Patent EP0066786A2 successfully refreshed from Google Patents.
EP0066786A2
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Method of producing a colon specific antigen P tryptic peptide and anti-colon specific antigen P antibodies
Assignee: Goldenberg Milton David
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Filing: 1982-05-27
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Granted: —
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Expires: 2002-05-27
Abstract
Abstract A tryptic peptide is produced by partial tryptic digestion of purified, high molecular size colon-specific antigen-p (CSAp), to produce a lower molecular size antigen carrying the CSAp antigenic determinant. The tryptic peptide is used to produce monospecific anti-CSAp antibodies.
Inventors
- 👤 Milton David Goldenberg
- 👤 Dan Shochat
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 06:12
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/575; G01N33/5753; C; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K16/00; C07K16/18; A; A61; A61K; A61K39/00
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
118 words
A method for producing a purified immunologically active tryptic peptide having colon-specific antigen-p (CSAp) antigenic activity, comprising the steps of: (a) homogenizing CSAp-containing tissue in cold 0.3-3M aqueous salt solution: (b) centrifuging the resultant homogenate, removing the floating lipid layer, and recovering the aqueous supernatant; (c) fractionating the recovered aqueous supernatant by gel filtration, and recovering and concentrating at least one fraction containing a CSAp having a molecular weight of about 4 million; (d) enzymatically partially digesting the concentrated CSAp fraction with trypsin; (e) fractionating the partial tryptic digest, and recovering at least one fraction containing a tryptic peptide having CSAp antigenic activity, having a molecular weight of about 120,000 and having substantially no absorbance at 280 nm.
Claim 2
Independent Claim
118 words
A method for producing a purified immunologically active tryptic peptide having colon-specific antigen-p (CSAp) antigenic activity, comprising the steps of: (a) homogenizing CSAp-containing tissue in cold 0.3-3M aqueous salt solution: (b) centrifuging the resultant homogenate, removing the floating lipid layer, and recovering the aqueous supernatant; (c) fractionating the recovered aqueous supernatant by gel filtration, and recovering and concentrating at least one fraction containing a CSAp having a molecular weight of about 4 million; (d) enzymatically partially digesting the concentrated CSAp fraction with trypsin; (e) fractionating the partial tryptic digest, and recovering at least one fraction containing a tryptic peptide having CSAp antigenic activity, having a molecular weight of about 120,000 and having substantially no absorbance at 280 nm.
Claim 3
Dependent Claim
31 words
The method of claim 1, wherein the CSAp-containing tissue is normal human colon, human colonic tumor, human intestinalized gastric tumor, human pancreatic tumor, or cyst fluid from human ovarian mucinous cystadenocarcinoma.
Claim 4
Dependent Claim
14 words
The method of claim 1, wherein in step (a), the salt is sodium chloride.
Claim 5
Dependent Claim
18 words
The method of claim 3, wherein the aqueous salt solution further contains 0.05 - 0.2 M ammonium bicarbonate.
Claim 6
Dependent Claim
26 words
The method of claim 1, wherein in step (f), the centrifugation is effected at about 48,000 x g, at a temperature of 1-6°C, for 30-60 minutes.
Claim 7
Dependent Claim
32 words
The method of claim 1, wherein in step (d), the partial tryptic digestion is effected in an aqueous pH 7.5 - 8.5 buffer solution at about 37°C for 4.5 - 5.5 hours.
Claim 8
Dependent Claim
49 words
The method of claim 6, wherein the aqueous buffer is 0.01 - 3 M in NaCl and 0.05 - 0.5 M in NH 4 HC0 3 ; and the trypsin is present at a relative concentration of 0.5 - 7% by weight relative to the CSAp to be digested.
Claim 9
Dependent Claim
46 words
The method of claim 1, wherein in step (e) the fractions containing a tryptic peptide having CSAp antigenic activity are pooled and concentrated by ultrafiltration through ultrafiltration membranes or in dialysis membranes using a hygroscopic compound to absorb water passing to the outside of the membrane.
Claim 10
Dependent Claim
25 words
An immunologically active tryptic peptide having colon-specific antigen-p antigenic activity and having a molecular weight of about 120,000, produced by the method of claim 1.
Claim 11
Dependent Claim
134 words
In a method for producing substantially mono-specific antibodies to colon-specific antigen-p (CSAp), comprising the steps of: (a) raising antibodies in an animal by challenging the animal with an antigen having CSAp antigenic activity, and recovering antiserum from the animal; (b) purifying the antibodies by affinity chromatography on a human colon cancer immunoabsorbent column, and then on immunoabsorbent columns containing bound antigens from human spleen, lung and plasma extracts, from hamster liver, kidney and plasma extracts, and from phenol-water extracts of GW-39 tumor; thereby removing species-specific and crossreactive antibodies against normal tissue antigens, and antibodies against non-phenol-labile colon-specific antigens; and (c) freeing the resultant antibodies from any antibody aggregates formed during the purification procedure, and recovering monospecific anti-CSAp antibodies, the improvement wherein said antigen is a tryptic peptide produced by the method of claim 1.
References Cited (Backward Citations)
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