456 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 72, No. 5, 1978

Standardization

of the indirect

fluorescent

antibody

test for malaria

BINGUMAL R. ~&INAWAJXJ* AND ALISTER VOLLER London School of Hygiene and Tropical Medicine and Nuflield Institute of Comparative Medicine, The Zoological Society of London, Regents’ Park, London NWI, England Summary Methods are described whereby results of malarial immunofluorescence tests can be evaluated objectively. The IFA test was quantitated by standardizing the physical system against a fluorescent standard and preparation of biological standards of malarial antisera and fluorescein labelled conjugates. Using these known standards the reactivity of antigens was characterized. It was found that antigen preparations are best when they include mature schizonts, and keep best when they are stored in a dry condition at or below -20°C. However, even under carefully controlled conditions of storage, antigens showed considerable variation of reactivity between individual batches. Introduction The indirect fluorescent antibody (IFA) test is now firmly established as one of the most suitable techniques for the detection and measurement of malarial antibody (COLLINS & SKINNER, 1972). Many papers have been published showing that it can be useful in detailed investigations on the antibodv responses of individuals and in enidemiological progmmmes (VOLLER, 1975; ~BROISETHOMAS. 1974: DRAPER et al.. 1972X For these

purposes the antigen is preparid as thick or thin smears of infected blood from a defined source and reacted with the test serum and fluorescein-labelled conjugate, and examined under an ultra-violet (U-V) fluorescence microscope. The subjective estimation of end points by minimal fluorescence of serially diluted sera has been used to quantitate the antibodies in the sera in terms of titres. Although the detection of the fluorescent antibodies is reproducible, the quantitation in terms of titres is arbitrary and variable for the same serum tested by different laboratories, or even the same laboratory at different times. One of the most important sources of variability lies in the fact that the end results of the immunofluorescence tests are judged by eye, and so are extremely subjective. Although results obtained by a single individual can be quite consistent, this subjective element in the reading of the tests hampers comparability between laboratories. Another factor which hampers the comparability of the IFA test is the extreme variability of each batch of antigens and fluoresceinlabelled conjugates. In addition to these biological variables, the physical system, consisting of the U-V source and microscope, shows variations from day to day. Therefore any attempt at standardization would need the definition and specification of all the above factors. The work of TA~OR et al. (1971) on using

a fibre-optic probe to measure fluorescence of bacteria reacted in the IFA test suggested to us that the application of this apparatus might lead to a new level of precision in malarial immunofluorescence. The present communication deals with our experience in achieving standardization of the malarial immunofluorescence test. Materials and Methods Equipment The photometric equipment consisted of a fibreootic nrobe of 50 urn diameter fitted into a 10 X f&us&g microscopic eyepiece. The probe was connected to a flexible fibre-optic probe, which was in turn connected with a photomultiplier. The photomultiplier was coupled to a digital photometer, and the results were either read manually or continuously recorded using a chart recorder (the complete equipment was manufactured by Gamma Scientific Inc., United States of America). The above equipment was used in connection with a standard microscope (Zeiss, Federal Republic of Germany) for transmitted light fluorescence or with a fluorescence microscope (Zeiss, Federal Republic of Germany) adapted for epi-illumination studies. The light source was on Osram H.B.O. 200 with mercury vapour lamp. Excitor filters BG 38, BG 12 and an interference filter Balzer FITCS were used together with barrier filters Zeiss 50, Zeiss 44 and a red suppression filter Zeiss-65. All measurements were made with the 100 X oil immersion objective. The equipment was housed in a dark room. This was essential for accurate readings. Before any readings could be made it was necessary to standardize the complete physical system, i.e. microscope, light source and electronic equipment. For this purpose a uranyl glass block 15 mm X 10 mm X 10 mm was used. This block, which fluoresced green when illuminated with UV-blue, was placed on the microscope stage, and the oilimmersion objective was focussed on a carefully marked area. .A reading of fluorescence intensity was then made on that spot with the fibre-optic probe described above, and the photometer was adjusted to read 1000 fluorescence units (arbitrary units) by means of the high voltage control. This procedure was repeated on each day when the equipment was used, and it was found to be an effective way of compensating for slight daily Correspondence to : B. R. Manawadu, M.D., Ph.D., Department of Anesthesiology (B 113), University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, Colorado 80262, USA.

B.R.MANAWADUANJJ

routinely was sheep antihuman IgG, labelled with fluorescein isothiocyanate (Wellcome Reagents) at a dilution of 1:lO in PBS (phosphate buffered saline 0 *15M pH 7 ‘2, Fluorescein/Protein molar ratio 3 *0, Antibody protein 1.6 mg/ml).

variations in the equipment and for the longer term decay of intensity of the light source. The necessity for such standardization was clearly shown by the fact that the excitation produced by an HBO 200 which had been used for 200 hours was only 45% of that of a new bulb. In later studies, an HBO 50 lamp was used and it proved to be a more stable source of light.

Experiments

The indirect fluorescent antibody was used following the procedures of VOLLER (1964) for thin blood film antigens and SULZER et al. (1969) for thick smear antigens. The antigen blood smears were made from Aotus trivirgutus (owl monkeys) which had been experimentally infected with one of the adapted strains of the human parasitesPlasmodium falciparum, P. malariae or P. vivax. At the end of the IFA procedure the slides were dried and the test areaswere covered with Polarfluor B mounting medium (Polaron Ltd., United Kingdom) under a coverslip. This mountant was found to preserve the fluorescence best and to give minimal light scatter. The fluorescent antiglobulin conjugate used frequency homologous

and Results

(a) Dilution of test serum An IFA test was set up with I’. falciparum antigen containing many schizonts and with an antiserum obtained from a patient recently in&ted with P. falciparum. Fluorescence measurements were made on 100 schizonts on each of three antigen slides which were reacted with either undiluted serum or serum diluted 180 or 1:1280. The frequency distribution of peak fluorescence readings is shown in Table I. A second series of IFA tests was set up with P. vivax antigen containing many schizonts and an antiserum obtained from a patient who had recently recovered from an infection of P. vivax. Fluorescence measurements were again made on 100 schizonts for each serum dilution. The mean values are shown in Table II.

Immunofluorescence tests

Table I-The reacted with

457

A.VOLLER

distribution of the peak antisera in an IFA test

fluorescence

of the schixonts

of P. falciparum

Fluorescence units Mean, Antiserum fluorescence dilution units,fSD O-49 50-99 loo-149 150-199 200-249 250-299 300-349 350-399 400-449 Numbers of each 3 15 schizonts4F youp

Undiluted

252 *70

0

0

1:80

121k38

1

21

66

8

4

94

6

0

0

0

1:1280

29flO

Table II-The fluorescence intensity at various dilutions in an IFA test

of schixonts

>450

11

6

2

2

0

0

0

0

0

0

0

0

0

0

of P. vivax reacted

with

homologous

antiserum

Intensity of fluorescence of schizonts (fluorescence units) Serum dilutions

Mean values for 100 schizonts at each dilution

1 :lO

1:20

1:80

1:320

1:640

2722

1965

936

520

270

zk86

&40

f413

S.D. Table

III-The

rate of fading

5285

of fluorescence

of malaria

&I22 parasites

reacted

in an IFA test

Fluorescence (in arbitrary units) Sample

Initial reading

After 1 minute

%

After 5 minutes

1

10,000

7,000

(70)

3,800

:

2,800 360

2,500 340

2,000 320

y0

After 10 minutes

“/!

2,480 (89)

1,550 310

036)

458

STANDARDIZATION OF IFAT FORmARIA

It can be seen that the intensity of fluorescence is directly correlated with the concentration of the test serum. The range encompassing most fluorescence readings became narrower with increasing dilution. An important feature noted in this experiment was the very rapid fading of the parasites once they were subjected to U-V illumination. As Table III shows, the fading was most pronounced and fastest in the specimens with the highest initial readings. When the fluorescence of a malaria parasite in a field is being measured or observed, all the other parasites in the same field are fading, as may be expected. During this study all fluorescence readings were taken on parasites in previously unilluminated fields, and the peak reading recorded (at about 20 seconds after illumination) was used. This was very important as a delay of even a few minutes could minimize the differences between stronger and weaker reactions. Table

IV-The

differential

reactivity

(b) Stage of development of the parasite Although many workers have noted in passing that the mature forms of malaria parasites react better in the IFA, only TARGETT (1970) attempted to measure this. He found that titres obtained when using schizonts of P. falciparum as antigen were two or three dilutions higher than those observed with ring forms of trophozoites. In the present experiment an owl monkey was infected with P. falciparum and the asexual cycle was monitored by the examination of Giemsastained blood films. During a single asexual cycle sets of unstained blood films were made for use as antigens when the peripheral blood contained mostly (i) ring forms of trophozoites, (ii) mature trophozoites or (iii) schizonts. These three antigens were then reacted in an IFA test using a homologous P. falciparum antiserum and an anti-IgG conjugate. The results are shown in Table IV. The schizont stages were, by far, the most

of different

stages of P. falciparum

in an IFA test

Fluorescence intensity (in arbitrary units) Antiserum dilutions Antigen Ring forms of trophozoites Mature trophozoites

1:1280

1:5120

1:20480

201 (dc 54)

116 (zt 35)

-

-

776 (&lW

624 (f113)

324 65)

-

-

(i

1,632 (f239)

1,530 (f185)

1,030 (&150)

1:80

449 ( i 120)

(*

1,002 f 191)

Schizonts

Table

1:320

1:20

3,685 (f655)

V-The

effect of storage,

under

different

268 68)

conditions,

438 (XII 66)

on P. falczparum antigen

(*

E)

reactivity

Fluorescence intensity of schizonts (arbitrary units) Days of storage

Storage temperature 1

7

14

- 70°C

1,060 f136

1,065 1198

1,170 f219

f

-20°C

830 f 62

1,160 f207

+ 4°C

1,020 f117

Room temperature, dry Room temperature, humid 37OC

30

62

118

960 10

1,060 f 194

990 f 144

980 1181

1,060 f 85

790 f139

1,070 f186

1,050 f210

960 &158

1,120 f 192

870 &151

770 f 138

960 f142

840 f 178

i

760 93

510 f141

620 f 136

420 f 65

880 *133

180 zk 20

170 f 20

-

-

-

720 f140

290 f 32

315 f 74

160 f 72

92; zt

90 + 22

B. R. MANAWADU

reactive and gave readings four times higher than trophozoites and eight times higher than ring forms of trophozoites when tested with the least diluted antiserum, All subsequent readings were made on schizonts since these were the most sensitive antigens. (c) Storage of antigen

For most practical purposes it is necessary to make malaria antigen slides in bulk and then to store them until the day of use. This experiment was designed to indicate the factors affecting reactivity during storage. P. falciparum antigen slides were prepared from an infected owl monkey, making thick and thin blood films. All the thick tihns and half of the thin films were air-dried, the remainder of the thin films were tixed with acetone. The slides were then dried over silica gel and stored in the dry condition in plastic bags at -7O”C, -2O’C, +4”C, room temperature, and +37”C. One additional set of slides was kept humid at room temperature. The antigens were tested on the day of preparation and again on days 1, 7, 14,30, 62 and 118 of storage, in an IFA test using a standard homologous antiserum at 1:20 dilution, and a fluorescein labelled anti-IgG conjugate at 1: 10 dilution. The antigens stored under humid conditions rapidly deteriorated and, within one week, became quite useless yielding only a non-specific over-all green fluorescence of low intensity. The patterns of degradation of the air-fixed thick and thin films and the acetone-fixed thin films were very similar. The Table VI-The and various conjugate

459

AND A. VOLLER

findings with the air-dried thick films are given as an example in Table V. The antigens stored at 37°C were rapidly degraded. Similarly, the antigens stored at room temperature deteriorated, but more slowly. The slides kept at -7O”C, -2O”C, and +4”C remained reactive throughout the experimental period of four months. (d) The conjugate and its dilution In the second stage of any IFA test the crucial factor is the reactivity of the labelled antiglobulin conjugate, but this has attracted relatively little attention from malaria serologists. Since we had the means to measure the fluorescence intensities reached in the IFA test very precisely, we examined a series of commercial conjugates and compared them with the fluorescein isothiocyanate labelled antihuman IgG conjugate research standard 68/45. This research standard was prepared by the National Institute for Biological Standards and Control, Holly Hill, London, NW3 6RB, and may be requested from that Institute by workers requiring it. This research standard conjugate 68/45 was obtained by the courtesy of the then named Division of Biological Standards, National Institute for Medical Research, London, and dilutions were made to contain 100,50,25,12*4,6*3, 3.1 and 1.6 units of the conjugate. These various dilutions were used as the indicator reagent in an IFA test using P. vivax antigen and a P. vivux antiserum at I:20 dilution. The results are shown in Table VI.

mean fluorescence intensity of P. vivax schizonts dilutions of research standard 68/45 antihuman

reacted with homologous antisera IgG fluorescein isothiocyanate

Units of conjugate 68/45

Fluorescence of P. vivax schizonts (arbitrary units)

Mean

100

50

25

12.5

6.3

3.1

1.6

920

680

485

360

270

150

75

90

60

78

50

45

10

5

of schizonts of P. oivux batches of conjugate

reacted

f SD.

Table VII-The fluorescence intensity homologous antiserum but with different

in

an IFA

test

Batches of conjugates 1 Antiserum dilution l/10

2

3

4

5

Intensity of fluorescence of schizonts (arbitrary units) 1,180 ( It 130)

Q55%

l/M

688 (zk 46)

512 (=t 45)

l/160

362 (=t 39)

238 (=k 5%

824 97)

488 (k 77)

398 (fk 51)

556 (zt 82)

416 kt 32)

328 (h 31)

212 (zt 31)

116 (=k 5)

(i

with

460 Table VIII-Fluorescence antisera in the malaria

STANDARDIZATION

measurements test

OF IFAT

FOR MALARIA

of P. falciparum

antigens

reacted

with

homologous

and

Fluorescence intensity* Plasmodium falciparum

antigens

ABCDEFGHIJ

Mean SD

Antisera P. falciparum

132

90

420

214

P. vivax Ratio vivax/falciparum

0.;:

0.;:

0%

P. malariae Ratio malariae/fakiparum

0.45 60

O-55 50

O-35 147 0.;;

0-g

110

350

256

470

244

228

254

60 65 O-24 0.28 O’&Ol,

0-i:

0-i;

0-Z;

0%

O-52 90

0.24 85

0.52 135 O-46 220

O-46 110 0-i:

0’~~&0*12

* Expressed in arbitrary units. Each figure represents mean of 10 readings.

Table IX-Fluorescence heterologous antisera

measurements on P. vivax in the malaria IFA test

antigens

reacted

with

homologous

and

Fluorescence intensity* Plasmodium vivax antigens

ABCDEFGHIJ

Mean&SD

Antisera

P. vivax

878

774

650

295

784

223

361

912

788

635

630

Ratio falciparum/vivax

P. falciparum

363 210 368 158 243 107 215 401 227 273 315 O-41 0.27 O-57 O-54 0.32 O-49 0.60 O-68 0.67 0.43 0.50fO.14

P. malariae Ratio malariae/vivax

524 224 286 170 121 90 154 283 517 263 295 0.62 0.28 0.44 0.58 O-40 O-40 0.43 O-48 O-66 O-41 0.47f0.12

* Expressed in arbitrary units. Each figure represents mean of 10 readings.

Table X-Fluorescent heterologous antisera

measurements in the malaria

on P. malariae. antigens IFA test

reacted

with

homologous

Fluorescence intensity* Plasmodium malariae antigens

A

B

C

D

E

P. malariae

418

332

460

390

520

P. falciparum

140 0.33

Mean f SD

Antisera

Ratio falciparum/malariae P. vivax

Ratio vivaxJmalariae

95

o-22

425

0.;;

,tE

142 O-36

213 0.41

145 0*34fO-07

70 o-21

115 O-25

108 O-27

135 O-26

106 0.25 10.03

* Expressed in arbitrary units. Each figure represents mean of 10 readings.

and

461

B.R. MANAWADU AND A. VOLLER

Five different batches of antihuman IgG conjugate were then compared. These were reconstituted according to the manufacturer’s insuuctions and used as the indicator reagents in an IFA test with P. wiwax antigen reacted with homologous antiserum. The results are shown in Table VII: There were bie differences in the results obtained by the use of diserent conjugates, even though the first four samples were all from one manufacturer. It is clear that for comparable results to be obtained by different workers with the malarial IFA, the conjugates must be standardized against the available research standard. If this is done it is possible to dilute the commercial reagent so that a predetermined number of “units” measured in terms of the research standard can be used. (e) The malaria parasites used as antigen

The biological variables of the IFA are the conjugate, the antiserum and the antigen. If any two of these are fixed then the third, the unknown, can be measured or standardized. In the previous section we have shown how the conjugate may be standardized and as reference preparations of antisera are available it is possible to compare antigens and to measure their homologous and heterologous reactivity. The reference antisera were collected from patients who each had had one single exposure leading to malaria infection with a single infection of P. falciparum, P. v&ax or P. malffriae. These sera were divided into aliquots and stored at -70°C. The sera were reacted in IFA tests at 1:20 dilution and in each test the same antihuman IgG conjugate was used at one dilution (= 75 units of research standard 68/45). The fluorescence of the malaria parasites (obtained from owl monkeys) used as antigens in these tests was measured using the fibre-optic probe. The results of readings on ten different batches of P. falciparum antigen are shown in Table VIII. on ten different P. vivax antigens in Table IX, *and on five different P. mal&icx antigens in Table X. On each table the ratios of heterologous to homologous reactivity of the three reference sera are given. These results indicate that individual batches of antigen made from one and the same species of malaria parasites showed considerable variation, even though we were careful to control conditions of storage and to ensure that all antigens contained mature schizonts. In this particular series of tests the P. vivax antigens were the most reactive over-all. For any particular batch of antigen the homologous serum always gave the highest titre and heterologous sera produced mean values of one quarter to one half of those of the homologous serum. Discussion

In this paper we have dealt with the various factors which can influence the end result of malarial immunofluorescence tests. The use of the fibre-optic system and a fluorescent glass standard allows compensations to be made for the all-important day-to-day variations in the illumination score and the optical system. Using such a standardized measuring system, it is clear that all the three biological parameters, antigens, antisera and conjugates, have to be

standardized. The provision of reference preparations of the second two of these variables allows standardization of the antigens. Our results confirm those of TARGETT (1970) who showed that the stage of the malarial parasite used as antigen is of the utmost importance. It is essential that large trophozoites or schizonts be used as antigen if maximum sensitivity and discriminatory cauacitv of the malarial IFA is to be achieved, However, even initially good antigens must be stored correctly at a temperature of -20% or below if they are to be of use on later occasions. We share the view of SULZER &WILSON (1971) that many of the reported anomalies of malaria immunofluorescence tests have their foundations in the inadequacy of the antigens used. A surprising amount of variability was found between different commercial antiglobulin conjugates. No two batches even from the same manufacturer gave comparable results, and it is evident that every batch of conjugate must be used in a standardized malaria IFA test and its reactivity explored in terms of the reference coniuaate now . available. When a complete series of reference antimalarial sera (that is. P. vivax, P. malariae. P. ovale and P. fakiparukz) is available, it should be possible using the techniques we have outlined to obtain comparable results in different laboratories and at different times. Acknowledgements

We wish to acknowledge the help given by Dr. C. C. Draper, Ross Institute, London, and the encouragement by Dr. L. G. Goodwin and Dr. D. Bidwell of the Nuffield Institute of Comparative Medicine. The technical assistance of Mrs. Daphne Green and clerical help of Ms. Carolyn Stewart is gratefully acknowledged. This work was supported by a grant of the Ministry of Overseas Development and World Health Organization. References Ambroise-Thomas, P. (1974). The immunofluorescence reaction in the sero-immunology study of malaria. Bulletin of the World Health Organization, 50(3-A), 267-276. Collins, W. E. & Skinner, J. C. (1972). The indirect fluorescent antibody test for malaria. American Journal of Tropical 690-695.

Medicine

and Hygiene,

21

Draper, C. C. & Voller, A., with Carpenter, R. G. (1972). The epidemiological interpretation of serologic data in malaria. American Journal qf Tropical Medicine and Hygiene, 21, 696-703.

Sulzer, A. J., Wilson, M. 8~ Hall, E. C. (1969). Indirect fluorescent antibody tests for parasitic disease. V. An evaluation of a thick-smear antigen in the indirect fluorescent antibody test for malaria antibodies. American Journal of Tropical Medicine and Hygiene, 18, 199-205. Sulzer, A. J. & Wilson, M. (1971). The fluorescent antibody test for malaria. In: The fluorescent antibody test for malaria. Critical Reviews in Clinical Laboratory Sciences, 2, 601-619.

462

STANDARDIZATION

Targett, G. A. T. (1970). Antibody response to Plasmodium falciparum malaria. Comparisons of immunoglobulin concentrations, antibody titres and the antigenicity of different asexual forms of ~;;~~i7Clinical and Experimental Immunology, Ta;lor, C. E: D., Heimer, G. V. & Lidwell, 0. M. (1971). Use of a fibre-optic probe for quantitative immunofluorescence studies. Lancet, i, 785. Voller, A. (1964). Fluorescent antibody methods and

OF IFAT

FOR MALARIA

their use in malaria research. Bulletin of the World Health Organization, 30,343-354. A. (1975). Application of immunoVoller, fluorescence to the seroepidemiology of malaria. Annals of the New York Academy of Sciences, 25A, 326-330.

Accepted for publication

New Fellows 15th June 1978 AL-ABYAD, s. A. A., M.B., B.CH., D.M., D.T.M. & H., Kuwait BEATO, Jr., U.P., B.S., M.D., M.P.H., U.S.A. ERASMUS, V. P., M.B.B.s., India GUPTA, B. S., M.D., M.B.B.s., India KHAN, M. A. u., B.SC., M.B.B.S., D.P.H., M.P.H. & T.M., F.A.P.H.A., Pakistan KIRKMAN, M. F., M.B.CH.B., D.T.M. & H., Britain KWA, B. H., PH.D., M.SC., B.SC., Malaysia MALLA, F. B., B.SC., M.B.B.s., D.T.M. & H., Nenal NOORUNNOBI, S.-T. I., M.B.B.s., Bangladesh SCHARLAU. Gerlinde. M.D.. Germanv * SINGH, R. I& M.B.B.s:, Nebal SIVAGURU, K. S., M.B.B.s., Sri Lanka SOOD, J. J., M.D., M.B.B.s., F.c.G.P., India SUBHAN, S. M. A., M.B.B.s., Bangladesh TORRENCE III, W. L., B.SC., U.S.A. TUKEI, P. M., M.B., CH.B., M.SC., Kenya WHITTLE, Susan H., B.SC., Britain

2nd March,

1978.