Derivative of alpha' 1'-fetospecific serum protein and process for its manufacture

Claim 2

A DERIVATIVE OF THE ALPHA1-FETOSPECIFIC SERUM PROTEIN, WHICH A. IS FORMED BY THE ACTION OF THE ENZYME NEURAMUNIDASE ON THE ALPHA,-FETOSPECIFIC SERUM PROTEIN, B. IS PRECIPITATED BY AN ANTISERUM ACTING AGAINST THE ALPHA,FETOSPPECIFIC SERUM PROTEIN, AND C. UPON INJECTION IN VERTEBRATES CAUSES THE FORMATION OF ANTIBODIES WHICH ARE SPECIFIC AGAINST BOTH THE ALPHA,FETOSPECIFIC PROTEIN AND AGAINST SAID DERIVATIVE.

Claim 3

A process for the manufacture of a derivative of the alpha1-fetospecific serum protein as claimed in claim 1, which comprises isolating a fraction containing the alpha1-fetospecific serum protein from body fluids or tissue extracts, treating it with neuraminidase and isolating the derivative of the alpha1-fetospecific serum protein in pure form by preparative electrophoresis.

Claim 4

A process as claimed in claim 2, wherein the fraction containing the alpha1-fetospecific serum protein is isolated from body fluids or tissue extracts, preferably from fetal blood, plasma of new-borns, plasma of patients suffering from primary liver carcinoma, from Ascites liquid of extracts from the livers of patients suffering from primary liver carcinoma.

Claim 5

A process as claimed in claim 2, wherein the fraction containing the alpha1-fetospecific serum protein is separated from accompanying proteins by a method of salt fractionation, advantageously using ammonium sulfate in a saturation concentration of the salt of more than 45 %.

Claim 6

A process as claimed in claim 2, wherein undesired proteins are separated from body fluids or tissue extracts at a pH-value of from 6 to 9 by an addition of ammonium sulfate until a final concentration of 1.8 to 2.2 M is reached, the salt content of the supernatant solution is reduced, water-soluble derivatives of an acridine base are added to this solution until a concentration is reached which does not yet precipitate the alpha1-fetospecific protein, further amounts of a water-soluble derivative of the acridine base are added to the supernatant at pH 7 - 10 after separation by centrifuging or filtering off the resulting precipitate until a concentration is reached which precipitates the alpha1-fetospecific protein from the solution, the residue is dissolved either in a weekly acid aqueous solution and the protein is separated from the precipitating agent by means of a molecular sieve method or by adding an adequate amount of chloride ions until a concentration of an about 5 % chloride-containing aqueous solution is reached, the precipitating agent is precipitated and the protein is set free in solution, the precipitate is centrifuged or filtered off, the protein solution is dialysed against a buffer solution at a pH-value of from 8 to 9, at an ion strength of from 0.05 to 0.15 and, at a protein concentration of about 2 %, the dialysate is subjected to a preparative carrier electrophoresis, whereupon the alpha1 -fetoprotein-containing zone is withdrawn and thus separated from remaining contaminations.

Claim 7

A process as claimed in claim 2, wherein the alpha1-fetospecific serum protein is treated with a microbial neuraminidase at a pH-value optimum for the reaction of the enzyme, until an almost quantitative conversion of the protein into the derivative as claimed in claim 1 is achieved.

Claim 8

A process as claimed iN claim 6, wherein the alpha1-fetospecific serum protein is treated with neuraminidase obtained from Vibrio cholerae, at a pH of 5.0 to 5.8 in a ratio of 0.5 to 50 units of neuraminidase per mg of protein at a temperature of 0* to 25*C over a period of 5 to 20 hours, a long incubation time requiring low temperatures and a short incubation time requiring accordingly higher temperatures.