Immunoglobulin level in donor blood reactive for antibodies to human immunodeficiency virus

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0095-1137/87/020364-06$02.00/0

Immunoglobulin

Level in Donor Blood Reactive for Antibodies

to

Human

Immunodeficiency

Virust

NRAPENDRA NATH,l* CARA

WUNDERLICH,'

FRED W. DARR II,2 DEBORAH K.DOUGLAS,3 AND ROGER Y. DODD'

TransmissibleDiseases Laboratory, American Red Cross Biomedical Research andDevelopmentLaboratories, Bethesda,

Maryland

208141;

American Red Cross BloodServices, Washington, D.C. 200062; and American Red Cross Blood

Services, Baltimore, Maryland212183

Received 7 August1986/Accepted 24 October 1986

Bloodsamples from98asymptomaticvolunteerblooddonors, including55 that were reactive forantibodies to humanimmunodeficiency virus(HIV)in Western blot(WB)assay,weretested for levels ofimmunoglobulin G(IgG), IgM, and titer ofantibodies toHIV,cytomegalovirus,andherpes simplexvirus. Levels ofIgGwere significantly elevated (P c 0.001)in donors with specificanti-HIV reactivity. A total of 69% of donors with anti-HIV had IgG levelsof .12 mg/ml, and 44% hadIgG levels of .14.5 mg/ml. Levels ofIgM were not

significantly

different among WB-reactiveand nonreactive donors. The titer of anti-HIV wassignificantly (P< 0.02) correlated withIgG levels amongdonorsreactive in the WBassay.Elevation ofIgG, however, wasnot significantly associated withthepresence ofanticytomegalovirusoranti-herpes simplexvirus antibodies. The datashow thatelevation ofIgG may representanearlymanifestationof HIV infection before thedevelopment of clinicalsymptoms ofacquiredimmunodeficiency syndrome.

The human T-cell

lymphotropic

virus type III or lymph-adenopathy virus (1, 3, 10, 12), recently renamed human

immunodeficiency virus (HIV), isgenerally accepted asthe

etiologic agent of acquired immunodeficiency syndrome

(AIDS). Testsforantibodies tothis virus are now routinely

used to detect and eliminate from the donor pool blood donors who might be asymptomatic carriers of HIV.

Anti-bodiesto HIVweredetected in the vastmajorityofcasesof AIDS (6, 7, 9) compared withhealthy heterosexual individ-uals. The long-term prognosis of donors with anti-HIV reactivity is not clearly defined. It has been shown that at least someindividualswho transmitted AIDStorecipientsof

their blood eventually develop AIDS or related clinical conditionsthemselves (3). It isnotknown whatproportions of infected individuals remain

asymptomatic

fortherest of

their lives.

Significant changes in immunological parameters such as theratioofT4/T8cells, natural killer cell activity, and levels ofimmunoglobulinhave been reported among patients with

AIDS (6, 7), pre-AIDS, and certain populations that are at riskofcontracting infection with HIV (13). It is not known,

however, how soon after exposure to HIV changes in

immunologicalmarkers become evident.

Inthispaper, wedescribe the levels of immunoglobulin G (IgG) and IgM among apparently healthy blood donors who arebelievedtohave been infected with HIV, on the basis of

thepresence of specific antibodies in their blood.

MATERIALS AND METHODS

Serum specimens. A panel of 98 serum samples from

selected individual blood donors was assembled from blood

donatedtothe American Red Cross Blood Services Regions

located in Washington, D.C., and Baltimore, Md., over a

*Correspondingauthor.

tContribution no.699.

period ofoneyear. All samples were reactive for anti-HIV on routine testing in the regions, and all donors were

asymptomatic at the time of blood donation and met all

specific criteriarequiredof all donors bythe American Red Cross. The criteria for acceptance ofablood donor include normalbodytemperature,hemoglobinlevels, orhematocrit.

Additionally,tobeacceptedadonor maynothaveahistory of hepatitis, drug use, travel to a malaria-endemic area

duringthe 6 monthsbefore donation, or show any physical sign of parenteral drug abuse. All donors are alsoadvised and encouragednot to donate blood ifthey belongtoAIDS risk categoriesasdefinedby the Centers for DiseaseControl (2). Serawerestored frozenat -20°C orkeptat4°Cwithout anypreservative.

Testfor anti-HIV. Allsera weretestedforthe presence of anti-HIV by an enzyme immunoassay (EIA) manufactured

by Abbott Laboratories (North Chicago, 111.). Sera were tested at 1:400 dilution following the instructions of the

manufacturer. Allinitiallyreactive samples were retested in

duplicate intheregionsandwereincluded in this study only if one orboth were reactive in retests. All sera were retested athird time for anti-HIVby the Abbott EIA and by Western

blot (WB) assay (14) when received by the Transmissible DiseasesLaboratory. A total of 13 samples in the panel were

determined to be nonreactive for anti-HIV when tested at theTransmissible Diseases Laboratory; all were also nonre-activebyWBfor anti-HIV. The EIA test in the regions may have been false-positive, not an uncommon occurrence. A samplewascalled WB reactive if it had antibodies to two or more peptides specific to HIV that included peptides or glycopeptides of 120,000 (gp-120), 64,000 (p-64), 53,000 (p-53), 41,000 (gp-41), and 24,000 (p-24) molecular weight. Anti-HIV titers were determined by testing serial log dilu-tions of samples, using Abbott EIA kits for the assay.

Test for immunoglobulin level. Quantitations of IgG and IgM in sera were done by a solid-phase fluorescence im-munoassay with Fiax immunoglobulin G and M test kits

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TABLE 1. ElevatedIgG levels relative to the presence of anti-HIV andthe sex of the donor

Anti-HIVa No. No. of No. with IgG

tested each sex -14.5 mg/ml (%)

Reactive 55 46male 20(44)

9female 4 (44)

Nonreactive 43 26male 1(4)

17female 0

Total 25(25)

aAnti-HIVreactivityasdeterminedinWBassay.

manufactured by MA Bioproducts (Walkersville, Md.). In brief, a 1:50or higher dilution ofsera was incubated with

membrane-bound antibodies specific for IgG or IgM.

Cap-tured IgG or IgMwas detected by reacting it with specific

antibodies labeled with fluorescein isothiocyanate. Data

were interpreted and quantified by comparisonwith a

stan-dard curvebyusing the Fiax fluorometer.

Test for other antibodies. Antibodies to cytomegalovirus (CMV) and herpes simplex virus (HSV)weredetermined by

using specific Fiaxtestkits.

RESULTS

AssummarizedinTable 1, 55 of 98 (56%) blood donors in thisstudy hadantibodiestoHIVasconfirmed by WBassay.

Asignificant (P<0.02) majority (83%) of the positiveswere men. Overall, the mean age of all donors in this study

populationwas33.5years. Inmenthemean age was32and

36 years among WB reactives and nonreactives,

respec-tively; thecorrespondingmeanagesforwomen were31and

33 years, respectively. Among those found reactive on the

WBassay, menranged inagefrom 17to50yearsandwomen

from17to44years.Nosignificant difference inage was seen

among men and women when data were analyzed for the

2.5 5.5 0.5 11.5 14.5 17.5 20.5

TO TO TO TO TO TO TO

5.5 8.5 11.5 14.5 17.5 20.5 23.5

Ig ne/si

FIG. 1. Comparison of levels of IgG in asymptomatic blood donors whowerereactive(solid bars)ornonreactive(open bars)for

anti-HIV when testedbyWBassay.

TABLE 2. Sera withelevatedlevels of IgM

Donor Immunoglobulin

Sample Anti- level (mg/ml) identifi- WB HIV titer

cationno. Age Sex (103) IgG IgM

(yrs) SeIg g

88 17 Female + 100 16.7 >2.28

89 44 Male + 100 6.6 2.21

91 35 Male + 100 18.6 2.10

141 35 Male - NDa 10.9 2.10

aND, Notdetermined.

presence or absence of antibodies to HIV. Only 2 of 55

WB-reactive donors showedweak (-/+) reaction togp-41,

while four otherspecimenslackeddetectable levels of anti-body to p-24. No WB-reactive sample lacked reactivity to

both p-24andgp-41 simultaneously.

The IgG levelamongWB-reactivedonors ranged from 1.2 to >42.2mg/ml including five specimenswith IgGlevels less than 5

mg/ml

(Fig. 1). The mean value of IgG was 14.48

mg/ml(standard

deviation,

6.9mg/ml)inWB-reactiveblood

donors in this study. The corresponding values in

WB-negative donorswere

7.78-mg/ml

IgG anda standard

devia-tion of2.35 mg/ml. Data were furtheranalyzed to evaluate

the distribution of

IgG

levels relative to the presence of

anti-HIV

considering a level of IgG -14.5 mg/ml to repre-sent the elevated level. The IgG level of14.5 mg/ml was

arbitrarily selected based on the IgG level among

WB-nonreactivedonorsin thisstudy. Asignificantly(P< 0.001) greater numberofdonorswith anti-HIVhadelevatedlevels

(.14.5

mg/ml) of IgG in their blood (Table1).Ofa totalof 25

donors with IgG levels in excess of 14.5 mg/ml, 24 were

reactive for anti-HIV.

IgG

levelsof

.12

mg/mlwere

found

in 38 of55 (69%) donors with anti-HIV reactivity and 3 of43

(7%) in the absence of anti-HIV. The

difference

remains highly significant

(X2

= 38.2).

The mean levels of IgM were 1.15 and 1.17 mg/ml, respectively, among donors with and without antibodies to HIV.The

difference

isnotsignificant.Therewere, however,

foursampleswithelevatedIgM levels

(.2

mg/ml), including

three that were reactive for anti-HIV activity in the WB assay (Table 2). Two of the three WB-reactive donors

showed elevated

IgG

levels

aswell.

A subnormal level of IgG (c5

mg/ml)

was found in five

donors, three of which were

negative

on WB assay for anti-HIV. Only one had

anti-CMV activity,

and three had

anti-HSV activity. Twowere women. All had normallevels

of

IgM.

Thetitersof anti-HIVreactivity among

WB-positive

sam-TABLE 3. Titerofanti-HIVreactivityrelativetoelevated levels

ofIgGamong WBreactives

Anti-HIV N Maie

IgG

.14.5 mg/ml

3nti-N%

No. Male Female i

titer(10) No.

ffl)2

Male Female

1 2 1 1 0

10 18 15 3 5(28) 4 1

100 31 26 5 15(48) 12 3

1,000 3 3 0 3(100) 3 0

10,000 1 1 0 1(100) 1 0

Total 24(44)

aPercentageof total numberofsampleswithcorrespondinganti-HIV titer.

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40'

9

6

10 100

FIG. 2. Levels of IgGinrelationtothe titer of anti-HIVamongWB-reactive donors. Valuesshownare mean +standard deviation (bars).

ples aresummarizedinTable3.Overall,56% of theserahad atiter between 105 and 106. Two donors had a low titer of

103, while a 34-year-old man showed atiter ofat least i07. Significant (P< 0.02) positive correlationbetween the titer

ofanti-HIVandthe levels ofIgGis shown inFig. 2. Assummarized in Table 4,69and84%of 98 donorstested hadantibodies to CMV and HSV,

respectively.

Antibodies

to CMV and HSV were present in greater than 95% of all

samples positive for anti-HIV. Association between

anti-HIV, CMV, andHSV is statistically

significant

(P <0.01). Table 5 summarizes the distribution of antibodies to CMV and HSVin relationtoIgGlevels. The data indicate that the

presence

ofantibodies to CMV and HSV was not

signifi-çantly

associated with the elevated levels of IgG, even though 23ofthe 25(92%)donors who hadelevated levels of IgG also hadantibodies to CMV and HSV. Anti-CMV and

anti-HSVwerepresentin 44

(60%)

and60(82%) of73donor sera, respectively, among those with IgG levels <14.5

mg/ml.Theseprevalencesarenotsignificantlydifferentfrom

92%amongdonors with IgGlevels .14.5 mg/ml.

Anincreaseinthe level ofIgGwas notcorrelated withan increase in titer ofanti-CMV (Fig. 3) or anti-HSV (Fig. 4). Data suggest thatelevationof IgGoccurredindependentlyof CMV or HSV infection.

Only9donors among 98didnot haveantibodies toHIV,

TABLE 4. Presenceof antibodies to CMV and HSVrelative to

thepresenceof antibodies toHIV

No. with:

Anti-HIV No.tested Anti-CMVa Anti-HSVb

+ + _

Reactive 55 53 2 52 3

Nonreactive 43 15 28 31 12

aAssociation between presence ofantibodies to HIV and CMV, x2 = 42.9, Ps 0.001.

bAssociation between the presence of antibodies to HIV and HSV, x2

9.38,Pc0.01.

CMV,orHSV. Allweremen,witha meanageof30

(range,

17 to45) years.

DISCUSSION

The objective of the present study was to

identify

a

nonspecific

serological characteristic which may be associ-atedwith the earlystagesof

HIV

infection

and whichmaybe

useful

as a marker to

identify

blood donors

infected

with

HIV. To

investigate

this

possibility,

we studied

immuno-globulin levels in a panel of sera from 98 routine blood

donors whowere initially reactive for

anti-HIV

on routine testing withacommercialtestkit. Usingtheresults ofaWB assay as the basis of characterizing the

specificity of

anti-HIVreactivity,wedividedthe98seraintotwogroups: WB

positiveand

negative.

The 43

WB-negative

seraincluded 30

thatwererepeatably reactivewhentested withthe

commer-cial EIA test kit, and the remaining 13 may represent

false-positives

in

initial

testings. Similarhigh

proportions

of nonspecific

reactions

with commercial

kits

have been

re-ported

previously

(11). The

nonspecific

reactivity in EIAis generally considered due to

reaction

between naturally

oc-curring

antibodies

andresidual host-cell components in the

viral

antigen

preparation

used as thereagent inthe EIAkits. This reactivity is an inherent property ofthe test kit and

would

beexpectedto vary with the compositionof the test assay.

Our data show that levels of IgGincreased inassociation

with the increase in the titer of antibodies to HIV but not with the age of the donor. In other words, older men with

TABLE 5. AntibodiestoCMV and HSV relative to levels of IgG

IgG level No.anti-CMV No.anti-HSV

(mg/ml) N reactive reactive

214.5 25 23a 23a

<14.5 73 44 60

aStatistically there isnosignificant

difference

(x2

=0.76) in thedistribution ofanti-CMVandanti-HSVinrelation toelevated IgGlevels.

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£ Il

a i0

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O0 A o

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50 100 150 200 250 300 350 400 TI CMV lITI

FIG. 3. Levels of IgG in relation to the titer of anti-CMV in the Fiax test. Values <20 are nonreactive for anti-CMV. Samples reactive (E) andnonreactive(A)on WB for anti-HIV are shown.

specific antibodies to HIV did not necessarily have higher

levels of IgG or a higher titer of anti-HIV. A high titer of antibodies to HIV was the norm rather than the exception. All except two donors had titers

.10,

including a donor withatiter of107. All five individuals with subnormal levels ofIgG had normal levels of IgM, and two were negative for antibodiestoHIV, CMV, and HSV. Antibodies to CMV and

HSV were significantly more likely to occur in donors reactivefor antibodies to HIV. However, the antibodies to CMV and HSV were notassociated with elevatedIgGinour testpopulation.Thepractical significanceof this observation willbe further discussed later.

The level of IgM was not significantly different among donors reactive or nonreactive for anti-HIV in the WB

0

o ° o

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o o

Ab cinz

o

-A o A là

o o

o m

o i A

ci

^i

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20 40 80 i0 100 120 140 180 180 ATIH5VTIlI

FIG. 4. Level ofIgGin relationto the titer of anti-HSV in the Fiax test. Values <8 are nonreactive for anti-HSV.Samplesreactive(E)

and nonreactive(A) onWB for anti-HIVareshown.

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assay. Elevated levels of IgM occurred infrequently in the presentstudy and did not appear to beof immediatepractical significance. However,HIV-specific IgM could be of inter-est in determining the immediacy of infection.

Elevated levels of IgG in clinical cases of AIDS or

pre-AIDS have been reported previously (13) and arenot

particularly surprising in view of the multiple opportunistic infectionsassociated with this disease complex. However, it

should be pointed out that all subjects in this study were apparently healthy by virtue of the fact that they were

acceptedasblood donors after a routinemedical history and evaluation. Almost half (44%) of the 55 individuals who had

anti-HIV reactivity on the basis of the WB assay also had

IgGlevels in excess of 14.5 mg/ml. Fully 69% had IgG levels

.12 mg/ml among WB-reactive donors. The single individ-ual withelevated IgG (14.59 mg/ml) among the WBnegatives hadserumwhich was repeatablyreactive in the Abbott EIA.

This donor could have represented a seronegative HIV

carrier(5); however, no follow-up specimens wereavailable.

Of course, it is also quite possible that this individual had another undiagnosed condition or infection resulting in IgG

elevation. Elevated IgG levels .14.5 mg/ml were associated

withthe presence ofantibodies toHIV, CMV, or HSV in 44, 35, and 28% of the reactives, respectively. Even though all are sexually transmitted infections, the association between HIV and elevated IgG appears to beparticularly remarkable.

Our data suggest that the elevated levels of IgG found in

blood donors represent a certain host response to HIV infection, since elevation of IgG was associated only with

anti-HIV and not with anti-CMV and anti-HSV. There is also noevidencethat the increase in IgG levels generated a

concomitant increase in the titer of anti-CMV or anti-HSV. The data, however, do not show conclusively that the

elevationof IgG levels in blood donors was indeed a specific response to HIV infection.

IgG levels may increase in response to massive foreign

antigen load or in response to infection. High levels of antibodies (mode and median value of anti-HIV titer,

10')

may result from repeated stimulation of B cells by an

antigen(s) of HIV. Alternatively, the very high titers of

anti-HIV reactivity reported here in most sera positive in WB assays may be a result of loss ofimmunological control

resultingfrom infection of T-4 ceils withHIV. Inmost of the sera positive in the WB assay in this study, antibodies to

polypeptidesof HIV including gp-120, p-64, p-53, gp-41, and p-24 were detectable (data not shown). The presence of high-titer, multiple-specific antibodies to HIV, along with thefactthat some of these donors most certainly carried the viable virus in their blood (3), suggests the presence of

circulating antigen-antibody complexes. High titers of multispecific antibodies to HIV antigens may result from

multipleorpersistent antigenic stimulation of Bcells over a long period of time before any clinical symptoms become evident.

Significantlyelevated levels of IgG were found in a major-ity ofhomosexual menwith Kaposi's sarcoma (13). Several of these individuals also had a corresponding elevation in IgA and IgM levels. A similar pattern of simultaneous elevation of IgG, IgM, IgA, and IgE was also found in a few of 11 individuals withPneumocystis

carinii

pneumonia (8). In retrospect, it is likely that these reports actually described

individualswith AIDS, which is nowknown to be caused by HIV (1, 4, 10). In athorough study of B-cell

abnormalities

in

patientswith AIDS, healthyhomosexual

males,

and hetero-sexual controls, Lane et al. (6) found ample evidence of in

vivo polyclonal activation of Bcells andelevated levels of

IgG and IgA in patients with AIDS. Our data show that

hypergammaglobulinemia occurs in HIV infection

long

be-fore the clinical manifestation of AIDS becomes

evident,

suggesting that polyclonal activation of B

cells

occurs

early

in infection withHIV.

In conclusion, we present evidence that

hypergam-maglobulinemiaisanearlyresponsetoinfection byHIV.We also show that determination of IgG levels in the blood of

donors which was reactive for anti-HIV on routine

testing

may be a usefuladjunct in diagnosticevaluation ofexposure toHIV.

ACKNOWLEDGMENTS

We thank T. Mohanakumar for his helpful suggestions and critique of thismanuscript.

This workwas supported by the American Red Cross.

LITERATURE CITED

1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S.

Chamaret, J. Gruest, C. Daugnet, C. Axier-Blin, F.

Brun-Vezinet, C. Rouzioux, W. Rozenbaum, and L.Montagnier. 1983.

Isolation of a T-lymphotropic retrovirus from a patientat risk

for acquired immune deficiency syndrome (AIDS). Science

220:868-871.

2. Centers for Disease Control. 1983. Acquired immunodeficiency

syndrome (AIDS) update-United States. Morbid. Mortal. Weekly Rep. 32:309-311.

3. Curran, J. W., H. W. Jaffe, T. A. Peterman, and J. R. Allen.

1985. Epidemiologic aspects ofacquiredimmunodeficiency

syn-drome (AIDS) in the United States: cases associated with transfusions, p. 259-269. In R. Y. Dodd and L. F. Barker (ed.), Infection, immunity and blood transfusion. Alan R. Liss, Inc., New York.

4. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleski, B. Safai, G. White, P. Foster, and P. Markham. 1984. Frequent

detection and isolation ofcytopathicretrovirus(HTLV111) from

patients with AIDS and at risk for AIDS. Science224:500-503.

5. Groopman, J. E., P. I. Hartzband, L. Shulman, S. Z.

Salahuddin, M. G. Sarngadharan, M. F. McLane, M. Essex, and

R. Gallo. 1985. Antibody, seronegative humanT-lymphotropic

virus, type II (HTLV lll)-infected patients with acquired

immunodeficiency syndrome or related disorders. Blood

66:742-744.

6. Lane, H. C., H. Masur, L. C. Edgar, G. Whalen, A. H. Rook,

and A. S. Fauci. 1983. Abnormalities of B-cell activation and

immunoregulation in patients with the acquired

immunodefi-ciency syndrome. N. Engl. J. Med. 309:453-458.

7. Laurence, J., F. Brun-Vezinet, S. E. Schutzer, C. Rouzioux,D.

Klatzmann, F. Barre-Sinoussi, J. C. Chermann, and L. Montagnier. 1984. Lymphadenopathy-associated viral antibody

in AIDS. Immune correlation and definition of a carrier. N.

Engl. J. Med. 311:1269-1273.

8. Maur, H., M. A. Michelis, J. B. Greene, I. Onorato, R. A.

Stouwe, R. S. Holzman, G. Wormser, L. Brettman, M. Lange, H. W. Murray, and S. Cunningham-Rundles. 1981. An outbreak

of community-acquired Pneumocystis carinii pneumonia. N.

Engl. J. Med. 305:1431-1438.

9. Nicholson, J. K. A., J. S. McDougal, H. W. Jaffe, T. J. Spira, M. S. Kennedy, B. M. Jones, W. W. Darrow, M. Morgan, and M. Hubbard. 1985. Exposure to human T lymphotropic virus

type III Iymphadenopathy-associated virus and immunological

abnormalities in asymptomatic homosexual men. Ann. Intern.

Med. 103:37-42.

10. Popovic, M., M. G. Sarngadharan, E. Reed, and R. C. Gallo.

1984. Detection, isolation and continuous production of

cytopathicretrovirus (HTLV III)from patients with AIDsand

pre-AIDS. Science 224:497-500.

11. Schorr, J. B., A. Berkowitz, P. D. Cumming, A. J. Katz, and

on April 11, 2020 by guest

http://jcm.asm.org/

(6)

S. G. Sandler. 1985. Prevalence of HTLV II1 antibody in American blood donors. N.Engl. J. Med. 313:384-385. 12. Schupbach,J.,M. Popovic, R. V.Gilden,M. A.Gonda, M. G.

Sarngadharan,andR. C. Gallo. 1984.Serological analysis ofa

subgroup of human T-lymphotropic retroviruses (HTLV III)

associated withAIDS. Science224:503-505.

13. Stahl,R.E., A.Friedman-Kien, R.Dubin,M. Marmor, andS.

Zolla-Pazner. 1982. Immunologic abnormalities in homosexual

men. Relationship to Kaposi's sarcoma. Am. J. Med. 73: 171-178.

14. Towbin, H.,T.Staehelin,andJ.Gordon.1979. Electrophoretic

transfer of proteins from polyacrylamide gels tonitrocellulose sheets:procedure andsomeapplications. Proc. Natl. Acad. Sci. USA 76:4350-4354.