In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

Concerted evolution is often invoked to explain the diversity and evolution of the multigene families of major histocompatibility complex (MHC) genes and immunoglobulin (Ig) genes. However, this hypothesis has been controversial because the member genes of these families from the same species are not necessarily more closely related to one another than to the genes from different species. To resolve this controversy, we conducted phylogenetic analyses of several multigene families of the MHC and Ig systems. The results show that the evolutionary pattern of these families is quite different from that of concerted evolution but is in agreement with the birth-and-death model of evolution in which new genes are created by repeated gene duplication and some duplicate genes are maintained in the genome for a long time but others are deleted or become nonfunctional by deleterious mutations. We found little evidence that interlocus gene conversion plays an important role in the evolution of MHC and Ig multigene families.

https://www.pnas.org/content/pnas/94/15/7799.full.pdf

Evolution by the birth-and-death process in multigene families of the vertebrate immune system

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 x 10(9) members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody-antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

https://www.pnas.org/content/pnas/95/11/6157.full.pdf

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be ∼6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.

https://rupress.org/jem/article-pdf/188/11/2151/1117349/98-1478.pdf

The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus

Sequence and evolution of the human germline V lambda repertoire.

The maps of the human immunoglobulin heavy-chain and kappa light-chain loci have recently been completed. We have now completed a map of the human lambda locus (IGL) located on chromosome 22q11.2. We mapped 52 V lambda genes from 10 V lambda families and 7 J lambda and C lambda genes on a 1140 kb contig constructed from eight YACs and 129 cosmid clones. The V lambda genes are arranged within 800 kb. Genes of the different V lambda families are organized in three clusters, V lambda II and III families (cluster A); V lambda I, V, VII and IX families (cluster B); V lambda IV, VI, VIII and X families (cluster C), in contrast to the dispersed organization of the different VH and V kappa families within the human VH and V kappa loci. We note that the most frequently used V lambda families (V lambda II and III) are proximal to the J lambda and C lambda genes. The VpreB gene, encoding part of the surrogate light chain, the GGT2 gene and the BCRL4 pseudogene were also mapped within the lambda locus.

I M Tomlinson

Papers

Sequence and evolution of the human germline V lambda repertoire.

Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2