Enzyme immunoassay for intact human insulin in serum or plasma

1993 ◽  
Vol 39 (4) ◽  
pp. 578-582 ◽  
Author(s):  
L Andersen ◽  
B Dinesen ◽  
P N Jørgensen ◽  
F Poulsen ◽  
M E Røder
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Human Serum ◽  
Clinical Importance ◽  
Porcine Insulin ◽  
Microtiter Plates ◽  
Relative Response ◽  
Normal Individuals ◽  
Insulin Dependent ◽  
Murine Monoclonal Antibodies

Abstract We describe an enzyme-linked two-site immunoassay for quantitation of intact insulin in human serum and plasma. The method uses two murine monoclonal antibodies that bind to two different epitopes on the insulin molecule. The immunoassay is specific. Human proinsulin is not bound by the antibodies, and the binding of partially processed proinsulin intermediates is believed to be of minor clinical importance. The relative response of human, bovine, and porcine insulin is 1, 1, and 3, respectively. The assay is sensitive (detection limit 5 pmol/L), accurate (101% recovery with 50 pmol/L insulin added to samples, 95% with 100 pmol/L, and 89% with 300 pmol/L), and fast (results within 3 h), and has a high analytical capacity (done in microtiter plates). The working assay range selected is 5-600 pmol/L, corresponding to a clinically useful range. Because of its specificity, this two-site immunoassay gives results that are lower than those obtained by using a competitive radioimmunoassay, both in normal individuals and in non-insulin-dependent diabetics.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

1989 ◽  
Vol 35 (10) ◽  
pp. 2087-2092 ◽  
Author(s):  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Solid Phase ◽  
Sample Volume ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Peroxidase Conjugate

Abstract In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies

1993 ◽  
Vol 39 (10) ◽  
pp. 2146-2150 ◽  
Author(s):  
L L Kjems ◽  
M E Røder ◽  
B Dinesen ◽  
S G Hartling ◽  
P N Jørgensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Human Serum ◽  
Enzyme Immunoassay ◽  
Healthy Subjects ◽  
Sandwich Elisa ◽  
C Peptide ◽  
Peptide Antibody ◽  
Analytical Recovery ◽  
Highly Sensitive

Abstract A highly sensitive two-site sandwich ELISA measuring total proinsulin immunoreactive material in serum or plasma was developed. The assay was based on two monoclonal antibodies, an anti-C-peptide antibody bound to a microtest plate and a biotin-labeled anti-insulin antibody. The detection limit (3 SD above zero value) in buffer was 0.05 pmol/L, corresponding to 0.25 pmol/L in human serum (diluted 1:5). The linear calibrator range was 0.05-20 pmol/L. Interassay CVs were 4.7% at a median (range) of 2.3 pmol/L (1.4-2.8 pmol/L, n = 8), 6.7% at 5.1 pmol/L (3.3-8.0 pmol/L, n = 8), and 8.7% at 10.0 pmol/L (8-12 pmol/L, n = 10). Mean analytical recovery of added human proinsulin (hPI) (2, 5, and 10 pmol/L) to serum was 84% (range 68-128%, n = 9). Human insulin and human C-peptide did not cross-react at 5000 and 10,000 pmol/L, respectively. The four major proinsulin conversion intermediates reacted 65-99%: split(32-33)hPI 74%, des-(31,32)hPI 65%, split(65-66)hPI 78%, and des(64,65)hPI 99%. All serum values from 38 fasting healthy subjects were above the detection limit: median (range) 4.0 (2.1-12.6) pmol/L.

scholarly journals In vitro cytodestruction of human leukemic cells using murine monoclonal antibodies and human complement

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1120-1124 ◽  
Author(s):  
DE Stepan ◽  
RM Bartholomew ◽  
TW LeBien
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Classical Pathway ◽  
Normal Human ◽  
Human Leukemic Cells ◽  
Murine Monoclonal Antibodies

Abstract We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20- fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.

scholarly journals In vitro cytodestruction of human leukemic cells using murine monoclonal antibodies and human complement

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1120-1124
Author(s):  
DE Stepan ◽  
RM Bartholomew ◽  
TW LeBien
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Classical Pathway ◽  
Normal Human ◽  
Human Leukemic Cells ◽  
Murine Monoclonal Antibodies

We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20- fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.

Measurement of Urokinase-Type Plasminogen Activator (u-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies

1986 ◽  
Vol 56 (03) ◽  
pp. 411-414 ◽  
Author(s):  
V Darras ◽  
M Thienpont ◽  
D C Stump ◽  
D Collen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Microtiter Plates ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Murine Monoclonal Antibodies

SummaryAn enzyme-linked immunosorbent assay (ELISA) for the measurement of urokinase-type plasminogen activator (u-PA) was developed. Three murine monoclonal antibodies to single chain urokinase-type plasminogen activator (scu-PA) were isolated and shown to react with non-overlapping epitopes in scu-PA. Two of the three antibodies were coated onto microtiter plates and bound u-PA was quantitated with the third antibody conjugated to horseradish peroxidase. The assay was equally sensitive to Mr 54,000 u-PA in the single chain or two-chain form but did not respond to Mr 33,000 urokinase. The lower limit of sensitivity of the assay was 0.1 ng/ml in buffer and 1 ng/ml in plasma. Coefficients of variation of the assay at physiological levels of u-PA were 6.5 percent within assays and 13 percent between assays. The level of u-PA in normal resting plasma was 1.9 ± 0.66 ng/ml (mean ± SD, n = 54). The assay can be performed within one working day and provides an efficient, reproducible, and stable means for the measurement of u-PA in biological fluids. As such it may facilitate physiological and pharmacological studies of urokinase-type plasminogen activators in man.

The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

1992 ◽  
Vol 68 (04) ◽  
pp. 464-469 ◽  
Author(s):  
Y Fujimura ◽  
S Miyata ◽  
S Nishida ◽  
S Miura ◽  
M Kaneda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Normal Individuals ◽  
Rag 1 ◽  
The One ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

Monoclonal antibodies defined abnormalities of T-lymphocytes in type I (insulin-dependent) diabetes

Diabetes ◽  
1983 ◽  
Vol 32 (1) ◽  
pp. 91-94 ◽  
Author(s):  
P. Pozzilli ◽  
O. Zuccarini ◽  
M. Iavicoli ◽  
D. Andreani ◽  
M. Sensi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
T Lymphocytes ◽  
Insulin Dependent Diabetes ◽  
Type I ◽  
Insulin Dependent

scholarly journals Screening and development of monoclonal antibodies for identification of ferret T follicular helper cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenbo Jiang ◽  
Julius Wong ◽  
Hyon-Xhi Tan ◽  
Hannah G. Kelly ◽  
Paul G. Whitney ◽  
...  
Keyword(s):  
Animal Model ◽  
Monoclonal Antibodies ◽  
Lymph Node ◽  
Cross Reactivity ◽  
Tfh Cells ◽  
Follicular Helper ◽  
Lymph Node Cells ◽  
Human Viruses ◽  
Humoral Responses ◽  
Murine Monoclonal Antibodies

AbstractThe ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre‐clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret‐reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.

A receptor-antibody sandwich assay for teicoplanin.

1987 ◽  
Vol 33 (9) ◽  
pp. 1615-1618 ◽  
Author(s):  
A Corti ◽  
L Cavenaghi ◽  
E Giani ◽  
G Cassani
Keyword(s):  
Detection Limit ◽  
Dose Response ◽  
Response Curve ◽  
Synthetic Analog ◽  
Sandwich Complexes ◽  
Sandwich Assay ◽  
Receptor Antibody ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Biological Target

Abstract We have developed a new method for quantifying teicoplanin in complex matrixes, a receptor-antibody sandwich assay (RASA). The method is based on bioselective adsorption of teicoplanin onto microtiter plates coated with albumin-epsilon-aminocaproyl-D-alanyl-D-alanine, a synthetic analog of its biological target, and reaction with anti-teicoplanin antibodies. The sandwich complexes are detected by incubation with peroxidase-labeled goat antibodies to rabbit IgGs and chromogenic reaction with o-phenylenediamine. The dose-response curve was linear for teicoplanin concentrations in the range from 0 to 0.15 mg/L. We used the assay to measure teicoplanin concentrations in various biological matrixes. Analytical recovery from serum was 99.5%, the interassay CV was 5.1%, and the detection limit was 30 micrograms/L (P less than 0.01). Mean analytical recoveries from other biological specimens were 98% from ascitic fluid, 100% from pleuric liquid, 104.8% from prostate homogenate, and 98.5% from bronchial expectorate.

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