Soluble HLA Technology

Soluble HLA Class II molecules are glycoproteins as well, however, they have an α and β chain where the α1 and β1 regions of the chains come together to make a membrane-distal peptide-binding domain, while the α2 and β2 regions, the remaining extracellular parts of the chains, form a membrane-proximal immunoglobulin-like domain. The Class II trimeric protein complex is approximately 61kDa in size consisting of a 27 kDa beta chain and a 26 kDa alpha chain with a 2 kDa peptide (15 to 24mer) in the groove. Finally, it is important to note that Class II molecules have a Leucine zipper tail at the carboxy end of the α2 and β2 region to increase stability and support assembly of the molecule within the cell.

Since HLA Class II DQ and DP molecules function as dimers containing two protein subunits, the alpha chain and beta chain, four different combinations are possible, as each individual receives one set from the maternal chromosome and the other set from the paternal chromosome making such a person a double heterozygote. Naturally, that dramatically increases the number of possible protein combinations in each individual. To offer our customers a more satisfactory solution to solve this complexity, Pure Protein formulated combinatorial matrices consisting of a selection of specific alpha and beta chains with the goal to provide combinations where each, alpha or beta chain are represented at least twice. However, there are factors that limit this variation. Some combinations seem not to be made. Furthermore, haplotypes are naturally observed where a set of variations tend to be inherited or linked together, which further reduces the combinatoric possibilities. As of 2020, Pure Protein has over 270 established cell lines in inventory from which HLA proteins are produced from.

Because the genes encoding the HLA molecules are highly polymorphic, they require a systematic nomenclature. Each HLA allele name has a unique number corresponding to up to four sets of digits separated by colons. The length of the allele designation is dependent on the sequence of the allele and that of its nearest relative. Our sHLA alleles all receive a four-digit name, which corresponds to the first two fields. For researchers in the transplant field, it is important to note that the digits before the first colon describe the type, and often corresponds to the serological antigen carried by an allotype. Alleles whose numbers differ in the two fields naturally differ in one or more nucleotide substitutions that change the amino acid sequence of the encoded protein.

Although the number of individual HLA alleles that have been identified is large, approximately 40% of these alleles appear to be unique, having only been identified in single individuals while roughly a third of alleles have been reported more than five times in unrelated individuals. Because of this variation in the rate at which individual HLA alleles are detected, attempts have been made to categorize alleles at each expressed HLA locus in terms of their prevalence. The result is a catalog of common and well-documented HLA alleles, as well as a catalogue of rare and very rare HLA alleles. Rare alleles are defined as those that have been reported one to four times, and very rare alleles as those reported only once. We are displaying these frequencies varying by geographic region. Data were obtained from the Allele frequency net database 2020. http://www.allelefrequencies.net/hla6006a.asp

Using a special color tracking system, the presence of the Class II B chain constructs can be confirmed by tracking the co-expression of a green fluorescence protein (gfp), whereas the A chain expression is monitored through a Mcherry protein (mc) as the fluorescence tracker. These colored proteins are co-produced within the transfectant cells, but are not part of the final sHLA molecule, nor are they secreted.