J. clin. Path. (1965), 18, 193

Indirect fluorescent antibody technique in theserology of Toxoplasma gondi

S. FLETCHER

From the University Department ofPathology, Western Infirmary, Glasgow

SYNOPSIS The indirect fluorescent antibody technique has been used to titrate patients'antibody against Toxoplasma gondii. Rigorous tests of the immunological validity of the methodwere made. The reactions of the gamma globulin of human immune serum with Toxoplasma gondiiand fluorescent rabbit anti-human gamma globulin were thus shown to be specific. Thereafter thetechnique could be used with confidence to detect the reaction between doubling dilutions ofpatient's serum and smears of unfixed Toxoplasma gondii. In this way, titres were obtained with theindirect fluorescent antibody technique which agreed well with those of the dye test at both lowand high levels of antibody.Compared with the dye test, the indirect fluorescent antibody technique has many advantages.

The end-point is sharp and obviates the counting of stained and unstained organisms; supplies ofantibody-free accessory factor sera are not needed; prozones undetected by the dye test are stronglypositive at screening dilutions, the reagents keep indefinitely and lend themselves to preparation,standardization, and issue by a central reference laboratory.

Serological confirmation of Toxoplasma gondiiinfection is commonly provided by the cytoplasm-modifying (dye) test of Sabin and Feldman (1948)and by the complement-fixation test of Warren andSabin (1942) as subsequently modified by Sabin(1949). The more important dye test is based on arather unusual immunological phenomenon, namely,that toxoplasmata whose cytoplasm has beenmodified by exposure to specific antibody fail tostain with alkaline methylene blue. The dye test is adifficult technique to carry out. Constant intra-peritoneal passage of Toxoplasma gondii in mice isrequired and the living organisms must be usedalmost immediately after collection. The end-pointof the titration is obtained by a laborious count ofunstained and stained toxoplasma. An additionaldisadvantage is that the reaction proceeds only inthe presence of a serum accessory factor which mustbe supplied by fresh antibody-free human serum.Such accessory factor sera may only be obtainedafter screening perhaps as many as 100 donors.Even taking all the precautions derived from theexperiences of Beverley and Beattie (1952) there stillseem to be uncontrollable influences which make itsperformance more difficult. Thus Frenckel, in thediscussion of Eichenwald's contribution (1956),Received for publication 6 May 1964.

records the prevalence of false positive reactions dueto modification of the toxoplasma by antibodyderived from the mice in which they are passaged.He emphasizes the frequency of prozones, dis-courages 'screening' at low serum dilutions, andurges a complete titration by doubling dilutions tobeyond I in 1,024 in all cases. Frenckel also quotesJacobs and Cook (1954) who observed that patient'santibody may be blocked by soluble antigen in thetoxoplasma exudate and thus lead to false negativeresults.

In view of the difficulties of the dye test it isunderstandable that only a few laboratories offer itas a diagnostic service. Although it is a very sensitivetest, it is sometimes thought to lack specificity. Itstitres rise early in acute infection, reach numericallyhigh values, e.g., 1 in 4,096, and decline slowly.The complement-fixation test, on the other hand,

is thought to be highly specific but it is much lesssensitive. It rises late in the infection, reachesnumerically low titres, e.g., 1 in 128, and declinesrapidly. It is thus sometimes taken as evidence ofacute infection. Recently it has been discontinuedby the reference laboratories and now only the dyetest titres are reported.There is thus apparently much to be said in favour

of Ludlam's (1960) observation that there is a need193

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S. Fletcher

for 'a serological test as sensitive as the dye test butless exacting'.

In this paper, I present evidence that the indirectfluorescent antibody test may well fill the need.Smears of Toxoplasma gondii are incubated withserial dilutions of patient's serum. After adequatewashing to remove unreacted serum components,specific reaction may be detected up to titre byapplying fluorescein-conjugated anti-human gammaglobulin. On dark-ground ultra-violet microscopy,the toxoplasma fluoresce in the green with anintensity determined by the amount of patient'santibody which was available to react with them.Previous workers have applied this principle totoxoplasma serology. Thus, Kelen, Ayllon-Leindl,and Labzoffsky (1962) found that titres with theindirect fluorescent antibody test closely paralleledthose of the complement-fixation test and were of thesame numerical order. Mandras, Vanini, andCiarlini (1962), on the other hand, found highervalues for these titres which were approximatelycomparable with those of the dye test.

There is much in favour of retaining serologicaltitres which are of the same order as the dye test andthus familiar to clinicians. I therefore consideredthat the aim of this work must be to obtain titreswith the indirect fluorescent antibody test as close aspossible to those of the dye test without jeopardizingstrict immunological validity. Accordingly, thetechniques finally adopted are described and resultsare given of several tests which sufficiently establishthe immunological specificity of the method. There-after a technique suitable for routine use is appliedto the titration of a range of sera of known dye testtitre and the results are discussed.

MATERIALS AND METHODS

ORGANISM Toxoplasma gondii (RH strain) was keptpassaged in white mice of the Porton strain at three-to-four-day intervals. The peritoneal exudates so obtainedwere diluted five times with saline and 0 1 ml. was usedto inject healthy mice intraperitoneally.

TOXOPLASMA GONDII SMEARS A washed Toxoplasmasuspension was prepared from mouse peritoneal exudatesby the method of Goldman (1957) but formalin fixationwas omitted. As far as possible, to avoid the presence ofmouse antibody, young (8-10-week-old) mice were usedand their peritoneal exudates were harvested at three days(Beverley and Beattie, 1952).From the suspension, air-dried smears not more than

1 cm. diameter with 300-400 toxoplasma per high-powerfield, were made. They were stored over CaCI2 in adesiccator at -20°C. The smears showed no tendencyto wash off and were quite safe as the toxoplasma dierapidly. Since their antigens were undamaged byformalin, the organisms fluoresced more brilliantly thanafter fixation.

E. COLI PERITONEAL EXUDATES E. coli N.C.T.C. 8196 wasgrown in 15 ml. of broth for 24 hours. One ml. of theculture was injected intraperitoneally in each of a dozenmice. The mice were killed after 24 hours and the peri-toneal exudates harvested with the help of saline flushing.The collected exudates were made up to 30 ml. withsaline, spun hard, and the sediment of E. coli andperitoneal macrophages was stored at -20°C.

RAT LIVER POWDER The method of Nairn (1962) wasused to prepare an acetone-dried rat liver powder.Haemoglobin was removed by thorough washing withseveral large volumes of cold saline. When the powderwas still damp from its final acetone wash it was removedfrom the filter paper and thoroughly rubbed with thefingers. In this way the material dried as a fine lightpowder of good activity in relation to its weight, andgrinding in the mortar was avoided.

HUMAN SERA AND SERUM PRODUCTS Sera of known dyetest titre, normal sera, and sera with antibody titresagainst a variety of heterologous antigens were obtainedand stored at -20°C. until used.One serum with a dye test titre of 1 in 4,096 was used

as a reference standard throughout the work.Human gamma globulin (as supplied for measles

prophylaxis) and human serum albumin (freeze dried)were obtained from the Blood Transfusion Service andstored at 4°C. until used.

PREPARATION OF HUMAN GAMMA GLOBULIN Humanserum, 10 ml., was diluted to 100 ml. with 0-02 M phos-phate buffer, pH 6-3, and made into a slurry with 10 g. ofDEAE Sephadex A-50 (Pharmacia, Uppsala) previouslyequilibrated with the same buffer according to themakers' instructions. The mixture was stirred overnightat 4°C. and then centrifuged. The supernatant whichcontained the gamma globulin fraction was removed andconcentrated at 4°C. by pressure dialysis against saline.A Visking membrane of 24/32 in. diameter operated at80 cm. Hg was used and pressure was maintained untilthe volume was approximately 2 ml. A typical total yield(estimated spectrophotometrically at 280 mix) was 150 mgof gamma globulin. The purity of the preparation waschecked by immuno-electrophoresis on agar gel againsta trough of rabbit anti-whole human serum.

IMMUNIZATION OF RABBITS WITH HUMAN GAMMA GLOBULINRabbits were immunized with gamma globulin-Freund's complete adjuvant mixture by two 1-5 ml.injections one week apart. After five weeks a boosterof 2 ml. was given and after 10 days the indirect Coombstest (Boorman and Dodds, 1957) was performed on therabbit serum. If the titre was unsatisfactory, a boost with1 ml. of an alum-absorbed vaccine prepared from BloodTransfusion Service gamma globulin after the method ofProom (1943) was used. Gamma globulin, 750 mg., wassubstituted for the 25 ml. of serum quoted in the originalmethod. The rabbit antiserum obtained after boostinghad an anti-human gamma globulin titre of 1 in 128,000+ in the indirect Coombs test.

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Indirect fluorescent antibody technique in the serology of Toxoplasma gondii

CONJUGATION OF RABBIT IMMUNE GLOBULIN From therabbit anti-human gamma globulin serum just described,a preparation of immune gamma globulin was obtainedusing the DEAE Sephadex A-50 method described above.The final yield after pressure dialysis was 185 mg. in avolume of 2 5 ml.

Conjugation was carried out as described by Nairn(1962). Rabbit gamma globulin was diluted to a level ofapproximately 10 mg. per ml. with the recommendedpH 9 0 buffer.To avoid large amounts of unreacted fluorescent

material fluorescein iso-thiocyanate was used at a con-centration of only 20 mg. per gram of gamma globulin.Before use each 2 mg. required was dissolved in 1 ml. ofacetone.When conjugation was over unreacted fluorescent

material was removed on a Sephadex G-50 columnprepared according to Nairn (1962) and the manu-facturer's literature. The conjugated gamma globulinwas eluted by the recommended buffer and concentratedby pressure dialysis to a final volume of 3 ml. Thematerial was stored at -20°C. in 0-5 ml. lots and used atdilutions between 1/20 and 1/35 according to the needsof particular experiments.

NON-SPECIFIC STAINING At the dilutions used for variousapplications non-specific staining was minimal, but, asan extra precaution, all conjugates were treated for onehour at 4°C. with previously moistened rat liver powder inthe proportion of one small knife point of powder per30 drops of diluted conjugate.

TITRATION OF PATIENT'S SERA All dilutions were made insaline by dropping methods, using the same pipette foradditions of diluent and reagent. The method used forthe titration of patient's sera and in the tests of immuno-logical specificity was as follows.

1 Three or four drops of each dilution of patient'sserum were applied to a separate toxoplasma smear. Theslides were put in 6 in. diameter Pyrex Petri dishes alongwith moist cotton wool to prevent drying. Incubationat 37°C. for 15 minutes followed.

2 The smears were momentarily rinsed under a gentlestream of tap water and then washed for 15 minutes insaline-filled Coplin jars on a shaking machine.

3 As much saline as possible was removed from

around the smears with a soft cloth so that the fluorescentantibody subsequently added was not unduly diluted.4 To the still wet smear, one large drop of diluted

fluorescent conjugate was added and the smears wereincubated for 15 minutes at 37°C.

5 The smears were once more gently rinsed under thetap and immediately washed by agitation in fresh salinefor 15 minutes.

6 The smears were thoroughly but gently rinsed in tapwater to remove saline. The surround was carefully driedwith a soft cloth and the region of the smear left to air-dryslowly. Glycerol mounting and cover glasses were foundto be unnecessary and when dry the preparations wereready for microscopy.

Titration of patient's sera may be expedited inroutine use by gently blotting the smears with cleanblotting-paper at stages 3 and 6 instead of wiping roundthem with a cloth: there is no tendency for the smears torub off.

MICROSCOPY The apparatus used for ultra-violet darkground microscopy was essentially that of Young (1961).

RESULTS

IMMUNOLOGICAL VALIDITY OF THE INDIRECT FLUOR-ESCENT ANTIBODY TECHNIQUE In the experiments tobe described in this section, a 1 in 20 dilution ofconjugate treated with rat liver powder and found togive no non-specific staining of control smears wasused except where otherwise stated.Immune nature of the reactions Two reactions

must be shown to be of immune nature: 1 thatbetween fluorescent conjugate and human immunegamma globulin (which has combined with Toxo-plasma gondii); 2 that between Toxoplasma gondiiand the gamma globulin of human immune serum.The reaction between the conjugate and human

immune gamma globulin which had combined withToxoplasma was first tested by the techniques ofabsorption and blocking.

Specific absorption tests A 1 in 10 saline dilutionof rat liver powder-absorbed conjugate was divided

BLE IIMMUNOLOGICAL SPECIFICITY OF THE REACTIONS BETWEEN TOXOPLASMA GONDII, HUMAN ANTISERUM, AND FLUORESCENT

ANTIHUMAN GAMMA GLOBULIN

Smears of Toxoplasma Treated for 1S Minutes at 37°C. with 1/256 Human Toxoplasma Antiseru-n

A. Staining by Conjugate Absorbed forI Hour at 37'C. with:-

Human Gamma Human SerumGlobulin Albumin(12-5 mg. in (12 5 mg. in01 ml.) 01 ml.)

Saline(Control)(0 1 ml.)

B. Staining by Conjugate of SmnearsBlocked for 30 Minutes at 37°C. with:-

Rabbit Anti- Rabbit Non- Salinehuman immune (Control)Gamma Serum (1/8)Globulin (1/8)

C. Staining by Conjugate of SmearsTreated with 11256 Toxoplasma AntiserumAbsorbed with:-

Toxoplasma Escherichiagondii coli.

Saline(Conitrol)

_ ++i++ +±++- = auto-fluorescence only

+ +++ brilliant green fluorescence.

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into three six-drop amounts and absorbed withhuman gamma globulin, human serum albumin, andan equivalent volume of saline. The absorbedpreparations were tested for their ability to react withToxoplasma gondii previously incubated with a 1 in256 dilution of standard high titre patient's serum.The details of the experiment and the results areshown in Table I, section A.

Blocking tests Toxoplasma smears previouslysensitized with a 1 in 256 dilution of standard serumwere 'blocked' by 1 in 8 saline dilutions of uncon-jugated rabbit anti-human gamma globulin serum oftitre 1 in 128,000 + in the indirect Coombs test andnon-immune rabbit serum. The results of fluorescentmicroscopy after treatment with conjugate are shownin Table I, section B.

Conjugated low Coombs titre rabbit globulin Con-jugates made from rabbit anti-human serum of poorCoombs titre failed to stain toxoplasma previouslytreated with human immune sera.The reaction between fluorescent rabbit anti-

human gamma globulin and human immune globulinhas thus satisfied controlled tests of absorption andblocking and the failure of low titre conjugates tostain has been observed. It therefore seemed fair toassume that the reaction was of a true immuno-logical nature.The reaction between human immune serum and

Toxoplasma gondii smears was next examined.Absorption tests Ten-drop portions of diluted

standard patient's serum were absorbed by theequivalent of three peritoneal exudates from miceinfected with E. coli and Toxoplasma gondii. Aftercentrifuging, the absorbed serum was applied totoxoplasma smears and the standard fluorescenttechnique was carried out. Table 1, section C, showsthe details of the absorptions and the results.

Sera with high heterologous titres Several humansera with high titres against a selection of hetero-logous viral and bacterial antigens were set up in thestandard method for the titration of patient's serum.The dilutions of rat liver powder absorbed conjugatewas 1 in 30 in all cases. The titres obtained are shownin Table II and are typical of those of a 'normal'population. There is no significant cross-reactionwith any of the sera investigated.

Cross-reaction of patient's immune sera withdifferent organisms This was carried out to see if ahigh-titre anti-toxoplasma human serum wouldcross-react with a number of organisms especiallyprotozoans, some of which the donor could notreasonably have encountered. It thus sheds additionallight on the specificity of the method and has animportant bearing on an occasional use of thetechnique, the identification of Toxoplasma gondii inunfixed infected tissues.

TABLE IITOXOPLASMA TITRES DETERMINED BY INDIRECT FLUORESCENT

ANTIBODY TEST OF SERA WITH HIGH TITRES AGAINSTHETEROLOGOUS ORGANISMS

Nature of Antigen Agent Toxo-plasma

Agglutin- Comple- Indirect Dyeation Titre ment- Fluorescent Test

fixation Antibody TitreTitre Titre

Virus1 Mumps S

v2 Mumps S

V3 Mumps S

VChickenpoxHerpes zoster

Leptospira1 Leptospira canicola2 Leptospira canicola

Salmonella1 S. paratyphi B

S. typhi2 S. typhi

3 S. paratyphi B

H00H0H0

1/641/5121/641/5121/161/2561/1281/512

1/8

< 1/8

1/16

1/81/32

1/512 < 1/81/256 1/32

1/801/1601/20Nil1/160Nil1/160

ControlsNormal serumToxoplasmosis

1/8

1/8

1/8

Intdirect fluorescent antibody technique in the serology of Toxoplasma gondii

probably combined with immune globulin, either inthe form of low titres of their appropriate antibody,or, in a non-specific fashion.The staining of Staphylococcus aureus and beta

haemolytic streptococcus by the indirect and directtechniques brought to light some interesting findings.Both organisms stained well by the indirect anddirect methods. However, in the controls for theindirect method where saline was substituted for thestandard anti-toxoplasma serum the beta-haemolyticstreptococcus still stained although Staphylococcusaureus did not. Such findings suggest that thestandard anti-toxoplasma serum contains bothstaphylococcal and streptococcal antibodies andthat the conjugate contains streptococcal antibodyin addition to that directed against human gammaglobulin.

In conclusion, the absorption of the standardanti-toxoplasma serum with toxoplasma and E. coliand the tests of specificity with heterologous seraand heterologous organisms collectively establishthe specificity of the reaction between a typicalhigh-titre patient's serum and Toxoplasma gondii.

Since the specific nature of the reaction betweenconjugate and reacted human immune globulin has

also been demonstrated it seemed fair to regard theoverall response of the indirect fluorescent antibodytechnique as immunologically specific for Toxo-plasma gondii.

APPLICATION OF INDIRECT FLUORESCENT ANTIBODYTECHNIQUE TO TITRATION OF PATIENT'S SERA Onlywhen the specificity of the indirect fluorescentantibody technique had been established was itsapplication to the titration of human anti-toxo-plasma sera considered. Certain difficulties wereencountered. When normal serum or very lowdilutions of normal serum were used, non-specificfluorescence of the toxoplasma was found even afterprolonged absorption of the conjugate with rat liverpowder. Saline controls invariably showed nostaining at all. Inefficient washing contributed to theeffect but the most important cause was the use ofabsorbed conjugate in needlessly high concentration(see Nairn, 1962). The correct concentration ofconjugate must therefore be found. A compromisehas to be made between a concentration of conjugatehigh enough to detect patient's antibody with thesensitivity of the dye test, and one low enough toavoid significant fluorescence with normal serum at

TABLE IIICOMPARISON OF INDIRECT FLUORESCENT ANTIBODY AND DYE TEST TITRES OF PATIENTS' SERA

Designation of Fluorescence at Reciprocal Seruim Dilutions of:- Indirect Fluorescent Dye TestSerum Antibodv Titre Titre

BETSMcP.WRI81/OrWR21024/Or

J.McG.256/OrHAN128/OrMrs. McG.HUTAPD

722/Or776/Or624/OrTG16/Or8/OrBNJ. McG.WN6S2SN

r3753192

Normal sera 4 930424

< 'A'

512 1,024+ + +

+ 4- +

32

++

8

-+ ++++4-4---4-

,i ++

64±4+4+4++4+4++4-4+±+

++16

++++4+ +-

197

4,0964,0962,0482.0482,0481,024512512

256

2,048

++

128

+4

++

32+

+4-

++

±.

2561282561283212864

64+-4+4+

++

4.0964,0964,0962,0482.0481,0241,024

256256256128646432

323232321688

the chosen starting dilution of 1 in 8. Accordingly,different concentrations of conjugate from 1 in 20to 1 in 35 were made up and treated with rat liverpowder. They were then applied to toxoplasmasmears previously incubated with dilutions of anormal serum of negative dye test and standardserum of dye test titre 1 in 4,096. The standardprocedure was rigorously followed and that con-centration of conjugate was found which gave nofluorescence at 1 in 8 with the negative serum butthe highest end-point with the standard positive.The latter dilution was usually equal to, or onedoubling dilution lower, than the dye test titre. Forthe particular conjugate used throughout the workto be described, a dilution of 1 in 30, absorbed withrat liver powder, was found to be most suitable.Every new lot of conjugate must be titrated in thisway to find the optimum concentration for use inthe titration of patient's sera.When batches of patients' sera were put up for

titration by the indirect fluorescent antibodytechnique, a few low dilutions of normal serum(1/8, 1/16, 1/32) and a few limiting dilutions ofstandard positive serum (1/1,000, 1/2,000, 1/4,000)were always included. The intensity of fluorescencewas graded from + + + + to ± and the latter wastaken as the end point of the titration. The results ofthe titration by this technique of human sera withhigh, medium, low, and negative dye test titres aregiven in Table III.

DISCUSSION

Much of the present work has been devoted to arigorous inspection of the specificity of each stepin the technique and it is considered desirable tosubject new batches ofconjugate to the tests describedin part I of the results (with the exception of thosewith heterologous organisms).

In order to obtain indirect fluorescent antibodytechnique titres approximating to those of the dyetest every batch of conjugate must be titrated tofind the concentration which gives the highestsensitivity with the least non-specific staining. Oncethis concentration has been found the routine con-duct of serology by the indirect fluorescent antibodytechnique is very easy indeed.

Table III shows a good agreement between dyetest and titres by the indirect fluorescent antibodytechnique. In particular, highly immune sera giveby this technique titres which are equal to, or onedoubling dilution less than, those of the dye test.It was with one member of this group that a mostinteresting observation was made. The serumdesignated McP. came from a youth of 18 who hada febrile illness with lymphadenopathy. Lymph

node biopsy showed typical appearances of toxo-plasmosis as described by Stansfeld (1961). On twooccasions the titre by the indirect fluorescentantibody technique was 1 in 2,048 and thus confirmedthe clinical impression. However, the dye test wasinitially reported as less than 1 in 8 on routinescreening up to a final dilution of 1 in 128. In view ofthe clinical and histological findings and the indirectfluorescent antibody titration, I considered that thediscrepant results were probably due to a prozonein the dye test. The reference laboratory very kindlyrepeated the dye test, this time extending thedoubling dilutions to 1 in 8,192. In this way aprozone was observed which gave negative dye testresults up to a dilution of 1 in 128. At 1 in 256 thereaction was feebly positive: thereafter, it remainedstrongly positive up to a final titre of 1 in 4,096which was in good agreement with the indirectfluorescent antibody technique titre of 1 in 2,048.Throughout the dilutions affected by the prozone,the indirect fluorescent antibody technique gavebrilliant fluorescence. How common such sera areis not known but it seems certain that the indirectfluorescent antibody technique will detect at leastsome of them at low screening dilutions.With the group of sera of only moderate dye test

titre the agreement is again good and equal to, orwithin one doubling dilution of, the dye test titre.The lower part of Table III shows the results withsera of low dye test titre. The tendency here is forthe indirect fluorescent antibody technique to read alittle high-perhaps by one or even two doublingdilutions. This is illustrated clearly by the four seraof dye test titre less than 1 in 8 of which three arepositive with the indirect fluorescent antibody tech-nique.The lowermost part of Table III shows the

titres of a small group of normal individuals ofunknown dye test titre. By combining the resultswith those of Table II for high titre heterologoussera further similarities to the dye test emerge.In the combined group of 17 sera none exceeds adye test titre of 1 in 32 and five (29%) are positiveat 1 in 16 or over. This proportion agrees well withthe common finding of reference laboratories thatbetween 20 and 40% of normal people have dyetest titres between 1 in 8 and 1 in 128.

It is interesting to study the results of previousworkers with normal and low titre human sera.Kelen et al. (1962) used the indirect fluorescentantibody technique to titrate 92 sera which werepositive in the dye test at titres from 1 in 16 to 1 in 64.Only eight were positive in the indirect fluorescentantibody technique at 1 in 8 or more, thus showingthe relative insensitivity of the method. Yet anotherfinding emphasizes the insensitivity. Normal serum

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Indirect fluorescent antibody technique in the serology of Toxoplasma gondii

is used as a negative control by Kelen et al. (1962)but no mention is made of diluting it. If it is, in fact,used neat some fluorescence of toxoplasma wouldbe expected due to the prevalence of low-titreantibody. In addition, non-specific attachment ofimmune globulin to the toxoplasma may take placewith neat serum. My experience with three differenthigh potency conjugates was that neat normalserum nearly always gave fluorescence. The inactiva-tion of all sera by Kelen et al. is unlikely to explainthe discrepancy. The sera in the present experimentswere not inactivated since early experiments showedthat it had no effect on the indirect fluorescenceantibody technique titres of low-titre dye test sera.Mandras et al. (1962) followed the technique of

Kelen et al. very closely but obtained a goodcorrelation between the indirect fluorescent antibodyand dye test titres especially with high-titre sera.With low-titre sera, however, the sensitivity waspoor. Thus of 51 normal sera only one gave apositive reaction with the indirect fluorescentantibody technique at 1 in 10. This is far below theexpected figure.

It is difficult satisfactorily to account for thedifference between the present results and those ofKelen et al. and Mandras et al. However, neither ofthe previous publications mentions the need toobtain toxoplasma from 8 to 10-week-old micekilled early on the third day to avoid mouse antibody(Beverley and Beattie, 1952). Mouse antibody causesfalse positive reactions in the dye test but mayexert a blocking effect and lead to false negatives inthe indirect fluorescent antibody technique. It maythus entirely obscure the reaction with low-titresera but diminish the titre of highly immune seraby only one or two doubling dilutions. In this waythe discrepancy between the indirect fluorescentantibody technique and dye test at low titres may beexplained.A further influence operates equally at all titres

and serves to decrease the sensitivity of the indirectfluorescent antibody technique as performed pre-viously. Both groups of workers used formalin-fixedtoxoplasma whose fluorescence is inferior to that ofthe unfixed immunologically-intact preparationsused throughout the present work.

It would seem that the good agreement with thedye test which I have found depends upon the use ofunfixed toxoplasma free from mouse antibody and apurified conjugate of high potency and specificity.The indirect fluorescent antibody technique has

only two disadvantages: 1 Each batch of conjugatemust be tested for immunological validity. Since,however, this technique is very economical ofconjugate, such tests are infrequent. 2 If animalsera are to be titrated, the appropriate species-7

specific conjugated gamma globulin has to beprepared. The dye test must be used as a commonreference standard and the conditions of the indirectfluorescent antibody technique have to be adjustedso that comparable titres may be obtained fromspecies to species.On the other hand the advantages of the indirect

fluorescent antibody technique are simplicity inroutine use, indefinite storage of reagents, economyof mice, sharp end point, avoidance of numericalcounts and accessory factor, absence of prozonespermitting screening procedures, and, finally, ease ofstandardization.The last point is very important indeed. Dye test

results may vary greatly from one laboratory toanother. This need not be so with the indirectfluorescent antibody technique. The reagents notonly keep indefinitely but lend themselves to bulkpreparation and standardization in a central issuinglaboratory. In this way, uniform results of greatvalue in epidemiological work could be obtained byperipheral laboratories scattered over a very widearea.The acceptance of a new laboratory test is greatly

assisted if the results of the old and the new aresimply related. The relation between the indirectfluorescent antibody technique and dye test titrescould not be simpler and it is hoped that it will playsome small part in hastening the adoption of thiselegant and reliable method.

I should like to thank Drs. J. K. A. Beverley and C. N.Iland for letting me have mice infected with the RH strainsof Toxoplasma gondii and Dr. G. B. Ludlam who per-formed the dye tests. I am also grateful to Professor P. C.C. Garnham who made available preparations oftrypanosoma, leishmania, and plasmodium, to Drs.N. R. Grist and R. J. Fallon for supplies of sera, and toDr. H. E. Hutchison for carrying out the Coombstitration.

REFERENCES

Beverley, J. K. A., and Beattie, C. P. (1952). J. clin. Path., 5, 350.Boorman, K. E., and Dodds, B. E. (1957). Blood Group Serology,

p. 81. Churchill, London.Eichenwald, H. F. (1956). Ann. N. Y. Acad. Sci., 64, 207.Goldman, M. (1957). J. exp. Med., 105, 549.Jacobs, L., and Cook, M. K. (1954). Amer. J. trop. Med. Hyg., 3, 860.Kelen, A. E., Ayllon-Leindl, L., and Labzoffsky, N. A. (1962). Canad.

J. Microbiol., 8, 545.Ludlam, G. B. (1960). Proc. roy. Soc. Med., 53, 113.Mandras, A., Vanini, G. C., and Ciarlini, E. (1962). Igiene mod.,

55, 636.Nairn, R. C. (1962). Fluorescent Protein Tracing. Livingstone, Edin-

burgh.Proom, H. (1943). J. Path. Bact., 55, 419.Sabin, A. B. (1949). Pediatrics, 4, 443.-, and Feldman, H. A. (1948). Science, 108, 660.Stansfeld, A. G. (1961). J. clin. Path., 14, 565.Warren, J., and Sabin, A. B. (1942). Proc. Soc. exp. Biol. (N. Y.),

51, 11.Young, M. R. (1961). Quart. J. micr. Sci., 102, 3rd series, 419.

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