Nagaradona Harindranath

Half-life of polyreactive antibodies

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The halflife of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.

Genetic analyses of restriction fragment length polymorphisms using high molecular weight DNA from sperm or lymphocytes embedded in agarose

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.

Generation of human monoclonal antibody-producing cell lines by Epstein-Barr virus (EBV)-transformation of B lymphocytes and somatic cell hybridization techniques

The use of Epstein-Barr virus to immortalize human B cells and generate monoclonal antibody-producting B cell lines is described.

Genomic sequence and organization of rabbit Tcrb constant region genes

We previously reported that some rabbits have three different copies of T-cell receptor b (Tcrb) constant region genes unlike man, mice, and rats who generally have two copies. Two of theseCß genes were found on an ≈ 14 kilobase (kb) and one on an ≈ 6 kbEco RI fragment. The gene on the 6 kb fragment is of ß2 type. A previously described portion of the 14 kb fragment appeared to have sequences characteristic ofCß1. We have now shown that the 6 kb fragment is adjacent to and 3′ of the 14 kb fragment. Furthermore, the second linked sequence ofCß gene present on the 14 kb fragment resembles to a large extent theCß2 gene present on the 6 kb fragment. Moreover, this secondCß gene has a 5′ cluster ofJß sequences resemblingJß2 of other species. However, exon 4 and the 3′ unranslated region (3′UT) are of the ß1 type. Mapping studies using southern analyses of both genomic DNA and the 14 kb clone have identified another cluster ofJß2 sequences 5′ of the third tandemCß2 gene present on the 6 kbEco RI fragment. Thus, the second gene on the 14 kb fragment appears to be a chimeric genomicTcrb gene that may have arisen by an unequal crossing-over event analogous to that which may have deletedCß1,Dß2, andJß2 in NZW mice.

Linked genetic markers of the rabbit ? light chain are not linked to the Tcr ? chain genes

In order to investigate linkage, we used serum allotypes of the two rabbit Cκ isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for Vκ, Cκ, and T-cell receptor Cβ. The inheritance of these genetic markers was studied through backcross and F2 matings. Southern analysis and hybridization of genomic DNA with a Cκ probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the κ1bbas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the κ2bas2 gene and lack of expression of the K2bas1 allotype. A Vκ probe detected a 1.3 kb Eco RI fragment linked to the presence of the κ1bbas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (Cβa or Cβb) detected with a Tcrβ chain probe segregated independently from Cκ allotypes and RFLPs. It has previously been found that Cκ and Cβ are also unlinked in man, whereas in the mouse they are linked at a distance of ≈8 centimorgans.