Optimal fluorescein to protein ratios of bacterial direct fluorescent antibody reagents

0095-1137/81/030498-05$02.00/0

Optimal

Fluorescein-to-Protein

Ratios of Bacterial Direct

Fluorescent-Antibody Reagents

G.ANN HÉBERT,* BERTIEPITTMAN, AND ROGERM. McKINNEY

Bureauof Laboratories, CentersforDisease Control, Atlanta, Georgia 30333

Anumber of bacterial systems were studied with specific direct fluorescent-antibody reagents prepared from rabbit antiserum fractions and having a wide range offluorescein-to-proteinratios. Thesesystemsincluded Bacteroides,

Bor-detella, Clostridium, Escherichia, Legionella, Listeria, Salmonella, Shigella,

and Streptococcus. For all systems studied, a fluorescein-to-protein ratio of 30 was optimal for conjugates prepared from ammonium sulfate fractions (>75% gamma globulin) and pure immunoglobulin G desorbed from the Sepharose-bound protein A of Staphylococcus aureus. A pepsin digestion procedure is described that yielded the F(ab')2 piece of pure immunoglobulin G; this was labeled and studied at twofluorescein-to-proteinratios.

Ourlaboratory has been involved in fluores-cent-antibody methodology research, training, and consultation for many years.Throughoutall of these activities the most frequently asked question was, and still is, "What

fluorescein/

protein (F/P) ratio shouldIhave?" Theanswer has always been "It depends on the intended

application,butforall bacterial systemswe pre-ferahigh F/Pratio."

An optimal F/P ratio is that which provides the maximumspecific stainingtiterwitha min-imum ofnonspecific(non-immunological)

stain-ing.This paperdeals with the directrelationship

between specific staining and F/P ratios for a

number of bacterial immunofluorescence sys-tems. The data presented have been

accumu-lated over anumber of years. Some new

tech-niqueshavebeenintroducedfromtimetotime, but the resultsareremarkablysimilar.

MATERIALS AND METHODS

Bacterialsystemsrepresenting9generaand13 spe-cies were examined during this study. These were Bacteroidesfragilis, Bordetella pertussis, Clostrid-iumsepticum, Escherichia coli, Legionella pneumo-phila (2),Legionella bozemaniisp.nov. (1), Legion-ella micdadei sp. nov. (12), Legionella dumoffii sp. nov. (1), Listeria monocytogenes, Salmonella, Shi-gella dysenteriae, Streptococcusmutans, and Strep-tococcuspyogenes.

Allof the datapresentedwereobtained from rabbit antisera. Most of the antisera were fractionated by three successiveprecipitations in35% saturated am-moniumsulfate, asdescribed previously (9). Immu-noglobulin G (IgG) was isolated from some of the antisera byaffinitychromatography on a column of proteinA (Staphylococcus aureus) covalentlybound toSepharoseCL-4B(PharmaciaFineChemicals, Inc., Piscataway, N.J.),aspreviously described (7). Some

ofthe IgGwasfurtherfractionated toF(ab')2by pepsin digestionof the Fcfragment; the method described by Nisonoff (16) was modified, and the procedure fol-lowed is describedbelow.

(i) The pH of the IgG solution(22 mgof IgGper ml in borate saline)wasloweredto 4.5 byadding 2 parts of0.1 Maceticacid, in 0.15 Msodiumchloride, to 3 partsofprotein.

(ii) Apepsinsolution (0.5 mg/ml)was prepared by rehydrating crystallized pepsin in 0.2M sodium ace-tate-1.0M aceticacid-0.15M sodiumchloride reagent, pH3.8.

(iii) The pepsin solutionwasadded toantibodyfor afinal ratioof 1 mgof pepsin for each50mgofIgG desorbed from protein A; the pHwas checkedtobe sureitwasbetween3and 4.

(iv) Themixture wasplacedin an incubator at37°C overnight (16 to 18 h).

(y)Afterincubation,themixture wascentrifugedto removetheacid-insoluble glycoproteins.

(vi) The clear supernatant antibody mixture was carefully decanted, and itspHwas raisedto 10with concentrated sodiumhydroxidetoinactivate the pep-sin.

(vii) The total volume of theantibody mixturewas measured, and anequal volume of saturated ammo-niumsulfatewas added toprecipitate and recover the F(ab')2.

(viii) Aftercentrifugation to pack the precipitate, the supernatant was discarded; the precipitate was dissolved in distilled water and then reprecipitated withammonium sulfate to wash theprotein.

(ix)Aftercentrifugationtorecoverthe second sul-fateprecipitate,theproteinwasredissolved indistilled water and then dialyzed in 0.05 M borate-0.15 M sodium chloride(pH 7.4)to removethe sulfate before columnchromatography.

(x)Thesulfate-free antibodywasapplied to a col-umn ofproteinA-Sepharose CL-4B and eluted with the borate-saline solution. The final F(ab')2 product didnotbind toproteinAandcontainedapproximately one-half the totalproteinof theoriginalIgGsample. 498

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Nootherproteinwasobtained duringacid desorption of thecolumn.

The total protein concentrations of the globulin fractions were measured by the biuret method (8). The protein concentrations of the IgG and F(ab')2 samples were estimated by absorption at 280 nm, assuming El

./ml

- 1.4. Allantibody fractions were

then labeled with fluoresceinisothiocyanate (FITC) by the direct method, in which various dye weights were used to achieve the desired F/P ratios (11). Unreacted FITCwasremoved bydialysis inpH9.0to 9.5phosphate-bufferedsaline.

Ail of theconjugatesforagiven antigenwere pre-pared from thesamelot of antiseraandwereadjusted to approximately equal protein concentrations. All conjugatesweretitrated with theirhomologous anti-gens.Thetestantigenswerebacterial cellsuspensions in 0.85% sodium chloride containing 1% Formalin. Smears were preparedand then stained with serial twofold dilutions of the conjugates according to a standard directfluorescent-antibody procedure (14). Thestained smears were examined by fluorescence microscopy,and theconjugate titerswereexpressed

asreciprocalsof thehighestdilutions,relativetothe initialadjusted proteinconcentration, that retaineda 3+orgreaterstaining of the bacterialcells. All of the conjugates were also examined for physicochemical characteristics. Protein concentrationsweremeasured by the biuret method withoptical density readingsat 560 nm, FITC concentrations were determined by absorbance in 0.1 N sodium hydroxide at Am. (493 nm),F/P ratioswereexpressedasmicrograms of FITC per milligram ofprotein, and protein compositions wereexaminedby celluloseacetate strip electropho-resis(11).

RESULTS

Allof the globulin preparations obtained by fractionation with ammonium sulfate were at

least 75% gamma

globulin

and contained no more than 1% albumin. The characteristics of

theconjugates

prepared

fromthesefractionsare

shown in Table 1. With each of these eight bacterial systems, the

highest

specific

staining

titer wasobtainedatthe

highest

F/P ratio

pre-pared and tested. Most of thesemaximum F/P ratioswere between27 and 31,ug of FITC per

mg of

protein.

Anotherrelationship calculated

wastheF/T ratio,ormicrograms of FITCatthe

specific

titerdilution.Inall systemsstudied,the

lowestF/Tratiowasassociated withthe

maxi-mum

specific staining

titerand the

highest

F/P

ratio.

The first seven

conjugates

in Table 2 andall

oftheconjugatesinTable 3 werepreparedfrom the same poolof L.pneumophila antisera ob-tained by combining the sera ofthe last three

bleedings from a rabbit immunized with the

Philadelphia 2 strain (4) of serogroup 1. The

characteristics ofconjugates preparedwithIgG

fractions desorbed from proteinAareshown in

Table 2. Again, the specific staining titer

in-creased andthe F/Tratiodecreasedas the F/P ratio increased from below 20 up to approxi-mately 30. Above an F/P ratio ofapproximately

32, thespecific staining titer decreased and F/T ratioincreasedas the F/P ratioincreased.

TheF(ab')2, IgG, and globulinconjugates

pre-pared from L. pneumophila antisera were all examined at aproteinconcentrationof

approxi-mately 5 mg/ml (Table 3). The labeling

effi-ciency oftheF(ab')2 preparationswasextremely

low compared to the IgG and globulin (88% IgG)

fractions. Thesame high

specific

staining titer wasobtainedwith all threepreparations,butthe

globulin, IgG, andF(ab')2 conjugates with this 1: 320titer had quitedifferentF/P ratios of 30,28, and 8,respectively.

DISCUSSION

The

optimal

F/P ratio forglobulin and IgG

preparationswithall of the systems studied was

approximately 30; although this is higherthan most reports recommend, it is necessary with

bacterial systems in order to obtain high titers

and bright staining. The nonspecific staining expected withhigh F/Pratioswas not aproblem withanyofthese systemsbecause theresulting high titers allowedgreaterdilution without

sac-rificing

specific

stainingintensity,and dilution is very effective in reducing nonspecific staining

(10). This relationship is expressed inthe F/T

ratio,which isthemicrogramsofprotein-bound

FITC in 1 ml of

conjugate

at thespecific titer dilution.Nonspecific stainingisdirectlyrelated

totheFITCconcentration (10),sotheF/T ratio

is ameasure of thenonspecificstaining

potential

of areagent. In this study, the lowest F/Tratios

werealways associatedwith thehighestspecific staining titers and thehigh F/P ratiosnear30,

but further increasesinF/P ratio caused lower

specific

staining titers andhigher F/T ratios. Underidentical labeling conditionsat 5mgof protein per ml with 40 ,g of FITC per mg of

protein, the

globulin,

IgG, and F(ab')2

prepara-tions hadlabelingefficiencies (microgram ratio

of bound FITC to original reaction mixture weight) of75, 70, and 20%forfinalF/Pratiosof

30, 28, and 8, respectively

(Table

3). Despite these

differences,

all three

conjugates

had the

samehighspecific staining titer of 1:320. Since

IgGisonly 50% F(ab')2 byweight, the remainder

being Fcpiece,theIgG

conjugate

andthe

glob-ulin

conjugate

of this antiserum appear to be

about twice as effective as theF(ab')2conjugate

onamolar basis for directfluorescent-antibody staining. This can be

readily

accounted for on the basis of the higher degree of labeling ob-tained with intactIgGrelative to that obtained

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50)0 HÉBERT, PITTMAN,

TABLE 1. Characteristicsofconjugates prepared from ammoniumsulfate-fractionatedrabbit antisera F/Pratio Specific Proteinconcn FITCconcn F/Tratio Labeledantibody

(pg/mg)

titerf

(mg/ml)b

(pg/ml)

(jLg/ml)C

Escherichia coli 018ac:B21:H7 3 32 9 27 0.84

10 128 9

17 256 9

29 512 9

90 153 261

0.70 0.60 0.51 Bordetella pertussis

Streptococcus pyogenes (group A)

Listeria monocytogenes(type4b)

Salmonella(polyvalent)

Shigella dysenteriae(Ai)

Clostridiumsepticum

Bacteroides fragilis

5 20 6

18 80 6

23 160 6

27 320 6

30 108 138 162

3 <1 1.6 4.8

6 10 1.6 9.6

9 50 1.6 14.4

16 100 1.6 25.6

28 500 1.6 44.8

8 16 5

14 32 5

27 256 5

il 10 14

17 20 13

37 160 9

14 31

16 il

64 6

il 64 7

31 256 7

10 20 3

16 32 3

21 40 3

28 100 3

40 70 135 154 221 333 154 186 77 217 30 48 63 84

aSpecific stainingtiter =reciprocal ofthehighestdilution that retained a3+ orgreaterstainingofthe homologous cells.

bMeasuredbythe biuretmethod.

F/Tratio=FITCconcentrationatthespecific stainingtiter.

with the F(ab')2 preparation.Ifwe assume the same 20% labeling efficiency with the F(ab')2 portion of the IgG in the IgG conjugateas was

observedinlabeling F(ab')2thatwasfreed of the

Fc piece,then approximately 86% of the FITC

wasboundtothe Fcpiece in the IgG conjugate. Itappears,therefore, that the Fcpieceserves as an areaforattachingaheavyload offluorescein

without interfering with the antigen-antibody reaction.

Theuse ofF(ab')2 conjugates eliminates the

well-known reaction of intact IgG molecules with S. aureus strains that contain cell-bound

StaphylococcusproteinA. Thisundesired activ-ity can be blocked, however, by using

rhoda-mine-labeledpreimmunization IgG (5) asa

dil-uentforthe FITC-IgG conjugate. In this labo-ratoryweprefertouserhodamine-labeled

pre-immunization whole serum asthe diluent. The

added benefit oftherhodamine counterstain has been welldocumented (6, 17) and is particularly useful whenexaminingdeparaffinized tissue

sec-tions and sputum specimens (3). Although Danielsson and Forsum (6) reported no

signifi-cantdifference between IgG and F(ab')2 conju-gates atcomparable F/P ratios, several

differ-ences in methods ofpreparation, labeling, and

analysismake itdifficulttodirectlycompareour

results. Further studiesare,therefore, desirable, usingIgG and F(ab')2 conjugatestostain micro-bial antigens in diseased tissue sections where background andnonspecificfluorescenceare

di-agnostic problems.

Conjugationprocedureshave beendeveloped toproducereagentswithanydegreeoflabeling (F/P ratio) desired.ThechosenF/Pratio is best

1.50 1.35 0.86 0.51 <5

0.96 0.29 0.26 0.09 2.5 2.2 0.5 15.4 11.1 2.1 9.6 2.9 1.2 0.8 1.5 1.5 1.6 0.8 J.

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TABLE 2. Characteristicsof conjugates preparedfrom rabbit antibody (IgG) desorbed from protein A-Sepharose CL-4B

F/P ratio Speciflcstain- Proteinconcn FITCconcn F/T ratio Labeledantibody (j~g/mg) ingtitera

(mg/mI)b

(Ag/ml)

()Ag/ml»

Legionellapneumophila 7.6 20 5.0 38 1.90

Philadelphia2 14.7 80 5.1 75 0.94

21.2 160 5.0 106 0.66

27.9 320 5.1 143 0.45

31.3 320 5.3 166 0.52

37.7 160 5.3 200 1.25

44.3 80 5.1 226 2.83

L.pneumophilaTogus1 18.0 320 3.8 68 0.21

26.0 640 4.1 107 0.17

L.bozemanùi (WIGA) 30.1 1,024 10 301 0.29

L.micdadei (TATLOCK) 28.4 1,024 10 284 0.28

L.micdadei (HEBA) 31.0 1,024 10 310 0.30

L.dumoffli(Tex-KL) 29.0 1,024 10 290 0.28

Streptococcusmutans(typed) 29.8 2,048 12.6 375 0.18

31.7 2,048 12.6 400 0.20

aSpecific staining titer =reciprocal of the highest dilution that retained a 3+ or greater staining of the

homologouscells.

bMeasuredby the biuret method.

CF/T

ratio=FITC concentrationatthe specific staining titer.

TABLE 3. CharacteristicsofF(ab',h,IgG, andglobulinconjugatespreparedfromthesamepoolofL. pneumophila antisera

Reaction Labeling F/Pratio Specific Protein FITC

concn

F/T ratio Labeledantibody mixture efficiency staining concn

ratio' M)b (Pg/mg) titerc (Mg/Ml)d (Ag/ml)

(Alg/ml),

F(ab')2 20 il 2.1 5 4.9 10 2.0

IgG 20 74 14.7 80 5.1 75 0.94

F(ab')2 40 20 7.9 320 4.9 39 0.12

IgG 40 70 27.9 320 5.1 143 0.45

Globulin(88%IgG) 40 75 30.0 320 5.1 153 0.48

a Reaction

mixture

ratio=microgramsofFITC permilligramofproteinduringlabelingat25°C, pH9.5.

b

Labeling

efficiency

=

[bound

FITC (microgramspermilligramof

protein)/reaction

mixtureFITC

(micro-grainspermilligramofprotein)]x100.

cSpecific staining titer = reciprocal of the highest dilution that retained a3+ or greater staining of the

homologousceils.

dMeasuredby the biuret method.

'F/T ratio=FITC concentrationatthespecific staining titer. achieved by controlling the labeling reaction

conditionsand

using

FITCofknown

high purity.

The Tabelingreaction is mostefficient

chemi-cally at 25°C with a

pH

of 9.5 in 0.05 M

Na2HPO4.

The pH is critical to achieve the

higher F/P ratios: for example, a volume of

globulin was given 40 ,ug of FITC per mg of

protein; one-half ofthe mixture was removed and its pH was adjusted to 7.0; theremaining

one-halfwasadjustedtopH9.5, and then both

preparations were set aside to react. The final F/P ratio of the pH 7samplewas only 11, but

thepH9.5sample had the desired F/P ratio of 31. The pH is also critical during dialysis and storage of conjugates with high F/P ratios. These reagents must bekept at pH 9.0 to 9.5 to avoidprecipitation. Ifsomeprecipitate develops

during dialysis or storage, the pH should be checked and adjusted if indicated.

Studies of viral and rickettsial reagents rec-ommend mediumF/P ratios of 10 to 15 ,ug/mg because conjugates with higher ratios exhibit a prohibitive degree of background fluorescence

(13, 15, 18). The data in this report show why

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502

we canrecommend a high F/Pratio of 30 ,ug/ mg forbacterialconjugates; the high ratio gives ustheoptimal conditions of maximum specific staining titer with a minimum of nonspecific staining, and that allows full utilization of the potential ofagivenlot ofantibodyas a diagnos-tic reagent for fluorescent-antibody identifica-tionof bacteria.

ACKNOWLEDGMENTS

Wegratefullyacknowledge theparticipationand support

of W. B. Cherry, P. P. Harris, G. L. Jones, and B. M. Thomason in various aspects of these studies.

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