Pediat. Res. 11: 138-143 (1977)
Letters to the Editor Electrophoretic Analysis of Serum Proteins in Cystic Fibrosis JOHN M . THOMAS.'!" A. 1)ONALI) MERRITT. AND M . E . HODES
A considcrnhle amount of inclircct evidence suggests that fluids from cystic fihrosis (('F) patients ar~clobligate hctcrozygotcs contain a tictor o r factors ~ h i c hma! he relevant to the pathophysiology of ('F ( 7 ) . Much of the evidence suggests that the factor is a low 11ioIccu1;1r\\eight. c i ~ t i o ~ l iprotein c which in serum is ;~ssociatcdwith i m m u ~ l o g l o b u l iG~ ~( l g G ) ( 7 ) . Recent reports utilizilig electrophoretic technitlucs claim identification of a "C'F serum factor" ancl tlcvclopmc~itof assays to c ~ l a b l cthe
detection of ('F honlozygotes ant1 hetero~!gotes ( I . 5 , 0 ) . T h e stud! by W i l s o ~ l o(11. ~ ( 0 ) in 1073. ;~nnouncingthe dcmo~lstri~tion of serum protein differences in C'F by isoclectric focusing ( I E F ) in thin laycr poly;~cryl:~~~~irIe gcls (][:FAG). was follo\vcd in 1975 by a report claiming the cstension of this tcchnitluc as a "s!andardizcd biophysical assay for the rapicl detection of inclivicluals homozygous o r hctcrozygous for C'F" ( 5 ) . In thcir analyses, itlcrltific:~tion of the C'F genotypes \\.:IS basccl o n the ohscrvatioli of a protein bancl with a n isoclcctric point (PI) of 8.4-8.5 (5). In 1075 Altlancl 1.1 01. ( I ) statcrl that they could not identify this band \\ith a n ilnproved 1L:FAG tech~litluc but \\ere able to rlcmonstratc the presence of a relatively Io\v molecular weight. cationic protein ( p l 8 - 0 ) in the serum of a fc\r honlozygotes and hctcrozygotes for C F that they tcstcd hy tho-step IEFAG/thin layer electrophoresis ( I ) . Altlantl c8/ (11. ( I ) implied that thcir tcchnicluc Inay cnahle the unequivocal detection of paticllts and carriers by virtue of the fact t l i : ~ t thcir proceclurc separates thc factor from the IgCi fri~ctionand thus overcomes any v:lri:~tion in resolution at the I E F A G step.
Fig. I . ('omparison of !he alk;tli~icisoclcctric focusi~igpatterns of serum proteins ohtailled hy two methods: A : the technique of Wilsoncv ul. ( 5 ) ;B: isoclcctric focusing i n t h i n laycr polyacrylnniidc gcls according to one of our methods (3). which cxpancls the gradient in thc pH X- I0 region. Only the rclcva~itportions of the gels :ire shown. 'l'hc samples. ~ h i c hwere the same for c;rch run. wcrc from one cystic fihrosis ( ( ' F ) ,one ('F hctcrozygotc ( I f ) . and one normal control ( N ) suhjcet. 2nd each ct)ntoi~~cJ 3 0 0 pg i~ii~~~unoglobulin G . Urc;~colicclitratio~~ was 4 M. The ~ilcasurcdpH gradicrlts arc s h o ~ 1 to 1 the sicic of each gcl.
We havc ;~ttcmptcdto reproduce the IEFAG met hocl spccificd by Wilson ct (11. ( 5 ) in our labor~rtory( 7 ) . Since the resolution obtained with thcir proccclurc appeared to be inadccluate for identifying single protein bancls it1 the relevant region. n c have incorporated several methodologic iniprovcmcnts into the isoelectric focusing technicluc ( 3 ) . 'l'hesc improvcrncnts have significantly enhanced the resolution anel have thuscn;~l>ledus to demonstrate a greater degree of heterogeneity of serum y-globulins than is visible in the paper of Wilson ct (11. ( 5 ) . Figure I is it comparison of the alkaline I[-F patterns of serum proteins ohtaincd in our laboratory by the tncthod of Wilson (,/ crl. ( 5 ) and by one of our methods (3). I>espitc the significant incrci~scin the number of bands resolveel t ~ your niethocl. a cliffcrencc among C'F, hcterozygotc. and control scra in thc pH 8.4-8.5 region coulcl riot be detected. In aclclition. the t\\o-step Ilr tlic purpose. o f il~.t~.rniining po\\il>lc reasons for thcir inability to dctect C'FP by electrofocusing. Key STANDARIIIZA'I'ION O F ASSAY points made during these conversations are reitcrated below in detail in a firm attempt to alleviate future problems by incxperiAs originally reported (1 1). all scrurn samples should be enccd investigators in their attempts to use analytic electrofocus- analyzed using a volume of serum standardized to contain 300 ing to detect C'FP. p g IgG. We havc previously shown that C'FP is a srnall molecule (9) associated with the scrum lgG fraction (12). Based on the assumption that C'FP has a stoichiomctric relationship to IgG, we determined a level of IgG (the proposed carrier of C'FP in F
Letters to the Editor Electrophoretic Analysis of Serum Proteins in Cystic Fibrosis JOHN M . THOMAS.'!" A. 1)ONALI) MERRITT. AND M . E . HODES
A considcrnhle amount of inclircct evidence suggests that fluids from cystic fihrosis (('F) patients ar~clobligate hctcrozygotcs contain a tictor o r factors ~ h i c hma! he relevant to the pathophysiology of ('F ( 7 ) . Much of the evidence suggests that the factor is a low 11ioIccu1;1r\\eight. c i ~ t i o ~ l iprotein c which in serum is ;~ssociatcdwith i m m u ~ l o g l o b u l iG~ ~( l g G ) ( 7 ) . Recent reports utilizilig electrophoretic technitlucs claim identification of a "C'F serum factor" ancl tlcvclopmc~itof assays to c ~ l a b l cthe
detection of ('F honlozygotes ant1 hetero~!gotes ( I . 5 , 0 ) . T h e stud! by W i l s o ~ l o(11. ~ ( 0 ) in 1073. ;~nnouncingthe dcmo~lstri~tion of serum protein differences in C'F by isoclectric focusing ( I E F ) in thin laycr poly;~cryl:~~~~irIe gcls (][:FAG). was follo\vcd in 1975 by a report claiming the cstension of this tcchnitluc as a "s!andardizcd biophysical assay for the rapicl detection of inclivicluals homozygous o r hctcrozygous for C'F" ( 5 ) . In thcir analyses, itlcrltific:~tion of the C'F genotypes \\.:IS basccl o n the ohscrvatioli of a protein bancl with a n isoclcctric point (PI) of 8.4-8.5 (5). In 1075 Altlancl 1.1 01. ( I ) statcrl that they could not identify this band \\ith a n ilnproved 1L:FAG tech~litluc but \\ere able to rlcmonstratc the presence of a relatively Io\v molecular weight. cationic protein ( p l 8 - 0 ) in the serum of a fc\r honlozygotes and hctcrozygotes for C F that they tcstcd hy tho-step IEFAG/thin layer electrophoresis ( I ) . Altlantl c8/ (11. ( I ) implied that thcir tcchnicluc Inay cnahle the unequivocal detection of paticllts and carriers by virtue of the fact t l i : ~ t thcir proceclurc separates thc factor from the IgCi fri~ctionand thus overcomes any v:lri:~tion in resolution at the I E F A G step.
Fig. I . ('omparison of !he alk;tli~icisoclcctric focusi~igpatterns of serum proteins ohtailled hy two methods: A : the technique of Wilsoncv ul. ( 5 ) ;B: isoclcctric focusing i n t h i n laycr polyacrylnniidc gcls according to one of our methods (3). which cxpancls the gradient in thc pH X- I0 region. Only the rclcva~itportions of the gels :ire shown. 'l'hc samples. ~ h i c hwere the same for c;rch run. wcrc from one cystic fihrosis ( ( ' F ) ,one ('F hctcrozygotc ( I f ) . and one normal control ( N ) suhjcet. 2nd each ct)ntoi~~cJ 3 0 0 pg i~ii~~~unoglobulin G . Urc;~colicclitratio~~ was 4 M. The ~ilcasurcdpH gradicrlts arc s h o ~ 1 to 1 the sicic of each gcl.
We havc ;~ttcmptcdto reproduce the IEFAG met hocl spccificd by Wilson ct (11. ( 5 ) in our labor~rtory( 7 ) . Since the resolution obtained with thcir proccclurc appeared to be inadccluate for identifying single protein bancls it1 the relevant region. n c have incorporated several methodologic iniprovcmcnts into the isoelectric focusing technicluc ( 3 ) . 'l'hesc improvcrncnts have significantly enhanced the resolution anel have thuscn;~l>ledus to demonstrate a greater degree of heterogeneity of serum y-globulins than is visible in the paper of Wilson ct (11. ( 5 ) . Figure I is it comparison of the alkaline I[-F patterns of serum proteins ohtaincd in our laboratory by the tncthod of Wilson (,/ crl. ( 5 ) and by one of our methods (3). I>espitc the significant incrci~scin the number of bands resolveel t ~ your niethocl. a cliffcrencc among C'F, hcterozygotc. and control scra in thc pH 8.4-8.5 region coulcl riot be detected. In aclclition. the t\\o-step Ilr tlic purpose. o f il~.t~.rniining po\\il>lc reasons for thcir inability to dctect C'FP by electrofocusing. Key STANDARIIIZA'I'ION O F ASSAY points made during these conversations are reitcrated below in detail in a firm attempt to alleviate future problems by incxperiAs originally reported (1 1). all scrurn samples should be enccd investigators in their attempts to use analytic electrofocus- analyzed using a volume of serum standardized to contain 300 ing to detect C'FP. p g IgG. We havc previously shown that C'FP is a srnall molecule (9) associated with the scrum lgG fraction (12). Based on the assumption that C'FP has a stoichiomctric relationship to IgG, we determined a level of IgG (the proposed carrier of C'FP in F
