Characterization of a monoclonal antibody (HB-22) and development of an ELISA for human apolipoprotein A-I

1995 ◽  
Vol 41 (8) ◽  
pp. 1150-1158 ◽  
Author(s):  
T C Rothwell ◽  
V S Kamanna ◽  
F Y Jin ◽  
E Koren ◽  
T Foley ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
High Density Lipoproteins ◽  
Tween 20 ◽  
Major Protein ◽  
Binding Studies ◽  
Very Low Density Lipoproteins ◽  
Human Apolipoprotein

Abstract We describe the production and characterization of a high-affinity monoclonal antibody, HB-22, for apolipoprotein (apo) A-I, a major protein of human high-density lipoproteins (HDL). Including Tween 20 in the reaction mixture increased the binding capacity of HB-22 to apo A-I. HB-22 showed monospecific reactivity with HDL or apo A-I, displaying no cross-reactivity with apo A-II, intermediate-, low-, or very-low-density lipoproteins. Immunoaffinity columns with HB-22 (in the absence of Tween 20) showed an immunosorbent capacity of 80 micrograms of apo A-I per milligram of antibody. The immunosorbent capacity of HB-22 for apo A-I was similar in plasma samples from normolipidemic, hypercholesterolemic, or hypertriglyceridemic patients. Comparative binding studies demonstrated that compared with other available monoclonal apo A-I antibodies, HB-22 had the greatest apparent affinity for binding to HDL. A competitive ELISA developed by utilizing HB-22 could detect as little as 20 ng of apo A-I in the reaction mixture. The intra- and interassay CVs of the ELISA were 5.4% and 9.5%, respectively.

Characterization of G12 Sandwich ELISA, a Next-Generation Immunoassay for Gluten Toxicity

2012 ◽  
Vol 95 (2) ◽  
pp. 372-376 ◽  
Author(s):  
Elisabeth Halbmayr-Jech ◽  
Elisabeth Hammer ◽  
Richard Fielder ◽  
Jacqueline Coutts ◽  
Adrian Rogers ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Celiac Disease ◽  
Working Group ◽  
Sandwich Elisa ◽  
Extraction Methods ◽  
Cross Reactivity ◽  
Next Generation ◽  
Preliminary Results ◽  
Sample Extraction

Abstract In this work, a monoclonal antibody called G12, raised against the most immunotoxic peptide to celiac disease patients, was used to develop a sandwich ELISA. Preliminary results on cross-reactivities, recoveries, and extraction methods of the new assay are presented. The assay calibration was performed using material from the Prolamin Working Group. The antibody's specificity was determined by cross-reactivity studies on different grains, nuts, oils, and starches. Recovery of the assay was determined by spiking experiments on common food matrixes, as well as on problematic matrixes. Furthermore, sample extraction methods using ethanol, cocktail solution, and a proprietary buffer have been compared.

Characterization of the growth hormone-binding protein of human serum using a panel of monoclonal antibodies

1989 ◽  
Vol 123 (2) ◽  
pp. 327-332 ◽  
Author(s):  
R. Barnard ◽  
P. Quirk ◽  
M. J. Waters
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Binding Capacity ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Rabbit Liver ◽  
Rabbit Serum ◽  
Gh Receptor ◽  
Hormone Binding Protein

ABSTRACT A panel of monoclonal antibodies (MAbs) reactive with distinct epitopes on the rabbit liver GH receptor and rabbit serum GH-binding protein (GHBP) were tested for cross-reactivity with the GHBP from human serum. Four of seven MAbs reacted with the human serum GHBP. Immunoprecipitation of the human binding protein enabled hormonal specificity identical to that previously reported for human GH receptors to be demonstrated. Scatchard analyses of 125I-labelled human GH binding to the serum GHBP were carried out with correction made for endogenous human GH which was measured by radioimmunoassay of each serum sample. This approach yielded the first reliable estimates of the affinity and capacity of the human GHBP. The binding capacity (mean ± s.e.m.) of female sera (804±126 pmol/l; n= 6) was greater than that of male sera (505 ± 36 pmol/l; n=9; P < 0·02). The affinity of the GHBP was 0·91 ±0·10 litres/nmol (n= 15). The presence of multiple epitopes common to the human serum GHBP and the rabbit liver GH receptor is consistent with identity between the extracellular domains of the human GHBP and the human GH receptor, as is the case for the rabbit GHBP and GH receptor. Journal of Endocrinology (1989) 123, 327–332

Human apolipoprotein A-I: structure determination and analysis of unusual diffraction characteristics

1999 ◽  
Vol 55 (12) ◽  
pp. 2013-2021 ◽  
Author(s):  
David W. Borhani ◽  
Jeffrey A. Engler ◽  
Christie G. Brouillette
Keyword(s):  
Structure Determination ◽  
Density Lipoprotein ◽  
High Density ◽  
High Density Lipoproteins ◽  
Major Protein ◽  
Data Set ◽  
Cholesterol Acyl Transferase ◽  
Cell Parameters ◽  
Human Apolipoprotein ◽  
Apolipoprotein A

The crystallization and structure determination of recombinant human apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, is described. The fragment crystallized, residues 44–243 of native apo A-I [apo Δ(1–43)A-I], is very similar to intact native apo A-I in its ability to bind lipid, to be incorporated into high-density lipoproteins and to activate lecithin–cholesterol acyl transferase. Apo Δ(1–43)A-I crystallizes from 1.0–1.4 M sodium citrate pH 6.5–7.5 in space group P212121, with unit-cell parameters a = 97.47, b = 113.87, c = 196.19 Å (crystal form I). The crystals exhibit unusual diffraction intensity spikes and axial extinctions that are discussed in the context of the 4 Å crystal structure. When flash-cooled to 100 K, the crystals diffract synchrotron radiation to 3 Å resolution. Radiation sensitivity and crystal-to-crystal variation have hindered the assembly of a complete 3 Å data set.

scholarly journals Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced byBacillus cereus

1999 ◽  
Vol 65 (10) ◽  
pp. 4470-4474 ◽  
Author(s):  
R. Dietrich ◽  
C. Fella ◽  
S. Strich ◽  
E. Märtlbauer
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Bacillus Cereus ◽  
Cell Lines ◽  
Cross Reactivity ◽  
Uncharacterized Protein ◽  
Hybridoma Cell Lines ◽  
Different Strains ◽  
Hemolysin Bl

ABSTRACT A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereuswere established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L2 component, and antibody 1C2 was specific for the L1 protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains ofB. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity withB. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.

Isolation and preliminary characterization of a monoclonal antibody that interacts preferentially with the liver isoenzyme of human alkaline phosphatase.

1985 ◽  
Vol 31 (3) ◽  
pp. 381-385 ◽  
Author(s):  
G M Lawson ◽  
J A Katzmann ◽  
T K Kimlinger ◽  
J F O'Brien
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Human Liver ◽  
Clinical Laboratory ◽  
Bone Alkaline Phosphatase ◽  
Cross Reactivity ◽  
Immunological Methods ◽  
Enzymic Assay ◽  
Murine Monoclonal Antibodies

Abstract We have prepared murine monoclonal antibodies against isolated human bone alkaline phosphatase (ALP, EC 3.1.3.1). Hybridoma supernates were separately screened for reactivity against both human liver and bone ALP. Although most antibody-positive hybrids showed similar reactivity against both isoenzymes, one hybridoma produced an antibody that interacted preferentially with liver ALP. This antibody was purified and used to establish an immunoassay to differentiate liver ALP from bone ALP. When equal activities of the two isoenzymes (as determined by a conventional enzymic assay) were measured by the immunoassay, a fivefold greater response was obtained with liver than with bone ALP. The immunoassay can be used to measure the proportions of the bone and liver isoenzymes in mixtures of them. Cross reactivity with human placental and intestinal ALP is less than 3% relative to liver ALP. These findings support the feasibility of developing immunological methods to differentiate these isoenzymes in the clinical laboratory.

scholarly journals The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout

1978 ◽  
Vol 173 (2) ◽  
pp. 507-520 ◽  
Author(s):  
E R Skinner ◽  
A Rogie
Keyword(s):  
Apolipoprotein B ◽  
Evolutionary History ◽  
Unsaturated Fatty Acids ◽  
Salmo Gairdneri ◽  
High Density Lipoproteins ◽  
Antigenic Determinants ◽  
Low Density ◽  
Lipoprotein Class ◽  
Human Apolipoprotein

1. VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins were isolated from the serum of trout (Salmo gairdneri Richardson). 2. Each lipoprotein class resembled that of the human in immunological reactivity, electrophoretic behaviour and appearance in the electron microscope. Trout LD lipoprotein, however, was of greater density than human LD lipoprotein. 3. The trout lipoproteins have lipid compositions which are similar to those of the corresponding human components, except for their high contents of long-chain unsaturated fatty acids. 4. HD and LD lipoproteins were immunologically non-identical, whereas LD lipoproteins possessed antigenic determinants in common with VLD lipoproteins. 5. VLD and HD lipoproteins each contained at least seven different apoproteins, whereas LD liprotein was composed largely of a single apoprotein which resembled human apolipoprotein B. 6. At least one, and possibly three, apoprotein of trout HD lipoprotein showed features which resemble human apoprotein A-1.7. The broad similarity between the trout and human lipoprotein systems suggests that both arose from common ancestral genes early in evolutionary history.

scholarly journals Isolation and characterization of a myeloma–spleen-cell hybrid producing antibody to phenylalanine hydroxylase

1980 ◽  
Vol 191 (3) ◽  
pp. 777-783 ◽  
Author(s):  
R G H Cotton ◽  
I G Jennings ◽  
K H Choo ◽  
K Fowler
Keyword(s):  
Monoclonal Antibody ◽  
Spleen Cell ◽  
Phenylalanine Hydroxylase ◽  
Mammalian Species ◽  
Electrophoretic Analysis ◽  
Cross Reactivity ◽  
Double Immunodiffusion ◽  
Isolation And Characterization ◽  
A Cell

Application of the technique of myeloma–spleen-cell fusion [Kohler & Milstein (1975) Nature (London) 256, 495–497] has allowed the isolation of a cell colony that produced a monoclonal antibody against monkey liver phenylalanine hydroxylase. The antibody exhibited cross-reactivity against hepatic phenylalanine hydroxylase from other mammalian species, including human, rat and mouse. Cross-reactivity was established by (a) enzyme-inhibition assay, (b) double-immunodiffusion reaction, and (c) two-dimensional polyacrylamide-gel-electrophoretic analysis of immunoprecipitate. The various properties of the monoclonal antibody and its use in the study of mammalian phenylalanine hydroxylase are presented.

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