Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase

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

N. A. Kaushal & D. C. Kaushal558

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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


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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


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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.


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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.


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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.


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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%.


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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


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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.


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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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Documents

Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase