Journal
of Biological
Standardization
1975 3, 365-373
Measurement of rabies antibody : comparison of the mouse neutralization test (MNT) with the rapid fluorescent focus inhibition test (RFFIT) R. E. Louie, M. B. Dobkin, P. Meyer, B. Chin, R. E. Roby, A. H. Hammar and V. J. Cabasso-f
A rapid fluorescent focus inhibition test (RFFIT) was compared with the mouse neutralization test (MNT) for measuring rabies antibody. The RFFIT was found to be the more sensitive, reproducible and accurate of the two. Considering these attributes and the convenience of the test, the conclusion is reached that RFFIT should be substituted for MNT for measurement of rabies antibody.
INTRODUCTION In recent years a number of procedures have been described for detecting or measuring rabies antibody. These include the complement fixation test, the hemagglutination inhibition test, the passive hemagglutination test, the plaque reduction test and a radioimmune assay, all of which have been described in a World Health Organization (W.H.O.) publication (1973). There has been no general acceptance of any one test, and at the present time the mouse neutralization test (MNT) is specified by the W.H.O. Expert Committee on Rabies (1960) and by the United States Bureau of Biologics of the Food and Drug Administration as the official potency test for antirabies serum. Reproducibility of results with the MNT, however, is still questionable despite all efforts to standardize various parameters. The need to replace the MNT is clear. The recently described rapid fluorescent focus inhibition test (RFFIT) of Smith, Yager & Baer (1973) appeared promising because of its economical use of reagents and test sample, its completion in 24 hours, and its suitability for testing large numbers of samples. * Received for publication t Microbiology Research, 25
28 May 1975. Cutter Laboratories,
Inc., Berkeley,
California
94710, U.S.A.
365
R. E. LOUIE
ET AL.
Preliminary investigations in this laboratory have indicated that the reliability and reproducibility of RFFIT are dependent on meticulous attention to technical details. For this reason the test as performed by us is described below in some detail. In support of our proposal that RFFIT is a more than suitable replacement for MNT a comparison has been made of the reproducibility of both tests for the titration of a reference serum, U.S. Standard Antirabies Serum Lot 4,* and for monitoring the processing of a rabies immune globulin, human (RIGH).
MATERIALS
AND
METHODS
RFFIT Stationary cultures of BHKsJ13S cells, overlaid with growth medium consisting of BHK medium? supplemented with 10% heat-inactivated foetal calf serum, 10% tryptose phosphate broth, 50 units/ml of Polymyxin B and 50 pg of Neomycin, were grown in 75 ems flasks.$ Three- to four-day-old monolayers were detached with trypsin-versene and suspended in growth medium containing 0.02 mg/ml of DEAE-Dextran$ at a concentration of 6 x 104 cells per O-2ml. The cell suspension was held at room temperature for 10 minutes before use in the test. The strain of rabies virus used was the challenge virus standard (CVS ll)j/ adapted to BHK,,/lSS cells. The passage history of the virus has been described (Smith et al., 1973). To prepare the virus stock, a monolayer of BHKsJlSS cells grown in a 150 cm2 bottle was split 1 : 3 and resuspended in 150 cm2 bottles containing 60 ml of growth medium. The suspended cells were infected with virus at a multiplicity between 0.001 and O*OOOl mouse LD,,/cell and incubated at 35 “C for 3 days. The monolayers were dispersed with trypsin-versene and split 1 : 3 as before. Following another 3-day incubation at 35 “C the growth medium was discarded and replaced with maintenance medium which differed from growth medium by substituting 0.5% (w/v) human serum albumin for the foetal calf serum and by the addition of 0.01 M HEPES buffer. The cultures were further incubated for 24-48 hours before pooling the virus-containing fluids and storing at -70 “C. The titer of the virus was 1O5’sto 106.s LD,,/ml as determined by intracerebral inoculation of weanling mice. The level of working virus used in the test was the highest dilution of stock virus which produced 20 microscopic fields containing fluorescing foci out of 20 random fields examined (see below). In addition to the working dilution of virus, two successive ten-fold dilutions were also prepared. Separate 200-lambda “To Contain” (TC) pipettes were used to make the serial transfers. The tips of the pipettes were wiped with sterile gauze before delivery to the appropriate volume of growth medium. The three dilutions of virus were held at 4 “C until needed. Serum and plasma samples were inactivated by heating at 56 “C for 30 minutes. The U.S. Standard Antirabies Serum and RIGH samples were tested without prior * Obtained from the Bureau of Biologics (BOB), Food and Drug Administration, Rockville, Maryland, U.S.A. t GIBCO, Grand Island, New York, U.S.A. 1 Falcon Plastics, Division of BioQuest, Los Angeles, California, U.S.A. 5 Pharmacia, Uppsala, Sweden. 11Received from Jean S. Smith, Center for Disease Control, P.O. Box 363, Lawrenceville, Georgia, U.S.A.
MEASUREMENT
OF
RABIES
ANTIBODY
heat-inactivation. Serial two-fold dilutions of serum or plasma were made in growth medium using separate l-ml serological pipettes. The U.S. Standard Antirabies Serum Lot 4 was reconstituted according to instructions. An initial stock 1 : 128 dilution was prepared by filling a 500~lambda TC pipette with the reference serum, wiping the tip with sterile gauze and adding it to 63.5 ml of diluent (measured with appropriate volumetric pipettes). The lambda pipette was rinsed ten times in the serum-diluent mixture. Further two-fold dilutions were made with 0*5-ml volumes contained in separate l-ml serological pipettes. The remainder of the stock 1 : 128 dilution was frozen at - 20 “C and used for subsequent tests. Dilutions of RIGH were similarly carried out starting with an initial 1 : 20 dilution of globulin, and making subsequent two-fold serial dilutions. This meticulous procedure was required for accurate dilutions of the reference serum and minimizing dilution errors due to the viscosity of undiluted globulins. The RFFIT procedure was similar to the one described by Smith et al. (1973). Onetenth ml of each dilution of test sample was delivered to separate chambers of a Lab-Tek slide.* This was followed immediately by the addition of 0.1 ml of the working dilution of virus. At the same time, the virus control titration was prepared by the addition of O-1ml of the working virus dilution and two successive ten-fold dilutions to three other chambers. The plastic covers were placed over the chambers; the slides were tilted back and forth to ensure thorough mixing of serum and virus. The slides were incubated for 90 minutes at 37 “C in a CO, incubator containing a humidified atmosphere of 95% air and 5% CO, (commercial grade, bone dry). Two-tenths ml of cell suspension was added to each chamber with a Bi0pette.t A cell control containing O-2 ml of cell suspension and O-2ml of growth medium was also prepared. Again the slides were tilted to mix the contents and returned to the CO, incubator for 24 hours. At the end of the incubation period the medium was discarded and the plastic chambers were carefully detached from each slide without disturbing the gasket. The slides were rinsed for 5 minutes at 4 “C in phosphate-buffered saline (PBS), pH 7.4 (Dulbecco & Yogt, 1954), containing 0.1% sodium azide. This was followed by a l-minute rinse in acetone at -20 “C. The slides were fixed in fresh acetone at -20 “C for 5 minutes, and air-dried for 15 minutes in a -20 “C freezer. The slides were stained immediately thereafter, or stored in the freezer for later staining. The slides were stained with fluorescein isothiocyanate-conjugated antirabies globulin prepared from a pool of sera from hamsters hyperimmunized according to the method of Johnson (1969). Staining was carried out at 37 “C in a humidified chamber for 50 minutes. The slides were then drained, and washed for 10 minutes in PBS (0.017 1c1 phosphate, 0.15 M sodium chloride, 0.1% sodium azide), pH 7.6, stirred magnetically. Following a 2-minute rinse in distilled water, the slides were air-dried, the gaskets peeled off, and the preparations mounted with cover slips over a solution consisting of equal parts of PBS, pH 7.4 and glycerin. The slides were read with a Zeiss standard BA38 fluorescent microscope using BG 38 and BG 3 exciter filters and nos 47 and 65 barrier filters. An Osram HBO 2OOW/4 high pressure mercury vapor lamp provided the ultraviolet light source. Each section of a slide previously encompassed by the chambers was examined under x 250 magnification. * Cat. no. 4808, Lab-Tek Products, Division of Miles Laboratories, 60540, U.S.A. t Schwarz-Mann, Orangeburg, New York 10962, U.S.A.
Inc., Naperville,
Illinois
367
K. E. LOUIE
ET
AL.
Twenty evenly distributed microscopic fields were examined. Any field with less than 20% of its area covered with cells was not counted and another field examined. A field was considered positive for fluorescence if there were at least two fluorescing foci in the field. The score for each section of the slide was recorded as per cent positive =
no. of fields with fluorescing foci x 100. 20 fields examined
In an acceptable test the score of the virus control titration was recorded as follows: working dilution : 100% positive
0.1 x working dilution : 25555% positive
0.01 x working dilution : 100% negative
The 50% endpoints of virus and antibody titrations were calculated by the SpearmanKarber method (Spearman, 1908; KIrber, 1931). MNT
For the MNT, the virus stock was prepared from a passage of CVS-31B rabies virus obtained from the BOB. A pool of infected mouse brains from ll- to 14-g female Swiss albino mice was homogenized as a 10% suspension in sterile distilled water containing 5% heat-inactivated normal horse serum. The suspension was clarified by light centrifugation, dispensed into glass ampoules, flame-sealed, quick-frozen in a mixture of dry-ice and alcohol and stored at -70 “C. Serum, plasma and globulin samples were treated in the same manner as described in the RFFIT procedure, except that Dulbecco’s saline containing 0.2% gelatin was used as diluent. The initial dilution of the U.S. Standard Antirabies Serum Lot 4 was 1 : 10 and for RIGH samples was 1 : 8. The MNT was performed according to the procedure described by Atanasiu (1966) with the following modifications : Dulbecco’s saline containing 0.2% gelatin was used as diluent for the virus as well as for the rabies antibody samples; the stock virus dilution was calculated to contain a final concentration of 250 LDJO.03 ml. The virus-antibody mixtures were incubated for 1 hour in a 37 “C water bath. Swiss albino mice weighing 1l-14 g were injected intracerebrally with 0.03 ml of inoculum using a 0*5-ml disposable syringe fitted with a s-inch, 26-gauge needle. Ten mice were used for each dilution of antibody, and for each dilution of the virus control titration. Deaths occurring between the 6th and 14th days after injection were considered to have resulted from rabies. Antibody and virus titers were calculated according to the method of Spearman (1908) and KHrber (1931). Expression of antibody levels in international
units
Antibody titers of serum, plasma or globulin were expressed in international units (i.u./ml) (Cabasso, Dobkin, Roby & Hammar, 1974). Each antibody assay, therefore, included a titration of the U.S. Standard Antirabies Serum Lot 4 which has a stated potency of 2 i.u./ml. Antibody titers were converted to international units according to the following formula: titer of sample i.u./ml of sample = . x 2. titer of U.S. Standard Antirabies Serum Lot 4 368
MEASUREMENT
OF
RABIES
ANTIBODY
RESULTS
Reproducibility of RFFIT
and MNT
To compare the reproducibility of RFFIT and MNT, the U.S. Standard Antirabies Serum Lot 4 was titrated repeatedly by both methods. Because the multiplicative nature of serial dilutions results in a skewed distribution of titer values, the data were transformed to log,, values for all statistical analyses. Figure 1 shows the distribution of the log,, SN,, titers of the U.S. Standard Antirabies Serum from 61 MNT and 64 RFFIT titrations. The titers determined by MNT have a range of 1~1~2.09 whereas the titers from RFFIT are in a narrower range of Z-20-2.89. The distribution of titers from RFFIT approximates a normal distribution more closely than those from the MNT which shows two peaks, one at log,, SN,, values of 1.60-1.69 and another at 1+30-l-89. The geometric mean titer of the U.S. Standard Antirabies Serum calculated from titrations by RFFIT was 286 as compared to a mean titer of 55 from titrations by MNT (Table 1). Standard deviations of the log,, SN,,, values were 0.114 and 0.186 for RFFIT
=I24 22 20 ln
f 2 Ti
16 14 12 IO 0 6 4
2
III
n
10
Loglo reciprocal of SN,,
MNT
RFFIT
titer
Fig. 1. Titration of U.S. Standard Antirabies Serum Lot 4 by mouse neutralization test (MNT) and by rapid fluorescent focus inhibition test (RFFIT). Distribution of log,,, SN,, titers. TABLE
1. Titration
fluorescent
Method of titration
RFFIT MNT
of the U.S.
focus inhibition
No. of titrations 64
61
Standard
Serum Lot 4 by rapid and mouse neutralization test
Antirabies
test (RFFIT) (MNT) Geometric mean of SN,, titer*
Standard deviation of log,, S&s
286 (2.456)$ 55 (l-740)$
0.114 0.186
* Titers expressed as reciprocal of serum dilution. t Range = Anti-log (log,, geometric mean + standard $ ( ) = Log,, geometric mean.
Range of SNm1220-372 36-85
deviation).
369
R. E. LOUIE
ET
AL.
and MNT, respectively. Thus, the data from RFFIT show the range of log,, SN,, titers to be narrower with a smaller standard deviation than the corresponding data from MNT. These data, together with the higher geometric mean titer of the U.S. Standard Antirabies Serum from RFFIT, attest to the greater reproducibility and sensitivity of RFFIT over MNT. Criteria for acceptance of RFFIT
results
Based on the above data, and on the cumulative experience from 64 titrations of the U.S. Standard Antirabies Serum, two criteria were established for acceptance of RFFIT results. The first was the titer of the U.S. Standard Antirabies Serum Lot 4, which must fall within a range of 220-372. This range was calculated from the data contained in Table 1 by the following formula: range = anti-log (log,, geometric mean f one standard deviation). The second criterion was the titer of the virus control, which must fall within 5-11 fifty per cent focus-forming doses (FFD,,) per O-1ml. This range was established from the criteria described under the Materials and Methods section. There, the 25-55oi, variability in focus formation permitted for the lo- l’O dilution of the working virus dose in the control titration, was established from our cumulative experience with this test. That this choice was fortuitous may be seen from an examination of the virus titers obtained from the tests summarized in Table 2. In 64 tests by RFFIT method the challenge virus varied from 3.2 to 15.8 FFD,, with a geometric mean of 8.2 FFD,,. Using the above formula, the range of the challenge virus plus or minus one standard deviation was found to be 6-11 FFD,,. TABLE
2. Virus doses used in titration of the U.S. Standard Antirabies Serum Lot 4
Method of titration RFFIT MNT
No. of titrations 64 61
Virus dose (geometric mean) 8.2 FFD,, 90 LD,,
Actual range of virus dose 3.2-X.8 1047
FFD,, LD,,
In contrast with the relatively narrow range of variation of virus dose in RFFIT, the challenge virus varied from 10 to 447 LD,,, with a geometric mean of 90 LD,,, in 61 MNT titrations. The RFFIT data presented in the next section were obtained from tests in which both the U.S. Standard Antirabies Serum Lot 4 and the challenge virus doses were within the specified ranges of acceptance. Comparison of RFFIT and MNT for tztration of RIGH and plasma samples Rabies immune globulin is prepared from pools of plasma obtained from hyperimmunized donors. The globulin is a 16.5% (w/v) protein solution consisting of at least 95% IgG as determined by electrophoretic mobility. In actual experience, however, the IgG content is usually closer to 99%. An example is now presented of the use of RFFIT and MNT to monitor rabies antibody titers at various processing stages of a lot of RIGH. The lot of RIGH conformed to the specifications indicated above. 370
MEASUREMENT
OF
RABIES
ANTIBODY
The IgG content of the plasma pool from which this particular lot of RIGH was derived was 0.86% (WIv ) as determined by a single radial diffusion test (Mancini, Carbonara & Heremans, 1965) using a commercial kit. * The concentration of rabies antibody as IgG from the plasma pool to RIGH was calculated to be 16.5/O-86 = 19.2. Table 3 summarizes comparative titers of the starting plasma pool, of two dilution steps which occurred during processing, and of the immune globulin filled in the final container. Titers of the plasma pool by both methods were in fairly close agreement: 12.5 i.u./ml by MNT and 10.5 i.u./ml by RFFIT. The antibody levels of the various RIGH samples, however, were not as close between the two tests. TABLE
3. Titration of samples during processing of RIGH
Sample
antibody level* determined by A c \ MNT RFFIT
Plasma pool RIGH undiluted RIGH diluted 1 : 1.16 RIGH further diluted 1 : l-46$ RIGH in final container:
12.5 10.5 Not done Not done 351 133 158 102 223 114
Actual
Geometric means
antibody level minus dilution factor RFFIT MNT Actual
(240)t 407 267 377
172 193
345
172
* Antibody levels expressed as international units/ml. t ( ) = Expected titer of RIGH based on 19*2-fold concentration of the plasma pool. $ These two samples represent the same material, the first taken from bulk and the second from fmal filled containers. § Calculated from the three actual titers minus dilution factor.
In order to resolve these differences, the expected value of the undiluted RIGH was calculated by multiplying the plasma pool titer by the concentration factor, 19.2. The theoretical potency of this lot of RIGH, therefore, as projected from the plasma pool titer, was 240 i.u./ml according to MNT and 202 i.u./ml by RFFIT. To compare the theoretical RIGH value with the actual potency obtained by either method, the dilution factors have first to be taken into account. Columns 4 and 5 in Table 3 show these adjusted RIGH titers. The geometric mean of the adjusted titers was 345 i.u./ml by MNT and 172 i.u./ml by RFFIT, compared with expected values of 240 and 202 i.u./ml, respectively. The comparison between plasma pool and RIGH values does not take into account the likelihood that the rabies antibody content in the plasma pool could be heterogenous with respect to the immune globulin class. To test the possibility that not all antibody is IgG, the plasma pool was treated with 2-mercaptoethanol (2ME) to destroy any IgMrabies antibody which could be present. Parallel treated and untreated samples were titrated by RFFIT, and the results are seen in Table 4. The results suggest that 26.5% of the rabies antibody present in the untreated plasma pool could have been associated with IgM. This is in accord with Rubin, Gough, Gerlach, Dierks, Gregg & Sikes (1971), who showed a similar distribution of rabies antibody in persons 225 days after initiation of immunization consisting of 14 daily * IgG Tri-Partigen
Kit, Behring
Diagnostics,
Somerville,
New Jersey, U.S.A.
371
R.
ET AL.
E. LOUIE
TABLE
4.
Treatment
RFFIT test no.
Plasma untreated
1
2 3 Geometric
of rabies hyperimmune ethanol (2ME)
mean
plasma
Plasma 2ME-treated
pool with
2-mercapto-
Expected antibody level of undiluted RICH*
9.97 10.4 10-2
7.4 8.4 6.7
142 161 129
10.2
7.5
143 (172)$
* Calculated by multiplying antibody level of 2ME-treated plasma by 19.2. t Antibody levels expressed as international units/ml. $ ( ) = Geometric mean of actual antibody levels minus dilution factor. See Table 3.
doses of duck embryo vaccine followed by boosters 10 and 20 days after the 14th injection. Thus, the geometric mean titer of the treated plasma pool, multiplied by 19.2, now yields an expected RIGH potency of 143 i.u./ml which is a more valid figure for comparison with the actual adjusted value of 172 i.u./ml obtained by RFFIT. A similar comparison of IgM-free rabies antibody determinations by MNT was not possible since the geometric mean of 34.5i.u./ml was greater to begin with than the expected potency of 240 i.u./ml shown for the untreated plasma pool. This discrepancy can be attributed to the wide variation that MNT is known to be subject to. DISCUSSION In viva methods of antibody assay which utilize death of the host as an endpoint are inherently imprecise because of the many uncontrollable factors, such as non-specific deaths, or susceptibility or resistance to lethal infections. Such has been our experience with MNT for measurement of rabies antibody. To circumvent these problems, the in vitro RFFIT method was investigated as a possible substitute for MNT. A comparison of the sensitivity and reproducibility of two tests was carried out by repeated titrations of the U.S. Standard Antirabies Serum Lot 4. The geometric mean titer of this reference serum was found to be five-fold higher by RFFIT than by MNT. In 64 RFFIT titrations the log,, SN,, titers varied between 2.20 and 2.89 whereas a range of l-10-2.09 was seen in 61 MNT titrations. This narrower range of titers was also reflected in a correspondingly smaller standard deviation of the log,, SN,, values from RFFIT. Another indication of the greater reproducibility of RFFIT results was seen in the variations of the titer of virus used in the two tests. In the 64 titrations of the U.S. Standard Antirabies Serum by RFFIT the titer of the virus varied from 3.2 to 15.8 FFD,,, whereas the challenge virus in 61 MNT titrations varied from 10 to 447 LD,,. Thus, with this degree of reproducibility more stringent criteria could be established for acceptance of rabies antibody titration results. These were that (a) the titer of the U.S. Standard Antirabies Serum Lot 4 by RFFIT must be within one standard deviation of the geometric mean titer (220-372) and (b) the virus used in the test must be within the limits of 5-11 FFD,, Subsequent experience has shown that these limits are reasonable and are within the capability of the test.
372
MEASUREMENT
OF RABIES
ANTIBODY
A comparison of the accuracy of the two tests was carried out by monitoring the rabies antibody titer during the processing of a rabies hyperimmune plasma pool to IgG. By measuring the IgG concentration of the plasma pool and final IgG solution, the concentration factor of rabies antibody in the IgG solution could be determined. The titer of IgG rabies antibody in 2ME-treated plasma multiplied by the concentration factor would then give the expected rabies antibody titer in the IgG solution. When this calculation was performed on the rabies antibody titer of the 2ME-treated plasma, determined by RFFIT, and compared with the titers of the IgG solutions during various stages of processing, a value of 143 was obtained for the expected titer versus a geometric mean titer of 172 for the IgG after various processing stages. A similar comparison of the expected rabies antibody titer, based on comparing the antibody content of plasma, with that of the IgG at different stages of processing, yielded geometric means of 240 and 345 i.u./ml, respectively, by MNT. Thus, the comparative data from the two tests point to the greater sensitivity, reproducibility and accuracy of RFFIT over MNT. Considering the convenience of RFFIT along with these attributes, the conclusion is drawn that this test is more than suitable as a replacement for MNT. Addendum
While this paper was in preparation a publication by Cho & Fenje (1975) presented results of a comparison of RFFIT with MNT for titration of rabies antibody. REFERENCES Atanasiu, P. (1966). Quantitative assayand potency test of antirabies serum. In Laboratory Techniquesin Rubies, 2nd ed., pp. 167-172. Geneva: World Health Organization. Cabasso, V. J., Dobkin, M. B., Roby, R. E. & Hammar, A. H. (1974). Antibody response to a human diploid cell rabies vaccine. Applied Microbiology 27, 5.53-561. Cho, H. C. & Fenje, P. (1975). Rabies neutralizing antibody determination in tissue culture by direct fluorescent antibody technique. Journal of Biological Standardizat on 3, 101-105. Dulbecco, R. & Vogt, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. Journal of Experimental Medicine 99, 167-182. Johnson, H. N. (1969). Rabiesvirus. In Diagnostic Procedures for Viral and Rickettsial Infections, 4th ed., pp. 321-353. New York: American Public Health Association, Inc. Karber, G. (1931). Beitrag ziir kollektivin Behandhmg pharmakologischer Reihenversuche. Archiv fiir Experimentelle Pathologie und Pharmakologie 162, 480-483. Mancini, G., Carbonara, A. O., & Heremans, J. F. (1965). Immunochemical quantitation of antigens by a single radial immunodiffusion. Immunochemistry 2, 235-254. Rubin, R. H., Gough, P., Gerlach, E. H., Dierks, R. E., Gregg, M. B. & Sikes, R. ET. (1971). Immunoglobulin response to rabies vaccine in man. Lancet 2, 625-628. Smith, J. S., Yager, P. A. & Baer, G. M. (1973). A rapid reproducible test for determining rabies neutralizing antibody. Bulletin of the World Health Organization 48, 535-541. Spearman, C. (1908). The method of “Right and Wrong Cases” (constant stimuli) with Gauss’ formulae. British Journal of Psychology 2, 227-242. W.H.O. Expert Committee on Rabies (1960). World Health Organization Technical Report Series No. ZOI, 9-11. Geneva: W.H.O. W’.H.O. (1973). Special Diagnostic and Research Techniques. In Laboratory Techniques in Rubies, 3rd ed. World Health Organization Monograph Series No. 23, pp. 101-185. Geneva: W.H.O.
373
of Biological
Standardization
1975 3, 365-373
Measurement of rabies antibody : comparison of the mouse neutralization test (MNT) with the rapid fluorescent focus inhibition test (RFFIT) R. E. Louie, M. B. Dobkin, P. Meyer, B. Chin, R. E. Roby, A. H. Hammar and V. J. Cabasso-f
A rapid fluorescent focus inhibition test (RFFIT) was compared with the mouse neutralization test (MNT) for measuring rabies antibody. The RFFIT was found to be the more sensitive, reproducible and accurate of the two. Considering these attributes and the convenience of the test, the conclusion is reached that RFFIT should be substituted for MNT for measurement of rabies antibody.
INTRODUCTION In recent years a number of procedures have been described for detecting or measuring rabies antibody. These include the complement fixation test, the hemagglutination inhibition test, the passive hemagglutination test, the plaque reduction test and a radioimmune assay, all of which have been described in a World Health Organization (W.H.O.) publication (1973). There has been no general acceptance of any one test, and at the present time the mouse neutralization test (MNT) is specified by the W.H.O. Expert Committee on Rabies (1960) and by the United States Bureau of Biologics of the Food and Drug Administration as the official potency test for antirabies serum. Reproducibility of results with the MNT, however, is still questionable despite all efforts to standardize various parameters. The need to replace the MNT is clear. The recently described rapid fluorescent focus inhibition test (RFFIT) of Smith, Yager & Baer (1973) appeared promising because of its economical use of reagents and test sample, its completion in 24 hours, and its suitability for testing large numbers of samples. * Received for publication t Microbiology Research, 25
28 May 1975. Cutter Laboratories,
Inc., Berkeley,
California
94710, U.S.A.
365
R. E. LOUIE
ET AL.
Preliminary investigations in this laboratory have indicated that the reliability and reproducibility of RFFIT are dependent on meticulous attention to technical details. For this reason the test as performed by us is described below in some detail. In support of our proposal that RFFIT is a more than suitable replacement for MNT a comparison has been made of the reproducibility of both tests for the titration of a reference serum, U.S. Standard Antirabies Serum Lot 4,* and for monitoring the processing of a rabies immune globulin, human (RIGH).
MATERIALS
AND
METHODS
RFFIT Stationary cultures of BHKsJ13S cells, overlaid with growth medium consisting of BHK medium? supplemented with 10% heat-inactivated foetal calf serum, 10% tryptose phosphate broth, 50 units/ml of Polymyxin B and 50 pg of Neomycin, were grown in 75 ems flasks.$ Three- to four-day-old monolayers were detached with trypsin-versene and suspended in growth medium containing 0.02 mg/ml of DEAE-Dextran$ at a concentration of 6 x 104 cells per O-2ml. The cell suspension was held at room temperature for 10 minutes before use in the test. The strain of rabies virus used was the challenge virus standard (CVS ll)j/ adapted to BHK,,/lSS cells. The passage history of the virus has been described (Smith et al., 1973). To prepare the virus stock, a monolayer of BHKsJlSS cells grown in a 150 cm2 bottle was split 1 : 3 and resuspended in 150 cm2 bottles containing 60 ml of growth medium. The suspended cells were infected with virus at a multiplicity between 0.001 and O*OOOl mouse LD,,/cell and incubated at 35 “C for 3 days. The monolayers were dispersed with trypsin-versene and split 1 : 3 as before. Following another 3-day incubation at 35 “C the growth medium was discarded and replaced with maintenance medium which differed from growth medium by substituting 0.5% (w/v) human serum albumin for the foetal calf serum and by the addition of 0.01 M HEPES buffer. The cultures were further incubated for 24-48 hours before pooling the virus-containing fluids and storing at -70 “C. The titer of the virus was 1O5’sto 106.s LD,,/ml as determined by intracerebral inoculation of weanling mice. The level of working virus used in the test was the highest dilution of stock virus which produced 20 microscopic fields containing fluorescing foci out of 20 random fields examined (see below). In addition to the working dilution of virus, two successive ten-fold dilutions were also prepared. Separate 200-lambda “To Contain” (TC) pipettes were used to make the serial transfers. The tips of the pipettes were wiped with sterile gauze before delivery to the appropriate volume of growth medium. The three dilutions of virus were held at 4 “C until needed. Serum and plasma samples were inactivated by heating at 56 “C for 30 minutes. The U.S. Standard Antirabies Serum and RIGH samples were tested without prior * Obtained from the Bureau of Biologics (BOB), Food and Drug Administration, Rockville, Maryland, U.S.A. t GIBCO, Grand Island, New York, U.S.A. 1 Falcon Plastics, Division of BioQuest, Los Angeles, California, U.S.A. 5 Pharmacia, Uppsala, Sweden. 11Received from Jean S. Smith, Center for Disease Control, P.O. Box 363, Lawrenceville, Georgia, U.S.A.
MEASUREMENT
OF
RABIES
ANTIBODY
heat-inactivation. Serial two-fold dilutions of serum or plasma were made in growth medium using separate l-ml serological pipettes. The U.S. Standard Antirabies Serum Lot 4 was reconstituted according to instructions. An initial stock 1 : 128 dilution was prepared by filling a 500~lambda TC pipette with the reference serum, wiping the tip with sterile gauze and adding it to 63.5 ml of diluent (measured with appropriate volumetric pipettes). The lambda pipette was rinsed ten times in the serum-diluent mixture. Further two-fold dilutions were made with 0*5-ml volumes contained in separate l-ml serological pipettes. The remainder of the stock 1 : 128 dilution was frozen at - 20 “C and used for subsequent tests. Dilutions of RIGH were similarly carried out starting with an initial 1 : 20 dilution of globulin, and making subsequent two-fold serial dilutions. This meticulous procedure was required for accurate dilutions of the reference serum and minimizing dilution errors due to the viscosity of undiluted globulins. The RFFIT procedure was similar to the one described by Smith et al. (1973). Onetenth ml of each dilution of test sample was delivered to separate chambers of a Lab-Tek slide.* This was followed immediately by the addition of 0.1 ml of the working dilution of virus. At the same time, the virus control titration was prepared by the addition of O-1ml of the working virus dilution and two successive ten-fold dilutions to three other chambers. The plastic covers were placed over the chambers; the slides were tilted back and forth to ensure thorough mixing of serum and virus. The slides were incubated for 90 minutes at 37 “C in a CO, incubator containing a humidified atmosphere of 95% air and 5% CO, (commercial grade, bone dry). Two-tenths ml of cell suspension was added to each chamber with a Bi0pette.t A cell control containing O-2 ml of cell suspension and O-2ml of growth medium was also prepared. Again the slides were tilted to mix the contents and returned to the CO, incubator for 24 hours. At the end of the incubation period the medium was discarded and the plastic chambers were carefully detached from each slide without disturbing the gasket. The slides were rinsed for 5 minutes at 4 “C in phosphate-buffered saline (PBS), pH 7.4 (Dulbecco & Yogt, 1954), containing 0.1% sodium azide. This was followed by a l-minute rinse in acetone at -20 “C. The slides were fixed in fresh acetone at -20 “C for 5 minutes, and air-dried for 15 minutes in a -20 “C freezer. The slides were stained immediately thereafter, or stored in the freezer for later staining. The slides were stained with fluorescein isothiocyanate-conjugated antirabies globulin prepared from a pool of sera from hamsters hyperimmunized according to the method of Johnson (1969). Staining was carried out at 37 “C in a humidified chamber for 50 minutes. The slides were then drained, and washed for 10 minutes in PBS (0.017 1c1 phosphate, 0.15 M sodium chloride, 0.1% sodium azide), pH 7.6, stirred magnetically. Following a 2-minute rinse in distilled water, the slides were air-dried, the gaskets peeled off, and the preparations mounted with cover slips over a solution consisting of equal parts of PBS, pH 7.4 and glycerin. The slides were read with a Zeiss standard BA38 fluorescent microscope using BG 38 and BG 3 exciter filters and nos 47 and 65 barrier filters. An Osram HBO 2OOW/4 high pressure mercury vapor lamp provided the ultraviolet light source. Each section of a slide previously encompassed by the chambers was examined under x 250 magnification. * Cat. no. 4808, Lab-Tek Products, Division of Miles Laboratories, 60540, U.S.A. t Schwarz-Mann, Orangeburg, New York 10962, U.S.A.
Inc., Naperville,
Illinois
367
K. E. LOUIE
ET
AL.
Twenty evenly distributed microscopic fields were examined. Any field with less than 20% of its area covered with cells was not counted and another field examined. A field was considered positive for fluorescence if there were at least two fluorescing foci in the field. The score for each section of the slide was recorded as per cent positive =
no. of fields with fluorescing foci x 100. 20 fields examined
In an acceptable test the score of the virus control titration was recorded as follows: working dilution : 100% positive
0.1 x working dilution : 25555% positive
0.01 x working dilution : 100% negative
The 50% endpoints of virus and antibody titrations were calculated by the SpearmanKarber method (Spearman, 1908; KIrber, 1931). MNT
For the MNT, the virus stock was prepared from a passage of CVS-31B rabies virus obtained from the BOB. A pool of infected mouse brains from ll- to 14-g female Swiss albino mice was homogenized as a 10% suspension in sterile distilled water containing 5% heat-inactivated normal horse serum. The suspension was clarified by light centrifugation, dispensed into glass ampoules, flame-sealed, quick-frozen in a mixture of dry-ice and alcohol and stored at -70 “C. Serum, plasma and globulin samples were treated in the same manner as described in the RFFIT procedure, except that Dulbecco’s saline containing 0.2% gelatin was used as diluent. The initial dilution of the U.S. Standard Antirabies Serum Lot 4 was 1 : 10 and for RIGH samples was 1 : 8. The MNT was performed according to the procedure described by Atanasiu (1966) with the following modifications : Dulbecco’s saline containing 0.2% gelatin was used as diluent for the virus as well as for the rabies antibody samples; the stock virus dilution was calculated to contain a final concentration of 250 LDJO.03 ml. The virus-antibody mixtures were incubated for 1 hour in a 37 “C water bath. Swiss albino mice weighing 1l-14 g were injected intracerebrally with 0.03 ml of inoculum using a 0*5-ml disposable syringe fitted with a s-inch, 26-gauge needle. Ten mice were used for each dilution of antibody, and for each dilution of the virus control titration. Deaths occurring between the 6th and 14th days after injection were considered to have resulted from rabies. Antibody and virus titers were calculated according to the method of Spearman (1908) and KHrber (1931). Expression of antibody levels in international
units
Antibody titers of serum, plasma or globulin were expressed in international units (i.u./ml) (Cabasso, Dobkin, Roby & Hammar, 1974). Each antibody assay, therefore, included a titration of the U.S. Standard Antirabies Serum Lot 4 which has a stated potency of 2 i.u./ml. Antibody titers were converted to international units according to the following formula: titer of sample i.u./ml of sample = . x 2. titer of U.S. Standard Antirabies Serum Lot 4 368
MEASUREMENT
OF
RABIES
ANTIBODY
RESULTS
Reproducibility of RFFIT
and MNT
To compare the reproducibility of RFFIT and MNT, the U.S. Standard Antirabies Serum Lot 4 was titrated repeatedly by both methods. Because the multiplicative nature of serial dilutions results in a skewed distribution of titer values, the data were transformed to log,, values for all statistical analyses. Figure 1 shows the distribution of the log,, SN,, titers of the U.S. Standard Antirabies Serum from 61 MNT and 64 RFFIT titrations. The titers determined by MNT have a range of 1~1~2.09 whereas the titers from RFFIT are in a narrower range of Z-20-2.89. The distribution of titers from RFFIT approximates a normal distribution more closely than those from the MNT which shows two peaks, one at log,, SN,, values of 1.60-1.69 and another at 1+30-l-89. The geometric mean titer of the U.S. Standard Antirabies Serum calculated from titrations by RFFIT was 286 as compared to a mean titer of 55 from titrations by MNT (Table 1). Standard deviations of the log,, SN,,, values were 0.114 and 0.186 for RFFIT
=I24 22 20 ln
f 2 Ti
16 14 12 IO 0 6 4
2
III
n
10
Loglo reciprocal of SN,,
MNT
RFFIT
titer
Fig. 1. Titration of U.S. Standard Antirabies Serum Lot 4 by mouse neutralization test (MNT) and by rapid fluorescent focus inhibition test (RFFIT). Distribution of log,,, SN,, titers. TABLE
1. Titration
fluorescent
Method of titration
RFFIT MNT
of the U.S.
focus inhibition
No. of titrations 64
61
Standard
Serum Lot 4 by rapid and mouse neutralization test
Antirabies
test (RFFIT) (MNT) Geometric mean of SN,, titer*
Standard deviation of log,, S&s
286 (2.456)$ 55 (l-740)$
0.114 0.186
* Titers expressed as reciprocal of serum dilution. t Range = Anti-log (log,, geometric mean + standard $ ( ) = Log,, geometric mean.
Range of SNm1220-372 36-85
deviation).
369
R. E. LOUIE
ET
AL.
and MNT, respectively. Thus, the data from RFFIT show the range of log,, SN,, titers to be narrower with a smaller standard deviation than the corresponding data from MNT. These data, together with the higher geometric mean titer of the U.S. Standard Antirabies Serum from RFFIT, attest to the greater reproducibility and sensitivity of RFFIT over MNT. Criteria for acceptance of RFFIT
results
Based on the above data, and on the cumulative experience from 64 titrations of the U.S. Standard Antirabies Serum, two criteria were established for acceptance of RFFIT results. The first was the titer of the U.S. Standard Antirabies Serum Lot 4, which must fall within a range of 220-372. This range was calculated from the data contained in Table 1 by the following formula: range = anti-log (log,, geometric mean f one standard deviation). The second criterion was the titer of the virus control, which must fall within 5-11 fifty per cent focus-forming doses (FFD,,) per O-1ml. This range was established from the criteria described under the Materials and Methods section. There, the 25-55oi, variability in focus formation permitted for the lo- l’O dilution of the working virus dose in the control titration, was established from our cumulative experience with this test. That this choice was fortuitous may be seen from an examination of the virus titers obtained from the tests summarized in Table 2. In 64 tests by RFFIT method the challenge virus varied from 3.2 to 15.8 FFD,, with a geometric mean of 8.2 FFD,,. Using the above formula, the range of the challenge virus plus or minus one standard deviation was found to be 6-11 FFD,,. TABLE
2. Virus doses used in titration of the U.S. Standard Antirabies Serum Lot 4
Method of titration RFFIT MNT
No. of titrations 64 61
Virus dose (geometric mean) 8.2 FFD,, 90 LD,,
Actual range of virus dose 3.2-X.8 1047
FFD,, LD,,
In contrast with the relatively narrow range of variation of virus dose in RFFIT, the challenge virus varied from 10 to 447 LD,,, with a geometric mean of 90 LD,,, in 61 MNT titrations. The RFFIT data presented in the next section were obtained from tests in which both the U.S. Standard Antirabies Serum Lot 4 and the challenge virus doses were within the specified ranges of acceptance. Comparison of RFFIT and MNT for tztration of RIGH and plasma samples Rabies immune globulin is prepared from pools of plasma obtained from hyperimmunized donors. The globulin is a 16.5% (w/v) protein solution consisting of at least 95% IgG as determined by electrophoretic mobility. In actual experience, however, the IgG content is usually closer to 99%. An example is now presented of the use of RFFIT and MNT to monitor rabies antibody titers at various processing stages of a lot of RIGH. The lot of RIGH conformed to the specifications indicated above. 370
MEASUREMENT
OF
RABIES
ANTIBODY
The IgG content of the plasma pool from which this particular lot of RIGH was derived was 0.86% (WIv ) as determined by a single radial diffusion test (Mancini, Carbonara & Heremans, 1965) using a commercial kit. * The concentration of rabies antibody as IgG from the plasma pool to RIGH was calculated to be 16.5/O-86 = 19.2. Table 3 summarizes comparative titers of the starting plasma pool, of two dilution steps which occurred during processing, and of the immune globulin filled in the final container. Titers of the plasma pool by both methods were in fairly close agreement: 12.5 i.u./ml by MNT and 10.5 i.u./ml by RFFIT. The antibody levels of the various RIGH samples, however, were not as close between the two tests. TABLE
3. Titration of samples during processing of RIGH
Sample
antibody level* determined by A c \ MNT RFFIT
Plasma pool RIGH undiluted RIGH diluted 1 : 1.16 RIGH further diluted 1 : l-46$ RIGH in final container:
12.5 10.5 Not done Not done 351 133 158 102 223 114
Actual
Geometric means
antibody level minus dilution factor RFFIT MNT Actual
(240)t 407 267 377
172 193
345
172
* Antibody levels expressed as international units/ml. t ( ) = Expected titer of RIGH based on 19*2-fold concentration of the plasma pool. $ These two samples represent the same material, the first taken from bulk and the second from fmal filled containers. § Calculated from the three actual titers minus dilution factor.
In order to resolve these differences, the expected value of the undiluted RIGH was calculated by multiplying the plasma pool titer by the concentration factor, 19.2. The theoretical potency of this lot of RIGH, therefore, as projected from the plasma pool titer, was 240 i.u./ml according to MNT and 202 i.u./ml by RFFIT. To compare the theoretical RIGH value with the actual potency obtained by either method, the dilution factors have first to be taken into account. Columns 4 and 5 in Table 3 show these adjusted RIGH titers. The geometric mean of the adjusted titers was 345 i.u./ml by MNT and 172 i.u./ml by RFFIT, compared with expected values of 240 and 202 i.u./ml, respectively. The comparison between plasma pool and RIGH values does not take into account the likelihood that the rabies antibody content in the plasma pool could be heterogenous with respect to the immune globulin class. To test the possibility that not all antibody is IgG, the plasma pool was treated with 2-mercaptoethanol (2ME) to destroy any IgMrabies antibody which could be present. Parallel treated and untreated samples were titrated by RFFIT, and the results are seen in Table 4. The results suggest that 26.5% of the rabies antibody present in the untreated plasma pool could have been associated with IgM. This is in accord with Rubin, Gough, Gerlach, Dierks, Gregg & Sikes (1971), who showed a similar distribution of rabies antibody in persons 225 days after initiation of immunization consisting of 14 daily * IgG Tri-Partigen
Kit, Behring
Diagnostics,
Somerville,
New Jersey, U.S.A.
371
R.
ET AL.
E. LOUIE
TABLE
4.
Treatment
RFFIT test no.
Plasma untreated
1
2 3 Geometric
of rabies hyperimmune ethanol (2ME)
mean
plasma
Plasma 2ME-treated
pool with
2-mercapto-
Expected antibody level of undiluted RICH*
9.97 10.4 10-2
7.4 8.4 6.7
142 161 129
10.2
7.5
143 (172)$
* Calculated by multiplying antibody level of 2ME-treated plasma by 19.2. t Antibody levels expressed as international units/ml. $ ( ) = Geometric mean of actual antibody levels minus dilution factor. See Table 3.
doses of duck embryo vaccine followed by boosters 10 and 20 days after the 14th injection. Thus, the geometric mean titer of the treated plasma pool, multiplied by 19.2, now yields an expected RIGH potency of 143 i.u./ml which is a more valid figure for comparison with the actual adjusted value of 172 i.u./ml obtained by RFFIT. A similar comparison of IgM-free rabies antibody determinations by MNT was not possible since the geometric mean of 34.5i.u./ml was greater to begin with than the expected potency of 240 i.u./ml shown for the untreated plasma pool. This discrepancy can be attributed to the wide variation that MNT is known to be subject to. DISCUSSION In viva methods of antibody assay which utilize death of the host as an endpoint are inherently imprecise because of the many uncontrollable factors, such as non-specific deaths, or susceptibility or resistance to lethal infections. Such has been our experience with MNT for measurement of rabies antibody. To circumvent these problems, the in vitro RFFIT method was investigated as a possible substitute for MNT. A comparison of the sensitivity and reproducibility of two tests was carried out by repeated titrations of the U.S. Standard Antirabies Serum Lot 4. The geometric mean titer of this reference serum was found to be five-fold higher by RFFIT than by MNT. In 64 RFFIT titrations the log,, SN,, titers varied between 2.20 and 2.89 whereas a range of l-10-2.09 was seen in 61 MNT titrations. This narrower range of titers was also reflected in a correspondingly smaller standard deviation of the log,, SN,, values from RFFIT. Another indication of the greater reproducibility of RFFIT results was seen in the variations of the titer of virus used in the two tests. In the 64 titrations of the U.S. Standard Antirabies Serum by RFFIT the titer of the virus varied from 3.2 to 15.8 FFD,,, whereas the challenge virus in 61 MNT titrations varied from 10 to 447 LD,,. Thus, with this degree of reproducibility more stringent criteria could be established for acceptance of rabies antibody titration results. These were that (a) the titer of the U.S. Standard Antirabies Serum Lot 4 by RFFIT must be within one standard deviation of the geometric mean titer (220-372) and (b) the virus used in the test must be within the limits of 5-11 FFD,, Subsequent experience has shown that these limits are reasonable and are within the capability of the test.
372
MEASUREMENT
OF RABIES
ANTIBODY
A comparison of the accuracy of the two tests was carried out by monitoring the rabies antibody titer during the processing of a rabies hyperimmune plasma pool to IgG. By measuring the IgG concentration of the plasma pool and final IgG solution, the concentration factor of rabies antibody in the IgG solution could be determined. The titer of IgG rabies antibody in 2ME-treated plasma multiplied by the concentration factor would then give the expected rabies antibody titer in the IgG solution. When this calculation was performed on the rabies antibody titer of the 2ME-treated plasma, determined by RFFIT, and compared with the titers of the IgG solutions during various stages of processing, a value of 143 was obtained for the expected titer versus a geometric mean titer of 172 for the IgG after various processing stages. A similar comparison of the expected rabies antibody titer, based on comparing the antibody content of plasma, with that of the IgG at different stages of processing, yielded geometric means of 240 and 345 i.u./ml, respectively, by MNT. Thus, the comparative data from the two tests point to the greater sensitivity, reproducibility and accuracy of RFFIT over MNT. Considering the convenience of RFFIT along with these attributes, the conclusion is drawn that this test is more than suitable as a replacement for MNT. Addendum
While this paper was in preparation a publication by Cho & Fenje (1975) presented results of a comparison of RFFIT with MNT for titration of rabies antibody. REFERENCES Atanasiu, P. (1966). Quantitative assayand potency test of antirabies serum. In Laboratory Techniquesin Rubies, 2nd ed., pp. 167-172. Geneva: World Health Organization. Cabasso, V. J., Dobkin, M. B., Roby, R. E. & Hammar, A. H. (1974). Antibody response to a human diploid cell rabies vaccine. Applied Microbiology 27, 5.53-561. Cho, H. C. & Fenje, P. (1975). Rabies neutralizing antibody determination in tissue culture by direct fluorescent antibody technique. Journal of Biological Standardizat on 3, 101-105. Dulbecco, R. & Vogt, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. Journal of Experimental Medicine 99, 167-182. Johnson, H. N. (1969). Rabiesvirus. In Diagnostic Procedures for Viral and Rickettsial Infections, 4th ed., pp. 321-353. New York: American Public Health Association, Inc. Karber, G. (1931). Beitrag ziir kollektivin Behandhmg pharmakologischer Reihenversuche. Archiv fiir Experimentelle Pathologie und Pharmakologie 162, 480-483. Mancini, G., Carbonara, A. O., & Heremans, J. F. (1965). Immunochemical quantitation of antigens by a single radial immunodiffusion. Immunochemistry 2, 235-254. Rubin, R. H., Gough, P., Gerlach, E. H., Dierks, R. E., Gregg, M. B. & Sikes, R. ET. (1971). Immunoglobulin response to rabies vaccine in man. Lancet 2, 625-628. Smith, J. S., Yager, P. A. & Baer, G. M. (1973). A rapid reproducible test for determining rabies neutralizing antibody. Bulletin of the World Health Organization 48, 535-541. Spearman, C. (1908). The method of “Right and Wrong Cases” (constant stimuli) with Gauss’ formulae. British Journal of Psychology 2, 227-242. W.H.O. Expert Committee on Rabies (1960). World Health Organization Technical Report Series No. ZOI, 9-11. Geneva: W.H.O. W’.H.O. (1973). Special Diagnostic and Research Techniques. In Laboratory Techniques in Rubies, 3rd ed. World Health Organization Monograph Series No. 23, pp. 101-185. Geneva: W.H.O.
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