Process for isolating a fibrin stabilizing factor from human placenta

Claim 1

A PROCESS FOR ISOLATING A FIBRIN-STABILIZING FACTOR WHICH COMPRISES: A. EXTRACTING HUMAN PLACENTAS WITH A DILUTE AQUEOUS SODIUM CHLORIDE SOLUTION AT A TEMPERATURE BETWEEN 0*C. AND 10*C. AND REMOVING SOLID CONTAMINANTS FROM THE EXTRACT, B. ADDING A DILUTE AQUEOUS SOLUTION OF DIAMINO-ETHOXYACRIDINE LACTATE TO SAID EXTRACT AT A TEMPERATURE OF 10*C. AND AT A PH BETWEEN 5.0 AND 7.5 IN AN AMOUNT FURNISHING ABOUT 6 TO 10 GRAMS OF SAAID LACTATE PER 100 GRAMS OF PROTEIN CONTENT IN SAID EXTRACT TO FORM A FIRST PRECIPITATE, AND ISOLATING SAID FIRST PRECIPITATE, C. DISSOLVING SAID FIRST PRECIPITATE AT A TEMPERATURE BETWEEN 10*C. AND 25*C. BY TREATING IT WITH FROM 1.5 TO 3 PERCENT OF AN ALKALI MATEL CHLORIDE IN A NEUTRAL AQUEOUS MEDIUM, D. ADDING FROM 12 TO 20 PERCENT BY VOLUME OF A LOWER ALCOHOL TO SAID AQUEOUS CHLORIDE MEDIUM AT A TEMPERATURE FROM 10*C. TO 25*C. TO FORM A SECOND PRECIPITATE, AND ISOLATING AND DISCARDING SAID SECOND PRECIPITATE, E. CLARIFYING THE REMAINING SUPERNATANT LIQUID, DILUTING IT WITH WATER TO AN ALKALI METAL CHLORIDE CONCENTRATION BELOW 2 PERCENT, ADDING THERETO FROM 12 TO 20 PERCENT BY VOLUME OF A LOWER ALCOHOL AT A TEMPERATURE BETWEEN -5*C. AND 5*C. AT A PH FROM 5.5 TO 6.5 TO FORM A THIRD PRECIPITATE, AND ISOLATING SAID THIRD PRECIPITATE, F. SUSPENDING SAID THIRD PRECIPITATE AT ABOUT 0*C. IN A NEUTRAL ETHYLENE DIAMINE TETRAACETIC ACID/ALKALI METAL HYDROXIDE BUFFER CONTAINING FROM 0.5 TO 3 PERCENT OF SODIUM CHLORDE, 3.2 TO 2 PERCENT OF GLUCOSE, AND 0.05 TO 0.2 PERCENT OF SODIUM AZIDE, AND REMOVING ANY INSOLUBLE MATTER, G. GEL FILTERING THE RESULTANT SOLUTION IN A CHROMATOGRAPHIC COLUMN FILLED WITH A GEL FILTRATION MEDIUM WITH A FRACTIONATION RANGE FOR LINEAR MOLECULES BETWEEN MOLECULAR WEIGHTS OF 1000 TO 150,000 AND COLLECTING THE ACTIVE FRACTIONS HAVING AN ACTIVITY OF MORE THAN 10 UNITS OF FIBRIN-STABILIZING FACTOR AND COMBINING THEM, H. CHROMATOGRAPHING SAID COLLECTED ACTIVE FRACTIONS ON DIETHYLAMINOETHYL CELLULOSE USING A NEUTRAL 0.3 TO 1.0 PERCENT SOLUTION OF SODIUM CHLORIDE CONTAINING FROM 0.05 TO 0.5 PERCENT OF EPSILON-AMINO-CAPROIC ACID AS THE ELUANT, AND I. DIALYZING THE ELUATE AGAINST A 0.01 TO 0.02 PERCENT NEUTRAL DODIUM PHOSPHATE BUFFER CONTAINING 0 TO 2 PERCENT OF GLUCOSE AND RETAINING THE DIALYZED ELUATE.