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

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.

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Immunoradiometric method and electrophoretic system compared for quantifying bone alkaline phosphatase in serum

Clinical Chemistry ◽

10.1093/clinchem/41.6.853 ◽

1995 ◽

Vol 41 (6) ◽

pp. 853-857 ◽

Cited By ~ 16

Author(s):

V O Van Hoof ◽

M Martin ◽

P Blockx ◽

A Prove ◽

A Van Oosterom ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Malignant Disease ◽

Screening Method ◽

Bone Alkaline Phosphatase ◽

Cross Reactivity ◽

Osteoblastic Activity ◽

Alp Activity ◽

Agarose Electrophoresis ◽

End Stage ◽

Rule Out

Abstract Agarose electrophoresis (Isopal, Beckman) and an immunoradiometric assay (IRMA) involving specific monoclonal antibodies (Ostase, Hybritech), two methods for the quantification of serum bone alkaline phosphatase (ALP, EC 3.1.3.1), a marker of osteoblastic activity, were compared in 293 patients: 79 with end-stage renal failure treated with hemodialysis and 214 with malignant disease. Overall correlation between the two methods was good (r = 0.92), except (a) for low values of bone ALP and (b) in some samples with high total liver ALP activity--both due to considerable cross-reactivity of the anti-bone ALP antibodies of the Ostase kit with liver ALP. This interference was not constant and was not evenly distributed across all concentrations of bone ALP. Low bone ALP determined with the IRMA (< or = 5 micrograms/L) was confirmed by electrophoresis (< or = 21 U/L), but bone ALP activity determined by electrophoresis to be low (< or = 21 U/L) was not correlated with the IRMA results. After standardizing our results by computing z-values for bone ALP, delta z (= zOstase - zIsopal) was significantly correlated with liver ALP activity (r = 0.73, P < 0.0001). We conclude that the IRMA for quantifying bone ALP is acceptable as a screening method. However, when high values for bone ALP are found with the Ostase method, confirmation by electrophoresis remains mandatory to rule out cross-reactivity with high amounts of liver ALP. For detecting low bone ALP activities, electrophoresis remains the method of choice.

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Generation and Characterization of a Murine Monoclonal Antibody Specific for the Human T1-Cd5 Molecule

The International Journal of Biological Markers ◽

10.1177/172460088700200302 ◽

1987 ◽

Vol 2 (3) ◽

pp. 143-150 ◽

Cited By ~ 2

Author(s):

Federico Genzano ◽

Ada Funaro ◽

Massimo Alessio ◽

Lucia B. De Monte ◽

Graziella Bellone ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

T Cell ◽

T Lymphocytes ◽

Membrane Glycoprotein ◽

Functional Features ◽

Specific Membrane ◽

Murine Monoclonal Antibodies ◽

Selection Of

Murine monoclonal antibodies (MoAbs) have found widespread applications in the characterization of the molecular and functional features of lymphocyte differentiation antigens. The present paper summarizes the results of our work dealing with the production and selection of a murine MoAb recognizing a molecule expressed during the whole differentiative life of T lymphocytes. The MoAb CB01 resulted to be specific for an apparently unique epitope of the T-cell specific membrane glycoprotein T1-CD5.

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Immunoadsorption assay for bone alkaline phosphatase compared with wheat germ agglutinin precipitation assay in serum from (pre)pubertal girls

Clinical Chemistry ◽

10.1093/clinchem/42.12.1970 ◽

1996 ◽

Vol 42 (12) ◽

pp. 1970-1974 ◽

Cited By ~ 3

Author(s):

A A Bouman ◽

C M de Ridder ◽

J H Nijhof ◽

J C Netelenbos ◽

H A Delemarre-vd Waal

Keyword(s):

Alkaline Phosphatase ◽

Correlation Coefficient ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Bone Alkaline Phosphatase ◽

Cross Reactivity ◽

Reference Ranges ◽

Correlation Studies ◽

Deming Regression ◽

The Mean

Abstract The performance characteristics of two bone alkaline phosphatase (ALP; EC 3.1.3.1) assays, a wheat germ agglutinin (WGA) precipitation assay and a new immunoadsorption assay (IAA), were compared. The within- and between-run imprecision of the IAA (3.6-4.2% and 3.6-7.7%) was comparable with that of the WGA assay. The mean cross-reactivity with liver ALP appeared to be 4% in the WGA assay and 11% in the IAA. The reference ranges in a group of 155 healthy Caucasian (pre)pubertal schoolgirls were: 149-401 U/L (total ALP, 30 degrees C), 105-349 U/L (bone ALP, 30 degrees C, WGA assay), and 58-205 U/L (bone ALP, 25 degrees C, IAA). Comparison of the WGA assay (x) with the IAA (y) demonstrated a correlation coefficient of 0.95 [Deming regression equation: y = (0.56 +/- 0.01)x + (2.0 +/- 1.5); Sy[symbol: see text]x = 5.3 U/L]. Correlation studies of the WGA assay and the IAA results with total ALP demonstrated r = 0.98 and 0.96, respectively.

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Development of a monoclonal antibody specific for human bone alkaline phosphatase

Bone and Mineral ◽

10.1016/0169-6009(92)90733-t ◽

1992 ◽

Vol 17 (2) ◽

pp. 182-186 ◽

Cited By ~ 5

Author(s):

K. Masuhara ◽

S. Suzuki ◽

H. Yoshikawa ◽

T. Tsuda ◽

K. Takaoka ◽

...

Keyword(s):

Monoclonal Antibody ◽

Alkaline Phosphatase ◽

Bone Alkaline Phosphatase ◽

Human Bone

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Amplified Enzyme-Linked Immunoassay of Human Proinsulin in Serum (Detection Limit: 0.1 pmol/L)

Clinical Chemistry ◽

10.1093/clinchem/38.2.227 ◽

1992 ◽

Vol 38 (2) ◽

pp. 227-232 ◽

Cited By ~ 11

Author(s):

F J Dhahir ◽

D B Cook ◽

C H Self

Keyword(s):

Monoclonal Antibody ◽

Alkaline Phosphatase ◽

Cross Reactivity ◽

C Peptide ◽

Human Proinsulin ◽

Microtiter Plates ◽

Enzyme Amplification ◽

Fold Excess ◽

Amplification Procedure ◽

Serum Detection

Abstract We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.

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

Journal of AOAC International ◽

10.5740/jaoacint.sge_halbmayr-jech ◽

2012 ◽

Vol 95 (2) ◽

pp. 372-376 ◽

Cited By ~ 17

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.

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Schwann Cell Marker Defined by a Monoclonal Antibody (224?58) with Species Cross-Reactivity. II. Molecular Characterization of the Epitope

Journal of Neurochemistry ◽

10.1111/j.1471-4159.1986.tb12987.x ◽

1986 ◽

Vol 46 (2) ◽

pp. 435-439 ◽

Cited By ~ 25

Author(s):

C. Goujet-Zalc ◽

A. Guerci ◽

G. Dubois ◽

B. Zalc

Keyword(s):

Monoclonal Antibody ◽

Schwann Cell ◽

Molecular Characterization ◽

Cross Reactivity ◽

Cell Marker ◽

Species Cross

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Optimization and Characterization of Capture ELISA Methodology for Lp(a) Lipoprotein Quantification

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456329202900304 ◽

1992 ◽

Vol 29 (3) ◽

pp. 275-282 ◽

Cited By ~ 2

Author(s):

Karl M Doetsch ◽

Paul S Roheim ◽

James J Thompson

Keyword(s):

Monoclonal Antibody ◽

Alkaline Phosphatase ◽

Dose Response ◽

Reaction Conditions ◽

Elisa System ◽

Capture Elisa ◽

Antibody Recognition ◽

C Storage ◽

Time Periods

In order to better characterize and optimize a typical capture ELISA system for Lp(a) lipoprotein, we have analysed kinetic details of the reaction. Plate coating with polyclonal antibody, recognition of captured analyte with monoclonal antibody, and detection of monoclonal antibody with alkaline phosphatase-labeled antiglobulin were essentially complete after one hour, probably being driven forward by a relative excess of reagent. However, complete capture of the Lp(a) analyte required about 6 h at low input concentrations. Shorter time periods for capture might therefore result in decreased sensitivity and reproducibility. Deviations from linearity in the assay dose response were associated with incomplete capture of Lp(a) and significant depletion of the monoclonal recognition antibody. With the final reaction conditions described, no significant differences in immunochemical reactivity between samples were found by analysis of dose response slopes. Finally, interferences from plasminogen, −20°C storage, anticoagulants, LDL, haemolysis, and bilirubin were minimal.

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