Clin. exp. Immunol. (1983) 54, 135-142.

Characterization of the humoral immune response in

Plasmodiumfalciparum malaria. II. IgG subclass levels of
anti-P. fakiparum antibodies in different sera
M. WAHLGREN,*t K. BERZINS,* P. PERLMANN* & M. PERSSONJ *Department of
Immunology, University of Stockholm; tDepartment of Infectious Diseases, Karolinska Institutet,
Roslagstull Hospital, Stockholm and TDepartment of Clinical Immunology, Karolinska Institutet,
Huddinge Hospital, Huddinge, Sweden
(Acceptedfor publication 9 May 1983)

SUMMARY
The IgG subclass levels of anti-Plasmodium falciparum antibodies in human sera were
determined in ELISA with monoclonal mouse antibodies specific for the human IgG
subclasses as analytical reagents. The parasite antigen was a trophozoite/schizont
enriched preparation of in vitro cultivated P. falciparum. Serum samples were from
Swedish malaria patients and adult Liberians. Parasite specific antibodies were found in
all four subclasses. Relatively elevated levels of IgG1 antibodies were found both in
Swedish patients and in the Liberians. Relative to the Liberians, high IgG2 antibody levels
were seen in most Swedish patients. In Liberian sera but not in those from Swedish
patients elevated IgG3 levels were found. In two Swedish patients followed consecutively
for a period of 15 weeks, elevated levels of IgG 1 and IgG3 antibodies were seen after
relapse. No differences between the groups in regard to the levels of IgG4 antibodies were
noticed. The differences between the Swedish patients and the Liberians were also in part
reflected by differences in the total amounts of the four IgG subclasses.
Keywords Plasmodiumfalciparum malaria IgG subclasses ELISA

INTRODUCTION
In spite of high levels of anti-plasmodial antibodies in malaria patients sera, a protective immunity
is acquired only after multiple exposures to the parasite over a long period of time. The reasons for
this are not known and further knowledge of both the antigens involved and the antibodies elicited
during different phases of the disease is urgently needed. Since the biological effector functions of
the different IgG subclasses vary greatly (Spiegelberg, 1974) the serum concentrations of antibodies
belonging to a given subclass may reflect their clinical role in the course of an infection. In the
accompanying paper (Wahlgren, Berzins & Perlmann, 1983) we have described an ELISA for
measuring antibody levels in P. falciparum malaria. This assay has now been further developed to
determine the antibody subclass of anti-parasite antibodies.

Correspondence: Dr Mats Wahlgren, Department of Immunology, University of Stockholm, S-10691

Stockholm, Sweden.
'35
136 M. Wahigren et al.

MATERIALS AND METHODS

P. falciparum antigen and human red blood cell antigen. The antigen preparations used were
aliquots of those described in the preceding paper (Wahlgren et al., 1983).
Serum samples. Ten serum samples were from 10 in-patients with P. falciparum malaria at
Roslagstull Hospital, Stockholm. All gave strong reactions in the P. falciparum ELISA (Wahlgren
et al., 1983). Twelve consecutive serum samples were from two additional Swedish patients (HP and
CA) with relapsing chloroquine resistant malaria (Aronsson et al., 1981). Six sera were from six
adult Liberians, living in a malaria endemic area of Liberia (Hedman et al., 1979). Control sera were
from five healthy Swedish blood donors. All sera were absorbed with normal human erythrocyte
ghosts as described (Wahlgren et al., 1983).
Mouse monoclonal antibodies. Antibodies specific for human IgG subclasses were a kind gift of
Dr N.R. Ling, Department of Immunology, The Medical School, Birmingham, UK. The
antibodies were derived from the following clones: anti-IgGI, JL512; anti-IgG2, GOM1 and
GOM2; anti-IgG3, ZG4; anti-IgG4,RJ4. For further details see Lowe et al. (1982). Whole ascitic
fluids were used in all tests. The specificities of the monoclonal reagents were ascertained in an
ELISA in which the mircowells were coated with human myeloma protiens one with K and one with
A light chains of each of the IgG subclasses 1-4 respectively. No cross-reactivities were seen except
for a weak one obtained with anti-IgG3 when tested with the A IgG2 myeloma protein. Inhibition
experiments were performed in which different amounts of myeloma protein were added to the
monoclonal antibodies before the assay (Wahlgren et al., 1983). Complete inhibition was easily
obtained in the homologous inhibitor/antibody combinations and no cross-reactions were seen. All
monoclonal antibodies were stored at - 20°C in small aliquots until use.
Anti-mouse immunoglobulin (Ig) conjugate. Immunosorbent purified rabbit anti-mouse im-
munoglobulin antibodies were used. Antibodies cross-reacting with human immunoglobulin were
removed on a human IgG-Sepharose 4B column. Conjugation with alkaline phosphatase (ALP)
was performed as described (Wahlgren et al., 1983).
Analysis of P. falciparum antibodies of different IgG subclasses. Optimal dilutions of the
reagents were determined by titrations with a few known positive patients' sera and negative control
sera. The optimal dilution of the P. falciparum antigen stock solution (7 mg/ml) was 1: 1,000 and
that of the test sera was 1:200. For the monoclonal antibodies they were for anti-IgGl (4 mg
antibody/ml) 1: 500, for anti-IgG3 (4.4 mg antibody/ml) 1: 1,000 and for IgG4 (2 mg antibody/ml)
1: 1,000. For anti-IgG2, two different monoclonal antibodies (3-9 and 2 6 mg/ml, respectively) were
mixed and used at a dilution of 1: 1,000. The optimal dilution of the rabbit anti-mouse Ig conjugate
was 1: 3,000.
The procedure of coating the microtitre plates with P. falciparum antigen was as described
(Wahlgren et al., 1983). However, the incubation for aftercoating with bovine serum albumin (BSA)
at 4°C was extended to last overnight. After washing and addition of RBC absorbed test serum, the
plates were incubated at 40C overnight and washed again. Aliquots of the monoclonal antibodies
were then added to the wells. The plates were incubated overnight at room remperature, washed and
the conjugate was then added. After a final incubation overnight at 4°C the plates were washed and
supplemented with 0-1 ml/well of p-nitrophenylphosphate diluted in enzyme substrate buffer. The
precise incubation times were adapted on the basis of the reactions obtained with standard sera
included in each test. All sera were tested in duplicates.
Determination ofantibody concentrations. Microtitre wells were coated with different amounts (1
ng to 10 ig/ml) of myeloma protein of either one of the four IgG subclasses. Coating was done with
a mixture (1: 1) of one K and one A myeloma protein of each IgG subclass, respectively. An ELISA
was then performed as described above. A standard curve was constructed for each subclass by
plotting the OD405 against the different concentrations of the myeloma protein used for coating. In
order to estimate the amount of myeloma protein actually adsorbed to the wells, proteins ofeach of
the four subclasses were labelled with 10 mCi/mg Na'251 by the chloramine-T procedure (Hunter &
Greenwood, 1962). The adsorbtion to the plastic of radioactive protein was determined after a
complete ELISA by cutting out the wells and counting the remaining radioactivity in a gamma
 i-umoral immune response in malaria
TV *_
I37
counter. On the average, 46% (range 29-69%) of the amount of myeloma protein, added at four
different concentrations, was found to adhere to the wells, regardless of subclass. The IgG subclass
concentration of the anti-P. falciparum antibodies was determined by comparing the OD readings
obtained in the antibody ELISA described in the preceding paragraph with those of the myeloma
standards. The IgG subclass concentrations so obtained were multiplied with a factor of 0*46 in
order to correct for adherence to the plastic and by a factor 200 to correct for the dilution of the
serum.
Total IgG and IgM levels. The levels of certain test sera were determined by single radial
diffusion in agarose (Tri-Partigen, Behringwerke AG, Marburg, FRG).
IgG subclass concentrations. The total concentration of different IgG subclasses present in some
sera was determined in a similar way. Antisera were subclass specific rabbit antisera from the Red
Cross Blood Transfusion Service, Amsterdam, Holland and the test systems were standardized
using a WHO serum pool (67/97).

RESULTS
Fig. 1 shows four standard curves obtained in the subclass ELISA by coating the microwells with
either one of the myeloma proteins as described in the Materials and Methods section. Similar
curves were obtained with all subclasses although longer incubation times had to be used in the
IgGI and IgG2 assays due to a lower binding capacity of the monoclonal antibodies. In the IgG2
assay this was partly compensated for by using a mixture of two monoclonal antibodies, directed to
different epitopes on the Fc fragment of IgG2 (Lowe et al., 1982).
The OD readings obtained with the subclass specific monoclonals in the P. falciparum ELISA
varied greatly for individual sera. For the 10 Swedish patients and six Liberians from a malaria
endemic area, the serum concentrations of the anti-parasite antibodies, calculated from the
standard curves, ranged from < 1-115 pg/ml for IgGl, 1-33 pg/ml for IgG2, < 1-50 pg/ml for IgG3
and < 1-6 5 ug/ml for IgG4. For the Swedish controls, no reactions were obtained when they were
tested for anti-P.falciparum antibodies in the IgG 1, IgG3 or IgG4 assays. However, with anti-IgG2,
the OD readings were slightly elevated, corresponding to serum concentrations of < 8-5 pg/ml.
Individual antibody levels are shown in Fig. 2a-d. In both the Swedish and Liberian group a few
donors had elevated levels of antibodies of the IgGl subclass. Many of the Swedish patients with
primary infections also had elevated levels of IgG2 antibodies. In contrast, the IgG2 antibody levels
of the Liberians were low. For IgG3, this was different in that the Liberians tended to show higher

2-2

10 1.0 0 0 01 0-001
Antibody coated (ug/mI)
Fig. 1. Standard curves obtained in the ELISA when myeloma proteins of the four subclasses (IgG = 0;
IgG2 = 0; IgG3 = 0; IgG4 = *) were coated on the plastic at different protein concentrations.
I38 M. Wahlgren et al.
120 - (a) I Ib)
100 _ 30 _
_
E
80
o 0
.0 _0 20
60 a

. 40_
C\J 10 *

20 _

Patients Controls Patients Controls

S L S S L S

50 (c) 8 (d)

N 10 N
0 -o
Eo E0

Patients Controls Patients Controls

S L S S L S
Fig. 2. The levels of anti-P. falciparum IgG antibody subclasses (expressed in yg/ml) in sera. Patients: L = sera
from six adult Liberians, S = sera from IO Swedish patients with P. falciparum malaria. Controls: S = sera from
five Swedish healthy blood donors. (a) IgG I, (b) IgG2, (c) IgG3 and (d) IgG4.

antibody levels (one donor very high) while the IgG3 antibody level of the Swedish patients was low.
Both groups had low levels of IgG4 antibodies. However, one Liberian and two Swedes also had
relatively high levels of such antibodies.
Consecutive studies of antibody levels were performed on serum samples from two Swedish
patients (not included in Fig. 2) who had acquired chloroquine resistant P.Jfalciparum malaria. As
shown in the accompanying paper (Wahlgren et al., 1983) for one of these patients (HP), there was
an early peak in parasitemia during the first week after onset of the disease and recrudescences at 6
and I11 weeks. Her anti-P. falciparum antibodies showed a first peak during the third week after
onset and rose again strongly after the second relapse, with a high peak during weeks I11- 13. As
shown in Fig. 3 aliquots of the same serum samples from this patient have also been studied in the
subclass ELISA. Both the IgG I and IgG3 responses showed major peaks after the second relapse.
Relative to IgGl, IgG3 appeared to be even more enhanced than found in other Swedish sera (cf.
Fig. 2a and c). Also the IgG2 antibodies showed a narrow peak after the second relapse but the level
of this peak was low (cf. Fig. 2b). The IgG4 antibodies remained at a low level throughout. Similar
results were obtained with the sera of a second patient (CA; data not shown).
The total IgG level in some sera, studied by radial immunodiffusion, ranged from 14 20 mg/ml
which was slightly above normal (normal range for Europeans, 8-18 mg/ml as determined by the
Tri-partigen assay.) This elevation was the same for IgM, which ranged from 1 0-43 mg/ml
(normal range for Europeans 0-6-2 5 mg/ml). In order to obtain a more complete picture, most of
these sera were also analysed for the total concentration of each IgG subclass. The results are
summarized in Table I which also gives the corresponding values of the IgG subclass levels for the
anti-P. falciparum antibodies present in these sera.
 Humoral immune response in malaria I39
125F- (a)

100o_

501-
-
25
"I
I klI
>1 10 7( *hi n
7.11

" 7-5-
I -'J
_

50O_

2-5_

a. YI
a 5 10 15
Weeks after onset of disease
Fig. 3. The levels of anti-P. falciparum IgG antibody subclasses (expressed in pg/ml) in eight sera from patient
H.P. during a 15 weeks period. The patient had chloroquine resistant P. falciparum malaria with parasite
relapses (indicated by arrows). (a) IgGl (0), (b) IgG2 (0), IgG3 (-) and IgG4 (a).

Table 1. Immunoglobulin and antibody levels in nine sera from patients exposed to P. falciparum malaria

Total (mg/ml) Parasite specific (pg/ml)

Patient IgG IgM IgGI IgG2 IgG3 IgG4 IgGI IgG2 IgG3 IgG4
M.D.-L 20 2-3 13 2-9 2-3 0.1 50 2-5 7-5 2-00
P.J.-L nt nt 90 40 1-5 0-2 16 19 3-7 0-25
P.B.-L nt nt 11 3-0 1-6 03 nt nt nt nt
T1 154-L 20 17 nt nt nt nt 10 9-5 3-0 0-15
C.J.-S 19 10 8-8 50 03 0-2 5 23 19 020
K.P.-S 19 4-3 76 39 04 0 1 16 10 000
E.H.-S 15 2-2 7-8 4-0 1-2 01 10 35 3-7 0-15
B.P.-S 14 1-4 6-7 3-4 02 04 115 5 10 0-25
B.J.-S 14 2-8 nt nt nt nt 45 5 10 0-25

L = Liberian, S = Swedish, nt= not tested.

DISCUSSION
In the preceding paper (Wahlgren et al., 1983) we described an ELISA for measuring the levels of
antibodies to P. falciparum in patients' sera. This assay has now been developed further for
estimating the IgG subclass levels of anti-P. falciparum antibodies. These determinations were
performed by including monoclonal mouse antibodies specific for the human IgG subclass into the
assay system. Thus by using a three layer antibody assay it was possible to measure the
concentration of specific antibodies of a given subclass in a single test.
When the total amount of IgG anti-P. falciparum antibodies present in different sera was
140 M. Wahigren et al.
estimated by summation of the concentrations of the different subclasses, low values were obtained
(cf. Table 1) in comparison to what has been found by others (Curtain et al., 1964). Moreover, in the
few sera tested, the amount of P. falciparum antibodies of IgG 1, 2, 3 or 4 subclass was a relatively
minor fraction of the total amounts of the subclasses present. However, the total subclass
concentrations were measured by radial immunodiffusion with polyclonal rabbit antibodies and the
values for the absolute concentrations given are not comparable. The subclass ELISA may be
expected to give low values since the mouse antibodies were monoclonal and their avidities as well as
those of the anti-P. falciparum antibodies were unknown. Since the assay includes many washing
steps, antibody losses are bound to occur. It may further be argued that the anti-P. falciparum
antibodies belonging to different subclasses may compete with each other at the level of the antigen,
resulting in false negative or low estimates for antibodies of the less frequent subclasses. However,
the sensitivity of ELISA made it possible to minimize this competition by using high serum dilutions
and, therefore, to work in antigen excess. Our results indicate that comparison of the levels of
antibodies belonging to different subclasses are meaningful in the P. falciparum system.
Anti-P.falciparum antibodies were found within all four IgG subclasses. As was to be expected,
antibodies of the IgGl subclass were frequent and relatively elevated levels were seen both in
Swedish and Liberian sera. High IgG3 antibody levels relative to the Swedish sera were primarily
seen in the Liberian sera. However, when consecutive serum samples from patients H.P. were
analysed, a pronounced increase was found of the IgG3 level after the second relapse (Fig. 3). In
contrast, the Swedish patients had strikingly high levels of IgG2 antibodies as compared with the
Liberians (Fig. 2b). No distinct trends for the two groups could be discerned in respect to IgG4
antibodies. The differences in antibody levels were partially reflected by differences in the total
amounts of the different IgGs as seen in a small number of sera available for these tests. Summation
of the concentrations of the four subclasses for each individual patients gave similar values as the
determination of total IgG. Considering the fact that different standards were used in these tests (see
Materials and Methods), the values agreed reasonably well. Both the Swedish patients and the
Liberians had high concentrations of total IgG. For the Swedish patients, IgG I was 61O% of the total
IgG (sum of IgGl-4) which is close to normal, while their total IgG2 was high (32%) (Morell,
Skvaril & Barandun, 1976). The corresponding proportions for the Liberians were 67% for IgGl
and 20% for IgG2. However, the three Liberians had a very substantial elevation in IgG3 (1 1%)
while the total IgG3 value for the Swedish patients was normal (4.5%) (Morell et al., 1976). Both
groups had normal and similar levels of IgG4. Not much information is available regarding IgG
subclass levels in malaria patients. However, Salimonou, Williams & Osunkoya, (1982) recently
reported that Nigerian patients with acute malaria had elevated levels of IgG1 while all other
subclasses were slightly diminished.
The reasons for relatively elevated IgG3 anti-P.falciparum antibody levels in Liberian sera and,
in particular, for the high IgG2 antibody levels in most Swedish patients are not known. Genetic
differences in allotypic restriction between the donor groups may be considered and are further
investigated by Gm typing of antibodies and total IgG. Polysaccharides such as dextran or levan are
known to give rise to antibodies of the IgG2 subclass (Yount et al., 1968). Since such substances are
also known to be polyclonal B cell activators (Coutinho, Moller & Richter, 1974), parasite derived
material of a similar nature could well be responsible for both IgG2 antibody formation and a
general elevation of total IgG2 in acutely infected patients. Since the Liberian sera included in this
study were from donors with few or no parasites in their blood, this could explain their lower IgG2
antibody levels but not their relative elevation of IgG3. Most likely, the explanation of the
differences in IgG subclass expression between the two donor groups are more complex and involve
combined effects of polyclonal B cell activation and a T cell regulated switch in immunoglobulin C
gene expression, dependent on the immune status of the serum donors (Martinez-Alonso, Coutinho
& Augustin, 1980; Mongini, Stein & Paul, 1981).
Several interdependent factors such as mitogenicity of the parasite (Greenwood & Vick, 1975),
antigenic variation (M. Hommel, personal communication), imbalances in the T cell system
(Troye-Blomberg et al., 1983) as well as others (for review see Playfair, 1982; Allison & Eugui, 1982)
may be responsible for the delayed onset of acquired immunity in P. falciparum malaria. The
present findings might imply that the subclass of the antibodies formed in different phases of
 Humoral immune response in malaria 141
infection or after different length of parasite exposure may be instrumental in providing protection.
Restrictions in the IgG subclasses involving increased relative levels of IgG3 has been observed in
several instances (Devey & Voak, 1974; Mortimer & Widdowson, 1979; Beck, 1981). The IgG
subclasses also vary considerably in their capacity to activate various effector cells participating in
immune protection (Spiegelberg, 1974). Thus, while IgG I and IgG3 mediate phagocytosis or target
cell lysis by monocytes or Fc receptor bearing lymphocytes, IgG2 or 4 are inactive or less efficient in
this respect (Spiegelberg, 1974; Perlmann & Cerottini, 1979). Antibody-dependent cellular effector
mechanisms have also been suggested to play a protective role in malaria (Brown & Smalley, 1980,
1981; Celada, Cruchaud & Perrin, 1982). Anti-P. falciparum antibodies of the IgGI and IgG3
subclasses may therefore induce or amplify (Allison & Eugui, 1982) cell-mediated protection which
IgG2 (and IgG4) could not do even if they were specific for the same target antigens. On the
contrary, in this case, antibodies of the latter two subclasses might be inhibitory.
This work was supported by grant No. B83-16X-06252 from the Swedish Medical Research Council, the
Swedish Agency for Research Co-operation with Developing Countries (SAREC) and the Rockefeller
Foundation. We wish to thank Inga Ponten and the staff at Roslagstull Hospital for their kind help and
co-operation.

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