Aftab A. Ansari

A double antibody radioimmunoassay for mouse hemoglobins: Use of polyethylene glycol in conjunction with the second antibody

Abstract A double antibody radioimmunoassay (RIA) system is described for detection of small quantities of hemoglobins. Mouse (C57BL/6) hemoglobin and horse anti-mouse hemoglobin antiserum were used to develop the system. The first phase of the RIA, i.e., the initial reaction between the antigen and the antibody, was found to be complete within 24 h. The reaction proceeded better at 4°C than at 25°C. The second phase, i.e., separation of bound from unbound antigen, was achieved by precipitation with a second antibody (goat anti-horse IgG) and polyethylene glycol (PEG). A 50 g/l concentration of PEG was found to be best suited for the assay. Mixing of all the reagents together was found to decrease the binding as compared to the system in which second antibody and PEG were added after completion of the first phase. Maximum precipitation of the complex took place within 30 min after the addition of the second antibody and 1 h after the addition of 50 g/l PEG. The RIA system described here combines the conventional double antibody RIA with the PEG method. This method has decreased the amount of time necessary for precipitation from 24 h (or longer) to 1 h. Large molecular weight antigens could not be estimated in the conventional PEG method because of their insolubility in 200 g/l PEG utilized in the assay. The use of a low concentration of PEG along with the second antibody in the method described here allows the estimation of large molecular weight antigens. This double antibody-PEG method has a general applicability for small as well as large molecular weight antigens.

Comparison of double antibody and solid-phase radioimmunoassays

Abstract Two commonly used radioimmunoassay (RIA) procedures, double antibody assay and solid-phase assay, have been compared using the same reagents and conditions. Mouse hemoglobulin—horse anti-mouse hemoglobulin system was chosen for this study. The antibody was found to have more binding capacity in soluble form than in the Sepharose-bound form. The inhibition curves in both the assay systems were shifted to higher inhibitor concentration at higher control binding levels. At 40% control binding level, the double antibody RIA was found to be 2.7 times more sensitive than the solid-phase RIA. The reason for this most probably lies in the lower binding capacity of the Sepharose-bound antibody which necessitates the use of higher amounts of the antibody to reach 40% control binding.

Immunochemical studies of the a allotypes of rabbit heavy chain variable regions—I.: Comparisons of a3 allotypic determinants on normal IgG and IgG of limited heterogeneity by radioimmunoassays with purified labeled anti-allotype antibodies

Abstract Radioimmunoassays using purified and labeled anti-a3 antibodies were developed and used to study a3 allotypic determinants on normal IgGs and anti-polysaccharide antibodies of restricted heterogeneity. Our results provide additional evidence for differences in fine specificity of al anti-a3 and a2 anti-a3. Insights into the causes and nature of allotypic heterogeneity have come from the recognition that some a3 allotypic determinants on normal IgGs and restricted IgGs may bind anti-a3 antibodies with different average association constants. A purified fraction of a2 anti-a3 which cross-reacts with a1 IgG was found advantageous for assaying an a3 IgG of restricted heterogeneity. The use of this fraction (a2 anti-a3 CRI) for assays of peptides derived from the a3 heavy chain is described in a subsequent paper.

Immunological relatedness of two isozymes of 3-phosphoglycerate kinase from the mouse

Two isozymes of 3-phosphoglycerate kinase (ATP:3-phospho-D-~ycerate 1 -phosphotransferase, EC 2.7.2.3, PGK) are known to be present in mammalian tissues. PGK-I ** is found in all somatic tissues, and its structural locus is X-linked in humans [l] , kangaroos [2] and mice [3] .PGK-2 has been detected only in the sperm and testis of a number of mammals; however, there are exceptions where it has been found in somatic cells (kangaroo, dog, fox [4] ). Alleles determining electrophoretic variation map to chromosome 17 in the mouse [.5] . These two isozymes were purified in our laboratory, and their biochemical properties compared [6]. They were highly similar in their biochenlical properties even though the two loci must have been separated for over 100 million years [7]. To further explore the relatedness of these isozymes, antisera against the two PGK isozymes were produced separately, and their immunological properties were compared. The results of these studies suggest some antigenic relatedness between these two PGK isozymes.

An evaluation of effectiveness of bio-glas in molecular sieving of polypeptides in guanidine hydrochloride

Abstract The effectiveness of Bio-Glas 200 and 500 for separation and molecular weight estimation of polypeptide chains in 6 m guanidine hydrochloride was investigated. The distribution coefficients of 10 proteins after reduction and alkylation were determined. We found that the columns gave good calibration curves when pure proteins were passed individually. But the resolving power of Bio-Glas 200 and 500 in this solvent was very poor, apparently because of their high matrix volume and low inner volume.

Immunological comparison of mouse hemoglobins.

Immunological properties of several mice hemoglobins bearing α chains 1 or 4 and β chains s, dmaj or pmin were compared using radioimmunoassays that involved inhibition by these hemoglobins of a reaction between125I-labeled C57BL/6 hemoglobin (α chain 1, β chain s) and horse antibody against C57BL/6 hemoglobin. SJL hemoglobin (α chains 1 and 4. β chains) and C57BL/6 hemoglobin were found to have identical relative association constants,K0(rel). The dmaj hemoglobins from DBA/2 and AU/Ss mice (α chain 1, β chain dmaj) were 3.3 to 3.6 times less reactive while the pmin hemoglobin from AU/Ss mice was found to be 17 times weaker than C57BL/6 hemoglobin. These immunological differences correlate with the amino acid sequence differences in the β chains.

Studies of mouse immunoglobulin allotypes by reverse plaque assay: detection of spleen cells secreting immunoglobulins with specific allotype in C57BL/6J mice.

Abstract A reverse hemolytic plaque assay for the detection and enumeration of mouse spleen cells secreting immunoglobulins bearing a particular allotypic specificity is described. Sheep red blood cells (SRBC) coated with protein A or anti-mouse gamma globulin antibody were employed as indicator cells and an anti-allotype antibody was used as developer. A comparison of the efficiency of protein A, goat anti-mouse or rabbit anti-mouse gamma globulin antibody-coated SRBC as indicator cells in the plaque assay indicated that the rabbit anti-mouse gamma globulin-coated SRBC gave the best results in terms of number and morphology of the plaques. The number of indicator cells in the assay mixture also significantly affected the quality of the plaques formed. When the mouse spleen cells were assayed with the indicator cells and an anti-allotypic antibody as developer in presence of complement in a liquid medium, only those cells secreting the immunoglobulin of the given allotypic specificity formed hemolytic plaques.

Purification of fluorescein-labeled specific anti-hemoglobin antibody using cross-linked immunoabsorbent

Abstract Fluorescein-labeled monospecific anti-hemoglobin antibodies are useful tools for studying hemoglobin expression and somatic point mutation. The yield of monospecific antibodies is extremely low and the purification of these antibodies using hemoglobin immunoabsorbents is further complicated by the fact that hemoglobin ‘leaks’ out from the immunoabsorbent during elution of the antibody with acid medium contaminating the eluted antibody. Two improvements are reported in this paper for the purification of fluorescein-labeled goat anti-mouse hemoglobin antibody specific for DBA/2J hemoglobin that does not react with C57BL/6J mouse hemoglobin: (1) The entire gamma globulin fraction of anti-hemoglobin antiserum was labeled with the fluorochrome before proceeding for the absorption and purification steps (in contrast to the conventional methods where the antibody is first purified followed by the fluorochrome labeling). This modification was expected to yield only the functionally active labeled antibody preparation devoid of molecules that may have undergone denaturation during the labeling procedure. Also, this modification eliminated the need of carrying out the labeling procedure with a small amount of the precious purified antibody thus decreasing the antibody loss. (2) Instead of conventional immunoabsorbents (hemoglobin coupled to CNBr-Sepharose), immunoabsorbents cross-linked with glutaraldehyde were used. The eluted antibody was free of any hemoglobin contamination. The antibody thus purified was found to specifically react with fixed red blood cells from DBA/2J mice but not cells from C57BL/6J mice. The method described in this paper should be of general applicability in the preparation of monospecific antibodies.

Lactate dehydrogenase-C mRNA: Its isolation and invitro translation

Abstract Lactate dehydrogenase-C (LDH-C) mRNA was purified from DBA 2 mouse testes and translated in vitro . First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Immunochemical studies of the a allotypes of rabbit heavy chain variable regions--II. Immunological properties of peptides from variable regions of heavy chains of limited heterogeneity and a3 allotype.

Abstract Immunological properties of peptides from two different heavy chains of a3 allotype and restricted heterogeneity were studied using radioimmunoassays that involved inhibition by these peptides of a reaction between 125 I labeled anti-a3 antibodies and Sepharose-bound a3 IgG. A purified fraction of a2-anti-a3 which cross reacts with at IgG was used for the assays of peptides from one of the heavy chains (AH80-5). The picomoles of various inhibitors required for 50% inhibition of the a3-anti-a3 reactions were determined and the corresponding Δ G 0 values were calculated. Citraconylation of the lysine side chains (CH) markedly affected the inhibitory activity of one of the two heavy chains (AH80-5) and had a lesser effect [Δ(Δ G 0 ) 1.4 kcal/mole] on the other. In both instances when the lysines of the tryptic peptides were deblocked by exposure to low pH, we observed gains in activity (1.3–1.7 kcal/mole). Although some of the primary structure which contributes to some a3-antigenic determinants may have been removed by tryptic digestion, the immunopeptides which we recovered inhibited at least 60% of the maximal binding of purified, labeled a 2 anti-a3 antibodies to a3 IgG. Thus major antigenic determmant(s) recognized by these particular anti-a3 antibodies, were retained on the portions of V H region recovered from the tryptic digests. It is probable that losses in conformation contributed to the changes in Δ G 0