Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

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PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

10.1055/s-0038-1642806 ◽

1987 ◽

Author(s):

M Philips ◽

A G Juul ◽

S Thorsen ◽

J Selmer ◽

L Thim

Keyword(s):

Monoclonal Antibody ◽

Plasminogen Activator ◽

Human Placenta ◽

Solid Phase ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Single Chain ◽

Plasminogen Activator Inhibitor 1 ◽

Sds Page ◽

Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

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

Clinical Chemistry ◽

10.1093/clinchem/35.10.2087 ◽

1989 ◽

Vol 35 (10) ◽

pp. 2087-2092 ◽

Cited By ~ 9

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.

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Sensitive, rapid procedure for time-resolved immunofluorometry of lutropin.

Clinical Chemistry ◽

10.1093/clinchem/34.8.1640 ◽

1988 ◽

Vol 34 (8) ◽

pp. 1640-1644 ◽

Cited By ~ 5

Author(s):

M J Khosravi ◽

R C Morton ◽

E P Diamandis

Keyword(s):

Monoclonal Antibody ◽

Solid Phase ◽

Detection System ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Practical Procedure ◽

Daily Monitoring ◽

Fluorescence Measurements ◽

Capture Antibody ◽

Europium Chelate

Abstract In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.

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Use of monoclonal antibodies to detect human placental alkaline phosphatase.

Clinical Chemistry ◽

10.1093/clinchem/29.1.115 ◽

1983 ◽

Vol 29 (1) ◽

pp. 115-119 ◽

Cited By ~ 24

Author(s):

G De Groote ◽

P De Waele ◽

A Van de Voorde ◽

M De Broe ◽

W Fiers

Keyword(s):

Monoclonal Antibody ◽

Alkaline Phosphatase ◽

Monoclonal Antibodies ◽

Solid Phase ◽

Enzymatic Reactions ◽

Placental Alkaline Phosphatase ◽

Human Placental Alkaline Phosphatase ◽

Human Sera ◽

Starch Gel ◽

Alkaline Phosphatase Isoenzyme

Abstract Convenient, sensitive, and specific solid-phase immunoassays involving monoclonal antibody are described for the determination of human placental alkaline phosphatase (hPLAP). An endogenous enzyme immunoassay combined the specificity of the immunological and the enzymatic reactions. Alternatively, a solid-phase "sandwich" radioimmunoassay involving immobilized polyclonal rabbit anti-hPLAP in combination with iodinated monoclonal antibody provided some additional advantages. Both tests can be used to detect hPLAP from various sources, e.g., in human sera during pregnancy or as a tumor marker. The radioimmunoassay detected an increase in hPLAP at nine weeks of gestation. We discuss the use of monoclonal antibodies for the differentiation of different alkaline phosphatase isoenzyme types by electrophoresis on starch gel.

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Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

Clinical Chemistry ◽

10.1093/clinchem/39.6.942 ◽

1993 ◽

Vol 39 (6) ◽

pp. 942-947 ◽

Cited By ~ 19

Author(s):

D A Monaghan ◽

M J Power ◽

P F Fottrell

Keyword(s):

Monoclonal Antibody ◽

Enzyme Immunoassay ◽

Solid Phase ◽

Sandwich Elisa ◽

Normal Subjects ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Standard Curve ◽

Microtiter Plates ◽

Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

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Characterization of Primate Antibody Responses to Administered Murine Monoclonal Immunoglobulin

The International Journal of Biological Markers ◽

10.1177/172460089000500403 ◽

1990 ◽

Vol 5 (4) ◽

pp. 177-187 ◽

Cited By ~ 4

Author(s):

D.E. Milenic ◽

B. Detrick ◽

J.C. Reynolds ◽

D. Colcher

Keyword(s):

Monoclonal Antibody ◽

Antibody Response ◽

Solid Phase ◽

Hypervariable Region ◽

Antigen Binding ◽

Post Inoculation ◽

Antigen Binding Site ◽

Antibody Treatment ◽

Cancer Management ◽

Monoclonal Antibody Treatment

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.

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Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

Clinical Chemistry ◽

10.1093/clinchem/48.3.526 ◽

2002 ◽

Vol 48 (3) ◽

pp. 526-532 ◽

Cited By ~ 87

Author(s):

Marius Ulleland ◽

Ingar Eilertsen ◽

Edward V Quadros ◽

Sheldon P Rothenberg ◽

Sergey N Fedosov ◽

...

Keyword(s):

Monoclonal Antibody ◽

Detection Limit ◽

Quantitative Determination ◽

Reliable Method ◽

Solid Phase ◽

Reference Interval ◽

Magnetic Microspheres ◽

Affinity Constant ◽

Capture Assay

Abstract Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r2 = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

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Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

The Journal of Cell Biology ◽

10.1083/jcb.107.1.163 ◽

1988 ◽

Vol 107 (1) ◽

pp. 163-175 ◽

Cited By ~ 28

Author(s):

D J Meyer ◽

C L Afonso ◽

D W Galbraith

Keyword(s):

Monoclonal Antibody ◽

Cell Wall ◽

Monoclonal Antibodies ◽

Plasma Membrane ◽

Solid Phase ◽

Culture Media ◽

Gradient Centrifugation ◽

Cell Suspension Cultures ◽

Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

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