Detection of Antibodies to Squalene III. Naturally Occurring Antibodies to Squalene in Humans and

24 Pages·2017·0.71 MB·English

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US Army Research U.S. Department of Defense
2004
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Gary R. Matyas
Department of Membrane Biochemistry, Walter Reed Army Institute of Research, 503 Robert Grant
Avenue, Silver Spring, MD 20910-7500, USA
Mangala Rao
Department of Membrane Biochemistry, Walter Reed Army Institute of Research, 503 Robert Grant
Avenue, Silver Spring, MD 20910-7500, USA
Phillip R. Pittman
Division of Medicine, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick,
MD 21702-5011, USA
Robert Burge
Department of Biostatistics, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, USA
Iris E. Robbins
Department of Membrane Biochemistry, Walter Reed Army Institute of Research, 503 Robert Grant
Avenue, Silver Spring, MD 20910-7500, USA
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Matyas, Gary R.; Rao, Mangala; Pittman, Phillip R.; Burge, Robert; Robbins, Iris E.; Wassef, Nabila M.;
Thivierge, Brandie; and Alving, Carl R., "Detection of Antibodies to Squalene III. Naturally Occurring
Antibodies to Squalene in Humans and Mice" (2004). US Army Research. 39.
https://digitalcommons.unl.edu/usarmyresearch/39
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Gary R. Matyas, Mangala Rao, Phillip R. Pittman, Robert Burge, Iris E. Robbins, Nabila M. Wassef, Brandie
Thivierge, and Carl R. Alving
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usarmyresearch/39
JournalofImmunologicalMethods286(2004)47–67
www.elsevier.com/locate/jim
Research Paper
Detection of antibodies to squalene III.
$
Naturally occurring antibodies to squalene in humans and mice
Gary R. Matyasa,*, Mangala Raoa, Phillip R. Pittmanb, Robert Burgec,
Iris E. Robbinsa,1, Nabila M. Wassefa,2, Brandie Thiviergea, Carl R. Alvinga
aDepartmentofMembraneBiochemistry,WalterReedArmyInstituteofResearch,503RobertGrantAvenue,
SilverSpring,MD20910-7500,USA
bDivisionofMedicine,U.S.ArmyMedicalResearchInstituteofInfectiousDiseases,FortDetrick,Frederick,MD21702-5011,USA
cDepartmentofBiostatistics,WalterReedArmyInstituteofResearch,SilverSpring,MD20910-7500,USA
Received26August2003;receivedinrevisedform7November2003;accepted11November2003
Abstract
AnELISA-basedassayisdescribedforthemeasurementofantibodiestosqualene(SQE)inhumanserumandplasma.The
assaywasadaptedfromthepreviouslydescribedassayformurineantibodiestoSQE(J.Immunol.Methods267(2002)119).
Like the murine SQE antibody assay,the human antibody assay usedsterile cell culture 96-well plates coated with SQE (20
nmol/well).Phosphate-bufferedsaline(PBS)–0.5%caseinwasusedasbothablockingagentanddilutionbuffer.Theassayhas
ahighthrough-putcapacityandisreproducibleandquantitative.Thisassaywasusedtoevaluatesamplesfromthreedifferent
human cohorts. The first cohort was retired employees of the United States Army Medical Research Institute of Infectious
Diseases (USAMRIID alumni). The mean age was 68 (N=40; range 58–82). Most were vaccinated with the U.S. licensed
anthraxvaccine(AVA)andmosthadreceivedseveralothervaccinesthroughaUSAMRIIDspecialimmunizationprogram.The
secondcohortwasofsimilarage(N=372;meanage67;range54–97)fromthenormalpopulationofFrederick,MDandwere
not vaccinated with AVA. The third cohort (N=299) was from Camp Memorial Blood Center, United States Army Medical
Department Activities, Fort Knox, KY. (No additional volunteer information is available.) Using this new ELISA method,
antibodies to SQE were detected in all three of the cohorts. IgG antibodies to SQE were detected in 7.5% and 15.1% of the
samples from the USAMRIID alumni and Frederick cohorts, respectively. These differences were not significantly different
(v 2=1.69, p=0.19). In contrast, no IgG antibodies to SQE were detected in the Fort Knox cohort which is significantly
(1)
differentthantheFrederickcohort(v 2=49.25,p<0.0001).IgMantibodiestoSQEweredetectedin37.5%and32.3%ofthe
(1)
samplesfromtheUSAMRIIDandFrederickcohorts,respectively,buttherewasnosignificantdifferencebetweenthecohorts.
Abbreviations:ABTS,2,2V-azino-di-(3-erthylbenzthiazoline-6-sulfonate);AVA,anthraxvaccineabsorbed(licensedU.S.anthraxvaccine);
BSA,bovineserumalbumin;FBS,fetalbovineserum;GEL,PBS–0.3%gelatin,pH7.4;ISP,isopropanol;LDL,lowdensitylipoprotein;PBS,
Dulbecco’sphosphatebufferedsalinewithoutcalciumandmagnesium,pH7.4;PVDF,polyvinyldienefluoride;SQE,squalene;USAMRIID,
UnitedStatesArmyMedicalResearchInstituteofInfectiousDiseases;VLDL,verylowdensitylipoprotein.
$Disclaimer: The information contained herein reflects the views of the authors and should not be construed to represent those of the
DepartmentoftheArmyortheDepartmentofDefense.
* Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](G.R.Matyas).
1Presentaddress:DepartmentofMedicine,WalterReedArmyMedicalCenter,Washington,DC20307,USA.
2Presentaddress:PathogenesisandBasicResearchBranch,DivisionofAIDS,NIAID,NationalInstitutesofHealth,Bethesda,MD20892,
USA.
0022-1759/$-seefrontmatter.PublishedbyElsevierB.V.
doi:10.1016/j.jim.2003.11.002
48 G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67
IntheFortKnoxcohort,19.4%ofthesampleswerepositiveforIgMantibodiestoSQE,whichwassignificantlydifferentfrom
theFrederickcohort(v2 =14.23,p=0.0002).AlthoughtheageofthevolunteersfromtheFortKnoxcohortisunknown,the
(1)
demographic of the donors at the blood bank volunteers is 85% 17–21 years of age. This suggested that the prevalence of
antibodies to SQE may increase with age. This was confirmed with mouse studies in which the presence of antibodies was
monitoredasafunctionoftime.NoantibodiestoSQEweredetectedinfemaleBALB/c,B10.BrandC57BL/6miceat2months
ofage,buttheyreachedamaximumprevalencewith100%and89%ofanimalstestingpositiveforIgGandIgMantibodiesto
SQE,respectively,intheC57Bl/6miceat18monthsofage.BALB/candB10.BrmicealsodevelopedantibodiestoSQEover
time, but were at a lower prevalence than those observed in the C57BL/6 mice. Thirty-five of the 40 volunteers in the
USAMRIIDwerevaccinatedwithAVA(meanno.doses=26;range3–47).ComparisonoftheprevalenceofantibodiestoSQE
fromtheAVAimmunizedgroupwiththeFrederickcohortrevealedthattherewasnostatisticaldifferencesforIgG(v2 =2.3,
(1)
p=0.13)orIgM(v2=0.33,p =0.56).WhenthedatafromtheUSAMRIIDandFrederickcohortswerecombinedandanalyzed
(1)
for the presenceof antibodies to SQEwith respect to the sex of thevolunteer,females (40.8%) were foundtohave ahigher
prevalence of IgM antibodies to SQE than men (28.4%) (v2 =6.59, p=0.01). No significant difference was observed in the
(1)
prevalence for IgG antibodies to SQE in females (17.7%) and males (12.5%). We conclude that antibodies to SQE occur
naturally in humans; have an increased prevalence in females; are not correlated with vaccination with AVA; and appear to
increase in prevalencewith age.
Published byElsevier B.V.
Keywords:Squalene;Antibodydetection;Enzyme-linkedimmunosorbentassay;Anthraxvaccineadsorbed
1. Introduction Mitchell et al., 2002; McFarland et al., 2001; Cun-
ninghametal.,2001),andmalaria(Stouteetal.,1997,
Squalene (SQE) is a triterpenoid hydrocarbon oil, 1998; Kester et al.,2001; Rickman et al., 1991) have
C H (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22- usedSQEemulsions.Vaccinereactionsweretypically
30 50
tetracosahexene) (Fig. 1), that is widely produced in mild. However, some moderate to severe reactions,
plants and animals and is present in the food supply. which can be attributed to other adjuvants in the
In mammals, SQE serves as a precursor for the formulations, were reported (Kahn et al., 1994; Rick-
synthesis of cholesterol and steroid hormones (Gran- man et al., 1991; Stoute et al., 1997; Kester et al.,
ner, 1996; Mayes, 1996). SQE is synthesized in the 2001). Furthermore, no adverse reactions were
liver and in the epidermis of the skin by sebaceous reported following intravenous injection of humans
glands(Stewart,1992).BothSQEandcholesterolare with chylomicron-like lipid emulsions containing
transported in the very low density lipoprotein SQE (Relas et al., 2001).
(VLDL) and LDL (Miettien, 1982; Koivisto and AntibodiestoSQEhavegeneratedgreatinterestin
Miettinen, 1988). the popular press and have been the subject of U.S.
Because it is a naturally occurring biodegradable congressional hearings. This was initiated by a report
oil, SQE, and its hydrogenated derivative squalane, that antibodies to SQE were present in the serum of
eachhasbeenproposedastheoilcomponentofoil-in- sick Gulf War veterans, but not in healthy veterans
water emulsions to be used as new adjuvants for (Asa et al., 2000). The assay used in the later report
vaccines. Although not included in any vaccines was severely criticized for technical reasons; includ-
licensedintheUnitedStates,aSQE-containingemul- inglackofpositiveandnegativecontrols(Alvingand
sion is part of an influenza vaccine licensed in Italy Grabenstein, 2000). The Institute of Medicine con-
and has been given without adverse effects to cluded that the reported results did not provide evi-
hundreds of thousands of people (Podda, 2001; Ott dence that antibodies to SQE were successfully
etal.,2000).Numerousotherhumanclinicaltrialsfor measured (Fulco et al., 2000). Using the same assay,
influenza (Ott et al., 2000; Nicholson et al., 2001; a report was published that claimed that military
Gasparinietal.,2001),cytomegalovirus(Drulaketal., servicememberswhoweresickfollowingvaccination
2000), hepatitis B (Heineman et al., 1999), papillo- with certain lots of the U.S. licensed anthrax vaccine
mavirus (Harro et al., 2001), HIV (Kahn et al., 1994; hadantibodiestoSQE(Asaetal.,2002).However,in
G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67 49
Fig.1.Structureofsqualene,cholesterolandpolystyrene.
blinded testing, no correlation was found between purchased from Sigma-Aldrich, St. Louis, MO. Iso-
individuals who received AVA and those in whom propanol (BAKER ANALYZEDR A.C.S. Reagent;
purported antibodies to SQE were observed (Asa et cat. # 9084-03) and casein were purchased from J.T.
al., 2002). In view of the above observations and Baker, Phillipsburg,NJ. Costar round bottom 96-well
controversies, we undertook a study to develop a sterile tissue culture plates (cat.# 3799) were pur-
standardized assay for detecting antibodies to SQE chased from Corning, Corning, NY. FBS was from
and to establish background levels, if any, for natu- GIBCO BRL Grand Island NYand was heated at 56
rally occurring antibodies to SQE. jCfor1hpriortouse.10(cid:1) Dulbecco’sPBSwithout
Inordertodevelopareliableassayforantibodiesto Ca2+andMg2+wasalsopurchasedfromGibcoBRL.
SQE, we developed murine monoclonal antibodies to HumanmyelomaswerepurchasedfromtheAmerican
SQE that could serve as a positive antibody control Type Culture Collection, Chantilly, VA and were
(Matyasetal.,2000).Ouroriginalassayused96well grown as described in the supplier’s instructions with
platescontaininghydrophobicPVDFmembranes.The culture medium and additives from Gibco BRL. The
assay was highly reproducible, but was very labor human antibody secreting myelomas were IM-9 (IgG
intensive. A new assay was developed that utilized of unknown specificity obtained from a myeloma)
96wellpolystyrenetissuecultureplates(Matyasetal., (Fahey et al., 1971), SA13 (IgG to tetanus toxoid)
2002).Theassayhadahighthrough-putcapacity,was (Larrick et al., 1986), 16M3F10 (IgG to diphtheria
reproducible and quantitative, and had increased sen- toxoid) (Gigliotti et al., 1984), C5 (IgM to lipid A of
sitivity. We now describe the adaptation of this latter Gramnegativebacteria(Tengetal.,1985),L612(IgM
assayforthemeasurementofhumanantibodiestoSQE to gangliosides GM3 and GM4) (Irie, 1995) and
incohortsofnonselectedmilitaryandcivilianpopula- RPMI 1788 (IgM to tumor necrosis factor beta)
tions,andinpopulationsselectedfortheirexposureto (Aggarwal et al., 1984). Mouse myeloma SQE #16,
numerousvaccines,includingAVA. which secretes a monoclonal antibody to SQE, was
grown as described (Matyas et al., 2000, 2002).
Human IgG and IgM and mouse IgM quantitation
2. Materials and methods kits were purchased from Bethyl Laboratories, Mont-
gomery, TX and were used on the culture super-
2.1. Materials natants containing mAbs as per the enclosed
instructions. Affinity purified and adsorbed peroxi-
Squalene oil and bovine serum albumin (BSA) dase-linked sheep anti-human IgG (g-chain specific),
(essentially fatty acid free; cat. # A-7030) were anti-human IgM, anti-mouse IgG (g-chain specific)
50 G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67
and anti-mouse IgM were purchased from The Bind- Fort Knox, KY. No donor information was provided
ing Site, San Diego, CA. ABTS substrate was pur- totheinvestigatorsofthisstudy.However,theoverall
chased from Kirkegaard and Perry Laboratories, donor population of the Fort Knox Blood Center is
Gaithersburg, MD. Female BALB/c, B10.Br and comprisedof85%basictrainees, ranginginagefrom
C57BL/6 mice were purchased from Jackson Labo- 17 to 21. Their immunization status with respect to
ratories, Bar Harbor, ME. Retired breeder mice were AVA administration is unknown. Pooled normal hu-
purchased at 10 months of age. Animal research was man serum, which on analysis was found to be
conducted in compliance with the Animal Welfare slightly positive for IgG and IgM antibodies to
ActandotherFederalstatutesandregulationsrelating SQE, was purchased from United States Biological,
to animals and experiments involving animals and Swampscott,MA.Alloftheaboveserumandplasma
adheres to principles stated in the Guide for the Care werethawed,aliquotedandrefrozenat (cid:2)80jCprior
and Use of Laboratory Animals, NRC Publication, to assay.
1996 edition.
2.3. ELISA assay for antibodies to SQE in human
2.2. Volunteer population and serum preparation serum and plasma
Volunteers were recruited according to U.S. Army SQEwasdilutedinISP(0.2Amol/ml;9.6AlSQE/
regulations under human use protocols approved by 100ml)and0.1ml(20nmol)wasplacedineachwell
the Walter Reed Army Institute of Research and the ofaCostar96-wellsterileroundbottomtissueculture
United States Army Medical Research Institute of plate. Control wells contained ISP alone. The plates
Infectious Diseases Human Use Review Committees wereplacedinabiologicalsafetycabinetandincubat-
andtheU.S.ArmyHumanSubjectsResearchReview edovernighttoallowtheISPtoevaporate.PBS–0.5%
Board. Written informed consent was obtained from boiledcasein,pH7.4(0.3ml)wasaddedtoeachwell.
all volunteers prior to enrollment into the study. U.S. After incubation at room temperature for 2 h, the
Army human use guidelines were strictly adhered to buffer was removed and the plates were tapped on
throughout the course of this study. paper towels to remove the residual blocker. Human
Three cohorts were studied. The first cohort con- serum was diluted serially in PBS–0.5% casein start-
sistedofretiredemployeesoftheUnitedStatesArmy ingata1:25dilution.Dilutedserum(0.1ml/well)was
Medical Research Institute of Infectious Diseases addedtotheplateintriplicate.Theplateswerecovered
(referred to as USAMRIID alumni). Most had re- with plastic wrap and incubated overnight at room
ceived numerous doses of AVA and other vaccines temperature.Theplateswerethenwashed4timeswith
through a USAMRIID special immunization program 0.5 ml/well of PBS, pH 7.4. Peroxidase-linked sheep
(Pittman et al., 2002). All individuals (N=40) were anti-human IgG (g-chain specific) and IgM (g-chain
greaterthan58yearsold(meanage68;range58–82; specific)wasdiluted1:1000inPBS–0.5%caseinand
87.5%male)andmosthadreceivedmultipledosesof 0.1 ml/well was added to the plate. Following a 1-
AVA (mean of 24 doses, range 0–47 doses). Three h incubation at room temperature, the plates were
individuals were not vaccinated with the anthrax washed as described above. ABTS substrate (0.1 ml/
vaccine and the vaccine status of 3 others was well)wasaddedandtheplateswereincubatedatroom
unknown. The second cohort was of similar age temperature for 1 h. Absorbance was read at 405 nm.
(mean age 67; range 54–97; 61.8% male) from the Each plate contained culture supernatant from the C5
normal population of Frederick, MD. 372 individuals cell line, pooled normal human serum and blocking
were enrolled, and none had been vaccinated with bufferas controls. It should be noted that polystyrene
AVA. Blood was collected by standard clinical labo- pipets, tubes, or other objects containing polystyrene,
ratory methods and the serum was removed and shouldnotbeusedinthisassay.Theuseofpolystyrene
frozen at (cid:2)80 jC. causedhighbackground(ISP)absorbancesandgreatly
For the third cohort, outdated fresh frozen plasma increasedthevariabilityoftheresults.Highlypurified
was purchased from Camp Memorial Blood Center, ISP also was required in order to ensure low back-
United States Army Medical Department Activities, ground absorbances.
G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67 51
2.4. ELISA assay for antibodies to SQE in mouse the dilution curve became horizontal. Statistical anal-
serum yses were performed using Minitab Statistical Soft-
ware, Release 13 and StatXact, Version 5, Cytel
The assay was performed as described by Matyas Software, Cambridge, MA. Continuous variables
et al. (2002) with 10 nmol SQE per well. Briefly, the werecomparedusingt-tests,andcategoricaldatawere
mouse serum assay was similar to the human assay compared using Pearson’s chi-squared tests. To esti-
except for the following: (1) PBS–3% BSA was mate the probability of antibodies to SQE, logistic
used as a blocker and diluent. (2) Culture superna- regressionwasusedtoassessthecohorteffectadjust-
tant from hybridoma SQE#16 containing a monoclo- edforthepossibleconfoundingeffectsofgender.The
nal antibody to SQE was included as a positive between cohort odds ratios for IgG and IgM anti-
control on each plate. (3) Diluted mouse serum bodies to SQE were estimated.
was incubated for 1 h on the plates. (4) Peroxi-
dase-linked sheep anti-mouse IgG (g-specific) and
IgM (A-chain specific) (The Binding Site) were used 3. Results
a secondary antibodies.
3.1. Effects of blocking agents on the ELISA
2.5. Interpretation of data and statistical analysis
Several different blocking and diluent agents
Sera were judged to be positive for antibodies to were investigated for use in developing the ELISA
SQE if two dilutions (i.e., 1:25 and 1:50 dilutions for for human samples. In the absence of primary
human serum and 1:50 and 1:100 for mouse serum) antibody, either 0.5% casein (CASEIN) or 0.6%
had absorbances that were greater than 3 times base- gelatin (GEL/CASEIN) as blockers followed by
line.Baselinewasdefinedastheabsorbanceatwhich 0.5% casein as the diluent, worked best for blocking
Fig.2.Comparisonofblockinganddiluentagentsonsecondaryantibodybindingtocontrol(ISP-treated)andSQE-coatedwells(20nmol/well).
AssayswereconductedinPBS,pH7.4containingtheblockeranddiluentproteinindicated.GEL/CASEINwellswereblockedin0.6%gelatin
andthesecondaryantibodywasdilutedin0.5%casein.Allothersampleswereblockedanddilutedintheproteinbufferindicated.Valueswere
themeanoftriplicatedeterminationsFstandarddeviation.
52 G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67
of binding of the peroxidase-linked anti-human IgG (ISP treatment alone) and then blocked with 0.6%
(Fig. 2A) and anti-human IgM (Fig. 2B) to the plate. gelatin or 0.5% casein. Human serum was diluted in
The binding of peroxidase-linked anti-human IgG 0.5%caseinandplatedonthewellsAcontrolhuman
and anti-human IgM to SQE-coated wells was only monoclonal IgG (IM-9) did not bind to control (ISP-
slightly elevated when 4% FBS was used as blocker treated) or SQE-coated wells when 0.6% gelatin or
and diluent. Because we were concerned that the 0.5% casein were used as the blocking agent (Fig.
SQE, which is known to be present in lipoprotein, 3A,B). 0.6% gelatin did not effectively block the
might inhibit the binding of anti-SQE antibodies to binding of human serum IgG antibodies to control
SQE-coated wells, FBS was not considered for (ISP-treated) wells (Fig 3A). Absorbance values for
further use. Based on these data, 0.5% casein was serum numbers 39, 43, 99, and 109 were >1.0, when
chosen as the best diluent for human serum and the incubated on control wells. Furthermore, when 0.6%
secondary antibodies. gelatin was used as the blocker, the absorbance of
Inordertodeterminethebestblockingagent,wells serum IgG antibodies binding to SQE-coated wells
either were coated with SQE or not coated with SQE was detectable, but was only slightly above that of
Fig.3.Comparisonofblockingagentsonantibodiesbindingtocontrol(ISP-treated)andSQE-coatedwells(20nmol/well).Wellswereblocked
withPBS–0.6%gelatin(A,C)orPBS–0.5%casein(B,D).TheserumandsecondaryantibodiesweredilutedinPBS–0.5%casein.Values
wereabsorbancesfromseradiluted1:25andassayedforIgG(A,B)andIgM(C,D)andarethemeanoftriplicatedeterminationsFstandard
deviation.CulturesupernatantsofcontrolhumanmonoclonalantibodiesIM-9(IgG)andRPMI1988(IgM)wereusedataconcentrationof
4Ag/ml.
G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67 53
controlwells(Fig.3A).Incontrast,when0.5%casein SQE (Fig. 3C,D). Control human IgM monoclonal
was used as the blocker, the binding of serum IgG antibody (RPMI 1788) did not bind to control (ISP-
antibodiestocontrolwellswasgreatlyreducedforall treated) orSQE-coated wells with eitherthecaseinor
sera studied (Fig.3B).The absorbancefor serumIgG gelatin blocker (Fig. 3C,D). The absorbances for
antibodiesincubatedonSQE-coatedwellswasreadily serumcontainingIgMantibodiesincubatedoncontrol
detectable when 0.5% casein was used as the blocker wells were essentially the same for gelatin and casein
(Fig. 3B). Consequently, 0.5% casein was used in all blocked wells (Fig. 3C,D; open bars). However, the
further experiments for the measurement of IgG anti- absorbance of serum containing IgM antibodies incu-
bodies to squalene in human serum. bated on SQE-coated wells was dramatically higher
Gelatin and casein were also tested as blocking when the casein blocker was used than when gelatin
agentsforthemeasurementofserumIgMantibodiesto was used asa blocker (Fig. 3C,D;solid bars). Conse-
Fig.4.Bindingofserumanti-SQEantibodiesasfunctionoftheamountofSQEaddedtothewell.WellswereincubatedwiththeamountofSQE
indicatedin100AlofISP.TheELISAwasperformedusingtheprotocoldescribedinSection2withPBS–0.5%caseinastheblockerand
diluent.ValueswerethemeanoftriplicatedeterminationsFstandarddeviation.
54 G.R.Matyasetal./JournalofImmunologicalMethods286(2004)47–67
quently,caseinwaschosenasthepreferredblockerfor range in which the absorbance increased with SQE,
theELISA assay on human IgM antibodies to SQE. reached a plateau, and then decreased (Fig. 4A,D).
The plateau region typically ranged from 5 to 50
3.2. Effect of the amount of SQE coated on the well nmol, but was dependent upon the serum assayed.
The second type was relatively independent of SQE
The binding of human serum antibodies to SQE- (Fig. 4B). The third type of curve continued to
coated wells was dependent upon the amount of increase in absorbance over the entire range of
SQE (Fig. 4). Three types of binding curves were SQE assayed (Fig. 4C). These 3 types of absorbance
observed. The first type had an optimal absorbance curves were observed for both IgG and IgM. Since
Fig.5.ELISAdilutioncurvesfromrepresentativehumanseraassayedintwodifferentexperimentsforIgGantibodiestoSQE.Samplespositive
ofantibodiestoSQEareserum#43and329.SamplesnegativeforantibodiestoSQEareserum#108and336.TheELISAassaywasperformed
using the protocol described in Section 2 with PBS–0.5% casein as the blocker and diluent. Values were the mean of triplicate
determinationsFstandarddeviation.SerawerejudgedtobepositiveforantibodiestoSQEiftwodilutions(i.e.,1:25and1:50)hadabsorbances
thatweregreaterthan3timesbaseline.Baselinewasdefinedastheabsorbanceatwhichthedilutioncurvebecamehorizontal.