Abstract An improved method for the preparation of 125 I-labelled Protein A ( 125 I PA) of high specific and functional activity. 125 I PA has been used in combination with purified rabbit IgG (immunoglobulin G) bound to a solid support to develop a competitive binding assay capable of detecting Protein A or human, rabbit and guinea pig IgG at the nanogram level. Additionally, 125 I PA may be used to detect methotrexate, leucovorin and similar substances. 125 I PA has also been used to detect IgG anti-Forssman antibody bound to sheep erythrocytes and to line-1 and line-10 tumor cells and as an indirect assay for tumor associated antigen in the ascitic fluid of tumor-bearing guinea pigs. Additionally, an improved method of preparation of iodination of Protein A is utilized. This procedure used the Bolton-Hunter (1973) reagent of radioactive iodine in benzene which carrier is evaporated. The PA is added or attached while in an amino acid mixture and separation is per column utilizing Sephadex G-25 and VBS-gel.
A method of making 125 I-labelled Protein A (PA) for use utilizing as a tracer for the quantitative determination of proteins and haptens in physiological fluids comprising the preparation of 125 I PA from 125 I which has been evaporated from benzene and attached to Protein A in an amino acid mixture, followed by gel separation.
Claim 2
Independent Claim
56 words
A method of making 125 I-labelled Protein A (PA) for use utilizing as a tracer for the quantitative determination of proteins and haptens in physiological fluids comprising the preparation of 125 I PA from 125 I which has been evaporated from benzene and attached to Protein A in an amino acid mixture, followed by gel separation.
Claim 3
Dependent Claim
14 words
125 I-labelled Protein A which has been prepared by the process of claim 1.
Claim 4
Dependent Claim
54 words
A method of use of the product of claim 2 wherein 125 I PA is used with purified rabbit IgG bound to a solid support to develop a competitive binding assay or measurement of a substance selected from the group consisting of Protein A, human, rabbit and guinea pig IgG at the nanogram level.
Claim 5
Dependent Claim
24 words
A method of use of the product of claim 2 or 3 wherein the competitive binding assay detects human IgG at the nanogram level.
Claim 6
Dependent Claim
26 words
A method of use of the product of claim 2 wherein the 125 I PA is utilized to detect IgG anti-Forssman antibody bound to sheep erythrocytes.
Claim 7
Dependent Claim
32 words
A method of use of the product of claim 2 wherein the 125 I PA is utilized to detect line-1 and line-10 tumor cells as an indirect assay for tumor associated antigen.
Claim 8
Dependent Claim
20 words
A method of use of the product of claim 2 wherein the 125 I PA is utilized to detect methotrexate.
Claim 9
Dependent Claim
20 words
A method of use of the product of claim 3 wherein the 125 I PA is utilized to detect leucovorin.