Rabies neutralizing antibodies determination by the modified counterimmunoelectrophoresis test and the rapid fluorescent focus inhibition test

Zbl. Bakt. Hyg. A 256, 1-6 (1983)

Rabies Neutralizing Antibodies Determination by the Modified Counterimmunoelectrophoresis Test and the Rapid Fluorescent Focus Inhibition Test Bestimmung (Nachweis) neutralisierender Tollwur-Antikorper mit Hille der modifizierten Gegenstromelektrophorese und des fluoreszenzserologischen Fokus-Inhibitions-Schnelltestes ANA MARIA DIAZ Pan American Zoonoses Center, Pan American Health Organization/World Health Organization, Casilla de Correo 3092, Correo Central, 1000 Buenos Aires, Argentina

With 4 Figures' Received April 22, 1983 . Accepted June 14, 1983

Abstract The correlation between the modified counterimmunoelectrophoresis and the rapid fluorescent focus inhibition tests for detecting rabies virus antibodies in human sera was studied, using as reference the standard mouse neutralization test. Statistical analyses showed good correlation among the three serological tests. There were no statistically significant differences in titers obtained by serum neutralization and rapid fluorescent focus inhibition tests and the estimated titers based on the results of the modified counterimmunoelectrophoresis test.

Zusammenfassung Es wurde die Korrelation zwischen der modifizierten Gegenstromelektrophorese und dem Schnell test zur Hemmung herdforrniger Fluoreszenz zur Feststellung von Tollwutvirus-Antikorpern in menschlichen Seren untersucht, wobei der Standard-Neutralisationstest in der Maus als Bezugsgrofse diente. Statistische Analysen zeigten eine gewisse Korrelation zwischen den drei serologischen Tests. Es gab keine statistisch signifikanten Unterschiede zwischen den durch Serum-Neutralisation und den aufgrund der modifizierten Immunelektrophorese geschatzren Titern.

Introduction

Protection against rabies virus infection has been correlated with the presence of serum neutralizing antibodies (3, 4, 5) and a number of procedures have been described for detecting or measuring them (2, 12, 18, 19, 20), to provide the phy1 Zbl. Bakt. Hyg. A 256

2

A.M.Diaz

sician with information on the immune status of a patient after rabies exposure and during pre- or post-exposure treatment. Recently, a modified counterimmunoelectrophoresis (CIE) technique has been developed for determining rabies antibodies (7), which is a sensitive, simple, inexpensive and relatively rapid (approximately 4 h) procedure. When sera from individuals undergoing postexposure rabies immunization were studied by this antigen-antibody-binding procedure, the results obtained correlated well with those of the standard virus neutralization (SN) test in mice (7) and the indirect fluorescent antibody test (8). Because of the correlation between the CIE and the SN tests, it is possible to estimate neutralizing titers on the basis of CIE results (7). Several methods for testing neutralizing activity in cell culture by fluorescent antibody have been described (6, 11, 14). Of these, the test of choice appears to be the rapid fluorescent-focus-inhibition (RFFI) technique of Smith et al. (17), which requires only 24 hours for completion; the results of thi s test correlate very well with those of the SN when sera from human or animal origin are tested (9, 10, 15). This study was undertaken to evaluate the sensitivity and specificity of the CIE and the RFFI tests as compared with the SN test, for the detection of antibodies to rabies virus.

Materials and Methods Virus: A strain of standard chall enge virus (CVS 3111) obtained from the National Institutes of Health, Bethesda, Md., U.S.A., was passed twice in suckling rabbit brain (CVSSRB ll) in our laboratory. The seed viru s was prepared and standardized as described previousl y (7). Antigen: The antigen was prepared basically as described by Dlaz and Myers (7) except that the 40% CVS-SRB/2 suspens ion was made in distilled water with sucrose and glycine at a final concentration of 5% and 0.0075% respectively, instead of 0.01 M phosphate buffered saline solurion. This antigen was stable for a period of more th an 24 months, in a liquid or fre eze-dried form (Table 1). Table 1. Stability of the counterimmunoelectrophoresis test antigen (liquid or lyoph ilized) Months of storage

CIE antigen Liquid (4 °C)

Lyophilized (- 20 °C) 1:40& 1:40 1:40 1:40

18

1:40& 1 :40 1:40 1 :40 1:20 1 :20 1 :20 1 :14

21 24

1:16 1:16

1:6 1:8

1 2 3

5 7 12 13

1:18 1:14 1:14 1:12

&T iter: optimal dilution wh ich produced a sharp prec ipitation band wi th the standard equine hyperimmune antirabies indicator serum.

Rabies Neutralizing Antibodies

3

Sera: Forty-five serum samples were obtained at various intervals during the course of post-exposure immunization of man (six doses) with human diploid cells vaccine (HDCV), kindly supplied by the Merieux Institute. All sera were collected and examined for neutralizing antibodies at 10, 21, 60, 90 and 180 days post-exposure. Ten serum samples from non immunized individuals were used as negative controls. Serological tests: CIE (7), RFFI (17), and SN (1) tests were carried out as described previously. A serum titer of 1: 2 or above was considered positive.

Results The titers obtained with sera collected on the above-mentioned days after vaccination are shown in Fig. 1. There was complete agreement between the eIE and RFFI tests in the 45 SN positive sera, whose titers ranged from 1:5 to 1:78125 (sensitivity 100%).

4

o

10 20 30 40 50 60 70 80 90

180

Days

Fig. 1. Mean titers of rabies antibodies in 45 sera of individuals vaccinated with HDCV. • - . SN test; X - X RFFI test; ,A-,A theoretical SN titers calculated from CIE test; • - • CIE test. SN

/25 25

5 .,

2

3

4

5

6

7

8

9 Log2

2

4

8

/6

32

64

/28

256

512 Titers

'----+~--+--_+_--+----:+_-.;-___t_--+---+___--

C/E

Fig. 2. Linear regression for values obtained by SN tests versus CIE tests for 45 serum samples (0 = 3;. = 4; indicate points superimposed on others). y = 1.6 + 0.47 x. Correlation coefficient, r = 0.83. Error standard of the estimate: 0.74.

4

A.M.Diaz

All 10 negative sera for SN were also negative in both eIE and RFFI tests (specificity 100%). Fig. 2 shows the results of plotting SN titers against CIE titers; the linear regression equation was y = 1.6 + OA7x and the correlation coefficient between the titers obtained by the two methods was r = 0.83. Standard error of the estimate: 0.74. Fig. 3 shows the results of plotting SN titers against RFFI titers; the linear regression equation was y = 0.19 + 0.90x. The correlation coefficient between titers obtained by the two methods was r = 0.97. Standard error of the estimate: 0.35. Fig. 4 shows the results of plotting RFFI titers against CIE titers; the linear regression equation was y = 1.9 + 0.39 x and the correlation coefficient was r = 0.79. Standard error of the estimate: 0.76. SN Titers

Log10

1.000.000 6 100000

5

10.000

4

1.000

3

100 2

-,

10

./ 10

Fig. 3. Linear regression for values obtained by SN tests versus RFFI tests for 45 serum samples (.A. = 2; D = 3; • = 4 indicate points superimposed on others). y = 0.19 + 0.90 x. Correlation coefficient r = 0.97. Error standard of the estimate: 0.35. RFFI

Titers

Log5

15.625 6 3./25

5

625

4

/25

3

25

2

5

I

.:

2

2

3

4

5

6

7

8

9

4

8

/6

32

64

/28

256

5/2

Titers

Fig. 4. Linear regression for values obtained by RFFI tests versus CIE tests for 44 serum samples. (.& indicates 2 points superimposed). y = 1.91 + 0.39 x. Correlation coefficient r = 0.79. Error standard of the estimate: 0.76.

Rabies Neu tralizi ng Antibo dies

5

Discussion The dat a obtained in th is study confirmed th e reliab ility of th e modified CIE technique for the dete ction of seru m ant ibodies to rabies virus. This quantitative test measures mainly immunoglobul in G (IgG) antibod ies and, due to the nature of th e antigen used, may recogn ize neutralizing activity (7, 8). Because of their stabilizing effect on rabies virus , sucros e and glycine have been emp loyed in th e production of vaccin es of high potency (13, 16). The use of the se pro ducts for the prepar ati on of the CIE antigen resulted in an increased sta bility, which makes possible th e prepar at ion of antigen lots in a volume appropr iate for estimated needs. Ho wever, t itration every three months is required to dete ct possible declines in activity. Wh en the data resulting from th e CIE and SN tests were subjected to st atistical ana lysis, a correlation coefficient of r = 0.83 was obtained. This correlation factor was better than those obtained earl ier (7, 8) and could be a reflection of the higher qu ality of the antigen. The relationship between CIE and SN titers was used to calcul ate a theoretical neutralizing antibody curve, which did not show statistically significant differences with the curve obtained by the SN test in mice when analyzed by th e Student test (Fig. 1). There were no sta tistically significant differences either with th e titers obtained by the RFFI test and the titers estimated on th e basis of the result s of th e CIE test. This is proba bly due to th e fact th at both techn iques measure th e ability of seru m neutr alizing ant ibodies either to block th e infection of BHK-21 cells with a BHK adapted CVS rabi es virus or to bind th e envelope antige ns of a betapropiolactone ina ctivated CVS rabies vir us (8, 17). The dat a pre sented here confirmed th e excellent relat ion ship between the SN and the RFFI test; the correlation coefficient obtained (r = 0.97) is identi cal with that rep orted by Guillemin et al., who worked with human and animal sera (9). In thi s labor atory, the use of th e CIE in place of other sero logic tests, has been very satisfacto ry. It could be of grea t value for countries which do not have an adequate supply of labor at or y anima ls or facilities fo r t issue culture. Acknowledgemen ts. Appr eciation is expressed to th e foll owing staff memb ers of th e Pan American Zo ono ses Center : Dr. Oscar P. Larghi, for his helpful suggestio ns, and Ana Nebel, Graciela Perdom o and Oscar Becco, for their efficient technical assista nce.

References 1. Atanasiu, P., M.Bahmayar, M . Baltazard, A. Komarou, H.Koprow ski, Pi Lepine, F.Perez-Gallardo, and M. Schaeffer: Rab ies neutralizing antib od ies response to different

schedules of seru m and vaccine inoc ulatio ns in non-exposed persons. Bull. WId Hlth Org . 14 (1956) 593 2. Aianasiu, P., V. Savy, and P. Perrin : Epreuve immunoenzy matique pour la detection rapide des anticorps antira biques. Ann. Micro bio l. (Par is) A 128 (1977) 489 3. Bahmayar, M ., A. Fayaz, S. Nour-Salchi, M. Mo hammadi, and H. Kop rowski : Successful prot ection of humans exposed to rabies infection. J. Amer. vet. med. Ass. 236 (1976) 2751 4. Baltazard, M. , M. Babm ayar, M. Ghoddsi, A. Sabeti, C. Gajdusek , and E. Rouzbeni :

6

A.M.Diaz

Essai pr atique du seru m antirabique chez les mordus par loups enrages. Bull. WId Hlth Or g. 13 (1955) 747 5. Dean, D. ] ., W. M. Evans, and W. R. Thompson: Stud ies on the low egg pa ssage Flury strain of modified live ra bies virus produced in embryonat ing chicken eggs and tissue culture. Amer. J. vet. Res. 25 (1964) 756 6. Debbie, [, G., ]. A. Andrulonis, and M . K. Abe/seth: Rabies ant ibod y determination by immunofluoresc ence in tissue culture. Infect. Immun. 5 (1972) 902 7. Dlaz, A. M. and D. M. Mye rs: Determination of serum neutralization anti bodies to ra bies virus by a mod ified counterimmunoelectrophor esis test. ] . Clin. Microb iol. 12 (1980) 175 8. Dlaz, A. M. and D. M. M yers: Comparison between a modified counterimmunoelectroph oresis test and th e indirect fluorescent antibody test for detection of antibodies to rab ies virus in hum an sera. ]. Clin. M icrob iol. 14 (1981) 446 9. Guillemin, F., G. T ixier, ]. P. Soulebot, and G. Chappuis: Comparison de deux methodes de titrage des ant icorps antira biques neutralisant s.]. BioI. Standard. 9 (1981) 147 10. Guillemin, F., G. T ixier.], P. Soulebot , and G. Chappuis: Resultats compares des titrages des anticor ps antira biques par deux methodes util isant l'i mmunofluorescence. J. BioI. Standard. 9 (1981) 157 11. King, D.A., D.L.Groghaw, and E. L. Shaw: A rapid quantitative in vitro seru m neutr alizati on test for rabies antibody. Canad. vet.]. 6 (1965) 187 12. Kuwert, E.: The compl ement fixat ion test. In: M .M.Kaplan and H.Koprowski (eds.), Laboratory techn ique in rabies, 3 rd ed., Wo rld Health Or gan izat ion (Mo nograph series 23), Geneva (1973) 13. Largbi, O. P. and A.E. Nebel: Rabies virus inactivatio n by bin ary ethylenimine: new meth od fo r inactivated vaccine productio n. J. Clin. M icrobiol. 11 (1980) 120 14. Lennette, E. M. and R. W. Emm ons: The laboratory diagnosi s of rab ies: review and prospective. In: Y. Nagano and F. M. Davenport (eds.), Rabies. University Park Press, Baltimore (1971) 15. Louie, R.E., M . B.Dobkin, P. Meyer, B. Chin, R. E.Roby, A.H. Hammar, and V. ]. Cabasso: Meas urement of rabies ant ibody : comparison of th e mouse neut ralizat ion test (MNT ) with the rap id fluorescent focus inhibition test (RFFIT) . J. BioI. Stan dard. 3 (1975) 365 16. Sikes, K.R. and O.P.Larghi: Pur ified rab ies vaccine : Development and comparison of potency and safety with two hum an rab ies vaccines. J. Immunol. 99 (1967) 545 17. Smith,]. S., P. A. Yager, and G. M. Baer: A rapid reproducible test for determ ining rabies neutralizing antibody. Bull. WId Hlth Org . 48 (1973) 535 18. Th omas, ]. B., R. K. Sikes, and A. S. Ri cker: Evaluation of indirect fluorescent antibody techniques for detection of rabies antibody in hum an sera. J. Immun ol. 91 (1963) 721 19. Wes ter, L. T. and]. R. Dawson : Earl y diagnosis of rab ies by mou se pr otection test. Pro c. Soc. expo BioI. (N. Y.) 32 (1935) 570 20. Wiktor, T.].: T issue culture methods. In: M.M. Kaplan and H. Koprowski (eds.), Laboratory techn iques in rab ies, 3 rd ed., World Health Organizat ion (Monograph Series 23), Geneva (1973) Dr. Ana M aria Dlaz, Pan Americ an Zoonoses Center, Pan American Health Organizatio n/World H ealth Organ izat ion , Casilla de Corr eo 3092, Correo Central , 1000 Buenos Aires, Argentina