Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein.

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.

Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: application in the semisynthesis of molecular variants of alpha-globin.

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the alpha-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37 degrees C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the alpha-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of alpha-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected alpha-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of alpha-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated alpha-globin. The complementary fragments from two natural mutant forms of alpha-globin, viz., alpha 1-30 (K16E) from Hb I and alpha 31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant alpha 1-141 (K16E;D47H).

Site-directed immobilization of proteins.

To determine if immobilization chemistry can be used to orient antibody on a support so that the bivalent binding potential can be fully utilized, we developed three activated matrices that couple to different functional groups on the molecule. When AminoLink Gel was used to couple antibody randomly through primary amino groups, the molar ratio of immobilized antibody to recovered antigen averaged 1:1. Iodoacetyl groups on SulfoLink Gel couple through sulfhydryls in the hinge region of the antibody molecule, in theory leaving the antigen binding site available. However, the antibody-to-antigen molar ratio was only slightly improved. Hydrazide groups on CarboLink Gel couple to aldehyde groups generated by oxidation of carbohydrate moieties that are located primarily on the Fc portion of the antibody molecule. The molar ratio of immobilized antibody to purified antigen using CarboLink Gel reached the optimum of 1:2. CarboLink Gel is most effective at orienting antibody for better antigen purification capability.

Measurement of protein using bicinchoninic acid.

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences.

An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.

Isolation of the Maclura pomifera hemagglutinin on a deoxymelibiotol affinity support and preliminary characterization by buffer electrofocusing and high-performance liquid chromatography.

Maclurin, the potent non-specific blood-group hemagglutinin present in extracts of Maclura pomifera, has been purified by a new biospecific affinity-chromatographic procedure. Additional studies have indicated that this hemagglutinin occurs as five closely related tetrameric protein isoforms derived from two non-covalently-linked polypeptide chains, mol. wts. ca. 10,000 and 13,000 respectively. Buffer electrofocusing fractionated the lectin into 12 components; the major isolectin exhibited an isoelectric point at pH 4.8.

The localization of retinol-binding protein in rat liver by immunofluorescence microscopy.

The localization of immunoreactive retinol-binding protein (RBP) in rat liver was studied by immunofluorescence microscopy. The study employed specific antisera to rat RBP prepared in a rabbit and in a sheep. The indirect, two-stage method of localizing tissue antigens was employed, and livers of both normal and vitamin A-deficient rats were examined. Fab' fragments of immunoglobulins were used, to minimize non-specific labelling of the frozen sections of liver. With these techniques, the specific immune staining of RBP was observed within liver parenchymal cells. This staining appeared as both particulate and diffuse within the cytoplasm of the parenchymal cells, and was not concentrated within one region of the liver cell or lobule. Staining for RBP was not observed in nuclei or in cells other than parenchymal cells. Similar particulate and diffuse immune staining for RBP was observed in liver sections from both vitamin A-deficient and normal rats. More intense immune staining appeared to be present in the sections of vitamin A-deficient animals, in good correlation with the expected higher levels of RBP in deficient as compared to normal liver. When liver sections were exposed to an antiserum to rat albumin, instead of one to rat RBP, immune cytoplasmic staining was observed which was entirely of a diffuse nature, and did not appear particulate or granular. The findings suggest that RBP, unlike albumin, is localized in part within cytoplasmic vesicles or granules which are large enough to be detected with immunofluorescence, and which are present in livers of both normal and vitamin A-deficient animals. The nature of these putative RBP-containing particles remains to be explored.

Metabolism of retinol-binding protein and vitamin A during hypervitaminosis A in the rat.

Vitamin A is normally transported in plasma as retinol bound to a specific protein, retinol-binding protein (RBP). Detailed studies were conducted to examine the effects of excess vitamin A on the plasma concentration and metabolism of RBP, and to obtain information about vitamin A transport in the hypervitaminotic state. Two separate experiments were conducted. In the first (Study I, 99 days), plasma RBP and vitamin A levels were compared in three groups of rats fed 0.14 mg (control), 7.3 mg (group 2), or 41 mg (group 3) of vitamin A per day. After day 50 of the study, the administration of excess vitamin A to hypervitaminotic rats (groups 2 and 3) was discontinued and the rats were allowed to recover from vitamin A toxicity. In the second, shorter experiment (Study II), serum vitamin A and RBP levels were compared in control and hypervitaminotic (34 mg of retinyl acetate per day) rats. The rats in this study were also given [3-H]retinyl acetate daily to determine the distribution of retinyl esters and retinol between the lipoprotein and nonlipoprotein protein fractions of plasma. In both studies, administration of large, excessive doses of vitamin A resulted in substantial and significant decreases in the levels of serum RBP. Excessive doses of vitamin A produced fatty liver in the rats, in association with a normal (group 2, Study I) or with a decreased (group 3, Study I) level of RBP in the liver. It is possible that excess vitamin A leads to decreased rates of RBP synthesis in, and of RBP secretion from, the liver. Administration of excessive doses of vitamin A also resulted in elevations of serum vitamin A levels, which were mainly due to large increases in the circulating levels of retinyl esters. In the hypervitaminotic rats, most of the serum vitamin A, and virtually all of the retinyl esters, was found in association with the serum lipoproteins of hydrated density less than 1.21. These results demonstrate that the serum lipoproteins play an important role in the transport of the vitamin A that accumulates in serum in hypervitaminosis A. We suggest that the toxic manifestations of hypervitaminosis A occur when vitamin A circulates in plasma and is presented to membranes in a form other than bound to RBP. Plasma lipoproteins may nonspecificially deliver vitamin A to biological membranes and hence lead to vitamin A toxicity.