BACKGROUND
phage display libraries consist of small antibody fragments cloned into a display phage vector, allowing efficient antibody screening and production in a bacterial system  <cit> .
traditional antibodies are composed of a heavy- and a light-chain that need to recombine in a tetramer for the formation of a functional antibody.
because most of these random recombinations will yield non-functional antibodies, when produced as recombinant fragments in e. coli, isolation of effective antibodies demands extremely large phage libraries.
camelidae have, next to conventional antibodies, dimeric heavy chain antibodies  that lack light chains  <cit> .
the variable domain of the hcab  has a single binding domain with a specificity and affinity similar to conventional antibodies  <cit> .
in a phage display library, each phage displays a different antigen-binding domain on its surface.
to isolate specific antibodies, phage particles from a library are bound to an antigen, recovered and used to infect fresh bacteria.
subsequently, phages go through several rounds of epitope binding and re-infection resulting in an enrichment of binding phages.
a perfect experiment will ultimately yield groups of phages, each encoding a different antibody directed against the starting antigen.
the set of phages can be used together as 'polyclonal phages', individual phages as 'monoclonal phages'.
after selection, individual vhh clones are characterized to determine their specificity by elisa and their diversity by fingerprinting/sequencing.
although ultimate identification is done using clone-insert nucleotide sequencing, pre-sequence fingerprinting is performed to reduce cost.
phage display clones are usually analysed using restriction digestion of pcr amplified vhh insert, followed by agarose gel-electrophoresis  <cit> .
however, this methodology is time consuming, labour intensive, has limited resolution and is not effective for the analysis of a large number of clones.
in the current study, we developed a protocol using high resolution melt curve analysis  to visualise clonal diversity and study enrichment of clones after vhh-selection from a llama non-immune phage display library.
unlike the traditional application for melt curve analysis, where  <dig> base pair differences are detected through a change in melt temperature of a fully base-paired hybrid and mismatched hybrids, the current study uses differences in melt curve shape and the tm of each melt curve to identify template nucleotide sequence similarities within a large group of unlike pcr fragments.
similar melt curve shapes represent similar dna sequences and melt curves can be automatically and efficiently grouped using the available hrma software.
RESULTS
after two rounds of selection against an epitope spanning the first  <dig> amino acids of the huntingtin protein  <cit> ,  <dig> phages were picked and elisa showed  <dig> positive and  <dig> negative wells.
an optical density of  <dig>  or higher was considered a positive result while the negative control was less than  <dig> .
clone diversity was investigated using both hrma and hinfi restriction digestion of pcr-amplified clone inserts.
as expected, since the pcr fragments had an average size of  <dig> bp, hrma showed a wide range of melt profiles often containing multiple melting domains per fragment representing differences in nucleotide sequence.
representative results from  <dig> independent hrma analyses are shown in figure  <dig>  a comparison of the elisa and hrma results are shown in figure  <dig>  only the elisa-positive clones are represented in this figure.
there was a complete agreement of elisa-positive and elisa-negative clones with hrma analysis.
the  <dig> elisa-positive clones belonged to  <dig> different groups, the largest group contained  <dig> clones, one group  <dig> clones, one group  <dig> clones, and  <dig> groups contained a unique clone.
of the remaining  <dig> clones that showed a negative elisa,  <dig> clones could not be analysed by hrma because of a low pcr yield and/or low quality melting.
the remaining  <dig> clones could be grouped into  <dig> different groups, consisting of one large group of  <dig> clones and three groups of  <dig>   <dig>  and  <dig> clones, respectively.
the remaining groups all contained  <dig> clones or less.
all groups identified by restriction digestion fingerprinting were also identified by hrma.
groups could overall be confirmed by nucleotide sequence analysis and pcr fragments were approximately  <dig> bp in length ranging from  <dig> to  <dig> bp with a tm of either  <dig> or 86°c and a g/c content of  <dig> or 54%.
the most common fragment length was  <dig> bp with a tm of 86°c and a g/c content of 54%.
fragments with these characteristics were present in the majority of groups assigned by the software.
as can be seen in figure  <dig>  hrma even identified one additional group that was not seen with fingerprinting.
however, as can be seen in the red hrma group in figure  <dig>  occasionally, elisa results and hrma results did not agree with restriction digestion results.
sequencing showed that the red clone with the different digestion pattern was  <dig> bp different from the other clones in this hrma group.
these changes were outside the vhh encoding region and thus did not affect vhh binding characteristics.
however, this  <dig> bp difference did change a hinfi restriction site resulting in a different restriction pattern.
discussion
hrma has been used routinely for mutation scanning on patient samples with small fragments of equal size  that differ only  <dig> or a few nucleotides in sequence  <cit> .
the temperature at which the probe-template hybrid melts, distinguishes between fully base-paired hybrids from mismatched hybrids.
in the current study, we developed a protocol using hrma to identify clonal origin of vhh fragments selected from a phage display library as an alternative for dna fingerprinting.
the protocol uses the differences in melt curve shape and tm of each melt curve to identify template nucleotide sequence similarities within a large group of samples.
although the software used for hrma was originally designed to detect  <dig> bp differences between two small fragments, our results demonstrate that it is also capable of accurately analysing melt curves from longer unlike pcr fragments.
hrma is a very efficient technique to obtain a quick overview and determine if clone selection was successful.
from a successful selection experiment one expects several recurring clones.
when all clones are different, no enrichment has taken place and clone selection has probably failed, when all clones are identical, the success is probably also doubtful.
when the elisa negative clones contain a large recurrent set, these clones might either be well-growing but have low-affinity/are non-specific  or high affinity clones that somehow fail to produce enough vhh in order to obtain a positive elisa signal .
hrma is also suitable to follow clone selection; initially one expects all clones to be different, with larger clone sets emerging in later rounds of selection.
when comparing hrma to digestion analysis of the vhh fragments, hrma analysis is a more sensitive and efficient method to determine clonal similarity.
all groups identified with restriction digestion fingerprinting could also be identified by hrma and were overall confirmed by sequence analysis.
it is a simple and rapid method taking only two and a half hours to complete after pcr amplification.
furthermore, it is inexpensive, requiring only pcr, a dna dye, and melting instrumentation.
in the current study we have used the lightcycler <dig> for the final pcr amplification and melt curve analysis, however, any block cycler can be used for amplification and the subsequent melt analysis could then be performed with other high-resolution melting devices such as the lightscanner .
multiple samples can be analysed simultaneously  and groups can be assigned in an automated manner.
CONCLUSIONS
hrma is very efficient to obtain a quick overview and determine if clone selection was successful.
similar groups of clones identified by restriction digestion fingerprinting were also identified by hrma and were confirmed by nucleotide sequence analysis.
hrma is suitable for a wide variety of applications where verification and/or analysis of clonal diversity is essential, including determining clone diversity in a single-chain  fv phage library  <cit> , analysis of clones obtained after in vitro mutagenesis  <cit> , cdna clones to determine allelic expression  <cit>  and clones to determine methylation status of genomic regions  <cit> .

 phage display libraries consist of small antibody fragments cloned into a display phage vector, allowing efficient antibody screening and production in a bacterial system. traditional antibodies are composed of a heavy- and a light-chain that need to recombine in a tetramer for the formation of a functional antibody. because most of these random recombinations will yield non-functional antibodies, when produced as recombinant fragments in e. coli, isolation of effective antibodies demands extremely large phage libraries. camelidae have, next to conventional antibodies, dimeric heavy chain antibodies that lack light chains. the variable domain of the hcab has a single binding domain with a specificity and affinity similar to conventional antibodies. in a phage display library, each phage displays a different antigen-binding domain on its surface. to isolate specific antibodies, phage particles from a library are bound to an antigen, recovered and used to infect fresh bacteria. subsequently, phages go through several rounds of epitope binding and re-infection resulting in an enrichment of binding phages. a perfect experiment will ultimately yield groups of phages, each encoding a different antibody directed against the starting antigen. the set of phages can be used together as'polyclonal phages', individual phages as'monoclonal phages'. after selection, individual vhh clones are characterized to determine their specificity by elisa and their diversity by fingerprinting/sequencing. although ultimate identification is done using clone-insert nucleotide sequencing, pre-sequence fingerprinting is performed to reduce cost. phage display clones are usually analysed using restriction digestion of pcr amplified vhh insert, followed by agarose gel-electrophoresis. however, this methodology is time consuming, labour intensive, has limited resolution and is not effective for the analysis of a large number of clones. in the current study, we developed a protocol using high resolution melt curve analysis to visualise clonal diversity and study enrichment of clones after vhh-selection from a llama non-immune phage display library. unlike the traditional application for melt curve analysis, where base pair differences are detected through a change in melt temperature of a fully base-paired hybrid and mismatched hybrids, the current study uses differences in melt curve shape and the tm of each melt curve to identify template nucleotide sequence similarities within a large group of unlike pcr fragments. similar melt curve shapes represent similar dna sequences and melt curves can be automatically and efficiently grouped using the available hrma software.  after two rounds of selection against an epitope spanning the first amino acids of the huntingtin protein, phages were picked and elisa showed positive and negative wells. an optical density of or higher was considered a positive result while the negative control was less than. clone diversity was investigated using both hrma and hinfi restriction digestion of pcr-amplified clone inserts. as expected, since the pcr fragments had an average size of bp, hrma showed a wide range of melt profiles often containing multiple melting domains per fragment representing differences in nucleotide sequence. representative results from independent hrma analyses are shown in figure a comparison of the elisa and hrma results are shown in figure only the elisa-positive clones are represented in this figure. there was a complete agreement of elisa-positive and elisa-negative clones with hrma analysis. the elisa-positive clones belonged to different groups, the largest group contained clones, one group clones, one group clones, and groups contained a unique clone. of the remaining clones that showed a negative elisa, clones could not be analysed by hrma because of a low pcr yield and/or low quality melting. the remaining clones could be grouped into different groups, consisting of one large group of clones and three groups of and clones, respectively. the remaining groups all contained clones or less. all groups identified by restriction digestion fingerprinting were also identified by hrma. groups could overall be confirmed by nucleotide sequence analysis and pcr fragments were approximately bp in length ranging from to bp with a tm of either or 86°c and a g/c content of or 54%. the most common fragment length was bp with a tm of 86°c and a g/c content of 54%. fragments with these characteristics were present in the majority of groups assigned by the software. as can be seen in figure hrma even identified one additional group that was not seen with fingerprinting. however, as can be seen in the red hrma group in figure occasionally, elisa results and hrma results did not agree with restriction digestion results. sequencing showed that the red clone with the different digestion pattern was bp different from the other clones in this hrma group. these changes were outside the vhh encoding region and thus did not affect vhh binding characteristics. however, this bp difference did change a hinfi restriction site resulting in a different restriction pattern. discussion hrma has been used routinely for mutation scanning on patient samples with small fragments of equal size that differ only or a few nucleotides in sequence. the temperature at which the probe-template hybrid melts, distinguishes between fully base-paired hybrids from mismatched hybrids. in the current study, we developed a protocol using hrma to identify clonal origin of vhh fragments selected from a phage display library as an alternative for dna fingerprinting. the protocol uses the differences in melt curve shape and tm of each melt curve to identify template nucleotide sequence similarities within a large group of samples. although the software used for hrma was originally designed to detect bp differences between two small fragments, our results demonstrate that it is also capable of accurately analysing melt curves from longer unlike pcr fragments. hrma is a very efficient technique to obtain a quick overview and determine if clone selection was successful. from a successful selection experiment one expects several recurring clones. when all clones are different, no enrichment has taken place and clone selection has probably failed, when all clones are identical, the success is probably also doubtful. when the elisa negative clones contain a large recurrent set, these clones might either be well-growing but have low-affinity/are non-specific or high affinity clones that somehow fail to produce enough vhh in order to obtain a positive elisa signal. hrma is also suitable to follow clone selection; initially one expects all clones to be different, with larger clone sets emerging in later rounds of selection. when comparing hrma to digestion analysis of the vhh fragments, hrma analysis is a more sensitive and efficient method to determine clonal similarity. all groups identified with restriction digestion fingerprinting could also be identified by hrma and were overall confirmed by sequence analysis. it is a simple and rapid method taking only two and a half hours to complete after pcr amplification. furthermore, it is inexpensive, requiring only pcr, a dna dye, and melting instrumentation. in the current study we have used the lightcycler for the final pcr amplification and melt curve analysis, however, any block cycler can be used for amplification and the subsequent melt analysis could then be performed with other high-resolution melting devices such as the lightscanner. multiple samples can be analysed simultaneously and groups can be assigned in an automated manner.  hrma is very efficient to obtain a quick overview and determine if clone selection was successful. similar groups of clones identified by restriction digestion fingerprinting were also identified by hrma and were confirmed by nucleotide sequence analysis. hrma is suitable for a wide variety of applications where verification and/or analysis of clonal diversity is essential, including determining clone diversity in a single-chain fv phage library, analysis of clones obtained after in vitro mutagenesis, cdna clones to determine allelic expression and clones to determine methylation status of genomic regions. 
in the current study we show that high-resolution melt curve analysis is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge. hrma results nicely matched those obtained with elisa and compared favourably to dna fingerprinting of restriction digested clone insert-pcr. applied to the analysis of clones obtained after phage display antibody selection, hrma facilitated a quick overview of the overall success as well as the identification of identical clones.
 methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. in the current study we show that high-resolution melt curve analysis is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.  hrma results nicely matched those obtained with elisa and compared favourably to dna fingerprinting of restriction digested clone insert-pcr. dna sequence analysis confirmed that hrma-clustered clones contained identical inserts.  using hrma, analysis of up to samples can be done simultaneously and will take approximately minutes. clustering of clones can be largely automated using the system's software within hours. applied to the analysis of clones obtained after phage display antibody selection, hrma facilitated a quick overview of the overall success as well as the identification of identical clones. our approach can be used to characterize any clone set prior to sequencing, thereby reducing sequencing costs significantly. 