Charles W. Todd

Radioimmune assay of carcinoembryonic antigen

Abstract A double antibody technique for the detection and quantitation of carcino-embryonic antigen (CEA) in nanogram quantities is described. This antigen is characteristic of adenocarcinoma of digestive organs. The assay employs three γ-emitting radioisotopes. A computer program has been written to enable rapid calculation of results. The assay is of value in following the isolation and characterization of CEA and may also prove useful as a test for the detection of certain forms of cancer.

Isolation and characterization for carcinoembryonic antigen

Abstract Carcinoembryonic antigen (CEA) has been isolated from several tumors of the digestive tract. Comparisons of the products with one another and with CEA obtained from Dr. Gold have been made. CEA of two molecular sizes (6·8 S and 10·1 S) has been obtained, but the 6·8 S material is the one usually found. Considerable variation in yield has been observedp indicating that tumorsp may differ markedly in their content of CEA. Comparison of CEA isolated by Dr. Gold with material prepared by us has shown the two products to be immunologically indistinguishable.

Heterogeneity of the carcinoembryonic antigen

Abstract Fractionation of carcinoembryonic antigen (CEA) by ECTEOLA-cellulose chromatography and by isoelectric focusing indicated that the antigen was heterogeneous. The fractions obtained were variable in their content of sialic acid, amino sugars, and amino acids. All fractions were antigenically active and exhibited immunologic identity with respect to one another. Removal of the sialic acid with neuraminidase altered the electrophoretic migration and slightly decresed the heterogeneity of CEA as revealed by isoelectric focusing, but did not alter the activity of the material in the radioimmune assay.

Rabbit immunoglobulin allotype A12: a new agglutinating specificity.

A new allotype, A12, present on rabbit IgG is described. This allotype is detected by inhibition-of-agglutination techniques similar to those employed for the previously described allotype A11. The specificity is on the H chain in the hinge region of IgG. It can be associated with any of the H chain group a allotypes. A11 and A12 are transmitted by codominant autosomal genes.

Gas chromatography of methyl glycosides as their trimethylsilyl ethers : The methanolysis and re-N-acetylation steps☆

Abstract Gas chromatographic procedures for the analysis of carbohydrates in glyco-proteins frequently include methanolysis and re-N-acetylation steps. The usual methanolysis conditions were found to result in almost complete conversion of the methanolic HCl into methyl chloride. A neutral solution of methyl chloride in methanol was found capable of releasing fucose, sialic acid, and a portion of the galactose from a1-acid glycoprotein. During the re-N-acetylation of the amino sugars O-acetylation of the alditols commonly employed as internal standards occurs. Addition of the standards after the re-N-acetylation step avoided the problem. The complete procedure for the determination of both neutral and amino sugars has been simplified to involve only one transfer step prior to final gas chromatography.

Comparison of the allotypic combining sites on H-chains of rabbit IgG and IgM

Abstract Allotypic markers originally found on rabbit IgG have been shown to reside also on both the heavy and light chains of rabbit IgM. The light chains of IgG and IgM are the same, but the heavy chains of these two immunoglobulins differ extensively. The extent to which the allotypes present on the heavy chains are duplicated on the two immunoglobulins has been investigated by inhibition of phage neutralization and by a radiotracer technique. The inhibition by anti-allotype sera of phage neutralization by IgM antibody was shown to be essentially completely removed by prior absorption of the anti-allotype sera with IgG of the same heavy chain allotype. A preparation of IgM containing allotype a1 exhausted extensively but not completely, the precipitating ability of anti-a1 serum for a1, b4 IgG labeled with I125. It is concluded that in the anti-allotype sera investigated, essentially all of the antibodies directed against the allotypic sites present on IgM react with IgG, but not all the antibodies directed against the allotypic sites present on IgG are capable of reacting with IgM. The implications of these findings with regard to the genetic control of immunoglobulin synthesis and the evolution of the immunoglobulin genome are discussed.

Sensitive detection of 5-methylcytosine and quantitation of the 5-methylcytosine/cytosine ratio in DNA by gas chromatography--mass spectrometry using multiple specific ion monitoring.

Abstract A method combining gas chromatography and mass spectrometry (GC-MS) with multiple specific ion monitoring has been developed for the detection of 5-methylcytosine and the quantitation of the ratio of methyleytosine to cytosine in DNA. The trimethylsilyl derivatives of cytosine and 5-methylcytosine obtained from DNA hydrolysates are separated by isothermal elution on an OV-225 column and detected by specific ion monitoring in a DuPont 321 mass spectrometer. As little as 1.6 pmol of 5-methylcytosine in Φ χ 174 DNA can be detected, corresponding to a tenfold improvement in sensitivity over that obtained by conventional techniques. The ratio of 5-methylcytosine to cytosine of DNA from φ χ 174, calf thymus, salmon sperm, and several mouse tissues has also been determined. The results agree well with those obtained by other methods.

Characterization of allotype A11 in rabbits: A specificity detected by agglutination☆

Abstract Inhibition of agglutination techniques were used to characterize a new allotypic determinant, A11, of rabbit immunoglobulin. Agglutinator antiserum specific for the A11 determinant was used to agglutinate rabbit type F erythrocytes sensitized by coating with anti-F antibody bearing the A11 determinant. Agglutinator was prepared by injecting rabbits lacking the A11 determinant with IgG of the same group a and group b allotypes but possessing the A11 determinant. Several sensitizors for detecting the A11 specificity were prepared by immunizing rabbits possessing the A11 determinant with type F rabbit erythrocytes. The determinant was found to be associated with the IgG class of immunoglobulins by filtration and by DEAE chromatography. Studies with enzymatic fragments and subunits of IgG show that the A11 determinant is carried on the heavy ( H ) chain. Although the determinant was detectable on the (Fab′) 2 fragments obtained by pepsin hydrolysis of IgG, the requirement for the integrity of the inter- H chain disulfide bond precluded its detection on isolated H or light ( L ) chains. The results further suggest that certain structures on both H chains of a given IgG molecule require proper orientation for interaction with the anti-A11 serum.

Avidin as a precipitant for biotin-labeled antibody in a radioimmunoassay for carcinoembryonic antigen

Abstract Avidin is used in conjunction with polyethylene glycol to coprecipitate biotin-labeled carcinoembryonic antigen (CEA) antibody and the CEA-antibody complex together with biotin-labeled normal serum proteins. The procedure provides a reliable separation technique for a CEA radioimmunoassay. The technique is broadly applicable to assays employing immune precipitation.

Protein sequencing: Thermal conversion of thiazolinone derivatives of amino acids to thiohydantoins

Abstract A method for converting thiazolinone derivatives of amino acids to their thiohydantoin derivatives is described. Because this method proceeds through a thermally induced intramolecular rearrangement, the amide groups of glutamine and asparagine are not hydrolyzed. Special procedures are not required for the recovery of the derivatives of arginine and histidine. Finally, the thermal method is more rapid than the conventional aqueous conversion procedure.