Kabat Database Research Articles

An scFv phage clone that binds to human β2-microglobulin

Using phage display technology, a novel mouse scFv phage clone D1 was generated from mice immunized with single-chain HLA-A2, the human MHC class I molecules. D1 recognized a determinant in β2-microglobulin. It reacted with β2-microglobulin associated with the heavy chain of HLA-A2, recombinant β2-microglobulin made in Escherichia coli or β2-microglobulin prepared from human urine. The heavy chain variable region of D1 belongs to gene family VII and the light chain variable region belongs to the kappa chain gene family XI according to the Kabat database.

Immunoglobulin variable regions usage by B-lymphocytes infiltrating a human breast medullary carcinoma

Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade. We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour. A single cell suspension was obtained from a tumor specimen by enzymatic digestion. VH, V κ, and V λ regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products. They were then cloned and sequenced. Analysis of 9 VH, 5 V κ, and 10 V λ sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor. The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences. Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.

Preferred CDRH3 lengths for antibodies with defined specificities.

The third complementarity determining regions of heavy chain variable regions (CDRH3) confer the fine specificities of antibodies. The CDRH3 length distribution of all human or all mouse antibodies appears to be more or less random, with the human distribution having a wider spread. Since the Kabat database of aligned amino and nucleotide sequences of proteins of immunological interest has grown at least 4 times during the past 7 years, we attempt to study CDRH3 length distributions of human and mouse antibodies with defined specificities, where enough complete and distinct CDRH3 sequences are available. For mouse antibodies against small haptens or carbohydrates, a preferred length usually dominates for a given specificity. There are not enough human sequences of small hapten or carbohydrate antibodies for a similar study. Both human and mouse anti-protein antibodies have wider ranges of CDRH3 length distributions, probably due to the presence of different antigenic epitopes on the protein molecules, and thus many different antibodies. For anti-DNA antibodies, both humans and mice exhibit even wider length distributions, since DNA can be double stranded, single stranded, supercoiled, etc.

A very limited number of keywords (main patterns) describes all sequences of the human variable heavy (VH) and kappa (Vkappa) domains.

Sequences of the variable heavy (VH) and kappa (Vkappa) domains of Ig structures were divided into 21 fragments that correspond to strands, loops, or parts of these structural units of the variable domains. Amino acid sequences of fragments (termed "words") were collected from the 1,172 human heavy and 668 human kappa chains available in the Kabat database. Statistical analysis of words of 17 fragments was performed (fragments that comprise the complementary determining regions' fragments will not be discussed in this paper). The number of different words (those with different residues in at least one position) ranged, for various fragments, from 11 to 75 in the kappa chains, and from 23 to 189 in the heavy chains. The main result of this study is that very few keywords, or main patterns of words, were necessary to describe over 90% of the sequences (no more than two keywords per fragment in the kappa and no more than five per fragment in the heavy chains). No identical keywords were found for different fragments of the variable domains. Keywords of aligned fragments of the VH and Vkappa domains were different in all but two instances. Thus, knowing the keywords, one can determine whether any given small part of a sequence belongs to a heavy or kappa chain and predict its precise localization in the sequence. In addition, by using all of the keywords obtained through analysis of the Kabat database, it was possible to describe completely the sequences of the human VH and Vkappa germ-line segments.

Cloning, expression and characterization of a single‐chain antibody specific for the herbicide atrazine

The antigen‐binding domain of an antibody specific for the herbicide atrazine was cloned from hybridoma 4063–21–1 and expressed as a single‐chain antibody (scAb) in the vector pPM1‐His. The observed heavy and light chain gene sequences were consistent with those in subgroup VH I (B) and VK TV, as defined by the Kabat database of sequences. Subsequent expression in Escherichia coli strain XL‐1 Blue produced up to 0.3 μg of functional single‐chain antibody, from periplasmic protein preparations, per millilitre of culture. The scAb was purified as a monomer by immobilized metal chelate affinity chromatography via a hexa‐histidine tail or as a dimer by antibody affinity chromatography. The functionality and specificity of the recombinant antibody was confirmed by binding to an atrazine‐bovine serum albumin conjugate and free atrazine and simazine in a competition ELISA. This is the first example of the production and purification of a recombinant antibody that retains antigen binding for a triazine herbicide.