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Serum antibody response to capsular polysaccharide, outer membrane, and lipooligosaccharide in children with invasive Haemophilus influenzae type b infections

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0095-1137/87/122339-05$02.00/0

Copyright © 1987, American SocietyforMicrobiology

Serum

Antibody

Response to

Capsular Polysaccharide,

Outer

Membrane, and

Lipooligosaccharide in Children with

Invasive

Haemophilus

influenzae

Type

b Infections

BO A. CLAESSON,1*TERESA LAGERGARD,2 BIRGERTROLLFORS,3 LEIF GOTHEFORS,4AND ULF JODAL3 Departments ofinfectious

Diseases,'

MedicalMicrobiology,2 andPediatrics,3 University

of Goteborg,

S41685 Goteborg,

and

Department

of

Pediatrics,

University of

Umea,

S-901 87

Umed,4

Sweden

Received2 June1987/Accepted 14 September 1987

Serum antibodies against capsular polysaccharide (CPS),outer membrane(OM), and lipooligosaccharide (LOS) fromHaemophilusinfluenza type b were measured byenzyme-linked immunosorbentassay in acute-andconvalescent-phase sera from 21 children between 3 months and 4 years of age with invasive H. influenza type b infections. As expected, the levels of anti-CPS antibodies intheacute-phaseserumsampleswerelow or not detectable, as were the levels of antibodies against LOS. Incontrast, allchildrenhaddetectable antibodies against the OM in the acute-phase

serumn

sample, indicating that they are of little or no importance for protection. Anantibody response to CPS was noted in 13 of the 21patients,mainly inthe older children. An antibody response to the OM was seen in 16patients, with no evidentrelation toage. Theantibodyresponse to the OMpreparation, whichconsisted of proteins andLOS, wasprobably directedmainly againstthe OM proteins, sinceonly six children showed a response, usually of low magnitude, of antibodies to LOS.

Itiswellknownthatchildrenunder the ageof24months have apoor serumantibodyresponse toHaemophilus influ-enzae type b capsular polysaccharide (CPS), both after infectionandafter immunization withthepurified CPS(7,8, 17-22). Despite this,recurrentinvasiveH. influenzatype b infections seem to be rare (4, 18, 28). Thus, it should be takeninto considerationthat othersurfaceantigens, notably outer membrane (OM) proteins, might induce protective serum

antibodies

in humans.

Antibody

responses to OM proteinshavebeenreportedin bothchildrenandadultsafter

H.

influenza

type b infections (3, 5,

11, 12, 27).

Serum

antibodies to certain OM proteins have been shown to be protective against infection intheinfantratmodel(6, 10, 16, 24). Furthermore, one study indicates that naturally ac-quired anticapsularserumantibodies areprotective against invasive disease at lower levels than vaccine-induced anti-bodies (9), suggesting thatantibodies againstother bacterial components are induced by the whole organismand act in synergywith the anticapsularantibodies. It is also possible that secretory anticapsular antibodies, which have been demonstrated in children under 24 months of age (22), contribute to naturally acquired immunity. The third major surface antigen ofH. influenza type b is the lipooligosac-charide (LOS), whichalso can inducean antibody response in serum after invasive infections in humans. However, theseantibodies areprobably notprotective (11, 25).

The aimof thisstudy was to comparethe serum antibody response to CPS with the response to OM and LOS in children withinvasiveH.

influenza

type binfectionsand to survey the prevalence of serumantibodies to CPS, OM, and LOS in healthy children andadults.

MATERIALS ANDMETHODS

Patients. The patient group included 21 children, 17with meningitisand 4 withepiglottitis due to H.

influenza

type b.

*Correspondingauthor.

Serum samples were collected between 1982 and 1984.The ages ranged from 3 months to 3 years for the children with meningitis and from 20 months to 4 years for the children with epiglottitis (Table1). There were 7 male and 14 female patients. One patient (no. 1) with H. influenza type b meningitis at the age of 3 months wasborn at agestational age of 30 weeks. None of the otherpatients was preterm, and nonehadany known conditionpredisposingtoinfection. All patients showed normal responses to infection, with leukocytosisinperipheral bloodandpleocytosis in cerebro-spinalfluid. Allpatients survived. H. influenza type b had been isolated from blood and cerebrospinal fluid in all patients with meningitis and from blood in those with epiglottitis.

AntibodiestoCPS, OM, and LOSwere also measured in serum from 11cord blood samples andin samplesfrom 69 healthychildren,0to 6 yearsofage, and from 20adult blood donors (Table 2).

All sera werestored at -20°Cuntil analyzed.

The study wasapproved bytheEthical Committee ofthe University of Goteborg, Goteborg, Sweden.

Serological methods. The immunoglobulin G (IgG), IgM, and IgA isotype composition of the H.

influenza

type b antibodieswas measuredby enzyme-linked immunosorbent assay (ELISA) withbiotinylated H. influenza type b CPS (23). Microtiter plates (Dynatech, Novakemi, Enskede, Sweden)were coated with avidinat 5 ,ug/ml(Sigma Chemi-calCo.,St. Louis,Mo.)overnightat24°C, followedby 5,ug of biotinylated CPS per ml for 2 h at 26°C. The serum sampleswere tested induplicate in serial 10-fold dilutions, and specific antibodies were demonstrated with alkaline phosphatase-coupled swineantibodies against humanIgG

(y

chain specific), IgM (,u chain specific), and IgA (a chain specific) (Orion, Helsinki, Finland). H. influenza type b antibody references, standard serum (70 ,ug/ml), and the immunoglobulin fractions IgG (170,ug/ml), IgM (1.4 ,ug/ml), andIgA (4.0,ug/ml) obtained fromthe FDA (Bethesda,Md.) were used for comparison. The serum antibody titer was 2339

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TABLE 1. Serum antibody responses to CPS, OM, and LOS inchildren withmeningitis orepiglottitisdue to H. influenzatype b

Days after AntibodiestoCPS AntibodiestoOM AntibodiestoLOS

Patient Disease (Amoé) symptoms IgG IgM IgA IgG IgM IgG IgM

1 Me 3 1 <1 <1 <1 1.1 1.3 <1 <1

13 1 <1 <1 1.8b 1.8 1.4 1.3

1 1 8 1.4 17 <1 38 <1 103 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

2.2 1.6 <1

2.6 2.8b 1.3

2.5 1.9 1.2

2.4 2.1 1.1

2.1 1.9 <1

3 <1 33 <1 75 <1 1.2 <1 1.4 <1 1.3 <1

O <1 <1 <1

10 <1 <1 <1

3 <1 <1 <1

10 <1 <1 <1

20 <1 <1 <1

2.7 1.5 <1

2.9 1.6 <1

2.9 1.1 1.0

1.2 2.3b 1.2 1.2 1.0 1.2 1.2 1.6 <1 1.6 <1

3.0 1.5 1.1 <1

3.0 1.7 1.3 <1

2.3 1.7 1.2 <1

1 <1 <1 <1 1.9

10 <1 <1 <1 2.9b

16 <1 <1 <1 3.0b

1.2 1.4 <1

1.2 2.lb <1

1.3 2.2 <1

<1 1 <1 1.6b <1 1.5b

2.3 2.0 1.3 1.1

2.5 1.7 1.5 <1

2.3 1.9 1.2 <1

<1 2.3b 1.9 <1 2.2 <1 1.2 1.1

Me 10 2

24 120 <1 1.0 1.0 <1 1.8b 1.8b <1 1.3 1.3

1.2 1.5 <1

3.lb 2.5b 1.2

3.2b 2.5b 1.2

Me il Me 13 0 1.1 8 <1 15 <1 30 1.0 2 <1 26 <1 <1 <1 2.2b <1 2.1 <1 1.5b <1 <1 <1 1.1 <1

1.7 1.7 <1

3.0b 2.6b 1.5b

3.0b 2.6b 1.5b

2.7b 2.4b 1.4

1.7 <1 3.1b 1.6b Me 19 Me 19 Ep 20 1 <1 8 <1 5 10 O 6 23 <1 l1.8b 2.6b <1 1.2 1.0 <1 <1 1.0 <1 <1 2.6b 2.3b

1.2 1.2 <1 <1

1.4 1.6 <1 <1

<1 <1 2.6 <1 2.8b 1.8b <1 <1 1.b 1.8 <1 1.8b 1.1 1.2 2.3b 2.5b <1 1.2 2.0b Me 22 Me 23 Me 26 4 il 17 1 9 23 29 168

1.2 <1 <1 1.2 <1 <1

1.2 15b <1

1.2 2.0 <1

1.6 2.2 1.5

1.7 1.9 1.7

1.2 1.7 <1

1.6 1.8 <1

1.gb 1.9 <1

3.1 2.4 <1

3.2 2.3 1.2

2.9 2.2 2.1

2.7

2.4

2.3

1.7 1.6 1.0 1.2

1.9 2.2b 1.2 1.1

2.2 1.9 1.2 1.1

1.5 1.7 <1

2.1b 1.9 1.0

2.5b 1.9 1.2

1.2 1.5 1.2 2 Me 3 Me 4 Me s 4 4 Me <1 1.3 1.0 1.6b 1.0

6 Me 6

Me 8 7 9 Me 2 10 18 O 8 1.4 <1 2.1b <1 1.8 <1 <1 1.2 1.7b 10 il 12 1.0 1.2 1.3 13 14 <1 1 .6b 1.7b 1.6b <1 1.1 <1 l1.6b 15 16 1.3 <1 1.9b 1.4 1.9b 1.2 <1 1.3 l1.5b <1 1.1 1.0 17 1.2 1.2 1.3 Continued

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TABLE 1-Continued

Patient Age Daysafter Antibodiesto

CPS

AntibodiestoOM Antibodies to LOS

Patient

Diseasea

Age onsetof

no. (mo) symptoms IgG IgM IgA IgG IgM IgG IgM

18 Ep 35 4 1.4 2.4 1.3 1.7 1.9 1.1 1.2

6 1.7 20 1.3 2.2 1.9 1.2 1.2 39 2.2 24 2.3b 2.8b 1.9 1.5 1.2

19 Me 37 0 1.3 1.6 <1 1.5 1.7 1.0 1.1

23 1.9b 22b 1.8b 2. lb 23b 1.2 1.2

20 Ep 50 0 <1 1.3 1.0 1.7 20 1.0 1.2

2 1.2 1.6 1.2 2.5b 25 <1 1.2

16 2.5b 25b 2.6b 2.5b 26b 1.2 1.7

21 Ep 56 0 <1 <1 <1 1.2 1.6 1.0 1.0

2 1.0 1.1 <1 1.2 1.6 1.0 1.0

14 1.4 1.7b 1.2 1.gb 1.8 <1 1.2

Abbreviations: Me,meningitis; Ep,epiglottitis.

bIndicates significant increasecompared with the first serum sample.

expressed as the negative log10 value ofthe highest serum dilutionshowinganextinction of0.5 above thebackground. Values below 1 wereconsidered negative.

Serumantibodies to theOM andLOSwere measured by ELISA with OM and LOS preparations from a non-capsulatedmutantofH.

influenza

type bstrainEagan (26), kindly provided by R. Moxon, Oxford, United Kingdom. The OM was prepared by warm EDTA extractions as described previously (2). The preparation consists of pro-teinsandLOS(2). Purified LOSwaspreparedby extraction with hot phenol-water (29), followed by treatment with ribonuclease andprotease andultracentrifugationat105,000 x g for 4 h. The protein content was estimated to 0.5% by theLowry method (13).Themicroplateswerecoated with20 ,ugof OM per ml,previouslysonicated at 20kilocycles per s for1min at4°Corwith50,gof LOSper ml(11). Theserum samples were tested induplicate inserial 10-fold dilutions, and specific antibodies were demonstrated with alkaline phosphatase-conjugated swineanti-humanIgG(ychain spe-cific) and IgM

(,.

chain specific) (Orion). The optimal con-centrations ofantigens andconjugates were determined by checkerboard titrations. One positive and two negative serum samples from adults were used as references. The serumantibody concentrationwasexpressedasthenegative log10 valueofthehighestserumdilution showingan extinc-tion value of 0.5 above

the

background value.

An increase in IgG, IgM, or IgA antibodies of0.6 (four-fold)was considereda significant antibody response, based on a control material of paired serum samples from 15 healthyindividuals. Forall isotypes, +3 standarddeviations were less

than

0.6. Sera from the same patient were always tested simultaneously.

RESULTS

The serumantibodyresponses to CPS, OM, and LOS are summarized in Table 1.

Antibodies to CPS. In theacute-phase serum samples from childrenless than 1yearold, low levels of antibodies to CPS were detected in 4 of 10children. In childrenolderthan 12 months,anti-CPS antibodies were detected in 6 of 11. With oneexception, theseantibody levels were low. The patient with the highest anticapsular antibody level (no. 18) had, however, not been bled until 4 days after the onset of symptoms.

AsignificantantibodyresponsetoCPSwas noted inonly 4 of10 children of less than 1 year of age. All 4 children respondedwith a smallincreasein IgMantibodies,andone also had asmall increase in theIgAantibodyclass. Noneof them responded with an increase in IgG antibodies. In contrast, 9of the 11 olderchildrenrespondedwith antibod-ies to CPS. IgG, IgM, andIgA antibodies increased signifi-cantlyin5, 6, and 5 children, respectively.

Antibodies to OM. All 21 patients had detectable IgG or IgM antibodiesagainst theOM in their first serumsample. Seventeen hadantibodiesofboth isotypes.

Asignificant antibodyresponse to OMwas observed in7 of 10 children of less than 1 year of age. Six showed increasesin IgG antibodies, and4 showedincreasesin IgM antibodies. An antibodyresponse to OM antigens in older children was noted in 9 of 11. Eight responded with IgG antibodies, and sixresponded with IgMantibodies.

Antibodies to LOS. Antibodies against LOS ofthe IgG or IgMisotypeweredetected in thefirstserumsamplein 6of10 childrenyounger than 12 months and in 8of11children older than 12 months. With one

exception

(no. 15), the antibody levels in the firstserum sample were very low(1.0to 1.2).

Significant

increasesin antibodiesagainst LOSwerefound

in 3 of 10 children below and 3 of 11 children above 12 monthsofage.

Control material.Serumantibodylevels toCPS, OM,and LOS inhealthy children and adultsarepresentedin Table 2. All serum samples from healthy adults had detectable amounts of antibodies toCPS and OM ofthe IgG and IgM classes.Allcord bloodsampleshaddetectable IgG antibod-ies. In samples from children 3 to 5 months of age, no antibodiestoCPSwerefound,exceptforonechild withvery low IgM levels. In contrast, all of the children of 3 to 5 months ofage had detectable antibodies to OM, as had all older childrenand adults. Three offive children in the age groupof3 to 5months had low antibody levels to LOS; one hadIgG antibodies, and two hadIgM antibodies. Both IgG andIgM antibody levels increased with age.

DISCUSSION

In thisstudy, serum antibodies against the CPS, OM, and

LOS

antigens of H.

influenza

type b were measured in

children withinvasiveinfections and in healthychildren and

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TABLE 2. Levels of serum antibodies to H. influenzatype b CPS, OM, and LOS in sera from cord blood, healthy children, and adult blood donors

Antibodies to CPS Antibodies to OM Antibodies to LOS Specimen source (n) IgG IgM IgG IgM IgG IgM

No. Median No. Median No. Median No. Median No. Median No. Median positive value positive value positive value positive value positive value positive value

Cordblood(11) il 2.3 0 <1 il 2.0 0 <1 il 1.3 0 <1

Children

3-Smo(5) 0 <1 1 <1 5 1.3 3 1.0 1 <1 2 <1

6-11 mo(12) 5 <1 5 <1 12 1.7 12 1.8 1 <1 10 1.2

12-23mo (23) 15 1.3 21 1.6 21 1.8 23 2.0 8 <1 20 1.3

2-3 yr(21) 18 1.5 19 1.6 21 1.8 21 1.9 8 <1 19 1.4

4-6yr(8) 8 1.8 8 1.8 8 2.1 8 2.1 5 1.2 8 1.5

Adultblood donors (20) 20 2.4 20 2.3 20 2.5 20 2.4 20 1.4 20 1.5

adults. Our study shows that children younger than 1 year have low or undetectable levels ofanti-CPS antibodies in serumandthat these antibodies increase with age, which is in accordance with previous reports (1, 8, 19-22). Further-more,the presentandprevious studiesshow thatthe young-estchildren donotrespond with anti-CPS serum antibodies even after invasive infections, such as meningitis, whereas olderchildrenand adultsusually have anantibodyresponse, although the magnitude varies(8, 11, 17, 18, 22).

Asmentioned above, there are dataindicatingthat serum antibodiesagainst certain OM proteinsprotectagainst

inva-siveH.

influenza

typebinfectioninthe infantratmodel(6,

10, 16, 24) and there are also data suggesting that serum antibodies against noncapsular antigens play a role in pro-tectionagainst diseaseinhumans(3, 9, 15). Inthis study,an

OM

preparation

from

a noncapsulated mutant

of

H.

influ-enzaetypeb Eagan, wasusedasantigenin an ELISA. The OMproteinpattern

of

this strain, tested by sodium dodecyl

sulfate-polyacrylamide

electrophoresis followedby

immuno-blotting, showed high serological cross-reactivity

with

H.

influenza

typeb strains isolated frompatientsin Goteborg

(T. Lagergard, unpublished results).

Thestudy showed that 16 of21 children with invasiveH.

influenza

typeb infections hadantibodyresponses in serum

to this OM

preparation.

The antibody responses were not age related. This indicates that the immune system of humans

is

exposed to

theOM of H.

influenza

type

b

during

invasive infections and that children below and above the age of 1 year are capable of reacting with an antibody response tothe OM. The OM

preparation

usedherecontains both proteins and LOS. Sinceanantibodyresponse(oflow magnitude)to

purified

LOS fromH.

influenza

type bstrain Eaganwas seenin only 6of the21

patients,

it seems

likely

that the antibody response to OM was mainly directed against the

proteins.

Eleven

patients

not

responding

with antibodiesagainst LOS had

significant

increasesin antibod-iesagainstthe OM. The poorantibody responseto LOSin thepresentstudy contraststoa

previous

report, inwhicha pronounced

antibody

response to LOS

prepared

fromboth homologous strains and strain Eagan was seen (25). An antibody response to LOS was also seen in 7 of10 adults

with

invasive H.

influenza type

b

infections

(11).

The results of the

antibody

determinations in healthy adultsand children show that serum antibodies

against

the

OMare

acquired

at anearlyage and that the

antibody

levels

increasegradually during childhood.Whether these antibod-iesaretheresultofexposure totheOM fromH.

influenza

type b, to theOM from noncapsulated

H.

influenza

strains, or tocross-reactingproteins fromotherbacteriais currently notpossibletodetermine. Murphy etal. have demonstrated that an epitope ofa 16,600-dalton OM protein is present in bothcapsulated and noncapsulated H.influenzastrains and that antibodies to this protein have been

found

in normal human sera(14).This studyshows that the serumantibodies against

the

whole-OM

preparations

of H.

influenza

type b

used

here areprobablynot protective alone, since preexist-ing anti-OM antibodies were detected in the first serum sample obtained fromall 21patients. Withafewexceptions, these initialserum sampleswereobtained duringthefirst48 h of disease. However, the OM

preparation

used is com-posed of severaldifferent proteins elicitingawidevariety of antibodies.Todeterminetherole ofserumantibodiestoOM

proteins for protection against

H.

influenza type b disease,

itwill benecessary toseparateandpurifytheseproteinsand determine antibodies against single purified proteins. At-tempts have been made to determine antibodies against proteins of different molecular weight from the OM of H. influenzatype bwith variousmethods in alimited number ofpatients (5, 12, 27), but many questions remain in the evaluation ofthe roleof individualproteins for protectionin humans.

ACKNOWLEDGMENTS

Thisstudywassupported bygrantsfrom thePetterSilfverskiold Memorial Foundation, the Goteborg Medical Society, and the Faculty ofMedicine, University of Goteborg, Goteborg,Sweden.

LITERATURECITED

1. Anderson, P., D. H. Smith, D. L. Ingram, J. Wilkins, P. F. Wehrle,and V. M. Howie. 1977.Antibodytopolyribophosphate ofHaemophilusinfluenzatype b in infantsand children: effect of immunization with polyribophosphate. J. Infect. Dis.

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2. Dahlberg-Lagergârd, T. 1982. Target antigens for bactericidal and opsonizing antibodies to Haemophilus influenza. Acta Pathol. Microbiol.Scand. Sect. C90:209-216.

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4. Edmonson, M. B., D. M. Granoff, S. J. Barenkamp, and P.J. Chesney. 1982. Outer membrane protein subtypes and investi-gation ofrecurrentHaemophilus influenza type bdisease. J. Pediatr. 100:202-208.

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Johnston,and E.J.Hansen. 1982.Antibody response of infants tocellsurface-exposed outer membraneproteins of Haemoph-i/us influenza type b after systemic Haemophilus disease. Infect. Immun. 37:82-88.

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19. Peltola, H., H. Kayhty, A. Sivonen, and P. H. Makela. 1977. Haemophilusinfluenza type b capsular polysaccharide vaccine in children: a double blind field study of 100,000 vaccinees 3 months to S yearsof age in Finland. Pediatrics 60:730-737. 20. Ramadas, K., G. M. Petersen, D. C.Heiner, andJ. I. Ward.

1986.Classandsubclass antibodiestoHaemophilusinfluenza type b capsule: comparison of invasive disease and natural exposure. Infect.Immun. 53:486-490.

21. Robbins, J. B., J. C. Parke, Jr., R. Schneerson, and J. K. Whisnant. 1973. Quantitative measurement of "natural" and immunization-induced Haemophilusinfluenza type b capsular polysaccharide antibodies. Pediatr. Res. 7:103-110.

22. Rosales, S. V., L. J. Lascolea, Jr., and P. L. Ogra. 1984. Developmentof respiratory mucosal toleranceduring Haemophi-lusinfluenza typebinfectionininfancy. J. Immunol. 132:1517-1521.

23. Schneerson, R., J. B. Robbins, J. C. Parke, Jr., C. Bell, J. J. Schlesselman,A.Sutton,Z.Wang, G. Schiffman,A.Karpas,and J.Shiloach. 1986.Quantitativeandqualitative analysesof serum antibodies elicitedin adults byHaemophilus influenza typeb and pneumococcus type 6A capsular polysaccharide-tetanus toxoidconjugates. Infect. Immun.52:519-528.

24. Shenep, J. L., R. S. Munson, Jr., S. J. Barenkamp, andD. M. Granoff. 1983. Further studies of the role ofnoncapsular anti-body in protection against experimental Haemophilus influ-enzae typeb bacteremia. Infect.Immun. 42:257-263.

25. Shenep, J. L., R. S. Munson, Jr., and D. M. Granoff. 1982. Humanantibody response to lipopolysaccharide after meningi-tis due to Haemophilus influenza type b. J. Infect. Dis. 145:181-190.

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27. VanAlphen, L.,T.Riemens,and H.C.Zanen. 1983. Antibody responseagainstoutermembrane components of Haemophilus influenzae type b strains in patients with meningitis. FEMS Microbiol. Lett. 18:189-195.

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The students watch the video while waiting for their physical, complete a pre and post survey assessing their knowledge and perceived ability to make informed healthy food

Compliance with the Health Act 2007 (Care and Support of Residents in Designated Centres for Persons (Children And Adults) With Disabilities) Regulations 2013, Health Act 2007

Functionalization of ε -Carbonyl Cation Equivalents Facilitated by Functionalization of -Carbonyl Cation Equivalents Facilitated by Lewis Acid Catalysts.. Lewis

Abstract: - The paper builds on the Theory of Reason Action (TRA) and Technology Acceptance Model (TAM) to develop a research model in exploring information technology

Supplementary Figure S4.4 - Characterization of Hg-IMAC selectivity for hYVH1 C115S peptide thiols. Both CAM-labeled and unlabeled hYVH1 C115S were trypsin digested,

the friction ; is small, it does so in a step-wise manner and depends exponentially strongly on ; and (iii) if the metastable part of the potential consists of more than one well,