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Hypergammaglobulinemiaand ErythrocyteAutoantibody ComplicateEnzyme Immunoassay ofAntimalarial AntibodyKenneth W. Hunter a , L. Patrick Smith a , G.Thomas Strickland a & Warren A. Blackburn aa Uniformed Services University of the HealthSciences, Departments of Pediatrics andMedicine , 4301 Jones Bridge Road, Bethesda,MD, 20014Published online: 05 Dec 2006.

To cite this article: Kenneth W. Hunter , L. Patrick Smith , G. Thomas Strickland& Warren A. Blackburn (1981) Hypergammaglobulinemia and ErythrocyteAutoantibody Complicate Enzyme Immunoassay of Antimalarial Antibody, Journalof Immunoassay, 2:2, 99-108, DOI: 10.1080/15321818108056970

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JOURNAL OF IMMUNOASSAY, 2(2), 99-108 (1981)

HYPERGAMMAGLOBULINEMIA AND ERYTHROCYTE AUTOANTIBODY COMPLICATE ENZYME IMMUNOASSAY OF

ANTIMALARIAL ANTIBODY

Kenneth W. Hunter, L. Patrick Smith, G. Thomas Strickland and Warren A. Blackburn

Uniformed Services University of the Health Sciences Departments of Pediatrics and Medicine

4301 Jones Bridge Road, Bethesda, MD 20014

ABSTRACT

Enzyme irnmunoassay of specific antimalarial antibody in sera from Plasmodium yoelii-infected mice was complicated by hypergammaglobulinemia and erythrocyte (RBC) autoantibody. Malarious serum had higher immuno- globulin levels which resulted in a substantial nonspecific adsorption of antibody to antigen uncoated microtiter wells even in the presence of a surfactant. Since it was possible that some of the antibody binding to solid phase-bound malarial antigen was also due to nonspecific adsorption, the antibody binding of nonimmune serum was compared to that of malarial immune serum diluted t o an equivalent immunoglobulin concentration. This allowed differentiation of specific antimalarial antibody binding from nonspecific adsorption of immunoglobulin. Although malarial antigen was partially purified from intraerythrocytic parasites, it bound a significant amount of rabbit anti-mouse RBC antibody, indicating the presence of re- sidual RBC antigens. Serum from immune mice, but not nonimmune mouse serum, also showed substantial binding to RBC stroma antigen, suggesting that a component of the observed antibody binding to malarial antigen was anti-RBC antibody. Hypergammaglobulinemia and the induction of RBC autoantibody may both be related to the polyclonal activating property of the malarial par- asite. The presence of RBC autoantibody in serum from malaria infected mice necessitates a more conservative interpretation of specific antimalarial antibody levels until a more purified malarial antigen is available.

INTRODUCTION

Since its introduction in 1971 (4,16), enzyme immunoassay (EIA) has

become a widely used tool for measuring antibody to specific antigens (19.3).

One of the earliest applications of ETA in infectious disease was for the

detection of antibodies to human malarial parasites (17.18). We have re-

99

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100 HUNTER ET AL.

c e n t l y explored t h e use of E I A t o s tudy t h e s p e c i f i c an t ibody response t o

Plasmodium y o e l i i i n mice. During t h e course of t h e s e i n v e s t i g a t i o n s w e

encountered two major t e c h n i c a l problems r e l a t e d t o t h e b i o l o g i c a l charac-

t e r i s t i c s of t h e m a l a r i a l p a r a s i t e . I n t h i s paper w e d e s c r i b e t h e E T A

procedure i n d e t a i l and d i s c u s s t h e problems encountered.

MATERIALS AND METHODS

Mice and P a r a s i t e s . Six t o 8 week-old female C57BL/6 mice were obta ined

from Jackson L a b o r a t o r i e s , Bar Harbor, ME. Mice were housed i n groups of 6

o r fewer and g iven food and water ad l ib i tum.

y o e l i i 1 7 X was s t o r e d a t -7O'C a s f r o z e n p a r a s i t i z e d mouse blood i n A l s e v e r ' s

g l y c e r i n . This m a t e r i a l was thawed and i n j e c t e d i n t r a p e r i t o n e a l l y i n t o donor

mice from which i n f e c t i n g inocula were prepared when p a r a s i t e m i a l e v e l s a t -

t a i n e d 10%.

A n o n l e t h a l s t r a i n of &

Antibodies . A f f i n i t y p u r i f i e d r a b b i t an t i -mouse IgG (RaMy) and IgM (RaMp)

a n t i b o d i e s were prepared as p r e v i o u s l y d e s c r i b e d (10). Alkal ine phosphatase-

conjugated goa t a n t i - r a b b i t IgG was obta ined from Miles L a b o r a t o r i e s , E l k h a r t ,

I D . Rabbit anti-mouse red blood c e l l (RaMRBC) stroma (see below) a n t i b o d i e s

were produced by repea ted immunizations wi th C57BL/6 RBC s t roma i n complete

and incomplete Freund's ad juvant . Serum immunoglobulin ( I g ) l e v e l s were

measured by t h e method of Mancini (13 ) .

Antigens. A p a r t i a l l y p u r i f i e d m a l a r i a l a n t i g e n (MAP) was prepared from

i n t r a e r y t h r o c y t i c P. y o e l i i accord ing t o Hunter e t a l . (10). To prepare

mouse RBC s t roma a n t i g e n , normal mouse blood was f i r s t passed over a column

of c e l l u l o s e powder t o remove leukocytes . After two washes i n phosphate

buf fered s a l i n e (PBS), t h e packed c e l l s were resuspended i n 20 volumes of

d i s t i l l e d water. The RBC s t roma was then washed 5 times i n PBS by c e n t r i -

f u g a t i o n i n a n Eppendorf microfuge (Brinkman Ins t ruments Inc.. Westbury.

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EIA OF ANTIMALARIAL ANTIBODY 101

NY). The protein concentration of MAP and mouse RBC stroma antigens were

measured by the method of Bradford (2).

Test Sera.

erythrocytes, followed by 3 similar inoculations at monthly intervals. After

the primary infection (peak parasitemia 25%), only transient low levels of

parasitemia were found indicating the development of solid immunity. A pool

of immune serum was obtained 3 weeks following the final infection. A pool

of nonimmune serum was obtained from age-matched C57BL/6 mice left unchal-

lenged.

C57BL/6 mice were infected with 1.0 x lo5 P. yoelii parasitized

Enzyme Immunoassay Procedure. Polystyrene microtiter plates (Dynatech Labora-

tories, Alexandria, VA) were coated with 50 p1 of MAP o r mouse RBC stroma

(10 pg/ml protein concentration) in 0.1 M NaHC03 buffer, pH 9.6, for 24 hrs

at 4OC. After removing the coating antigen, the wells were washed with the

coating buffer, emptied, and filled to the top with blocking buffer consisting

of 5% bovine serum albumin (BSA) in PBS. Preliminary experiments revealed the

need for blocking antigen uncoated sites on the polystyrene surface. After 1

hr incubation at 4OC the plates were washed with PBS containing 0.2% Tween 20

(washing buffer). Plates were used immediately, but other experiments (data

not shown) indicated that they could be stored in PBS-Tween containing 0.02%

sodium azide for up to 1 week without significant loss of activity. Test

serum diluted i n PBS-Tween was then added (50 pl) and incubated for 30 min

at 4'C.

well with washing buffer. The next step was to add 50 p 1 of either RaMy or

RaMp (10 ug/ml antibody protein in PBS-Tween) followed by a 30 min incuba-

tion at 4OC.

phosphatase-conjugated goat anti-rabbit IgG (used at 1:500 dilution of stock).

After 30 min at 4OC, the plates were washed as above and 100 u1 of enzyme

substrate (p-nitrophenyl phosphate; Sigma Chemical Co., St. Louis, MO) was

added. The reaction was allowed t o proceed for 30 min at room temperature,

The plates were again washed by 5 cycles of filling and emptying each

After another wash cycle each well received 50 p1 of alkaline

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102 HUNTER ET AL.

then each p l a t e was scanned a t 405 nm us ing a Mul t i scan micro-ELISA r e a d e r

(Flow L a b o r a t o r i e s , Rockvi l le , MD) . When p l a t e s could not be read immediately,

t h e enzymatic r e a c t i o n was s topped by adding 25 ~1 of 3N NaOH.

RESULTS AND DISCUSSION

T i t e r s of IgG ant i -P. y o e l i i an t ibody were c a l c u l a t e d from the d i l u t i o n

curves of t h e t e s t s e r a . I n t h i s procedure , a t least 4 d i l u t i o n s of test

serum were e v a l u a t e d and t h e absorbance p l o t t e d a g a i n s t t h e l o g r e c i p r o c a l

serum d i l u t i o n (Fig. 1 ) . T i t r a t i o n curves genera ted i n t h i s manner a l lowed

comparisons t o be made on t h e l i n e a r p o r t i o n of t h e curves where d i f f e r e n c e s

r e l a t e i n a l i n e a r manner. This a l s o avoided s i t u a t i o n s where an t ibody excess

would mask d i f f e r e n c e s between sera. An absorbance v a l u e f o r t i t r a t i o n was

chosen a r b i t r a r i l y , bu t always represented a p o i n t n e a r t h e middle of t h e

l i n e a r p o r t i o n of t h e m a j o r i t y of curves ( i n t h i s c a s e , 0.7 absorbance u n i t s ) .

The r e l a t i v e an t ibody t i t e r was def ined a s t h e r e c i p r o c a l ser1.n d i l u t i o n which

produced a n absorbance of 0.7 u n i t s . I n t h e c a s e of t h i s p a r t i c u l a r immune

serum, t h e r e l a t i v e t i t e r was 54. Nonimmune serum showed a curve which was

s u b s t a n t i a l l y below t h a t of immune serum, but t h e e x a c t t i t e r could only be

c a l l e d less than 1:lO s i n c e t h e maximum absorbance v a l u e was less than t h e

s e l e c t e d t i t r a t i o n v a l u e of 0.7. However, i f t h e nonimmune serum curve was

l i n e a r a t l:lO, i t was p o s s i b l e t o e x t r a p o l a t e back t o t h e p o i n t where t h e

l i n e i n t e r s e c t s t h e t i t r a t i o n curve f o r immune serum.

I n pre l iminary experiments i t was observed t h a t ant igen-uncoated w e l l s

bound Ig from d i l u t i o n s of t es t serum ( d a t a n o t shown), even i n t h e presence

of a s u r f a c t a n t l i k e PBS-Tween. This n o n s p e c i f i c a d s o r p t i o n could be reduced

s u b s t a n t i a l l y by b locking t h e wells w i t h BSA-PBS. Even so, immune serum

d i l u t i o n s incubated i n BSA-coated wells gave a lower but c o n s i s t e n t back-

ground absorbance which a c t u a l l y t i t r a t e d (F ig . 1). Since m a l a r i a l i n -

f e c t i o n s induce polyc lona l a c t i v a t i o n and t h e product ion of a n t i b o d i e s of

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EIA OF ANTIMALARIAL ANTIBODY 103

RECIPROCAL SERUM MLUTlON

Fig. 1 . (0-0) and nonimmune (0-0) sera. Each point represents the mean of triplicate determinations. The dotted line indicates the chosen titration point (0.7 units) near the mid point of the linear portion of the titration curve. The arrow indicates the relative IgG titer of 5 4 . The solid hor- izontal line represents background absorbance in MAP-coated wells not incubated with serum. Note also the apparent titration of immune serum incubated in BSA-coated wells ( A - A ) . BSA-coated wells incubated with nonimmune serum (A) o r without serum (0) were negative.

EIA titration curves for IgG antimalarial antibody in immune

many specificities ( 5 , 7 ) . it Is possible that a component of the low level

binding of Ig to BSA-coated wells was due to specific anti-BSA antibodies.

The mitogenic effect of malarial infections also leads t o hypergamma-

globulinemia ( 1 , 1 4 ) . If the higher Ig content of immune serum also resulted

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104 HUNTER ET AL.

in more nonspecific "stricking" to MAP-coated wells, a component of the

difference in antimalarial antibody titers between immune and nonimmune

serum would be artifactual. To approach this question, the total IgG con-

tent of immune and nonimmune sera was determined by single radial immuno-

diffusion.

nonimmune serum (1670 mg/dl VS. 580 mg/dl).

in total IgG, a comparison was made (on the titration curve) between the

absorbance of 1:lO nonimmune serum and immune serum diluted to an equiva-

lent IgG concentration (e.g., 1:lO nonimmune serum x fold increase in IgG

(2.9) = 1:29). At a dilution of 1:29, the absorbance of immune serum was

still higher than 1:lO nonimmune serum (Fig. l ) , indicating that at least

this difference was due to specific binding of antimalarial antibody. This

method was also successfully applied to IgM and subclasses of IgG. It is

important therefore to routinely ascertain whether the higher titration

curve observed in an immune serum is due to higher levels of specific anti-

malarial antibody or to greater sticking of Ig in a hypergammaglobulinemic

serum.

Immune serum had approximately 2.9 times the total IgG as

Based on the fold increase

We have endeavored to remove contaminating RBC stroma from MAP by anion

exchange chromatography (10). To demonstrate the immunological purity of

MAP, we compared the binding of RaMRBC antibody to MAP and RBC stroma antigen.

The results of this experiment indicated that although more antibody bound

to RBC stroma, MAP-coated wells bound a substantial amount of RaMRBC anti-

body (Table 1). It thus appeared that o u r efforts to remove RBC stroma from

MAP had not been totally effective. This finding also raised the question

of whether some of the observed antibody binding to MAP in malarial immune

serum was due to anti-RBC antibody rather than antimalarial antibody. To

test the specificity of the anti-MAP response, microtiter plates were coated

with MAP or RBC stroma antigen and tested with malarial immune serum. It

was discovered that IgG antibodies (IgM antibodies also bound, data not shown)

from immune serum bound to MAP and RBC stroma (Table 2). Although it i s

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EIA OF ANTIMALARIAL ANTIBODY 105

TABLE 1

Comparison of the Binding of RaMRBC Antibody to MAP and RBC Stroma Antigen

Relative IgG Titer (Reciprocal) Serum MAP RBC Stroma BSA

RaMRBC 50 185 <10 Normal Rabbit <10 <10 < 10

RaMRBC and normal rabbit sera were titered at 0.9 absorbance units.

TABLE 2

Comparison of the Binding of Antibody from malarious Serum to MAP o r RBC Stroma Antigen

Relative IgG Titer (Reciprocal) Serum MAP RBC Stroma BSA

Immune Nonimmune

65 27 <10 < 10 <10 <10

Immune and nonimmune sera were titered at 0.7 absorbance units.

difficult to assess quantitative relationships, it appeared that about half

as many antibodies bound to the stroma. These findings suggest that malarial

immune serum contains antibodies with specificities for both malarial anti-

gens and RBC stroma antigens, whereas nonimmune serum contains very little

antibody to either of these antigens. As mentioned above, malaria is a

polyclonal activator capable of inducing the proliferation of B cells bearing

receptors for a multitude of antigenic specificities (6.9). Malarial in-

fections have been demonstrated to induce the production of antibodies to

determinants on RBC which are exposed by bromelain treatment (15), similar

to the way bacterial lipopolysaccharide, a very potent B cell mitogen,

stimulates the production of anti-RBC antibodies (8). In addition, our

previous studies (11) and the studies of Lustig et al. (12 ) have documented

the binding of Ig to noninfected RBC during the course of murine malarial

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106 WNTER ET AL.

infections. It seems probable that the antibody in immune serum which binds

to RBC is indeed RBC autoantibody. Therefore, a component of the observed

antibody binding to MAP may represent RBC autoantibodies reacting with

RBC stroma contaminants.

The correlation between depressed antibody binding to MAP and increased

P. yoelli parasitemia levels and mortality in CBA/N mice with an X-linked

B lymphocyte defect (10) argues that this assay reflects protective immunity.

Moreover, we have recently shown that the IgM and IgG anti-MAP response is

higher and more prolonged in genetically resistant AKR/J mice than in sensi-

tive C57BL/6 mice (Hunter et al., unpublished observations). Antibodies

bound to the surface of parasitized RBC, whether specific for malarial para-

site determinants or RBC determinants, would still sensitize the cell f o r

destruction and thus contribute to the protective host immune response.

The nonspecific binding of Ig and RBC autoantibody would also compromise

specific antimalarial antibody determination using radioimmunoassay if the

antigens employed are derived from intraerythrocytic parasites. It is also

possible that other widely used serologic tests such as hemagglutination and

immunofluorescence would be influenced by RBC autoantibody.

In conclusion, two problems have been defined which affect EIA for

antimalarial antibody: hypergammaglobulinemia and RBC autoantibody. The

first is easily solved by testing malarious sera at dilutions which contain

Ig concentrations equivalent to control sera.

dealt with and necessitates a more restricted interpretation of antimalarial

antibody levels until a more purified antigen preparation is obtained.

The second is less easily

ACKNOWLEDGEMENTS

We thank Dominique M. Nau for expert editorial assistance. This work

was supported in part by USUHS protocol R08603. Reprint requests should be

made to Kenneth W. Hunter, Jr., Department of Pediatrics, Uniformed Services

University, 4301 Jones Bridge Road, Bethesda, MD. 20014.

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EIA OF ANTIMALARIAL ANTIBODY 107

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