PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

Na 1 Nachlass Max Horkheimer, 76 - Korrespondenzen u.a. mit Hermann Igersheimer (p. II 9, 89-222c)

1 Brief und Beilage vom Institut für sozialwissenschaftliche Forschung Darmstadt an Max Horkheimer, 1949; 82 Briefe zwischen Hermann Igersheimer und Max Horkheimer, 1941-1950; 5 Briefe zwischen Harold E. Jones von der University of California und Max Horkheimer, 1947; 1 Brief von Morris Janowitz an Max Horkheimer, 1948; 2 Briefe vom Jewish Community Center an Max Horkheimer, 1948; 7 Briefe zwischen Robert P. St.John und Max Horkheimer, 1945; 2 Briefe zwischen Erich von Kahler und Max Horkheimer, 1945; 2 Briefe und Beilage zwischen Anselm Kahn und Max Horkheimer, 04.07.1945, 19.07.1945;

Crystal structure of glutamate-1-semialdehyde aminomutase: An α2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an α2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.

Letter from James Winthrop to William Bentley, 1788 February 27

Two leaves containing a one-page handwritten letter from Winthrop to Bentley that mentions Rev. John Prince (1751-1836) and briefly discussing the state of Winthrop's microscope and five volumes presented to Harvard that were printed in Calcutta, India.

Agricultural development in Venezuela, report of the SCIPA, 1943-1946,

"List of selected references": p. 83-85.

Chemical shoreline cleaning agents for oil spills : update state-of-the-art on mechanisms of action and factors influencing performance /

"EPA contract no. 68-C8-0062, Work assignment no. 3-48, SAIC project no. 01-0895-03-1000."

Land application of wastewater under high altitude conditions /

Mode of access: Internet.

The history of freemasonry : its antiquities, symbols, constitutions, customs, etc., derived from official sources throughout the world /

Volume 1 with added illustrated title page.

The Bible-reader's companion /

Mode of access: Internet.

Woman : in all ages and in all countries.

"Astral Edition, printed on Japan vellum paper, limited to two hundred and fifty numbered and registered sets..."

Thermal conductivity of one-dimensional lattices,

Mode of access: Internet.

The Wolfe expedition to Asia Minor /

"American School of Classical Studies at Athens, January 1888 : [report]": p. [1]-11, 2nd set.

Lectures on justification /

At least two states of the t.p. exist: in the first, Newman's titles are Vicar of St. Mary the Virgin's, and Fellow of Oriel College, Oxford, and there is no printer's imprint on the verso; in the second, the order of the titles is reversed, and there is a printer's imprint on the verso.

An essay in aid of a grammar of assent /

Mode of access: Internet.

Newman as a man of letters,

Mode of access: Internet.