JournaloflmmunologicalMethod~', 106 (1988) 245-249 245

Elsevier

JIM 04625

An inhibition enzyme immunoassay for estimating relative

antibody affinity and affinity heterogeneity
S. Rath, C.M. Stanley and M.W. S t e w a r d
lmrnunologv Unit, Department of Medical Mwrohiologv, London School of Hygiene and Tropu'al Medicine, London W('I E 7ttT, U.K.
(Received 3 August 1987, accepted 21 September 1987)

A method to measure the relative affinity of antibodies using an inhibition enzyme immunoassay is
described. It is validated using monoclonal antibodies of defined affinity characteristics and by compari-
son with conventional methods of affinity measurement. The method allows measurement of the relative
affinity of low levels of antibody, and the calculation of an empirical estimate of the heterogeneity of
affinity in antibody populations.

K O. words'." Antibody affinity; Enzyme immunoassay, inhibition; Affinity heterogeneity

Introduction a modified Sips plot (Eisen and Siskind, 1964)

The affinity of an antibody response is a factor
of critical biological significance (Steward and
Steensgaard, 1983) and a variety of methods have
been devised to obtain an estimate of the 'average'
affinity of the antibodies in an antiserum (Ste-
ward, 1986). Most of these are aqueous-phase
radioimmunoassays, since the law of mass action
can be applied with the least difficulty in such
systems. One disadvantage of these methods is
that they require high concentrations of antibod-
ies, and therefore it is difficult or even impossible
to measure the affinities of antibodies in sera with
low antibody levels. Furthermore, estimates of the
heterogeneity of the affinities of the antibodies in
a polyclonal response are particularly difficult to
make. Such estimates may be made by employing
Correspondence to." M.W. Steward. Immunology Unit, De-
partment of Medical Microbiology. LSHTM, Keppel Street.
l.ondon WC1E 7HT, U.K.
0022-1759/88/$03.50 "," 1988 Elsevier Science Publishers B.V. (Biomedical Division)
which assumes a normal distribution of affinities
about the mean, but there is evidence that such an
assumption is unlikely to be generally valid
(Werblin and Siskind, 1972).
An estimate of the heterogeneity of affinity of
an antibody response may, however, be obtained
by the technique of inhibition of antibody
plaque-forming cells (PFC). The affinity of anti-
body from individual PFCs is estimated using the
principle of inhibition of plaque formation by free
antigen (Andersson, 1970). High-affinity plaques
are inhibited by lower concentrations of free anti-
gen than are low-affinity plaques. The system
allows the description of the antibody response of
spleen cells in terms of populations of PFCs in-
hibited by defined ranges of free hapten con-
centrations. A purely empirical measure of en-
tropy has also been used to calculate the hetero-
geneity of affinities in such PFC inhibition assays
(Goidl and Siskind, 1974).
In this paper we describe an adaptation of the
principle of this system to permit the measure-
246
ment of average affinity and estimation of the
heterogeneity of affinities of antibodies in solution
using an inhibition enzyme immunoassay. The
results obtained are compared with those de-
termined with a conventional globulin-precipita-
tion affinity assay (Steward, 1986).
Materials and methods
Antibodies
Four monoclonal antibodies to the hapten
2,4-dinitrophenol ( D N P ) described elsewhere
(Stanley et al., 1983), and a polyclonal mouse
antiserum to D N P were employed in the study.
Antigens
Conjugates of D N P with ovalbumin (DNP-OA)
and bovine serum albumin (DNP-BSA) at various
substitution ratios were prepared (Eisen et al.,
1953). DNP-lysine (Sigma, U.K.) was used as the
monovalent haptenic inhibitor.
Enzyme immunoassays (EIAs)
Wells of EIA polystyrene plates (Alpha, U.K.)
were coated overnight at 4 ° C with 50 # l / w e l l of a
solution of 10 ~tg/ml of the relevant DNP-protein
in carbonate-bicarbonate buffer 0.1 M, pH 9.6.
Plates were washed and blocked with 1% gelatin in
phosphate-buffered saline (PBS), pH 7.4, at 37 ° C
for 2 h, washed, dried and stored at 4 ° C.
Serial dilutions of antibody preparations were
made on DNP-protein-coated plates and in-
cubated at 3 7 ° C for 1 h. After washing, a goat
anti-mouse IgG-peroxidase conjugate (Jackson
Laboratories, U.S.A.) was added at a dilution of
1/2500 and the plates incubated as before.
After washing, H 2 0 _, and o-phenylenediamine
were added as substrates. The colour development
was stopped with 2 M HzSO 4 and absorbances
were read at 492 nm on a Titertek Multiskan
(Flow Laboratories, U.K.).
Serial dilutions of the relevant inhibitor were
made on DNP-protein-coated plates and a selected
dilution of each antibody preparation that yielded
an absorbance (A) of approximately half the max-
imum value in the titration EIA described above
(i.e., A = 0.7) was added. The EIA was performed
as already described. The percent inhibition at
each inhibitor concentration was calculated and
the molar inhibitor concentrations required for
50% inhibition (lo5) were determined. These val-
ues represent an estimation of 'average' antibody
affinity. The percentile inhibition contributed by
each serial inhibitor concentration was calculated
and the values used to analyse heterogeneity.
Shannon's coefficient (which represents an index
of heterogeneity) was calculated from these per-
centile inhibition intervals using the formula:
l = - k G y~ pj In p
where I -- Shannon's coefficient, k = 1 / I n 2, G =
number of inhibitor intervals used, pj = percentile
inhibition contributed by a given inhibitor con-
centration, expressed as a fraction (Brillouin,
1956).
Affini O' radioimmunoassavs
Aqueous-phase radioimmunoassays were per-
formed by equilibrium dialysis (using e-[3H]DNP-
lysine as antigen) and globulin precipitation (using
125I-DNP3.~-HSA as antigen) and the affinity val-
ues (K, with units of M - n) were calculated (Stan-
ley et al., 1983).
The concentrations of antibodies required for
the radioimmunoassay were determined and were
significantly higher than those required for the
inhibition EIA (Table I).
Results
Correlation between K and 1~ .~ t,alues ~f monoclonal
anti-DNP antibodies
Inhibition EIAs using DNPll-OA-coated plates
and the monovalent inhibitor DNP-lysine were
performed and 105 values calculated as described.
An inverse correlation was observed between lhe
K values of the monoclonal antibodies as mea-
sured by equilibrium dialysis (Stanley et al., 1983)
and the 105 values as determined by the inhibition
EIA (Table II).
 247

~,(", 55 55'59
TABLE 1
CONCENTRATIONS OF ANTIBODIES REQUIRED FOR
THE EFFECTIVE MEASUREMENT OF ANTIBODY AF-
FINITY BY RADIOIMMUNOASSAY AND THE INHIBI-
TION EIA "0

Antibody used Concentration (t~g/ml) required

33
for affinity measurement
RIA
I (X)
EIA
0.06
:it
274
'
~
I
39 100 0.14
53 100 0.10 k; - "

49 100 0.10 ;~ !~I. 7 5~

33 + 30 100 0.12
33 + 39 + 53 100 0.10
33+39+53+49 100 0.09
:]2,,
Mouse anti-DNP 1/2 dilution 1/240(X) dilution
(polyclonal) crn

" i.- -.2.

TABI.,E I1
MONOCLONAL ANTI-DNP ANTIBODY AFFINITY
VALUES ASSESSED BY EQUILIBRIUM DIALYSIS (K, qT, z " .'., ZO " " ", . r, r.. x ., [" .:, _',

_ (. tJ v ]',F-1%1;,~
M -~) AND EIA(105)

Monoclonal K by equilibrium 1o.5 (/xM DNP-lysine) Fig. 1. Histogram showing percentile inhibition of antigen-an-
used dialysis × 106 M - i by inhibition EIA tibody binding in the EIA over a range of inhibiting DNP-lysine
concentrations.
33 1.2 85.8
39 7.8 15.8
53 16.4 1.4 Affinity and heterogeneity measurements of defined
49 41.3 0.8 mixtures of monoclonal anti-DNP antibodies
Defined equimolar mixtures were made from
the a b o v e m o n o c l o n a l a n t i b o d i e s a n d the e n t i r e
p a n e l a n d the p o l y c l o n a l a n t i - D N P s e r u m w e r e
TABLE IIl a n a l y s e d b y the i n h i b i t i o n E I A a n d by the m e a -
s u r e m e n t o f a f f i n i t y by g l o b u l i n p r e c i p i t a t i o n ra-
AFFINITY VALUES (K AND 10~) AND HETEROGENE-
ITY INDICES FOR MONOCLONAL ANTI-DNPs AND d i o i m m u n o a s s a y . A n i n v e r s e c o r r e l a t i o n in the
THEIR MIXTURES r a n k o r d e r s o f K a n d 105 v a l u e s was o b s e r v e d
( T a b l e III).
An[ib~xJ)' used K(×106 M -~ ) Io5 " Shannon T h e p e r c e n t i l e i n h i b i t i o n c o n t r i b u t e d by e a c h
coefficient
i n h i b i t o r i n t e r v a l was p l o t t e d in the f o r m of histo-
33 0.33 85.4 25.2 g r a m s (Fig. 1), f r o m w h i c h it is a p p a r e n t t h a t as
39 0.34 15.8 26.0
the c o m p o s i t i o n o f the a n t i b o d y m i x t u r e i n c r e a s e s
53 1.09 1.4 25.7
49 1.89 0.8 24.0 in c o m p l e x i t y , the ' s p r e a d ' of the h i s t o g r a m in-
33 + 39 0.38 33.2 27.1 creases. T h e S h a n n o n c o e f f i c i e n t was c a l c u l a t e d in
33 + 39 + 53 0.41 30.0 28.4 an a t t e m p t to q u a n t i f y this ' s p r e a d ' ( T a b l e I l l ) ,
33+39+53+49 0.94 6.2 33.4 a n d the v a l u e s o f this c o e f f i c i e n t are seen to
Mouse anti-DNP
i n c r e a s e in a m a n n e r c o n s i s t e n t w i t h an i n c r e a s e
(polyclonal) 1.28 8.0 28.8
in the e x p e r i m e n t a l l y m a n i p u l a t e d c o m p l e x i t y of
" P < 0.02 (Kendall's rank correlation test). the a n t i b o d y p o p u l a t i o n u n d e r study.
248

I'ABI.I" IV Discussion
] t i E EFFEC'I" OF THE EPITOPE DENSITY OF SOLID-
PHASE A N T I G E N ON 1o~ A F F I N I T Y VALUES OB- Inhibition EIAs have occasionally been used to
T A I N E D IN THE EIA WITH DNP-LYSINE AS INIlIBI-
obtain estimates of antibody affinity (Nieto el al.,
TOR
1984). In this paper, we present a systematic vali-
Mono- Solid-phase antigen dation of the assay using defined monoclonal an-
chmal tibodies and compare it with established assays.
DNR- DNP~I- DNP>- DNP~-
used BSA BSA BSA BSA The data clearly' show that there is a significant
correlation between the present assay and estab-
33 1.2 60.1 27.7 550.0
39 1.1 3.6 2.2 4.6 lished methods, with the advantage that the con-
53 0.4 1.0 1.1 1.2 centrations of specific antibody' required are much
4t~ I).2 0.8 0.9 1.0 smaller than those needcd for conventional affin-
ity assays. The assay maintains a consistent rank
order of affinities over a wide range of solid-phase
The effect of epitope densitv of solid-phase antigen epitopc densities and of inhibiting antigen valen-
on inhibition EIAs cies, and therefore should bc applicable in a variety
Plates were coated with similar amounts of of situations where information concerning anti-
DNP-BSA at different substitution ratios ( I ) N R - body affinity would be of interest.
BSA, DNPll-BSA, DNPIg-BSA and DNp>-BSA). In addition, the mcthod can be used to obtain
Inhibition EIAs were performed with the four an empirical, comparative measure of affinity het-
monoclonal anti-DNP antibodies binding to each erogeneity in antibody populations, a value of
solid-phase antigen using DNP-lysine as the in- great interest in exploring B cell population dy-
hibitor. The results (Table IV) show that 1.5 namics in vivo. Such information cannot be ob-
values vary with changes in the cpitope density tained by conventional affinity assays without
and that the variation is greater for low-affinity either the use of laborious iterative computational
than for the high-affinity antibodies. However. the analyses (Werblin and Siskind. 1972) or the ap-
relative rank order remained consistent throughout plication of inappropriate assumptions concerning
this epitope-density-dependent variation. the distribution of affinities in a polyclonal serum
(Steward and Steensgard. 1983). However, a sin>
The effect of polyt,alent mhibitors on inhibition EIAs pier model using histograms representing fractions
l,,s values were obtained using DNP~-BSA. of occupied binding sites has recently been pro-
DNPtt-BSA, DNP>-BSA and DNP35-BSA as posed (Sciutto et al.. 1987). It must be noted here
polyvalent inhibitors. A greater change was ob- that. in keeping with the stochastic nature of the
served in the values for the low-affinity than for phenomenon of competition, even monochmal an-
thc high-affinity antibodies (Table V). but the tibodies are inhibited over a wide range of inhibi-
relative rank order again remains consistent. tor concentrations as shown in Fig. 1. The histo-
grams also show, however, that the interval of
maximum inhibition tends to shift to the right as
TABI.}" V the affinity' increases. This is true for the mixtures
THE EFFE("I OF POI.YVALI-NT INHIBITORS ON /,, 5 as well, but the histograms of antibody mixtures
AI-FINITY VALUES OBTAINEI) IN THE [:.IA show a broader "spread'. The Shannon coefficient
confirms this qualitative visual impression, and
Mono- Inhibitor antigen yields a value that can be subjectcd to statistical
clonal DN P- DN P,- DN P1 i- DN Pl,; I)N P~,~- analysis. However, increases in the complexity of
used lvsine BSA BSA BSA BSA the antibody mixture bring about only small in-
33 85.4 378.1 1.1 80.0 120.1 creases in the Shannon coefficient, and therefore it
39 15.8 283.2 1.0 26.7 46.7 may not be ','cry sensitive to small changes in
53 1.4 14.3 0.5 8.5 15.9 affinity heterogeneity. The polyclonal antiserum
49 0.8 2.0 0.1 1.1 6.4
has an intermediate average affinity, although the

fact that it has a l o w e r S h a n n o n c o e f f i c i e n t t h a n
the m i x t u r e of all f o u r m o n o c l o n a l s is, p e r h a p s ,
surprising. H o w e v e r , it is n e c e s s a r y to r e m e m b e r
t h a t the c o e f f i c i e n t d o e s not reflect the n u m b e r of
a n t i b o d y c l o n o t y p e s b u t o n l y the r a n g e o f their
affinities. T h u s in an in v i v o p o l y c l o n a l r e s p o n s e ,
a f f i n i t y d i s t r i b u t i o n c o u l d be m o r e r e s t r i c t e d t h a n
in a m i x t u r e o f f o u r m o n o c l o n a l s w i t h w i d e l y
d i f f e r i n g affinities.
In c o n c l u s i o n , the m e t h o d d e s c r i b e d h e r e c a n
be used to e s t i m a t e ' a v e r a g e ' a n t i b o d y a f f i n i t y as
well as the h e t e r o g e n e i t y o f a f f i n i t i e s at low a n t i -
b o d y levels. It m a y t h e r e f o r e be a useful a d d i t i o n
to the m e t h o d o l o g y for the e x p e r i m e n t a l assess-
m e n t of a n t i b o d y affinity.
Acknowledgement
S.R. t h a n k s the W e l l c o m e T r u s t for a V i s i t i n g
Research Fellowship.
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