Antigen In Radioimmunoassay Research Articles

Studies on Biologically Active Pteridines. IV. Synthesis of Several Biopterin Derivatives as an Antigen in Radioimmunoassay for Biopterin

Abstract The synthesis of biopterin, d(and l)-erythro-neopterins, and their derivatives having a substituent at the 2-amino group via the 2-methylthio analogues is described. The 2-(methylthio)pteridines were synthesized by condensation of 4,5-diamino-6-hydroxy-2-(methylthio)pyrimidine with 5-deoxy-l-arabinose and d(and l)-arabinose phenylhydrazones, followed by oxidation with K3[Fe(CN)6] and O2 in the presence of KI in an acidic solution, respectively. Aminolysis of these thioethers with ammonia and other amines in the presence of acetic acid gave the title compounds.

Double-antibody method and the protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay.

We compared the protein-A-bearing Staphylococcus aureus immunoadsorbent to the double-antibody precipitation method for separating bound and free radiolabeled antigen in a radioimmunoassay. With human albumin (antigen) and rabbit anti-human albumin (antibody) as a model, our results indicate that formalin-fixed, heat-killed S. aureus cells could be substituted for the double-antibody precipitation method. Ease of preparation and high adsorption capacity of protein-A-bearing S. aureus for most mammalian IgG make this method economical and time saving.

Application of the parallel line bioassay method to quantitative determination of the HBs antigen in radioimmunoassay.

Precise and reproducible quantitative determination of hepatitis B surface antigen by applying the parallel line bioassay method was proposed. In this method, the value of any given sample can be expressed in terms of a relative value to a fixed standard or reference preparation. Decrease in antigenic reactivity due to the lowered protein content could be prevented by using 0.02% human albumin in physiological saline solution as the diluent. The use of such stabilizer(s) should be considered in the dilution or storage of samples containing HBs antigen.

Probability analysis of the interaction of antibodies with multideterminant antigens in radioimmunoassay: application to the amino terminus of the β chain of hemoglobin S

A simple theory, based on probability, is developed for the analysis of the interaction of multideterminant antigens with multispecific antisera in radioimmunoassays. The theory is completely general except for the assumptions that the determinants be unique and bind antibodies independently of one another. The analysis shows that the shape of the curve of bound/free as a function of the antigen concentration is very sensitive to the multiplicity of determinants. The predictive ability of the theory is illustrated for the case of antibodies to subregions of the N-terminal third of the beta chain of sickle hemoglobin, studied using antisera fractionated on affinity chromatographic columns of synthetic peptides. The implications for obtaining quantitative binding data by radioimmunoassay for natural antigens, which almost universally have more than one antigenic determinant on the same molecule, are discussed.

The use of microfilters as an alternative to centrifugation in radioimmunoassay

A microfilter was designed, together with a mechanical filtration apparatus for the separation of “antibody-bound” and “free” antigen in radioimmunoassay. A comparison of the results obtained using both filtration and centrifugation was carried out and the optimum incubation times, reaction conditions and washing volume for use with the microfilter were studied. The use of microfilters in radioimmunoassay gave reproducible results and offered a convenient means of automating the separation procedure.

A comparaive study of the effects of chemical modification on the immunochemical and optical properties of human plasma low-density lipoprotein(s) and apoproteins.

1. The structure of human plasma low-density lipoprotein(s) [LD lipoprotein(s)] was investigated by several immunological and optical techniques. The effects of delipidation and of chemical modification by 3-carboxypropionylation, acetylation, diazotization and amidination were examined. A sensitive double-antibody radioimmunoassay for human LD lipoproteins is presented and is used to assess the extent of immunochemical modification. A computer best-fit analysis is used to analyse circular-dichorism (c.d.) spectra. These methods permit comparisons of the relative effects of chemical modification and delipidation of LD lipoprotein under similar experimental conditions. 2. 3-Carboxypropionylation, acetylation or diazotization produces qualitative and quantitative changes in the immunochemical properties of LD lipoprotein. Amidination causes minor changes detected by radioimmunoassay but not by double-diffusion experiments. In general, the order of effectiveness in displacing (125)I-labelled LD lipoprotein is amidinated>diazo or acetyl>3-carboxypropionyl derivatives. 3. The order of the extent of conformational alteration induced in apoLD lipoprotein and LD lipoprotein, as judged by c.d. analyses, was 3-carboxypropionylation>diazotization>acetylation or amidination. 4. Delipidation of LD lipoprotein results in immunological alterations that are qualitatively detected by antisera to LD lipoprotein. Four of five antisera to apoLD lipoprotein form precipitin lines of identity between native LD lipoprotein and apoLD lipoprotein in double-diffusion experiments. An anti-(apoLD lipoprotein) serum that forms precipitin lines of complete identity between LD lipoprotein and apoLD lipoprotein reacts differently with these two antigens in radioimmunoassay. ApoLD lipoprotein is only one-fourth to one-half as effective as LD lipoprotein, on a protein basis, in the displacement of (125)I-labelled LD lipoprotein from this anti-(apoLD lipoprotein). 5. Conformational analysis indicates that apoLD lipoprotein retains a high proportion of the structural integrity of the native lipoprotein. Delipidation induces a small decrease in the content of beta-structure and a small increase in disordered structure, without greatly affecting the alpha-helical content. Chemical modification produces more severe conformational changes of apoLD lipoprotein than of LD lipoprotein. Computer analysis of the c.d. spectra of apoLD lipoprotein indicates that addition of high concentrations of sodium decyl sulphate abolishes most of the beta-conformation concomitant with increases in alpha-helical and disordered structure. 6. There is parallelism between the alteration of the charge of LD lipoprotein and apoLD lipoprotein and the extent of immunochemical and conformational changes.