production and characterization of monoclonal antibodies against substrate specific loop region of ...
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Immunological Investigations, 2014; 43(6): 556–571! Informa Healthcare USA, Inc.ISSN: 0882-0139 print / 1532-4311 onlineDOI: 10.3109/08820139.2014.892962
Production and characterizationof monoclonal antibodies againstsubstrate specific loop region ofPlasmodium falciparum lactatedehydrogenase
Nuzhat A. Kaushal1 and Deep C. Kaushal2
1Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226001,
India and2Research Department, Amity University Uttar Pradesh, Lucknow Campus,
Lucknow 226010, India
Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been
shown to be biochemically, immunologically and structurally different from the
mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydro-
genase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug
targeting. In the present study, we have produced six monoclonal antibodies, which are
against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these
monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop
region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and
10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of
PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity
non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff
0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known
inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in
inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be
utilized for identification of compounds inhibitory to PfLDH (binding to substrate
specific loop region of parasite LDH) from combinatory chemical libraries or medicinal
plants extracts. The Microplate Sandwich ELISA has also shown potential for specific
diagnosis of malaria using finger prick blood samples.
Keywords Lactate dehydrogenase, malaria parasite, monoclonal antibodies,
Plasmodium falciparum, substrate specific loop region of PfLDH
INTRODUCTION
Malaria, a major public health problem, continues to occupy the top position
among parasitic diseases. About 135–287 million people are affected by the
disease, of which 0.47–0.79 million people die annually (WHO, 2013). The
worldwide resurgence of malaria is due to the failure of conventional methods
for its control. In addition to malaria vaccines, other approaches for malaria
control are development of better diagnostics and more efficacious drugs.
Correspondence: Prof. Nuzhat A. Kaushal, Ex-Senior Principal Scientist, Division of
Parasitology, CSIR-Central Drug Research Institute, Lucknow 226031, India. E-mail:
[email protected]; [email protected]
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A thorough understanding of the parasite enzymes in comparison to its host
may lead to the identification of marked differences between the parasite and
the host enzymes. Such differences can be exploited for designing novel
inhibitors/antimalarial compounds specific to parasite enzymes. Therefore, the
molecular characterization of parasite specific proteins/epitopes (peptides) is of
considerable importance for developing control measures against malaria
(Guerin et al., 2002; Mehlin, 2005).
The malarial parasites depend mainly on glycolytic pathway for their
energy needs due to the lack of a functional TCA cycle. The rate of glucose
utilization in parasite-infected erythrocytes is 50–100 times higher than in
normal erythrocytes. The role of malarial parasite lactate dehydrogenase
(L-lactate-NAD+-oxidoreductase; EC 1.1.1.27, LDH) in regenerating NAD is
well documented (Roth, 1990; Sherman, 1979). Biochemical characterization
revealed that the plasmodial LDH (pLDH) is different from the equivalent host
enzyme in its kinetic (Vander Jagt et al., 1981) and electrophoretic properties
(Carter & Walliker, 1977; Kaushal et al., 1985). Our studies on immunochem-
ical characterization of pLDH using polyclonal (Kaushal et al., 1988; Watts
et al., 1987) and monoclonal (Kaushal et al., 1995) antibodies have shown the
specificity of antibodies to LDH from Plasmodium spp.
The amino acid sequence of PfLDH was found to be very similar (90%) to that
of LDHs from other human malaria parasites but different from that of
mammalian and bacterial LDHs (Bzik et al., 1993; Kaushal et al., 1993; Singh
et al., 2012; Turgut-Balik et al., 2004). The plasmodial LDH has a unique five
amino acid insert in the loop region, which is conserved within Plasmodium spp.
and contributes to the substrate specificity of plasmodial LDH (Bzik et al., 1993;
Hewitt et al., 1997; Hurdayal et al., 2010; Singh et al., 2012). This enlarged
active site cavity of parasite LDH in comparison to mammalian enzyme could be
exploited for designing highly selective inhibitors for plasmodial LDH. Gossypol,
a di-sesquiterpene isolated from cotton seeds and certain azole-based com-
pounds have been shown to inhibit the PfLDH activity by binding to substrate
specific loop region of the parasite enzyme. The azole-based compounds have
also exhibited in vitro activity against P. falciparum and in vivo activity against
P. berghei parasites (Cameron et al., 2004; Conners et al., 2005).
In the present study, we have generated six monoclonal antibodies against
P. falciparum LDH. Out of these, two monoclonals (10C4D5 and 10D3G2),
have been shown to recognize its substrate specific loop region peptide and
inhibiting the activity of PfLDH. A Microplate Sandwich ELISA utilizing
monoclonal antibodies against different epitopes of PfLDH was developed and
used to study the effect of gossypol (LDH inhibitor) on binding of inhibitory
monoclonals to substrate specific loop region of PfLDH. This Microplate
Sandwich ELISA can be utilized for screening of pLDH inhibitors/antimalarial
compounds as well as for the detection of malaria parasites (based on pLDH
detection) in blood samples.
MATERIALS AND METHODS
MaterialsBlue Sepharose 6B, Freunds Complete Adjuvant, mammalian LDHs
and all other biochemicals used for the study were procured from
P. falciparum LDH Loop Region Peptide Monoclonals 557
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Sigma-Aldrich (USA). The LDHs of P. knowlesi, P. falciparum, P. vivax and
P. malariae, cloned and expressed in our lab, were used. The P. falciparum and
P. malariae LDH gene was cloned in BamH1 and Not1 sites of pGEX-6P1
expression vector. The GST-tag PfLDH fusion proteins were purified by
affinity chromatography on GSTrap-Sepharose 4B column and cleaved by
precision protease (Kaushal et al, in preparation). The P. knowlesi LDH was
also cloned in pGEX-6P1 expression vector at EcoR1 and NotI restriction sites
as described elsewhere (Singh et al., 2012). The P. vivax LDH was cloned in
pET28a expression vector and purified on Ni-NTA column (Singh et al., in
preparation). The BALB/c mice were from the Animal Facility of CSIR-CDRI,
Lucknow, India.
P. falciparum Parasites and Purification of Lactate DehydrogenaseAn Indian isolate of P. falciparum (Kaushal, 1988) was grown in in vitro
culture by the method of Trager & Jenson (1976). The parasites from infected
blood (25 ml packed cells) were prepared as per the method described by
Kaushal et al. (1995) and P. falciparum LDH (PfLDH) was purified using Blue
Sepharose CL-6B affinity column (Chandra et al., 1994). The purified enzyme
was used for immunization of mice to produce monoclonal antibodies. The
RBC-LDH was also purified from normal RBC lysate using Blue Sepharose
CL-6B column.
Assay of LDH ActivityThe activity of PfLDH was measured at 340 nm by following the oxidation of
NADH according to the method described elsewhere (Anwar et al., 1977). One
unit of enzyme activity is defined as the amount of enzyme that catalyzes the
conversion of 1 mmol of coenzyme per minute.
Substrate Specific Loop Region Peptide of P. falciparum LDHThe substrate specific loop region peptide of P. falciparum LDH
(AGFTKAPGKSDKEWNRC, amino acid residues 98-109), containing
an additional cysteine residue at C terminus for coupling with carrier
protein, was custom synthesized from Memotopes, Victoria, Australia.
The peptide was conjugated to ovalbumin using succinimidyl m-maleimido-
bezoate reagent according to the procedure described elsewhere (Peeters
et al., 1989).
Immunization of Mice with PfLDH and Production of HybridomaSix BALB/c mice were immunized subcutaneously with purified PfLDH (20 mg/
mouse) emulsified in Freunds Complete Adjuvant following the protocol
approved by the Institutional Animal Ethics Committee. A total of four
injections were given at 15 days’ interval, and the mouse showing the highest
antibody titer was given the booster intravenous injection 3 days before fusion.
The PfLDH sensitized mouse splenocytes were fused with myeloma cells
(Sp2/0-Ag14) according to the procedure of Kohler & Milstein (1975) with
certain modifications (Kaushal et al., 1995). Ten days after fusion, the culture
supernatants from hybridoma clones were screened against PfLDH, PfLDH-
slrPep and RBC-LDH in ELISA. The single cell clones, which showed
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consistent high reactivity with PfLDH, were expanded in in vitro cultures and
injected to pristane primed mice for production of monoclonal antibodies in
ascites (Potter et al., 1972).
Isotyping and Purification of Monoclonal AntibodiesIsotyping of monoclonal antibodies was done using culture supernatants and
isotype ELISA kit. The Protein-A Sepharose affinity column was used for
purification of IgG isotype monoclonals while IgM isotype monoclonals were
purified on Sephacryl S-200 column according to manufacturer’s instructions.
The conjugation of purified monoclonals to peroxidase was performed by the
procedure of Nakane & Kawoi (1974).
Blood SamplesThe blood samples from microscopically proven malaria cases (n¼ 120)
and endemic controls (n¼ 30) were collected from malaria endemic region
with consent of the individuals and under the guidance of a medical
practitioner according to the guidelines approved by the Institutional
Ethics Committee of CDRI. Twenty-five non-endemic control blood samples
(from normal healthy individuals) were collected from a malaria non-endemic
region.
Enzyme Linked Immunosorbent AssayThe enzyme linked immunosorbent assay (ELISA) was performed according
to the method described elsewhere (Singh et al., 2012). Briefly, wells of
the microtitre plates were coated with 100 ml of purified PfLDH (15 ng/well)
or PfLDH-slrPep -ovalbumin (20 ng/well) or normal RBC LDH (20 ng/well)
diluted in phosphate buffered saline (pH 7.4, PBS) by incubation at 37 �C
for 1 h and then overnight at 4 �C. After blocking with 3% non-fat milk for 2 h
at 37 �C, the plates were incubated with 100 ml of hybridoma culture
supernatant or appropriately diluted monoclonals for 2 h at 37 �C followed
by 1.5 h incubation at 37 �C with peroxidase-conjugated secondary anti-
body (1:2000). The plate was washed four times with PBS-Tween
(0.05% Tween-20) between each incubation step. The color was developed by
adding OPD solution (1 mg/ml O-phenylenediamine in citrate–phos-
phate buffer, pH 5.0, containing 1 ml/ml H2O2) and the absorbance was
read at 490 nm using Molecular Devices LLC UV 190 plus microplate ELISA
reader.
Effect of Monoclonal Antibodies on P. falciparum LDH ActivityThe monoclonals were tested for their effect on the activity of PfLDH as
described earlier (Kaushal et al., 1995). Briefly, 2 Units of PfLDH and normal
RBC LDH were incubated with different concentrations (0.04–10 mg) of the
purified monoclonals in a final volume of 100 ml and LDH activity was
measured after 30 min incubation at 25 �C. The percent inhibition of LDH
activity was calculated as follows:
% Inhibition of LDH activity ¼
LDH Activity in absence of Ab
�LDH activity in presence of Ab
� �
Total enzyme activity� 100
P. falciparum LDH Loop Region Peptide Monoclonals 559
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Competitive ELISAThe epitope mapping of monoclonals was done by competitive ELISA (Kaushal
et al., 1995). Briefly, the wells of microtitre plates were coated with purified
PfLDH (15 ng/well), blocked with 3% non-fat milk and incubated at 37 �C for
30 min with 50 ml of appropriately diluted (1:200) unlabelled monoclonals.
After 30 min, 50 ml each of peroxidase conjugated anti-PfLDH monoclonals
(1:500) were added to the wells of microtitre plate and further incubated at
37 �C for 1.5 h. The plate was washed four times with PBS-Tween (0.05%
Tween-20) between each incubation step and developed as described for
ELISA.
The competitive ELISA was also used to study the effect of PfLDH-slrPep on
binding of monoclonals to PfLDH. The wells of ELISA plate were coated with
PfLDH (15 ng/well) and incubated with 50 ml of monoclonals (1:500 dilution) in
presence of either 100 ng of PfLDH-slrPep (unconjugated) or 200 ng of PfLDH
(positive LDH control) or 100 ng of PfCSP (NANP)4 (negative peptide control)
or 200 ng of RBC-LDH (negative LDH control) for 2 h at 37 �C. Then 100 ml of
peroxidase conjugated secondary antibody (1:2000) were added and further
incubated for 1 h at 37 �C. The plate was washed with PBS-Tween (0.05%
Tween-20) after incubation step and developed as described for ELISA. The
effect of different concentrations (1–1000 ng) of PfLDH-slrPep on binding of
monoclonals to PfLDH was also studied.
Determination of Affinity Constant of Monoclonals by ELISAThe affinity constant (Kaff) of monoclonal antibodies were determined by
measuring the binding affinities of different monoclonals as described
elsewhere (Friguet et al., 1985). Briefly, various concentrations (0.12 ng–
4000 ng/ml) of antigen was mixed with fixed concentration of antibody in 0.1 M
sodium phosphate, 2 mM EDTA, 10 mg/ml BSA, pH 7.8 and incubated for 15 h
at 30 �C. The antigen-antibody mix was then transferred to micro-titer plates
previously coated with antigen at 100 ng/well in 50 mM sodium carbonate
buffer pH 9.6 and incubated for 1 h at 30 �C followed by incubation with
peroxidase conjugated secondary antibody (1: 2000) for 1 h at 30 �C. The
washing and developing of plate was performed as described for ELISA.
The affinity constants were calculated using the Scatchard-Klotz equation
(Friguet et al., 1985).
Microplate Sandwich ELISA for Studying the Effect of Gossypol on Bindingof Monoclonal to PfLDHMicroplate Sandwich ELISA was developed by using the 10A5H5 non-
inhibitory monoclonal antibody for capturing of PfLDH and peroxidase
conjugated 10C4D5 monoclonal as revealing antibody. Briefly, the wells of
the microtitre plate were coated with 100 ml (1 mg/well) of 10A5H5 monoclonal
by incubating for 1 h at 37 �C and then overnight at 4 �C. The monoclonal
antibody coated plates were blocked with 3% non-fat milk, washed four times
with PBS-T.
The PfLDH (5 unit) was pre-incubated with different concentrations
(0–10mM) of gossypol or test compound in 200 ml at 25 �C in a separate plate.
After 30 min incubation, 50 ml of incubation mixture was used for the assay of
LDH activity in microplate ELISA reader (Molecular Devices LLC UV
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190 plus) and 100 ml was transferred to a microtitre plate pre-coated with
10A5H5 monoclonal (capturing antibody) followed by incubation for 1.5 h at
37 �C. Then 100 ml of peroxidase conjugated 10C4D5 or 10A4E11 monoclonal
(revealing antibody) was added to the wells of microtitre plate and incubated
at 37 �C for 1 h. The plate was washed four times with PBS-Tween (0.05%
Tween-20) between each incubation step. The plate was finally washed and
developed using substrate solution as described for ELISA.
Microplate Sandwich ELISA for Diagnosis of MalariaThe Microplate Sandwich ELISA was performed as described previously
(Singh et al., 2012) by using 10A5H5 monoclonal as capturing and peroxidase
conjugated 10C4D5 as a revealing antibody. The blood samples were lysed by
mixing with 100 ml of 50 mM Tris-HCL, pH 7.5, containing 0.1% Triton X-100
and 100 ml of lysed blood samples were used in Microplate Sandwich ELISA
(Singh et al., 2012).
Protein EstimationThe protein contents of the samples were measured by the procedure of
Bradford (1976) and BSA was used as standard.
RESULTS
Generation and Characterization of Plasmodium falciparum LDHMonoclonal AntibodiesHybridoma clones that produce monoclonal antibodies to PfLDH were obtained
from two fusion experiments. Out of a total of 376 microtitre wells plated, the
culture supernatants from 216 wells were selected by the appearance of clones
macroscopically and screened against PfLDH and RBC LDH in ELISA. The
culture supernatants from 34 wells having high reactivity with PfLDH and no
reactivity with RBC LDH were selected and screened against PfLDH-slrPep.
The culture supernatants from two wells showed high reactivity with PfLDH-
slrPep in ELISA. The six hybridoma clones (10A4E11, 10A5H3, 10A5H5,
10B6D1, 10C4D5 and 10D3G2) producing monoclonals of high and consistent
ELISA reactivity with PfLDH but not with normal RBC LDH (including two
monoclonals against PfLDH-slrPep) were selected, recloned and characterized.
Some of the characteristics of these monoclonals are given in Table 1. Isotypic
analysis revealed that two monoclonal antibodies (10A4E11, 10A5H3) were
IgM isotype, three (10A5H5, 10B6D1 and10D3G2) were IgG2b isotype and one
(10C4D5) was IgG1 isotype. These monoclonal antibodies showed high ELISA
reactivity with recombinant LDHs from other species of malarial parasites (P.
vivax, P. knowlesi and P. malariae) and did not cross-react with mammalian
LDHs (data not shown). The 10C4D5, 10D3G2, 10A5H5, 10B6D1 monoclonals
have affinity constant values of 0.306 ± 0.011, 0.661 ± 0.037, 1.272 ± 0.057,
1.361 ± 0.197 respectively, while affinity constant of 23.368 ± 4.645 and
37.07 ± 1.374 were obtained for IgM isotype 10A4E11 and 10A5H3 monoclonals
(Table 1).
The epitope analysis of the monoclonal antibodies by competitive ELISA
revealed the presence of at least three epitopes on parasite LDH (Table 1). The
10A4E11 and 10A5H3 monoclonals compete for binding with each other and
P. falciparum LDH Loop Region Peptide Monoclonals 561
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not with other four monoclonals (10A5H5, 10B6D1, 10C4D5, 10D3G2) in
Competitive ELISA. Similarly, 10A5H5 and 10B6D1 monoclonals compete
with each other without having any effect on the binding of other four
monoclonal antibodies (10A4E11, 10A5H3, 10C4D5, 10D3G2). The inhibitory
monoclonal antibodies (10C4D5, 10D3G2) also compete with each other for
binding, but do not compete with other four monoclonals (10A4E11, 10A5H3,
10A5H5, 10B6D1) in competitive ELISA (Table S1). These results suggest that
the monoclonal antibodies 10A4E11, 10A5H3 are directed against an epitope
designated as epiotpe-1 and the monoclonals 10A5H5, 10B6D1 are against
another epitope designated as epitope-2 different from the target epitope of
inhibitory monoclonal antibodies. The target epitope of only inhibitory
monoclonals (10C4D5 and 10D3G2) was identified and both the monoclonals
were directed against the substrate specific loop region. The substrate specific
loop region of pLDH is conserved within Plasmodium species; however, this
region is different from mammalian LDH by 6 amino acid residues and a 5
amino acid insert (Table 2).
Effect of PfLDH Substrate Specific Loop Region Peptide on Binding ofMonoclonal to PfLDHIn order to confirm further that 10C4D5 and 10D3G2 monoclonal antibodies
are against PfLDH substrate specific loop region, the effect of PfLDH-slrPep on
binding of monoclonal antibodies to PfLDH was studied and the results are
shown in Figure 1. The binding of monoclonal antibodies 10C4D5 and 10D3G2
to parasite LDH was almost completely inhibited by PfLDH-slrPep, while the
same peptide did not have any significant effect on binding of other four
monoclonals (10A4E11, 10A5H3, 10A5H5, 10B6D1) to PfLDH. The binding of
all the six monoclonals was inhibited by PfLDH (positive control) whereas
RBC-LDH and PfCSP(NANP)4 peptide (negative controls) had no effect on
binding of all six monoclonals to PfLDH. The effect of different concentrations
Table 1. Characteristics of P. falciparum LDH monoclonal antibodies.
ELISA Reactivityd
MoAbs IsotypeaAffinityb
constant (nM) Epitopec PfLDH PfLDH-slrPep RBC LDH
10A4E11 IgM 23.368 ± 4.645 1 3.523 ± 0.099 0.212 ± 0.020 0.231 ± 0.00910A5H3 IgM 37.070 ± 1.374 1 3.231 ± 0.105 0.168 ± 0.012 0.243 ± 0.00710A5H5 IgG2b 1.272 ± 0.057 2 3.417 ± 0.131 0.156 ± 0.027 0.147 ± 0.01110B6D1 IgG2b 1.361 ± 0.197 2 3.321 ± 0.119 0.187 ± 0.011 0.291 ± 0.00310C4D5 IgG1 0.306 ± 0.011 3 3.611 ± 0.059 3.225 ± 0.027 0.189 ± 0.01410D3G2 IgG2b 0.661 ± 0.037 3 2.945 ± 0.118 2.873 ± 0.103 0.198 ± 0.011
aIsotyping of monoclonal antibodies was done using culture supernatants and isotypeELISA kit from Sigma.
bAffinity constant (Kaff) of Moabs were measured as described in materials andmethods.
cEpitope mapping was done by competitive ELISA where binding of HRP-labelledmonoclonal to PfLDH measured in presence of other unlabelled monoclonals.
dPfLDH or PfLDH-slrPep-ovalbumin or RBC LDH coated plate incubated with culturesupernatants and ELISA was done as described in materials and methods.
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of PfLDH-slrPep (1–1000 ng) was further studied on the binding of two
monoclonal antibodies (10C4D5 and 10D3G2) to PfLDH and the inhibition
curves are shown in Figure 2. The 7 ng and 9 ng concentrations of PfLDH-
slrPep exhibited 50 % inhibition of binding of 10C4D5 and 10D3G2 monoclo-
nals to PfLDH respectively. The PfCSP(NANP)4 peptide did not show any
inhibition of binding of monoclonal to Pf-LDH even at 1000 ng concentration.
Figure 1. Effect of PfLDH-slrPep on binding of monoclonals to PfLDH. The PfLDH coatedplate incubated with monoclonals in presence PfLDH-slrPep, PfLDH, (NANP)4 peptide ofPfCSP and RBC-LDH followed by addition of peroxidase conjugated secondaryantibody. Error bars indicate the standard deviations of the mean of three OD readings.Data were analyzed by One-way analysis of variance (ANOVA) followed by Tukey’s testusing statistical software PRISM 5. The difference between the data obtained forinhibition of binding of monoclonals by specific peptide (PfLDH-slrPep) compared tonegative control peptide [PfCSP(NANP)4]; inhibition of binding of monoclonals by PfLDHcompared to RBC-LDH (negative LDH control) was found to be highly significant (p value50.001) and marked as ***.
Table 2. Amino acid sequence alignment of loop region peptide of PfLDH and otherpLDH as compared to mammalian LDH.
LDH source aa no. Sequence aa no.
98 * * * * * * 109P. falciparum A G F T K A P G K S D K E W N RP. knowlesi A G F T K A P G K S D K E W N RP. vivax A G F T K A P G K S D K E W N RP. malariae A G F T K V P G K S D K E W N RP. ovale A G F T K A P G K S D K E W N RP. yoelii A G F T K A P G K S D K E W N RLDH-A A G A R Q Q E G E S – – – – – RLDH-B A G V R Q Q E G E S – – – – – RLDH-C A G A R Q Q E G E T – – – – – R
*Denotes amino acids different in Plasmodium species than mammalian LDH.
P. falciparum LDH Loop Region Peptide Monoclonals 563
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Effect of Monoclonal Antibodies on Parasite LDH ActivityThe effect of different concentrations of purified monoclonals on enzyme
activity of PfLDH and RBC LDHs is depicted in Figure 3. Two monoclonals
(10C4D5 and 10D3G2), which were against the PfLDH-slrPep, inhibited the
enzyme activity of PfLDH while other four monoclonals (10A4E11, 10A5H3,
10A5H5 and 10B6D1) did not have any inhibitory effect on PfLDH activity.
The 50% inhibition of PfLDH activity was obtained with only 320 ng of 10C4D5
while 630 ng of 10D3G2 monoclonal was required to achieve the same level of
inhibition (50%) of enzyme activity. Thus, 10C4D5 monoclonal was a more
potent inhibitor of PfLDH activity. None of these monoclonals had any effect on
the enzyme activity of mammalian LDHs (LDH-A4, LDH-B4 and LDH-C4,
data not shown).
Effect of Gossypol on P. falciparum LDH ActivityThe effect of different concentrations of gossypol on activity of P. falciparum
LDH was studied and results are given in insert of Figure 4. A 50% inhibition
of PfLDH activity was obtained at 0.625 mM concentration of gossypol.
Effect of Gossypol on Binding of Monoclonal to P. falciparum LDHTo confirm that 10C4D5 and 10D3G2 monoclonal antibodies and gossypol bind
to the same site of substrate specific loop region of PfLDH, a Microplate
Sandwich ELISA was developed using monoclonals against different epitopes
of PfLDH. The binding of inhibitory monoclonal antibodies (10C4D5 and
10D3G2) to PfLDH decreased with increasing concentration of gossypol, while
there was no significant effect of gossypol on the binding of non-inhibitory
monoclonal antibody (10A4E11) even at 10 mM concentration (Figure 4).
Figure 2. Effect of different concentrations of PfLDH-slrPep on binding of 10C4D5 and10D3G2 monoclonals to PfLDH. PfLDH coated plate incubated with monoclonalantibodies in presence of different concentrations (1–1000 ng) of PfLDH-slrPep and(NANP)4 peptide followed by addition of peroxidase conjugated secondary antibody.Error bars indicate the standard deviations of the mean of triplicates.
N. A. Kaushal & D. C. Kaushal564
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Figure 4. Effect of Gossypol on binding of monoclonals to PfLDH in Microplate assay.PfLDH (5 unit) preincubated with different concentrations of gossypol (0–10 mM) wastransferred to plate pre-coated with capturing monoclonal (10A5H5, 1 mg/well) followedby incubation with peroxidise conjugated 10C4D5 or10D3G2 or 10A4E11 monoclonals asdescribed in materials and methods. The inhibition of PfLDH activity at differentconcentrations (0–10mM) of gossypol is shown in Insert. Error bars indicate the standarddeviations of the mean of triplicates.
Figure 3. Inhibition of P. falciparum LDH activity by monoclonal antibodies. The PfLDHincubated with different concentrations (0.04–10 mg) of purified monoclonals and theenzyme activity was determined and the percent inhibition of enzyme activity wascalculated compared to the control as described in materials and methods. Error barsindicate the standard deviations of the mean of triplicates.
P. falciparum LDH Loop Region Peptide Monoclonals 565
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The 50% reduction in binding of 10C4D5 and 10D3G2 monoclonals to PfLDH
was obtained at 1.2 mM and 2.1 mM concentrations of gossypol respectively
(Figure 4). These results further confirm that 10C4D5 and 10D3G2 monoclo-
nals are specifically against substrate specific loop region of PfLDH and once
the gossypol binds, the 10C4D5 and 10D3G2 monoclonals could not bind to
substrate specific loop regionof PfLDH.
Microplate Sandwich ELISA for Diagnosis of MalariaThe potential of Microplate Sandwich ELISA for the detection of malaria
parasites (based on pLDH detection) in finger prick blood samples was
evaluated. The sensitivity of the Microplate Sandwich ELISA was done using
P. falciparum infected blood (1% parasitaemia) diluted two fold serially in
normal human blood and the Microplate Sandwich ELISA was positive up to
0.001% parasitaemia. Out of 120 malaria blood samples positive by microscopic
examination (parasitaemia 0.01–0.4%), 118 (98%) were found positive for
malaria by Microplate Sandwich ELISA. None of the endemic and non-
endemic controls samples was found positive by Microplate Sandwich ELISA
and microscopy (Table 3).
DISCUSSION
The monoclonal antibodies have been shown to be powerful tools in defining
structure-function relationship of parasite enzymes and also for identifying
subtle differences between the parasite and host enzymes (Goldman-Leikin &
Goldberg, 1983; Kaushal, 1994; Kaushal et al., 1995). The plasmodial lactate
dehydrogenase is known to be a potential target for chemotherapy (Dunn
et al., 1996) as well as for immunodiagnosis (Kaushal & Kaushal, 2002; Piper
at al., 1999) of malaria in view of its immunological (Kaushal et al., 1988;
Watts et al., 1987) and structural (Bzik et al., 1993; Kaushal et al., 1993; Singh
et al., 2012) differences from the analogous host enzyme.
In the present study, we have produced and characterized six monoclonal
antibodies against P. falciparum LDH. Two of these monoclonals (10C4D5 and
10D3G2) were found to be specific against the substrate specific loop region
of PfLDH and strongly inhibited the enzyme activity of parasite LDH.
Table 3. Detection of parasite LDH in malaria blood samples by Microplate SandwichELISA.
Positive for Malaria
Blood samples Number tested MicroscopyMicroplate
Sandwich ELISA
Malaria Cases 120 120 118Endemic Controls 30 0 0Non-endemic controls 25 0 0
Blood samples from malaria parasites (Positive for malaria parasites by microscopy),endemic controls and non-endemic controls were tested at 1:2 dilutions in MicroplateSandwich ELISA.
The parasitaemia of malaria positive blood samples was in the range of 0.01–0.4%.
N. A. Kaushal & D. C. Kaushal566
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The affinities of these two inhibitory monoclonals and the two non-inhibitory
monoclonals (10A5H5 and 10B6D1) for PfLDH were significantly higher than
those produced earlier against plasmodial LDH (Lee et al., 2011). Recently,
high affinity monoclonal antibodies have been produced against three
plasmodial recombinant proteins (dihydrofolatereductase-thymidylate syn-
thase, heme detoxification protein and glutamate rich protein) having
potential for malaria diagnosis (Kattenberg et al., 2012). In another study,
out of five monoclonal antibodies produced against P. falciparum1-Cys
peroxiredoxin, two monoclonals of high affinities were able to differentiate
P. falciparum from P. vivax and P. knowlesi (Hakimi et al., 2013).
In the present study, the reactivity of monoclonal antibodies with LDH from
different species of malaria parasites but the lack of their reactivity with
mammalian LDHs (A4, B4, C4) as well as LDH from other sources suggests the
malaria pan-specificity of these monoclonal antibodies. Hurdayal et al. (2010)
have shown the pan-specific nature of polyclonal antibodies against substrate
specific loop region peptide of P. falciparum. Molecular modelling studies
comparing substrate specific loop region of PfLDH with other LDHs
(plasmodial and mammalian LDHs) have shown that it is conserved within
Plasmodium and present on the surface of parasite LDH (Dunn et al., 1996).
However, this substrate specific loop region differed from mammalian LDH by
6 amino acid residues and a 5-amino acid insert in plasmodial LDH (Bzik et al.,
1993; Dunn et al., 1996).
The 10C4D5 inhibitory monoclonal was having more affinity for substrate
specific loop region than 10G3D2 monoclonal as evidenced by strong inhibition
of PfLDH activity by 10C4D5 monoclonal. This inhibitory effect was found to
be PfLDH specific as these monoclonals did not have any effect on the activities
of LDH from mammalian and bacterial sources. The inhibitory type polyclonal
antibodies against aldolase (Srivastava et al., 1990) and glucose phosphate
isomerase (Srivastava et al., 1992) of malaria parasites have been reported.
The monoclonals produced in the present study revealed the presence of three
epitopes on PfLDH and we were able to identify the target epitope of two
monoclonals (10C4D5 and 10G3D2) which are against substrate specific loop
region of PfLDH. The other four monoclonal antibodies, which have no
inhibitory effect on PfLDH activity, may be directed against other two epitopes
on PfLDH.
The potential of PfLDH for the development of novel anti-malarials has
been highlighted by a number of studies. Gossypol and its synthetic deriva-
tives have been reported to have antimalarial activity (Brady & Cameron,
2004; Dunn et al., 1996; Vander Jagt et al., 1984). The gossypol is shown to
exert its effect by binding to the substrate specific loop region and thus
inhibiting the activity of malaria parasite LDH. A series of azole-based
compounds have been shown by crystallographic studies to selectively
bind directly to the substrate binding loop region site of PfLDH. These
compounds inhibit PfLDH activity and kill the drug-resistant strain of
P. falciparum in vitro and suppress parasitaemia in P. berghei rodent model
(Cameron et al., 2004; Conners et al., 2005). In the present study, we have used
gossypol as a tool to confirm that inhibitory monoclonals are binding to the
substrate specific loop region site of PfLDH. The inability of 10C4D5 or
10G3D2 inhibitory monoclonals to bind to PfLDH-gossypol complex in
P. falciparum LDH Loop Region Peptide Monoclonals 567
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Microplate Sandwich ELISA suggests that both the monoclonals (10C4D5 and
10G3D2) and gossypol are binding to the same site of PfLDH substrate specific
loop region and once the gossypol binds, the inhibitory monoclonals
cannot bind.
This monoclonal antibody Microplate Sandwich ELISA can be used to
screen pLDH inhibitors for developing novel anti-malarial drugs targeting
plasmodial LDH. Monoclonal antibody-based immunoassays have also been
used for in vitro screening of antimalarial compounds selected by molecular
docking studies (Penna-Coutinho, 2011). The in silico approach for screening of
NADH analogs is purely qualitative and it is mandatory to know the structure
of the compounds to conduct molecular docking studies. Our Microplate
Sandwich ELISA is quantitative and can be used to screen the plant extracts
or compounds of unknown structures. Keluskar & Ingle (2012) have studied
the effect of crude plant extracts on recombinant Pf and Pv LDHs employ-
ing the spectrophotometric method, but the purified enzyme is required for
these studies. In our Microplate Sandwich ELISA, crude parasite extract/
bacterial lysate can be used since we capture the enzyme using the monoclonal
antibody.
In our earlier studies we have shown the potential of antibodies against
P. knowlesi LDH (both polyclonal and monoclonal) for the detection of
plasmodial LDH in malaria blood samples (Kaushal et al., 1995; Kaushal &
Kaushal, 2002). In the present study, the diagnostic potential of two high
affinity monoclonal antibodies (10A5H5 and 10C4D5) was determined in
Microplate Sandwich ELISA which could detect parasite LDH in malaria blood
samples. The monoclonal based Microplate Sandwich ELISA that we have
developed in the present study is for clinical diagnosis of malaria. This
Microplate Sandwich ELISA can detect 0.001% parasites which is equivalent
to 50 parasites/ml of blood and the detection limit is comparable with other
monoclonals used earlier for malaria diagnosis (Wilson, 2012). These mono-
clonal antibodies are pan-specific and can detect both Pf and Pv parasites.
Recently, Atchade et al. (2013) have used monoclonal antibody based
commercially developed Sandwich ELISA system for screening of blood bank
samples and their test can detect 1 parasite/ml of blood samples. However, they
have used two conjugates system (Avidin-Streptavidin) which may be costly.
Thus, in the present study we have successfully generated six monoclonal
antibodies directed against three epitopes of PfLDH. All the six monoclonal
antibodies are plasmodium specific as shown by their reactivity with LDHs
from different species of malarial parasites and no reactivity with mammalian
and bacterial LDHs. Two high affinity monoclonal antibodies (10C4D5 and
10G3D2) bind to substrate specific loop region peptide and inhibit the enzyme
activity of PfLDH. We have developed a Microplate Sandwich ELISA
employing the monoclonal antibodies against different epitopes of PfLDH
and our findings demonstrate that the inhibitory monoclonals (10C4D5 and
10D3G2) compete with gossypol (pLDH inhibitor) for binding to substrate
specific loop region peptide of PfLDH. The present study has provided a
reliable and specific Microplate Sandwich ELISA for high throughput screen-
ing of inhibitors/compounds binding to the substrate specific loop region of
PfLDH, which may enable a rational design and modification of anti-malarial
drugs from natural sources or from the combinatory chemical library.
N. A. Kaushal & D. C. Kaushal568
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This Microplate Sandwich ELISA can also be used for diagnosis of malaria
using finger prick blood samples.
ACKNOWLEDGEMENTS
The financial support provided by the Department of Biotechnology,
Govt. of India, New Delhi, India is acknowledged. The help of
Dr N.N. Mehrotra, a former Scientist from CSIR-CDRI is gratefully acknowl-
edged for editorial inputs in the manuscript. CSIR-CDRI Communication
No.: 8618.
DECLARATION OF INTEREST
The authors report no conflicts of interest. Both the authors equally conceived
and designed the experiments, performed experiments, analyzed the data and
wrote the paper.
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