New purified glycoproteins and use in diagnosing infectious mononucleosis

Claim 2

A mammalian erythrocyte glycoprotein selected from the group consisting of horse and sheep erythrocyte glycoprotein, the amino acid composition of the horse erythrocyte glycoprotein being about 8.1 mole % aspartic acid, about 10.6 mole % threonine, about 10.8 mole % serine, about 9.4 mole % glutamic acid, about 12.3 mole % proline, about 9.2 mole % glycine, about 11.3 mole % alanine, about 4.4 mole % valine, about 0.8 mole % methionine, about 3.5 mole % isoleucine, about 8.2 mole % leucine, about 1.1 mole % tyrosine, about 2.9 mole % phenylalanine, about 1.2 mole % histidine, about 1.3 mole % lysine and about 4.8 mole % arginine, and the amino acid composition of the sheep erythrocyte glycoprotein being about 5.6 mole % aspartic acid, about 8.1 mole % threonine, about 12.9 mole % serine, about 13.0 mole % glutamic acid, about 11.6 mole % proline, about 7.7 mole % glycine, about 9.6 mole % alanine, about 6.2 mole % valine, about 0.5 mole % methionine, about 4.6 mole % isoleucine, about 8.3 mole % leucine, about 4.6 mole % in tyrosine, about 1.2 % phenylalanine, about 1.6% histidine, about 3.2 mole % lysine, about 4.0 mole % arginine, and about 0.3% tryptophan.

Claim 3

A horse erythrocyte glycoprotein according to claim 1, the amino acid composition of which is about 8.1 mole % aspartic acid, about 10.6 mole % threonine, about 10.8 mole % serine, about 9.4 mole % glutamic acid, about 12.3 mole % proline, about 9.2 mole % glycine, about 11.3 mole % alanine, about 4.4 mole % valine, about 0.8 mole % methionine, about 3.5 mole % isoleucine, about 8.2 mole % leucine, about 1.1 mole % tyrosine, about 2.9 mole % phenylalanine, about 1.2 mole % histidine, about 1.3 mole % lysine and about 4.8 mole % arginine.

Claim 4

A sheep erythocyte glycoprotein according to claim 1, the amino acid composition of which is about 5.6 mole % aspartic acid, about 8.1 mole % threonine, about 12.9 mole % serine, about 13.0 mole % glutamic acid, about 11.6 mole % proline, about 7.7 mole % glycine, about 9.6 mole % alanine, about 6.2 mole % valine, about 0.5 mole % methionine, about 4.6 mole % isoleucine, about 8.3 mole % leucine, about 4.6 mole % tyrosine, about 1.2 mole % phenylalanine, about 1.6 mole % histidine, about 3.3 mole % lysine, about 4.0 mole % arginine, and about 0.3 mole % tryptophan.

Claim 5

A horse erythrocyte glycoprotein according to claim 2 which is a sialoglycoprotein comprised of about 55% carbohydrate residues in a composition of about 28 grams of N-glycolylneuraminic acid, about 11 grams of galactose, about 14 grams of N-acetylgalactosamine, about 1 gram of N-acetylglucosamine and about 1 gram of mannose per 100 grams of glycoprotein.

Claim 6

The horse erythrocyte glycoprotein of either of claims 2 and 4 which is essentially homogenous.

Claim 7

A horse erythrocyte glycoprotein according to either of claims 2 and 4, containing a label selected from the group consisting of radioisotopes, fluorogens, chromophores, enzymes, luminescent substances and other known labels used in immunoassay procedures.

Claim 8

The labelled horse glycoprotein of claim 6 which is labelled with 125 I.

Claim 9

A sheep erythrocyte glycoprotein according to claim 3 which is a sialoglycoprotein comprised of about 56% carbohydrate residues in a composition of about 17 grams of sialic acids, about 9 grams of galactose, about 14 grams of N-acetylgalactosamine, about 14 grams of N-acetylglucosamine, and about 2 grams of mannose per 100 grams of glycoprotein.

Claim 10

The sheep erythrocyte glycoprotein of any of claims 1, 3 and 8 which is essentially homogeneous.

Claim 11

A sheep erythrocyte glycoprotein according to any of claims 1, 3 and 8 containing a label selected from the group consisting of radioisotopes, fluorogens, chromophores, enzymes, luminescent substances and other known labels used in immunoassay procedures.

Claim 12

The labelled sheet glycoprotein of claim 10 which is labelled with 125 I.

Claim 13

A process for preparing the mammalian glycoproteins of claim 1 which comprises the steps of: (a) uniformly suspending dried, ground, hemoglobin-free stroma from mammalian erythrocytes in anhydrous acetone; (b) refluxing for from about 1 to about 6 hours, filtering and drying the residue; (c) suspending said dried residue in 100% anhydrous ethanol; (d) refluxing between about 1 and about 6 hours, filtering and drying the residue; (e) suspending the dried residue from step (d) in aqueous ethanol of between about 50% and about 80% strength, and repeating step (b); (f) dissolving the residue from step (e) in water and adding 90% aqueous ethanol, followed by incubating on ice until crystallization occurs, centrifuging and dialyzing the solid layer against a low pH, low ionic strength buffer; (g) passing the solid from step (f) through a cation exchange resin on a chromatographic column; (h) collecting the sialic acid containing fractions from the column and drying them; (i) treating the collected fractions from step (h) by extraction with a known lipid solvent, centrifuging, collecting the aqueous layer and drying it; (j) repeating step (i) on the product of that step, using a different lipid solvent; (k) recovering the product of step (j) in lyophilized form; (l) dissolving the product of step (k) in a low ionic strength buffer containing about 1% neutral detergent; (m) loading the solution from step (1) on an anion exchange chromatographic column; (n) washing the column thoroughly with low ionic strength buffer; (o) eluting the column with aqueous buffer to high salt concentration; and (p) dialyzing the product of step (o) against water and recovering the product in freeze dried form.

Claim 14

A process according to claim 12 wherein the cation exchange resin is phosphocellulose.

Claim 15

A process according to claim 12 wherein the lipid solvent of step (i) is diethyl ether and ethanol in a volume ratio between about 4:1 and about 1:1.

Claim 16

A process according to claim 12 in which the lipid solvent of step (j) is chloroform and methanol in a volume ratio of about 2:1.

Claim 17

A process according to claim 12 wherein the solvent in step (e) is about 75% aqueous ethanol.

Claim 18

A process for the inhibition of rosetting by human blood lymphocytes which comprises incubating said lymphocytes with the horse erythrocyte glycoprotein of either of claims 2 or 4.

Claim 19

A process for the inhibition of rosetting by human blood lymphocytes which comprises incubating said lymphocytes with the sheep erythrocyte glycoprotein of either of claims 3 or 8.

Claim 20

A process for monitoring or diagnosing human infectious mononucleosis the improvement which comprises utilizing the horse erythrocyte glycoprotein of either of claims 2 or 4 as an inhibitor of agglutination.

Claim 21

A process for monitoring or diagnosing human infectious mononucleosis the improvement which comprises utilizing the sheep erythrocyte glycoprotein of either of claims 3 or 8 as an inhibitor of agglutination.