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Functional recombinant MHC class II molecules and high-throughput peptide-binding assays.

Justesen S, Harndahl M, Lamberth K, Nielsen LL, Buus S - Immunome Res (2009)

Bottom Line: The affinities of the best binders were found to be in the low nanomolar range.Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately.We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. sjust@sund.ku.dk

ABSTRACT

Background: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged.

Results: We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately.

Conclusion: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

No MeSH data available.


Related in: MedlinePlus

Checkerboard titrations of DR4 α and β chains analyzed by LOCI. Various concentrations of urea denatured α and β chains incubated with (A) and without (B) 2 μM HA306–318 in refolding buffer pH 8. After 48 h of incubation, the assay was analyzed by LOCI using L243 antibody coupled to acceptor beads and streptavidin coupled donor beads. (C) Signal to noise ratios were calculated from samples with and without HA306–318. In this case, concentrations of 6.3 nM α chains and 1.3 nM β chains were determined to be optimal.
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Figure 7: Checkerboard titrations of DR4 α and β chains analyzed by LOCI. Various concentrations of urea denatured α and β chains incubated with (A) and without (B) 2 μM HA306–318 in refolding buffer pH 8. After 48 h of incubation, the assay was analyzed by LOCI using L243 antibody coupled to acceptor beads and streptavidin coupled donor beads. (C) Signal to noise ratios were calculated from samples with and without HA306–318. In this case, concentrations of 6.3 nM α chains and 1.3 nM β chains were determined to be optimal.

Mentions: We have recently developed a HTS assay for peptide-MHC class I binding using a non-radioactive bead-based homogenous proximity assay: Luminescent Oxygen Channeling Immunoassay [13]. As one tag, we used the biotin group engineered unto the β chain. As the other tag, we used conformation-dependent MHC class II specific antibodies (L243 for DR; 9,3F10 for DQ; B7/21 for DP and 14.4.4s for I-Ed). As for the development of the ELISA, two-dimensional titrations of MHC class II α and β chain concentrations were performed and diluted into refolding buffer containing, or not containing, an excess concentration of appropriate binding peptide. After 24 h incubation at 18°C, a mixture of streptavidin-donor beads and antibody coupled acceptor beads were added to the reaction wells and analyzed (see Figure 7). It was evident that peptide-MHC class II complexes required both MHC class II α and β chains, and were generated in a strictly peptide-concentration dependent fashion. In this case, optimal α and β chain concentrations were found to be around 6 nM and 1 nM, respectively. Optimal conditions in terms of whether long or short versions of the different molecules should be used, at what concentrations the α and β chain proteins should be used, and the optimal pH were subsequently determined (additional file 1, Table 2 and Figure 4). Strikingly, signal to noise ratios observed with the LOCI technique were consistently better than those observed with the more traditional ELISA technique (e.g. compare Figure 5C with Figure 7C).


Functional recombinant MHC class II molecules and high-throughput peptide-binding assays.

Justesen S, Harndahl M, Lamberth K, Nielsen LL, Buus S - Immunome Res (2009)

Checkerboard titrations of DR4 α and β chains analyzed by LOCI. Various concentrations of urea denatured α and β chains incubated with (A) and without (B) 2 μM HA306–318 in refolding buffer pH 8. After 48 h of incubation, the assay was analyzed by LOCI using L243 antibody coupled to acceptor beads and streptavidin coupled donor beads. (C) Signal to noise ratios were calculated from samples with and without HA306–318. In this case, concentrations of 6.3 nM α chains and 1.3 nM β chains were determined to be optimal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690590&req=5

Figure 7: Checkerboard titrations of DR4 α and β chains analyzed by LOCI. Various concentrations of urea denatured α and β chains incubated with (A) and without (B) 2 μM HA306–318 in refolding buffer pH 8. After 48 h of incubation, the assay was analyzed by LOCI using L243 antibody coupled to acceptor beads and streptavidin coupled donor beads. (C) Signal to noise ratios were calculated from samples with and without HA306–318. In this case, concentrations of 6.3 nM α chains and 1.3 nM β chains were determined to be optimal.
Mentions: We have recently developed a HTS assay for peptide-MHC class I binding using a non-radioactive bead-based homogenous proximity assay: Luminescent Oxygen Channeling Immunoassay [13]. As one tag, we used the biotin group engineered unto the β chain. As the other tag, we used conformation-dependent MHC class II specific antibodies (L243 for DR; 9,3F10 for DQ; B7/21 for DP and 14.4.4s for I-Ed). As for the development of the ELISA, two-dimensional titrations of MHC class II α and β chain concentrations were performed and diluted into refolding buffer containing, or not containing, an excess concentration of appropriate binding peptide. After 24 h incubation at 18°C, a mixture of streptavidin-donor beads and antibody coupled acceptor beads were added to the reaction wells and analyzed (see Figure 7). It was evident that peptide-MHC class II complexes required both MHC class II α and β chains, and were generated in a strictly peptide-concentration dependent fashion. In this case, optimal α and β chain concentrations were found to be around 6 nM and 1 nM, respectively. Optimal conditions in terms of whether long or short versions of the different molecules should be used, at what concentrations the α and β chain proteins should be used, and the optimal pH were subsequently determined (additional file 1, Table 2 and Figure 4). Strikingly, signal to noise ratios observed with the LOCI technique were consistently better than those observed with the more traditional ELISA technique (e.g. compare Figure 5C with Figure 7C).

Bottom Line: The affinities of the best binders were found to be in the low nanomolar range.Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately.We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. sjust@sund.ku.dk

ABSTRACT

Background: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged.

Results: We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately.

Conclusion: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

No MeSH data available.


Related in: MedlinePlus