A simple method for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli.

A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli. IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes. In the present method, gel filtration was required only once to separate the conjugate from unconjugated components in the final step, while gel filtration had to be repeated 3-4 times in the previous methods. The conjugate preparations obtained by the present method contained less nonspecific conjugate and gave a lower background by immunoenzymometric assay technique than those obtained by the previous method.

Large unilamellar lipid vesicles for use in therapeutic and diagnostic medicine.

Large unilamellar lipid vesicles are prepared by a detergent dialysis procedure using beta-D-octylglucoside as the detergent. This procedure is nondenaturing and allows for the encapsulation of sensitive biological molecules. Vesicles prepared with the composition of 2 mol phosphatidylcholine and 1 mol cholesterol have a mean diameter of 200 nm and allow for the encapsulation of 150 molecules of alkaline phosphatase per vesicle without loss of activity. Stability studies show that less than 4 per cent of the original enzyme leaks from the vesicles over a 250 day period upon storage at 4 degrees C. A mechanistic model for vesicle formation is described to provide a clear understanding of the events occurring during the encapsulation stages.

Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay.

A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.

An automated, affinity-column-mediated, enzyme-linked immunometric assay for digoxin on the Du Pont aca discrete clinical analyzer.

We describe an automated assay for digoxin that requires a 200-microL sample of serum. Total analysis time is 18 min. The method is extremely precise, with within-run CVs of 2.6, 1.6, and 4.9%, respectively, at 0.5, 1.5, and 3.5 micrograms of digoxin per liter (n = 20). The lower limit of detection is 0.2 micrograms of digoxin per liter. For patients' samples, the correlation with RIA (x) is excellent (r = 0.95; y = 0.95x - 0.14; standard error = 0.17 micrograms/mL). We saw no interferences in samples having high concentrations of rheumatoid factors, lipid, bilirubin, or hemoglobin. Cross reactivity with digoxin analogs and steroidal compounds is similar to that observed by RIA.

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy.

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.

Affinity-column-mediated immunoenzymometric assays: influence of affinity-column ligand and valency of antibody-enzyme conjugates.

We describe an affinity-column-mediated, enzyme-linked immunometric assay that is highly sensitive and adaptable to automation. Digoxin is the model test analyte. A comparison of digoxin with its analog, ouabain, for use as the immobilized ligand on the affinity column showed ouabain to be superior. We also report the effect of column elution rate. Antibody-enzyme conjugates prepared with the monovalent Fab'-fragment and the divalent F(ab')2-fragment coupled to beta-galactosidase are compared in terms of their overall assay performance. Although the monovalent Fab'--beta-galactosidase conjugate yields a more sensitive assay and dose-response curves that are linear over a wider range, the divalent F(ab')2--beta-galactosidase conjugate provides an assay with adequate sensitivity and extremely good precision, and is generally easier to synthesize reproducibly. This fully automated, rapid, and precise assay for digoxin is compatible with the Du Pont aca discrete clinical analyzer.

Highly sensitive immunoassays based on use of liposomes without complement.

We describe a novel liposome-based immunoassay in which covalently linked hapten-cytolysin conjugates are used instead of complement and surface-immobilized immunoreagents. Stable, unilamellar liposomes containing entrapped alkaline phosphatase as a marker enzyme were prepared by dialysis of octyl glucoside from suspensions of cholesterol and egg yolk lecithin. The resulting vesicles could be immediately lysed by addition of either bee venom melittin or hapten-melittin conjugates. Using ouabain, an analog of digoxin, we synthesized conjugates that were more lytic than mellitin alone but that were inhibited in the presence of antibody. This inhibition was affected by adding competing free digoxin at various concentrations to obtain standard curves. The same liposome preparations could be lysed with a biotin-melittin conjugate, which was inhibited by avidin. The latter system was affected by free biotin and might be used to couple this approach to various heterogeneous immunoassays.

Liposome-mediated immunoassays for small haptens (digoxin) independent of complement.

A lipid vesicle-mediated immunoassay for small haptens (digoxin) is described. Using a detergent removal procedure for vesicle formation, a water-soluble marker system like alkaline phosphatase is stably entrapped within 150-200 nm unilamellar lipid vesicles composed of egg yolk lecithin and cholesterol. Specific lysis of the lipid vesicles is achieved upon addition of a hapten-melittin conjugate. Inhibition or modulation of this lysis by the hapten-melittin conjugate can then be achieved by adding stoichiometric amounts of high affinity antibody. Finally, the antibody inhibition of hapten-melittin lysis can be modulated by the addition of competing amounts of free hapten. This assay approach is simple, fast, highly sensitive, and versatile such that it can be carried out in either a homogeneous or heterogeneous mode. Furthermore, unlike all other liposome-mediated immunoassays, complement is not required for lysis.

A highly sensitive affinity-column-mediated immunometric assay, as exemplified by digoxin.

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a porous affinity column containing immobilized analyte (or analog), present in vast excess. Only the labeled monovalent antibody that possesses an antigen in its binding site elutes from the column in the unbound fraction. The label present in this fraction is then quantified. Such an assay is extremely sensitive and obviates the limitations imposed by antibody affinity constants on homogeneous and competitive heterogeneous immunoassays. This assay can be performed rapidly and is readily amenable to automation.

The (Na+, K+)ATPase exhibits enzymic activity in the absence of the glycoprotein subunit.

Membrane-bound (Na+, K+)ATPase from avian nasal salt glands was exposed to limited papain digestion. Such treatment results in the selective removal of the beta-subunit rendering the alpha-subunit still membrane-bound and expressing full enzymic activity. With further exposure to papain the alpha-chain becomes fragmented into two major polypeptide components. The fragmented membrane-bound catalytic chain is extremely sensitive to detergent treatment and cannot be solubilized in an active state.

Isolation of type IV procollagen-like polypeptides from glomerular basement membrane. Characterization of pro-alpha 1(IV).

Type IV procollagen-like constituents of glomerular basement membrane were solubilized by reduction and alkylation of disulfide bonds under denaturing conditions. Four polypeptides were observed with apparent Mr = 185,000, 175,000, 164,000, and 152,000. The two largest chains correspond to pro-alpha 1(IV) and pro-alpha 2(IV), described in model systems which secrete a basement membrane-like matrix, while the smaller chains appear to be shortened forms of these polypeptides. Fractionation of the four polypeptides into two groups was achieved by ion exchange chromatography. Pro-alpha 1(IV) and 164,000 polypeptide are relatively acidic with respect to pro-alpha 2(IV) and 152,000 polypeptide, which is due in part to a relatively high content of arginine in the latter. Based on amino acid analysis of the collagenase-sensitive regions of these polypeptides, pro-alpha 1(IV) is the parent molecule from which alpha 1(IV) is derived on pepsin digestion of basement membranes and pro-alpha 2(IV) is the parent molecule of alpha 2(IV). Pro-alpha 1(IV) was isolated by gel filtration and ion exchange chromatography and characterized. It has a molecular weight of 194,000 as determined by sedimentation equilibrium. The polypeptide contains 14% carbohydrate in the form of both disaccharide, glucosylgalactosylhydroxylysine, and heteropolysaccharide units. The polypeptide backbone mass is calculated to be 167,000 daltons. Digestion of pro-alpha 1(IV) with bacterial collagenase resulted in two resistant segments of mass = 31,000 and 33,000 dalton, which make up approximately 30% of the polypeptide.

Physical studies on the rabbit hepatic galactoside-binding protein. Effects of calcium and ligands.

The rabbit hepatic galactoside lectin exhibited noncooperative binding of asialo-orosomucoid in four different nonionic detergents, Triton X-100, octyl glucoside, cetyl eicosaoxyethyleneglycol monoether (Brij 58), and dodecyl octaoxyethyleneglycol monoether (C12E8). The Brij 58-solubilized lectin chromatographed as a single peak upon gel filtration on Sepharose 4B and the molecular weight was determined to be 234,000 in the absence of calcium by sedimentation equilibrium ultracentrifugation. When calcium or calcium plus ligand was added, the molecular weight of the lectin increased to 612,000. A pronounced (15%) decrease in the intrinsic fluorescence of the galactoside lectin was observed upon addition of calcium. Based upon changes in fluorescence, the equilibrium dissociation constant for calcium and binding protein was 1.5 x 10(-3) M. No major changes were detected upon calcium addition by ultraviolet absorption or circular dichroism spectroscopy, nor were there any changes when a ligand such as lactose was added. The number of calcium ions bound, as determined by ultrafiltration, was 3.3 Ca2+/polypeptide chain in the absence of other divalent metals and 1.92 Ca2+/chain in the presence of 100 mM Mg2+. The equilibrium dissociation constant determined in this manner for Ca2+ was 3.5 x 10(-4) M.

Polypeptide molecular weights of the (Na+,K+)-ATPase from porcine kidney medulla.

The molecular weights of the polypeptide chains from (Na+,K+)-ATPase of porcine kidney medulla have been determined by analytical sedimentation equilibrium. The alpha-subunit molecular weight is 93 900, and the beta subunit is a glycoprotein with a polypeptide molecular weight of 32 300 (41 400 including protein and carbohydrate). Amino acid and carbohydrate compositions are presented together with related properties (i.e., partial specific volumes, extinction coefficients, and hydrophobic/hydrophilic amino acid content).

Physical properties of collagen--sodium dodecyl sulfate complexes.

Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.

Large scale preparation of bovine renal glomerular basement membrane in the presence of protease inhibitors.

A method is described for the isolation of large quantities of basement membrane from bovine renal glomeruli under conditions which minimize or prevent degradation by tissue-associated proteases. The method incorporates the use of moderately-high concentrations of the protease inhibitors: ethylenediamine tetraacetic acid, epsilon-amino caproic acid, N-ethymaleimide, and diisopropylfluorophosphate; and the maintenance of a temperature of 0-4 degrees C throughout the procedure. Glomeruli preparations are isolated by a modified sieving technique and are routinely of purity greater than 97%. Under optimum conditions, three to four grams of basement membrane can be isolated under safe and rapid conditions in one week.