.3%. The recoveries ranged from 85.5 to 106.6%, with a mean recovery of 102.2%. A method comparison was conducted with liquid chromatography with ultraviolet detection, using naturally contaminated scallop samples (n = 27) with DA levels at 0244 mg/kg. The overall correlation coefficient was 0.960 and the slope of the regression was 1.218, indicating a good agreement between the methods.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay Based on the Multiepitope Peptide for the Synchronous Detection of Staphylococcal Enterotoxin A and G Proteins in Milk

2015 ◽  
Vol 78 (2) ◽  
pp. 362-369 ◽  
Author(s):  
MINGYAN LIANG ◽  
TINGTING ZHANG ◽  
XUELAN LIU ◽  
YANAN FAN ◽  
SHENGLIN XIA ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Signal To Noise Ratio ◽  
Food Poisoning ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synchronous Detection ◽  
Standard Curve ◽  
Stability And Accuracy

Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milk-acquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra-and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk.

scholarly journals Quantitation of Indian Krait (Bungarus caeruleus) Venom in Human Specimens of Forensic Origin by Indirect Competitive Inhibition Enzyme-Linked Immunosorbent Assay

2006 ◽  
Vol 89 (5) ◽  
pp. 1360-1366 ◽  
Author(s):  
Ganneru Brunda ◽  
Beedu Sashidhar Rao ◽  
Rajendra Kumar Sarin
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Limit Of Detection ◽  
Linear Regression Analysis ◽  
Cross Reactivity ◽  
Assay System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Calibration Plot ◽  
Rat Tissues ◽  
Capture Assay

Abstract An indirect competitive inhibition enzyme-linked immunosorbent assay was reported to detect krait venom in human specimens of forensic origin. Polyclonal anti-krait venom antibodies were characterized by indirect antibody capture assay. The calibration plot was constructed based on linear regression analysis (y = 72.85 12.29x, r2 = 0.98) with concentration ranges from 0.013 to 1000 ng/well of krait venom with a limit of detection of 0.2 ng/mL in the assay system. The IC50 (inhibitory concentration at 50% displacement) value of krait venom was observed to be 70 ng. Spiking studies indicated recoveries of 95100% and 94100% when various concentrations of krait venom were spiked to rat tissues (skin, liver, and kidneys) and pooled human serum, respectively. Polyclonal anti-krait venom antibodies showed no cross-reactivity with cobra and viper venom when tested in the assay system. The coefficient of variation of various concentrations of working range in intra-assay (n = 6) was <5%, whereas in interassay (n = 6) it was observed to be 7%. Further, the method was used to quantitate krait venom in human autopsy and biopsy specimens of forensic origin. Concentration of krait venom was found to be in the range of 4172 ng/100 mg skin or skin scrapings and 64378 ng/mL blood or serum. The methodology may find application in forensic laboratories to assess the cause of death in the cases of krait-bite victims.

Use of a Monoclonal Antibody and Two Enzyme-Linked Immunosorbent Assay Formats for Detection and Quantification of the Substitution of Caprine Milk for Ovine Milk

1997 ◽  
Vol 60 (8) ◽  
pp. 973-977 ◽  
Author(s):  
ANA I. HAZA ◽  
PALOMA MORALES ◽  
ROSARIO MARTÍIN ◽  
TERESA GARCÍIA ◽  
GONZALO ANGUITA ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Goat’S Milk ◽  
Elisa Format ◽  
Ewe's Milk ◽  
Goat's Milk ◽  
Detection And Quantification

A stable hybridoma cell line (B2B) has been produced that secretes a monoclonal antibody (MAb) specific for goat's milk αS2-casein. The MAb B2B was used in two enzyme-linked immunosorbent assay (ELISA) formats for the detection and quantification of the presence of goat's milk in ewe's milk. In the indirect ELISA format the limit of detection was 0.5 to 15% (vol/vol) substitution of goat's milk for ewe's milk. Afterwards, a competitive indirect ELISA was successfully developed for the detection of 0.25 to 15% (vol/vol) of goat's milk in ewe's milk. This competitive indirect ELISA is a very sensitive assay; it can be performed in less than 5 h and is not influenced by the heat treatment of milk.

scholarly journals Rapid Detection of H5N1 Subtype Influenza Viruses by Antigen Capture Enzyme-Linked Immunosorbent Assay Using H5- and N1-Specific Monoclonal Antibodies

2009 ◽  
Vol 16 (5) ◽  
pp. 726-732 ◽  
Author(s):  
Hui-Ting Ho ◽  
Hong-Liang Qian ◽  
Fang He ◽  
Tao Meng ◽  
Milene Szyporta ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Rapid Detection ◽  
Conformational Epitope ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Influenza B ◽  
H5n1 Subtype ◽  
Antigen Capture

ABSTRACT Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype has caused devastating damage to poultry flocks and sporadic human H5N1 infections. There is concern that this virus subtype may gain transmissibility and become pandemic. Rapid diagnosis and surveillance for H5N1 subtype viruses are critical for the control of H5N1 infection. In this study, we report a robust antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) based on H5- and N1-specific monoclonal antibodies (MAbs) for the rapid detection of H5N1 subtype viruses. The H5 hemagglutinin (HA)-specific MAb (2D9) targets a conformational epitope which recognized multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8. The N1 neuraminidase (NA)-specific MAb (8H12) recognized a linear epitope comprising the sequence AELPF. This epitope was 99% conserved in the NA of 708 analyzed H5N1 viruses, while the epitope was absent in NAs of subtypes N2 through N9. The specificity of the AC-ELISA was examined by using 41 H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. The estimated detection limit was 1 to 2 HA titers. It is concluded that this H5N1 AC-ELISA can simultaneously detect H5 and N1 subtype antigens, eliminating the need for secondary testing for the NA subtype. Implementation of this assay in ELISA-like formats suitable for field use, such as dot ELISA, immunofiltration, or electrochemical biosensor technologies, would provide dual on-site detection of H5 and N1 in clinical or environmental specimens.

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