Archives of Virology 49, 199--205 (1975) © by Springer-Verlag 1975

Estimation of Hema991utination-lnhibition Titers By S. tIo~vJ~'r*¢ Department of Virolog7y~, Publie Health Institute of Budapest, Budapest, Hungary With 3 Figatres Aceepted July 10, 1975

Summary In the hemagglutination-inhibition test, simple relationships exist between units of hemagglutinating virus and hemagglutination-inhibition antibody tigers. Equations have worked out which allow estimating antibody titers for different virus doses, provided all other parameters are kept constant. They are applicable when the isofixation lines in log-log plots as obtained in box titrations either are positioned at angles of 45 ° or deviate from this value. Using lower or higher doses of hemagglutinin in the test, the coefficient of variation of the estimaged antibody tigers was found to be approximately 4 per cent. This procedure is useful in a number of virus-cell-serum ~ysgems and allows comparisons of antibody tigers obtained in different laboratories.

Introduetion Since 1941, m a n y viruses have been shown to cause hemagglutination (HA) (2, 3, 6), and during this time the hemagglutination-inhibition (HAI) test has been widely applied for the identification of viruses and the serological diagnosis of viral infections. If one wished go compare results obtained in different laboratories, one would have to standardize all variables affecting the t I A I test. In practice this is particularly difficult with regard to the antigen dose. If the employed quantity of H A is inadequate as revealed b y the control t i t r a t i o n - - w h i c h quite often is the c a s e - - t h e test must be repeated. In this communication it is shown t h a t a simple numerical relationship exists between antigen and antibody which allows estimating H A I tigers for a n y quantity of H A employed in the test.

Materials and Methods Strains o] Virus Used

The viruses used were the following: influenza virus (PR8, FM1, England 42/72 and B Hung. 138/73 strains), mumps virus (Enders strain), measles virus (Edmo~ston

200

S. H o ~ v £ T ~ :

s t r a i n ) , a n d r u b e l l a v i r u s ( J u d i t h strain). T h e r u b e l l a a n d t h e E n g l a n d 42/72 v i r u s s t r a i n s were k i n d l y s u p p l i e d b y Dr. M a r g u e r i t e S. P e r e i r a , C e n t r M P u b l i c H e a l t h L a b o r a t o r y , ColindMe. T h e i n f l u e n z a v i r u s e s were h a r v e s t s of v i r u s - i n f e e t e d fertile c h i c k e n eggs. T h e m e a s l e s v i r u s w a s c u l t i v a t e d in H e L a cell c u l t u r e s i n c u b a t e d a t 33 ° C. H A w a s p r e p a r e d f r o m t h e cells b y r a p i d l y freezing a n d t h a w i n g t h e c u l t u r e s t h r e e t i m e s w h i c h r e s u l t e d in H A t i t e r s of a b o u t 128. V i r u s w a s c o n c e n t r a t e d b y u l t r a e e n t r i f u g a t i o n for 60 m i n u t e s a t 25,000 × g. T h e r u b e l l a v i r u s was c u l t i v a t e d i n roller b o t t l e c u l t u r e s of B H K 2 1 cells a t 35 ° C, u s i n g k a o l i n - t r e a t e d s e r u m i n t h e m e d i u m (9). T h e B H K 21 cell line was k i n d l y s u p p l i e d b y Dr. J e n n i f e r M. Best, D e p a r t m e n t of Virology, St. T h o m a s ' H o s p i t M a n d MedieM School, L o n d o n . T h e H A was h a r v e s t e d d a i l y f r o m c h r o n i c a l l y i n f e c t e d cultures. T h e t i t e r of e x t r a e e l l u l a r H A was h i g h e s t w i t h a b o u t 256 o n t h e t h i r d d a y . T h e v i r u s s u s p e n s i o n was e o n e e n t r a t e d b y u l t r a e e n t r i f u g a t i o n for 120 m i n u t e s a t 5 0 , 0 0 0 × g a n d w a s e x t r a c t e d w i t h T w e e n - 8 0 a n d e t h e r (7). R u b e l l a v i r u s H A p r e p a r e d b y F l o w L a b o r a t o r i e s was also used. All m a t e r i a l s were clarified b y e e n t r i f u g a t i o n a t 1000 × g for 10 m i n u t e s a t 4 ° C a n d s t o r e d a t - - 4 0 ° C.

Antisera Prepared/or H A 1 Tests A f t e r p r e l i m i n a r y bleeding, c h i c k e n s r e c e i v e d 1 m l of c o n c e n t r a t e d i n f l u e n z a v i r u s i n t r a v e n o u s l y . One w e e k l a t e r a 2 n d a n d t w o weeks l a t e r a 3rd i n o c u l a t i o n were g i v e n i n t h e s a m e m a n n e r . T h e c h i c k e n s were e x s a n g u i n a t e d 10 d a y s later. Sera were h e a t e d a t 5 6 ° C for 30 m i n u t e s . T h e y were f u r t h e r t r e a t e d w i t h p o t a s s i u m p e r i o d a t e for r e m o v a l of non-specific s e r u m i n h i b i t o r s . A n t i - m u m p s v i r u s g u i n e a - p i g s e r u m p r e p a r e d b y F l o w L a b o r a t o r i e s was h e a t e d a t 56 ° C for 30 m i n u t e s , a n d t h e non-specific i n h i b i t o r s were i n a c t i v a t e d b y t r e a t m e n t with periodate. F o r m e a s l e s v i r u s a n t i b o d y h u m a n c o n v a l e s c e n t sera were collected. T h e H A in t h e i n a c t i v a t e d sera w a s a b s o r b e d o v e r n i g h t w i t h a few d r o p s of p a c k e d m o n k e y e r y t h r o c y t e s a t 4 ° C. F o r p r e p a r i n g i m m u n e sera a g a i n s t r u b e l l a virus, t h i s a g e n t was c u l t i v a t e d in l ~ K 1 3 cells u s i n g r a b b i t s e r u m i n t h e n u t r i e n t fluid. T h e h a r v e s t e d v i r u s was clarified b y e e n t r i f u g a t i o n a n d was i n o c u l a t e d i n t r a v e n o u s l y i n t o a r a b b i t 5 t i m e s w i t h i n t e r v a l s of s e v e n days. A f t e r 6 weeks t h e a n i m a l was e x s a n g u i n a t e d . H u m a n c o n v a l e s c e n t sera were also used. Sera were t r e a t e d w i t h H e p a r i n - M n C l ~ for r e m o v a l of non-specific i n h i b i t o r s (1). Diluents P h o s p h a t e - b u f f e r e d saline (PBS), p H 7.2 was e m p l o y e d i n t h e e x p e r i m e n t s e m p l o y i n g i n f l u e n z a a n d m u m p s viruses. I t was also u s e d i n t h e w o r k ~ d t h measles v i r u s . I{.hesus m o n k e y e r y t h r o e y ~ e s were s u s p e n d e d in a 3.3 p e r c e n t s o l u t i o n of MgSO4 • 7 H 2 0 (4). ttEPES-(N-2-hydroxyethylpiperazine-N'-2' e t h a n e s u l f o n i e acid) b u f f e r e d saline c o n t a i n i n g 0.5 p e r c e n t b o v i n e s e r u m a l b u m i n a n d 0.00025 p e r c e n t gelatin, p H 6.t, was e m p l o y e d as d i l u e n t for H A , s e r u m a n d r e d b l o o d cell s u s p e n s i o n i n t h e r u b e l l a v i r u s e x p e r i m e n t s (5). H A Test T i t r a t i o n s of H A were c a r r i e d o u t w i t h t h e m i e r o t i t e r s y s t e m (8). M i c r o t i t r a t i o n p l a t e s w i t h V - s h a p e d wells were e m p l o y e d . D r o p s of 0.025 m l of d i l u e n t were d e l i v e r e d t o e a c h well a n d serial t w o f o l d d i l u t i o n s of H A w e r e m a d e w i t h 0.025 m l d i l u t i n g loops. D i l u e n t (0.025 ml) a n d c h i c k e n r e d b l o o d cells (0.025 ml) were t h e n a d d e d t o e a c h well. F o r i n f l u e n z a a n d m e a s l e s v i r u s e s 1 p e r c e n t r e d b l o o d cell s u s p e n s i o n s were used, whJle i n t h e r u b e l l a v i r u s H A t i t r a t i o n s a 0.5 p e r c e n t e r y t h r o e y t e s u s p e n s i o n f r o m u n f e d 1-day-old chicks w a s e m p l o y e d . T h e p l a t e s were t h e n a g i t a t e d a n d s t o r e d a t r o o m t e m p e r a t u r e for 30 m i n u t e s i n i n f l u e n z a v i r u s t i t r a t i o n s . T h e y were i n c u b a t e d a t 37 ° C for 1 h o u r i n t h e measles v i r u s e x p e r i m e n t s , a n d a t 4 ° C for 1 h o u r in r u b e l l a v i r u s titrations.

Estimation of ttemagglutinatiowInhibition Titers

201

The last well showing 50 per cent ]{A was considered the titration end point, and that dilution in the reaction volume was taken to represent 1 unit HA.

H A I Test Diluent (0.025 ml) was dropped from a calibrated dropping tip to each well of the plate. Sera were diluted in serial twofold dilution with 0.025 ml diluting loops. Eight 50 per cent H A units in 0.025 ml were added. The plates were agitated and were incubated for 30 or 60 minutes at room temperature. Each well then received 0.025 mI of red blood eelI suspension. The plates were incubated as mentioned in the H A test. Appropriate erythrocyte and serum controls as well as control titrations of H A were included in each test. The H A I antibody titer was defined as the reeiproeM of serum dilution giving 50 per cent H A L

Results T h e t t A I a n t i b o d y t i t e r can be d e t e r m i n e d for different doses of H A in the test once t h e kinetics of the H A I reaction are known. T h e r e are two possibilities: the slope of t h e isofixation line of the H A I a n t i b o d y titers in the log-log plot runs either a t a n angle of 45 ° or d e v i a t e s from this value.

Estimation o/the H A l Antibody Titer, when the Slope o/the Iso/ixation Line is 45 ° Angle in the log-log Plot I m m u n e sera were t i t r a t e d w i t h a n u m b e r of different c o n c e n t r a t i o n s of the homologous influenza viruses ( P g S , F M 1 , E n g l a n d 42/72, B H u n g . 138/73). The results of t h e 50 per cent H A I were p l o t t e d in a log-log plot. The isofixation curves were s t r a i g h t lines a t angles of 45 °. Therefore t h e values of t a n g e n t s were t. T h e values were also 1 in t h e H A I tests carried o u t with m u m p s a n d measles viruses. The isofixation line for influenza F M 1 virus a n d the symbols used for calculation are d e m o n s t r a t e d in F i g u r e 1.

,o9 F

Virus control

t 7

t O

z @

, - 2 x, log2 d

/e

4 I

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2 1 0

0

I

2

3

4

5

6

7

8

9

10

n

-Log 2 serum dilution

Fig. 1. The isofixation line of the H A I test carried out with influenza FM 1 virus and homologous immune serum, and the symbols used for calculation

202

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The estimation of the H A I antibody tigers for a certain dose of 50 per cent HA units of virus is given by the formula

dh where

x~ -

i

(1)

xt is the quantity of the H A I antibody in the reaction volume of the undiluted immune serum under investigation which inhibited i 50 per cent H A units of the test virus. This is the H A I antibody titer of the immune serum. If the H A I antibody titer is to be estimated for example for 8 or 16 HA units of 50 per cent, the value of i is equal to 8 or 16, respectively. d is the reciprocal value of the serum dilution, at which 50 per cent H A I is observed using a certain quantity of 50 per cent I-IA units of virus. h is the dose of 50 per cent H A units employed in the test. I t is the virus tiger calculated from the control titration. i represents the dose of 50 per cent H A units for which the H A I antibody titer is estimated. For practical purposes the I~AI antibody titers are expressed as log2 units. Taking logarithms of Eq. (1) we obtain dh log2 x~ = log2 --=(2) and * log2 xi = log2 d + log2 h - - log2 i (3) Using the data of Figure 1 let us estimate the quantity of I-IAI antibody in the immune serum which inhibits 8 (log2 8 = 3) 50 per cent H A units of influenza FM I virus. The serum was diluted 4-fold for removal of non-specific inhibitors. Twofold dilutions were prepared and the H A I antibody was about 32. Thus the actual serum dilution was 1/128; log2 128 = 7. The 50 per cent HA tiger of the test virus in the control titration was equal to 16 (log2 16 ~-4). Therefore, according to Eq. (3) log2 xs = 7 + 4 - - 3 = 8. Taking the antilogarithm of 8, the HA1 antibody tiger was 256.

Estimation o/ the H A 1 Antibody Titer, when the Slope o/ the Iso/ixation Line is not 45 ° Angle in the log-log Plot The rabbit anti-rubella virus serum was titrated with different concentrations of rubella virus. The results were plotted in a log-log fashion (Fig. 2). The tangent (b) was higher than 1. I n this situation antibody tiger for a certain dose of HA can be eMeulated with the help of the formula xi =

d

(4)

I n practice I I A I antibody titers are expressed as log2 units. Taking logarithms, Eq. (4) becomes log2 x, = log2 d

(5)

and log2 xl = log2 d @

log2 h - - log2 i b

(6)

E s t i m a t i o n of H e m a g g l u t i n a t i o n - I n h i b i t i o n Titers

203

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/

/

6

~3 5

/

o

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2

./

/ 0 3

/

./

I

I

t

1

t

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6

7

8

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J

9

serum dilution

Fig. 2. The isofixation line of t h e H A I t e s t carried out w i t h t h e rubella v i r u s a n d homologous immune serum

The H A I experiments were performed with different rabbit and human convalescent rubella virus antisera. Antigens were the supernatant fluid of rubella virus-infected B H K 2 1 cells, concentrated virus suspension, and the rubella virus antigen prepared by Flow Laboratories. The values of tangents were calculated b y the method of least squares. The mean from 10 experiments was about 1.8 with a single standard deviation 0.27. I n practice the value of 1.8 can be used in rubella virus H A I tests. Eqs. (3) and (6) can also be employed when 100 per cent responses are used. I n this ease the I t A I titer is 2 or 3 log2 units lower than as compared with values --

h

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+

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8

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+

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50 per cent HA units 4 100 per cent HA units

+

, 9

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10

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Fig. 3. Difference between the I-IAI antibody titers using the same units of both i00 a n d 50 p e r c e n t H A doses of v i r u s for c a l c u l a t i o n

204

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obtained as 50 per cent responses. The difference of the tigers depends on the width of the transition zone of the H A reaction between 100 and 0 per cent responses (Fig. 3).

Reproducibility o] H A I Tigers Influenza PI~ 8 and rubella viruses, and their specific immune sera were used in the H A I experiments. After removing the non-specific inhibitor, the immune sera were titrated 60 times on different occasions against the homologous viruses with doses varying between 4 to 128 50 per cent HA units. The H A I titers were estimated for eight 50 per cent I-IA units and these were analysed statistically. For influenza PlY8 virus the mean of the antibody tigers was 2 :°-55 with single standard deviation 0.41 log2 units. The coefficient of variation was equal to 4 per cent. For rubella virus the mean of the H A I antibody titers was 29.22 with single standard deviation 0.33 log2 units. The coefficient of "variation was equal to 3.6 per cent.

Discussion The experiments were aimed at finding a simple relationship between virus dose and antibody titer. One of the variables which influences the outcome of a H A I test is the dose of HA. Using the "box" titration method (dilutions of antigen are tested against dilutions of serum) the slopes of the isofixation lines of the H A I reactions were found to be positioned either at 45 ° angles or deviated from it in the log-log plot. Since they were linear and constant in a given system t t A I antibody tigers can conveniently be estimated for different amounts of virus employed in the test. Two equations were worked out for estimating the H A I antibody titers for different doses of test virus, when all other parameters are kept constant. Using these equations the H A I antibody tigers can be determined with high accuracy. The low coefficients of variations demonstrate that the estimations of HAl antibody tigers with Eqs. (3) and (6) are suitable for practical purposes. This simple

estimation procedure is applicable in various virus-cell-serum systems, and facilitate comparisons of H A I antibody titers obtained in different laboratories.

Acknowledflments Tile author expresses his gratitude to Dr. Ernese Gy6rgy for preparing the specific immune serum and the hemagglutinin of measles virus, and to Dr. E. Sehulek for the mumps virus, and wishes to thank Miss Irene Kfi,llay and Mr. L. KSlt5 for their excellent technical assistance.

References I. FI~LI)~A:¢, I-I. A. : Removal by heparin-MnCl2 of nonspeoifie rubella hemagglutinin serum inhibitor. Prec. See. exp. Biol. Med. 127, 570--573 (1968). 2. I-IIRsT, G. K. : The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. Science 94, 22--23 (1941). 3. HIRST, G. K. : The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J. exp. Med. 75, 49--64 (1942).

Estimation of ttemagglutination-Inhibition Titers

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4. Hol~viT~, S., SzSLLSSY, E., IvXsrOVlCS, G. : Distribution of the receptor substance of the influenza and related viruses in the tissue-elements of different animal species. I. The virus adsorbing capacity and agglutinability of the erythrocytes of various vertebrates. Aeta Phys. Hung. 2, 77--86 (1952). 5. LI~BHAB~.R, H. : Measurement of rubella antibody by hemagglutination inhibition. I. Variables affecting rubella hemagglutination. J. ImmunoI. 104, 818--825 (t970). 6. McCLEnnAND, L., HARE, R.. : Adsorption of influenza virus by red cells and new i n vitro method of measuring antibodies for influenza virus. Canad. Publ. Health J . 32, 530--538 (1941). 7. NOR~BY, E. : Itemagglutination by measles virus. 4. A simple procedure for production of high potency antigen for hemagglutination-inhibition (HI) tests. Proc. Soc. exp. Biol. IVied. 11, 814---818 (1962). 8. SEVEg, J. L. : Application of a microtechnique to viral serological investigations. J. Immunol. 88, 320--329 (1962). 9. STEWAI~T,G. L., PARKMAN, P. D., Hot'I?s, H. E., DOUGLAS,1%. D., HAZCLILTON,J. P., MEYER, H. M., JR.. : Rubella-virus hemagglutination-inhibition test. New Engl. J. Med. 276, 554--557 (1967). Author's address : Dr. S. HOl~VX~i~,Department of Virology, Public Health Institute of Budapest., Vaci ut 172, 1138 Budapest, Hungary. l~eceived May 26, 1975

Arch. Virol. 49/2--3

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Estimation of hemagglutination-inhibition titers.

In the hemagglutination-inhibition test, simple relationships exist between units of hemagglutinating virus and hemagglutination-inhibition antibody t...
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