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Biomedical subjects

E A Kabat

Publications and source records attributed to E A Kabat.

At least 19 recordsLinked to original sources
PubMed1992

Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.

The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.

Antibody Specificity↗
PubMed1992

Subgroups of Tcr alpha chains and correlation with T-cell function.

T-cell receptor (Tcr) alpha chains are classified into four subgroups (I, II, III, and miscellaneous) based on the amino acid residues at positions 61 and 62. Subgroup I has Gly Phe at these positions, subgroup II has Arg Phe, subgroup III has Arg Leu, and subgroup miscellaneous has several other combinations. Variability plots for subgroups I, II, and III sequences show higher values around positions 93-103, 105, 108, 111, 113, and 115, suggesting that these positions may interact with the processed antigen molecules. Smaller peaks are present at various other regions which may bind the major histocompatibility complex class I or II molecules. The patterns of variability within one subgroup are similar for all species, for human alone, and for mouse alone. These subgroup patterns appear much less complicated than patterns for sequences in all subgroups taken together, implying that subgroups may be related to Tcr functions. Among 83 mouse chains, 15 are from cytotoxic cells and 40 from helper cells. Of the 15 from cytotoxic cells, 11, 2, 0, and 2 are in subgroups I, II, III, and miscellaneous; and of the 40 from helper cells, 9, 16, 12, and 3 are in subgroups I, II, III, and miscellaneous, respectively. Thus, a correlation between sequence and function of Tcr alpha chains seems possible.

Amino Acid Sequence↗
PubMed1992

Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-alpha(1----6)dextrans with groove- or cavity-type combining sites.

The variables regions of three syngeneic anti-idiotypic antibodies (Ab2s) were cloned and sequenced. They are encoded by different VL genes, two are from different members of V kappa-Ox1 superfamily. The H chains are encoded by VH genes belonging to three different VH families, J558, Q52 and 7183. Together with a previous report from this laboratory, the nucleotide sequences of four Ab2s to anti-alpha(1----6)dextrans have been presented. They are derived from a number of unrelated germline genes, and differ from similar studies in anti-NP, anti-GAT and anti-Ars systems. Three of four Ab2s in the anti-alpha(1----6)dextran system appear to have D-D fusions, which has also been reported in several other Ab2s.

Amino Acid Sequence↗
PubMed1992

Possible use of similar framework region amino acid sequences between human and mouse immunoglobulins for humanizing mouse antibodies.

We have previously noted that a specific amino acid sequence could form the second framework region of human, mouse and rabbit immunoglobulin light chains, suggesting that this sequence has been preserved for 80 million years. Through divergent evolution, each species has acquired a different set of framework region sequences; however, these sets still share a few similar or identical amino acid sequences. In the present study, we have identified such sequences for all four framework regions between human and mouse immunoglobulin light and heavy chains. They may be useful in humanizing or reshaping mouse or rat antibodies for therapeutic applications in human patients.

Amino Acid Sequence↗
PubMed1979

Biosynthesis of a blood-group-I determinant reacting with anti-I Ma serum (group 1).

A blood-group-I determinant reacting specifically with anti-I Ma serum (group 1) has been produced biosynthetically by the action of a beta-galactosyl transferase isolated from human milk on a precursor glycoprotein produced by formic acid hydrolysis and beta-D-galactosidase action on blood-group H substance prepared from hog gastric mucosa.

Animals↗
PubMed1979

Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions.

Two sets of apparently conflicting data on the genes coding for the variable region are being accumulated. One suggests that the sets of nucleotides coding for the framework segments of immunoglobulin light and heavy (VL and VH) chains assort independently and are therefore germ-line minigenes which, together with sets of nucleotides coding for the complementarity-determining regions (CDR) or segments assemble to form complete variable (V)-region genes (15, 16, 33). The other, based on the findings with clones from 12-d-old embryo and adult mouse coding for V-regions, infer that the first three frameworks and the three complementarity-determining segments are already assembled as germ-line V-genes (17-21). It is now generally accepted that the J segment, which in the one instance sequenced (21) is made up of nucleotides coding for framework (FR)4 plus two residues of CDR3, is a minigene. An examination of sequences of human, mouse, and rabbit V-regions, assuming the latter hypothesis, indicates that individual framework sets would have to be present in many copies. The FR2 segment found in one human, 20 mice, and 13 rabbits would have to be present in at least 10/14 copies in the NZB, and 5/6 in the BALB/c mouse, and 12/13 in the rabbit. The X-ray crystallographic data show this region to be a loop, projecting out from the V-domain, capable of accommodating many substiutions and 12 and 8 alternative sequences for this FR2 segment have been found in mouse and rabbit VK chains with substitutions possible at 13 of the 15 positions. These alternative sequences occur much less frequently than the preserved FR2 segment. Thus, there is no basis in the protein structure to account for evolutionary stability of this FR2 segment if it occurs in so many copies in germ-line genes coding for residues 1-96, but its stability is easily explained if it were coded for by a separate germ-line minigene present as a single copy; the alternative forms could then have arisen by duplication and mutation of this minigene. Somatic assembly of the minigene segments for the three framework and three complementarity-determining segments during differentiation would account completely for our assortment data from which FR4 was inferred to be a minigene.

Amino Acid Sequence↗
PubMed1979

Action of 2-acetamido-2-deoxy-beta-D-hexosidase from Turbo cornutus on periodate-oxidized and Smith-degraded, blood-group HLeb and Lea substances from human, ovarian-cyst fluids.

Immunochemical and chemical studies were used to monitor and evaluate the structural changes produced by an enzyme from Turbo cornutus in periodate-oxidized and Smith-degraded, human blood-group substances from ovarian cysts. After the first step of periodate oxidation and Smith degradation, two blood-group substances, JS (HLeb and N-1 (Lea), were precipitated by mouse-myeloma S117 serum, specific for terminal, nonreducing, beta-D-linked 2-acetamido-2-deoxy-D-glucosyl groups, but not by type XIV antipneumococcal horse serum specific for terminal, nonreducing, beta-D-linked D-galactosyl groups. An exoglycosidase, 2-acetamido-2-deoxy-beta-D-hexosidase (beta-N-acetylhexosaminidase) from Turbo cornutus, split off 2-acetamido-2-deoxy-D-glucose amounting to 22.5 and 20.4% of the total weight of JS and N-1 blood-group substances, respectively. After enzymic digestion, both blood-group substances precipitated with type XIV serum, and did not precipitate with S117 serum. The findings are in agreement with the structure propsed for the water-soluble, blood-group substances [Lloyd and Kabat, Proc. Natl. Acad. Sci. U.S.A., 61 (1968) 1477]. Specific enzymes can be of value in structural studies when used in conjunction with sequential periodate oxidation and Smith degradation.

Acetylglucosamine↗
PubMed1979

Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies.

A sensitive and rapid method for the detection of monoclonal antibodies secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immunized mice and directly cloned in soft agarose containing selective medium; hybrid clones can be seen after a week. Nitro-cellulose filters that have been coated with a specific protein antigen, with antigen-coupled erythrocyte ghosts, or with other cells used as antigens are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted antibodies specific for the filter-bound antigen, the filter is removed and overlaid with a suspension of antigen-coupled erythrocytes that react with the adsorbed antibodies; after unbound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which antibody-forming clones are present in the agarose. Alternatively, the filter may be treated with radiolabeled antigen followed by autoradiography. The reliability and sensitivity of the method are demonstrated with alpha-(1 leads to 3)-specific antidextran myeloma J558, and the method's applicability is established by detecting hybridomas with specificities for sheep erythrocytes and for alpha-(1 leads to 3) dextran.

Animals↗
PubMed1979

Some sequence similarities among cloned mouse DNA segments that code for lambda and kappa light chains of immunoglobulins.

A comparison between the cloned mouse DNA segments that were found to code for the lambda and kappa light chains of immunoglobulins established that there were seven short nucleotide sequences, two of which matched 6 out of 7, two 7 out of 8, two 8 out of 9, and one 9 out of 10 bases; these sequences were located either at homologous amino acid positions or at positions displaced by four amino acids or less. They all occurred in the framework regions (FRs), five next to the complementarity-determining regions (CDRs). Three of these were unique and did not occur elsewhere in the immunoglobulin nucleotides sequenced thus far or in DNA's of phage phi X174, phage G4, or simian virus 40. Five could serve as sites of joining by recombination or insertion of CDR to FR segments, and the invariant tryptophan that is the first residue of the second FR might serve as a sixth. These sites are consistent with the mini-gene or insertional hypotheses for the generation of antibody diversity but could also serve as points of recognition for a mutator enzyme or could serve to limit somatic mutation to the CDRs.

Amino Acid Sequence↗
PubMed1979

A versatile immunoadsorbent capable of binding lectins of various specificities and its use for the separation of cell populations.

A procedure for cell fractionation using lectin-affinity chromatography is described. It consists of a single affinity adsorbent, hog gastric mucin blood group A+H substance covalently coupled to Sephadex or Sepharose, to which lectins of various specificities can bind. The complex formed, lectin in equilibrium hog A+H substance-Sephadex, then serves as an affinity probe for isolating and fractionating cells. The lectins from Ulex europaeus, Lotus tetragonolobus, Helix pomatia, Dolichos biflorus, and Phaseolus lunatus were used with the same blood group substance as adsorbent. The affinity columns retained erythrocytes with blood group specificity for the adsorbed lectin and thus fractionate cells in mixtures. Cells as well as lectins are eluted by specific sugar inhibitors. Mixtures of two kinds of cells can be separated when the proportion of the adsorbed cells is not too low.

Animals↗
PubMed1979

Binding constants of NZB myeloma antidextrans for dextrans and isomaltose oligosaccharides determined by affinity electrophoresis.

Association constants of dextrans (Ka) and oligosaccharides (Kia) from NZB myeloma antidextrans (PC3858 and PC3936) were studied by affinity electrophoresis. With linear dextrans or with those with a low degree of branching, Ka ranged from 2.7 X 10(3) to 5.4 X 10(4) ml/g for PC3858 and from 1.3 X 10(4) to 2.6 X 10(5) ml/g for PC3936. Completely linear alpha-(1 leads to 6)-linked dextrans, LD7 and D3, showed relatively high affinities for the two NZB antidextrans. With oligosaccharides, the Kia value increased as the number oa alpha-(1 leads to 6)-linked glycosyl residues increased. Isomaltoheptaose (IM7) showed the highest Kia (1.9 X 10(4) M-1 for PC3858 and 1.63 X 10(4) M-1 for PC3936), whereas isomaltose (IM2) had the lowest Kia (2.36 X 10(2)M-1 for PC3858 and 1.32 X 10(2)M-1 for PC3936). Pullulan and glycogen showed very weak affinity for PC3936, but they did not react at all with PC3858. These findings indicate that NZB myeloma antidextrans, PC3858 and PC3936, are specific for internal chains of alpha-(1 leads to 6)-linked dextrans. Data on the precision with which Ka and Kia can be determined are presented.

Animals↗
PubMed1978

Immunochemical studies on blood groups LXVI. Competitive binding assays of A1 and A2 blood group substances with insolubilized anti-A serum and insolubilized A agglutinin from Dolichos biflorus.

Competitive binding assays using 3H-labeled blood group A substance and insolubilized Dolichos biflorus lectin or human anti-A were carried out, measuring competition by blood group A1 and A2 glycoproteins, and by unabsorbed anti-A sera, and with these sera absorbed with the A1 and A2 glycoproteins. With Dolichos lectin specific for (formula: see text) A1 substances had about 11 times as many determinants as did A2 substances, but the slopes of the lines in the competitive binding assays were the same. With insolubilized anti-A, A2 substances gave lines of lower slopes. Although individual A1 populations varied in the amounts giving 50% inhibition in the assays, as did A2 substances, the slopes of the lines for the A1 substances were the same and always higher than the slopes of the lines for the A2 substances. Competitive binding assays with unabsorbed anti-A sera and with these sera absorbed with insoluble polyleucyl A1 and A2 substances showed that partial absorption of polyleucyl A1 substances left antibodies of lower slope in the supernate, whereas absorption with polyleucyl A2 substance left antibodies (anti-A1) having the same or an even higher slope than the unabsorbed sera. The findings indicate that human A1 and A2 glycoproteins differ in their determinants, and that A2 specificity is determined by the type 2 chain in which the A trisaccharide (formula: see text) is linked beta 1 leads to 4 to DGlcNAc, whereas the A1 specificity is determined by the type 1 chain in which this trisaccharide is linked beta 1 leads to 3 to DGlcNAc; most of the determinants in the glycoproteins have a second LFuc linked alpha 1 leads to 3 and alpha 1 leads to 4 to the DGlcNAc of the type 2 and type 1 chains, respectively.

ABO Blood-Group System↗