Journal of Natural Medicines (2018) 72:32–42

https://doi.org/10.1007/s11418-017-1144-z

REVIEW

Enzyme‑linked immunosorbent assay for the quantitative/qualitative

analysis of plant secondary metabolites
Seiichi Sakamoto1   · Waraporn Putalun2 · Sornkanok Vimolmangkang3 · Waranyoo Phoolcharoen3 ·
Yukihiro Shoyama4 · Hiroyuki Tanaka1 · Satoshi Morimoto1

Received: 23 June 2017 / Accepted: 9 October 2017 / Published online: 21 November 2017
© The Author(s) 2017. This article is an open access publication

Abstract
Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoas-
says is based on specific antigen–antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic
studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to
develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for
which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoas-
says are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced
with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/
enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in
recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article
describes ELISA and its applications for the detection of plant secondary metabolites.

Keywords  Antibodies · Enzyme-linked immunosorbent assay (ELISA) · Hapten · Plant secondary metabolites

Introduction

Since the development of radioimmunoassay (RIA) in 1960,

there has been a rapid increase in immunoassay techniques
The original version of this article was revised due to a using radioactive labels [1]. However, radioactive labels
retrospective Open Access order. have been gradually replaced with enzyme labels because of
safety concerns associated with radioactivity since the study
* Seiichi Sakamoto
[email protected]‑u.ac.jp by Avrameas in 1969, who coupled antigens or antibodies
and enzymes using glutaraldehyde [2]. Currently, ELISA
* Hiroyuki Tanaka
[email protected]‑u.ac.jp has a higher number of immunoassays compared to RIA.
Plant secondary metabolites are plant-produced organic
1
Department of Pharmacognosy, Graduate School compounds that play an important role in the defense of
of Pharmaceutical Sciences, Kyushu University, 3‑1‑1 plants against herbivores, pests, and pathogens, as well as
Maidashi, Higashi‑ku, Fukuoka 812‑8582, Japan
in their adaptation to the environment, although they are not
2
Research Group for Pharmaceutical Activities of Natural directly involved in the growth and development of organ-
Products using Pharmaceutical Biotechnology (PANPB),
Faculty of Pharmaceutical Sciences, Khon Kaen University, isms [3, 4]. Because of their diverse functions, there has
Khon Kaen 40002, Thailand been a dramatic increase in their demand in pharmaceu-
3
Department of Pharmacognosy and Pharmaceutical Botany, ticals, cosmetics, and pesticides, as well as in food addi-
Faculty of Pharmaceutical Sciences, Chulalongkorn tives [5]. Quality control of these commercial products
University, 254 Phayathai Rd. Pathumwan, Bangkok 10330, containing secondary metabolites is crucial as the quality
Thailand directly affects their potential activity. In addition, Cragg
4
Department of Pharmacognosy, Faculty of Pharmaceutical and Newman recently reported that 34% of the currently
Sciences, Nagasaki International University, 2825‑7 Huis Ten used drugs originate from natural products [6]. Meanwhile,
Bosch, Sasebo, Nagasaki 859‑3298, Japan

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Journal of Natural Medicines (2018) 72:32–42 33

simple, selective, and sensitive analytical techniques are also produce p-nitrophenol, which can be detected at 405 nm
required in pharmacodynamic studies for monitoring effec- (yellow color), and HRP catalyzes the conversion of chro-
tive concentration, side effects, and metabolism, leading to mogenic substrates, e.g., 2,2′-azino-bis(3-ethylbenzothia-
a better quality of life for patients. Thus far, various analyti- zoline-6-sulfonic acid) diammonium salt, 3,3′,5,5′-tetra-
cal methods have been developed for such purposes, mainly methylbenzidine, and o-phenylenediamine into colored
based on high-performance liquid chromatography (HPLC). products. By using chemiluminescent substrates such as
However, ELISA exhibits several advantages over such tech- chloro-5-substituted adamantyl-1,2-dioxetane phosphate
niques because of its simplicity, selectivity, and sensitivity. and luminol for ALP and HRP, respectively, and fluoro-
The basic facts about ELISA and its practical use for genic substrates such as 4-methylumbelliferyl galactoside
measuring plant secondary metabolites are described in and nitrophenyl galactoside for β-galactosidase, even more
this review. sensitive detection can be achieved. These enzyme–sub-
strate reactions are typically completed within 30–60 min,
and the reaction stops with the addition of an appropriate
General principle of ELISA solution, e.g., sodium hydroxide, hydrochloric acid, sul-
furic acid, sodium carbonate, and sodium azide, for indi-
ELISA is based on the concept of antigen–antibody reac- vidual reactions [12, 13]. Finally, colored or fluorescent
tions, representing the chemical interaction between anti- products are detected using a microtiter plate reader.
bodies produced by the B cells of leukocytes and antigens.
This specific immune response plays an important role in
protecting the body from invaders such as pathogens and
toxins. Hence, by exploiting this reaction, ELISA permits Advantages and disadvantages of ELISA
the highly sensitive and selective quantitative/qualitative
analysis of antigens, including proteins, peptides, nucleic Advantages and disadvantages of ELISA are summarized
acids, hormones, herbicides, and plant secondary metabo- in Table  1. ELISA exhibits the following advantages:
lites. To detect these molecules, an antigen or antibody is (i) Simple procedure. (ii) High specificity and sensitiv-
labeled using enzymes, the so-called enzyme immunoas- ity, because of an antigen–antibody reaction. (iii) High
say, in which alkaline phosphatase (ALP) [7], horseradish efficiency, as simultaneous analyses can be performed
peroxidase (HRP) [8], and β-galactosidase [9–11] are com- without complicated sample pre-treatment. (iv) Gener-
monly used. The antigen in the fluid phase is immobilized ally safe and eco-friendly, because radioactive substances
on a solid phase, such as a microtiter plate constituting and large amounts of organic solvents are not required.
rigid polystyrene, polyvinyl chloride, and polypropylene. (v) Cost-effective assay, as low-cost reagents are used.
Subsequently, the antigen is allowed to react with a specific However, ELISA exhibits the following disadvantages:
antibody, which is detected by an enzyme-labeled second- (i) Labor-intensive and expensive to prepare antibody
ary antibody. The development of color using a chromog- because it is a sophisticated technique, and expensive cul-
enic substrate corresponds to the presence of the antigen. ture cell media are required to obtain a specific antibody.
For instance, ALP hydrolyzes p-nitrophenyl phosphate to (ii) High possibility of false positive or negative results

Table 1  Advantages and disadvantages of ELISA

Advantages Disadvantages

Simple procedure Labor-intensive and expensive to prepare antibody

 Easy to perform with simple procedure Sophisticated techniques and expensive culture media are required
High specificity and sensitivity High possibility of false positive/negative
 ELISA is based on antigen–antibody reaction Insufficient blocking of immobilized antigen results in false results
High efficiency Antibody instability
 Simultaneous analysis can be performed without complicated Refrigerated transport and storage are required as an antibody is a protein
sample pre-treatment
Generally safe and eco-friendly
 Radioactive substances and large amounts of organic solvent are not
required
Cost-effective assay
 Reagents are relatively low cost

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34 Journal of Natural Medicines (2018) 72:32–42

because of insufficient blocking of the surface of microtiter

plate immobilized with antigen. (iii) Antibody instability
because an antibody is a protein that requires refrigerated
transport and storage.

Types of ELISA

Direct ELISA

In 1971, Engvall and Perlmann [14] and Van Weemen and

Schuurs [15] were the first to develop direct ELISA (Fig. 1),
which was the base style for other types of ELISA. Primar-
ily, an antigen or an antibody is immobilized on the surface
of microtiter plate. After the surface is blocked with other
proteins (e.g., albumin, gelatin, casein, and skimmed-milk
[13]) to avoid the non-specific adsorption of other pro-
teins, the corresponding enzyme-labeled antibody or anti-
gen is allowed to react with the immobilized targets, fol-
lowed by color development with appropriate substrates.
With an increasing amount of targets, the signal increases.
Direct ELISA is suitable for the qualitative analysis of Fig. 2  Competitive ELISA to detect antigen (a) and antibody (b). (i)
macromolecules. Attach antigen/antibody to solid phase. (ii) Incubate antibody/antigen
with enzyme-labeled antibody/antigen. (iii) Wash unbound enzyme-
labeled antibody/antigen out. (iv) Develop color with substrate
Competitive ELISA

In 1973, Belanger developed competitive ELISA (Fig. 2) of indirect ELISA and sandwich ELISA [16]. The key
to detect rat α-fetoprotein, which involved the development event of competitive ELISA is the competitive reaction
between targets (antigen or antibody) in the sample and
enzyme-labeled targets (antigen or antibody) against cor-
responding immobilized antibody or antigen. To detect the
antigen in competitive ELISA, an enzyme-labeled anti-
gen is used to compete with the target antigens against
the immobilized antibody (Fig. 2b). Hence, the higher the
amount of antigen in the sample, the lower the amount of
enzyme-labeled antigen that binds to the antibody. That
is, with an increasing amount of target antigen, the sig-
nal decreases. In this case, competitive ELISA is suitable
for measuring macromolecules only because a labeling
enzyme is required to measure the antigen. If the antigen
is a low molecular weight compound (e.g., hapten), result-
ant hapten–enzyme conjugates are not recognized by the
immobilized antibody, leading to failure of the analysis.
To detect the antibody, the antigen is immobilized, and
the competition between the antibody in the sample and
enzyme-labeled antibody is observed (Fig. 2a). In this
case, both macromolecules and hapten can be detected
when hapten is exposed on the surface of the microtiter
plate.
Furthermore, detectable targets (antigen or antibody)
Fig. 1  Direct ELISA to detect antigen (a) and antibody (b). (i) Attach
can be changed depending on the competitors. When free
antigen/antibody to solid phase. (ii) Incubate with enzyme-labeled
antibody/antigen. (iii) Wash unbound enzyme-labeled antibody/anti- antigen is used as competitor instead of unlabeled antibody
gen out. (iv) Develop color with substrate in Fig. 2a, competitive reaction between free antigen and

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Journal of Natural Medicines (2018) 72:32–42 35

immobilized antigen against enzyme-labeled antibody can

be observed, enabling the detection of free antigen (mac-
romolecules and hapten) in the sample in this competitive
system, and vice versa when free antibody is used instead of
unlabeled antigen in Fig. 2b.
Direct and competitive ELISA methods are simple
because only one antibody is required. However, the labe-
ling step is required for each of the ELISA methods, possibly
leading to inactivation of the antibody (Table 2).

Indirect ELISA

Indirect ELISA systems have been developed on the basis of

direct ELISA to evaluate the presence of antibody in antisera
(Fig. 3) [17, 18]. The key step of this system is the two-
binding process of the primary antibody and enzyme-labeled
secondary antibody. That is, the target antigen is indirectly
detected by the secondary antibody, which is labeled with
the enzyme, or the so-called indirect ELISA. The antigen is
primarily immobilized on the surface of the microtiter plate,
which blocks the surface with blocking proteins as men-
tioned above. The primary antibody (in antisera) binding
to the immobilized antigen is then allowed to react with the Fig. 3  Indirect ELISA to analyze antibody. (i) Attach antigen to solid
enzyme-labeled secondary antibody, followed by the devel- phase. (ii) Incubate with primary antibody. (iii) Wash unbound pri-
mary antibody out. (iv) Incubate with enzyme-labeled secondary
opment of color. The signal increases with an increasing
antibody. (v) Develop color with substrate
amount of the immobilized target antigen. Indirect ELISA
is suitable for measuring macromolecules. With the use of
antisera as the primary antibody, the presence of a disease- The target antigen is immobilized on a solid phase of the
associated antibody in the antisera can be evaluated; thus, microtiter plate and is blocked. Subsequently, free target
indirect ELISA is effectively used to diagnose endocrine antigen and antibody are allowed to incubate and there is a
diseases [19, 20]. competition between the immobilized antigen and free anti-
gen against antibodies. The primary antibody that binds to
Indirect competitive ELISA the immobilized antigen is detected by the enzyme-labeled
secondary antibody. Similar to the case in competitive
Indirect competitive ELISA (icELISA) involves the combi- ELISA, in icELISA, the signal decreases with increasing
nation of indirect ELISA and competitive ELISA (Fig. 4). amount of the free antigen. icELISA can be applied for

Table 2  Characteristics of various types of ELISA

Direct ELISA Competitive ELISA Indirect ELISA Indirect competitive Sandwich ELISA
ELISA

Advantage Simple because only one antibody is used Higher sensitivity and versatility than direct High specificity as two
methods owing to usage of PAb that recognizes antibodies possessing
different epitopes of primary antibody different epitopes are
used
Disadvantage Labeling antibody is necessary for each ELISA, Nonspecific signal is induced through cross- To prepare two dif-
which may result in inactivation of antibody reactivity of secondary antibody ferent antibodies is
labor-intensive and
expensive
Target Macromolecules Macromolecules Macromolecules Macromolecules Generally macromol-
(Hapten) (Hapten) ecules
Signal (as Increase Decrease Increase Decrease Increase
target antigen
increase)

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36 Journal of Natural Medicines (2018) 72:32–42

Fig. 4  Indirect competitive ELISA to detect antigen. (i) Attach anti-

gen to solid phase. (ii) Incubate free target antigen with primary
antibody. (iii) Wash unbound free target antigen and primary anti- Fig. 5  Sandwich ELISA for specific detection of antigen. (i) Attach
body out. (iv) Incubate with enzyme-labeled secondary antibody. (v) capture antibody to solid phase. (ii) Incubate with target antigen. (iii)
Develop color with substrate Wash unbound target out. (iv) Incubate with enzyme-labeled anti-
body. (v) Develop color with substrate

measuring both the macromolecules and hapten when hapten

is exposed on the surface of the microtiter plate. adsorption of other proteins, the antigen in the sample is
The use of enzyme-labeled secondary antibodies in indi- allowed to react with the immobilized capture antibody,
rect methods (e.g., indirect ELISA and icELISA) exhibit and the antigen bound to the capture antibody is then sand-
advantages over direct methods (direct and competitive wiched with an enzyme-labeled antibody for color devel-
ELISA) with respect to sensitivity and versatility [16]. opment. This direct system can be modified to the indirect
Polyclonal antibody is a type of enzyme-labeled secondary system by using primary and enzyme-labeled secondary
antibody that recognizes different epitopes of the primary antibodies. The signal increases with increasing amount
antibody, leading to increased sensitivity as compared to of antigen. As two antibodies containing different epitopes
direct methods. In addition, a universal secondary antibody are required against the target antigen, sandwich ELISA
can be used if the original animal species of the primary is generally suitable for measuring macromolecules with
antibody are unified. Thus, the secondary antibody is com- some exceptions. Ciguatoxins, which are produced in the
mercially available, leading to high versatility. Indirect marine dinoflagellate Gambierdiscus toxicus, are the major
ELISA clearly exhibits disadvantages with respect to the causative toxins of ciguatera seafood poisoning. Cigua-
secondary antibody, i.e., the cross-reaction of the secondary toxins are structurally classified as ladder-like polyethers
antibody should be considered (Table 2). with a molecular weight of 1111 Da. Oguri et al. divided
these polyethers into two parts and prepared different
Sandwich ELISA monoclonal antibodies (MAbs) to individually recognize
each part for constructing a sandwich ELISA system to
In this system, the target antigen is detected via anchor- measure ciguatoxins [21]. More recently, Boscolo et al.
ing between two antibodies, which recognize different reported a sandwich ELISA method for marine biotox-
epitopes, or the so-called sandwich system (Fig. 5) [16]. ins, e.g., palytoxins with a molecular weight of 2680 Da
Sandwich ELISA starts from the immobilization of an [22]. The sandwich was formed by using two antibodies
antibody, called a capture antibody, on the microtiter plate. obtained from the same antigen with different antibodies,
After blocking the plate surface to avoid non-specific

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Journal of Natural Medicines (2018) 72:32–42 37

i.e., MAb and PAb, which were used as the capture and ELISA for plant secondary metabolites
primary antibodies, respectively.
A highly specific assay can be obtained via a sandwich sys- Specificity of antibody against hapten
tem because of the use of two antibodies. However, it is an
expensive and labor-intensive process to prepare two antibod- Most of the useful plant secondary metabolites are low
ies. In addition, one more step is required in the sandwich sys- molecular weight compounds (i.e., hapten) with immense
tem because immobilization is necessary for capture antibody, structural diversity, which are generally classified on the
which increases the assay time (Table 2). basis of their biosynthesis pathway [30, 31]. Hence, ELISA
used for their analysis is the competitive type (competitive
Open sandwich ELISA (OS‑ELISA) ELISA or icELISA) using MAb or PAb. When MAb is com-
pared with PAb against hapten, MAb tends to exhibit higher
Advances in DNA technology have enabled the develop- specificity because PAb recognizes several epitopes, while
ment of unique and interesting immunoassays based on the MAb recognizes only one epitope. In addition, hybridoma
interaction of variable regions of heavy ­(VH) and light ­(VL) cells secreting MAb exhibiting desirable characteristics can
chains, which are binding regions for antigens [23]. In the be screened. Pongkitwitoon et al. have prepared PAb against
presence of an antigen, the interaction between ­VH and ­VL bioactive isoflavonoids, daidzin (DZ), by immunizing rab-
regions is enhanced to form a ternary complex. In the afore- bits with DZ–bovine serum albumin (BSA) conjugates to
mentioned report, OS-ELISA started from coating of a solid- develop icELISA [32]. By comparing the cross-reactivity
phase microtiter plate with streptavidin. After blocking, the (CR), which is the factor of specificity calculated by the ratio
­VL region conjugated with biotin was allowed to react with of ­IC50 for DZ to that for the test compounds, of PAb with
streptavidin to immobilize the ­VL region. In the next pro- that of MAb obtained from the same DZ–BSA conjugates
cess, the phage-displayed ­VH region was incubated with hen [33], the specificity of MAb to DZ was greater than that of
egg lysozyme, which was used as an antigen. Finally, the PAb (Table 3).
phage-displayed ­VH regions forming a ternary complex were Apart from the types of antibodies, the design of hapten-
detected by the HRP-labeled antibody to develop color. The carrier proteins considerably affects the specificity of the
obtained signal increases with increasing amount of antigen. resultant antibody. The sodium periodate ­(NaIO4) oxidation
Currently, this OS-ELISA has been modified to be more method is the typical method for preparing the hapten-car-
easy and effective, and several studies on OS-ELISA for rier protein conjugates for glycosides, which involves the
measuring both the macromolecule and hapten have been oxidative cleavage of vicinal 1,2-diols of the sugar moie-
reported [24–27]. ties to form imides with the amino group of lysine residues
in the carrier proteins. Therefore, several anti-glycoside
Types of antibody antibodies are prepared by the conjugates obtained from
the ­NaIO4 oxidation method, which include paeoniflorin
In ELISA, any antibody can be used. In the first report of [34], solamargine [35], bacopaside I [36], saikosaponin a
immunoassay developed by Berson and Yalow, PAb present [37], liquiritin [38], and DZ [32, 33]. However, they tend
in the antisera of immunized guinea pigs was used to detect to exhibit broad CR, especially with compounds contain-
human insulin [1]. However, issues related to the specificity ing similar aglycone parts. To obtain an MAb specific to
of different batches were particularly concerning until the DZ, Yusakul et al. recently designed hapten-carrier protein
development of the MAb technology by Köhler and Milstein conjugates using the Mannich reaction, leading to the pro-
in 1975 [28]. In 1984, together with Niels Kaj Jerne, they duction of highly specific MAb to DZ (Table 3) [39]. Kitis-
were awarded the Nobel Prize in Physiology or Medicine. ripanya et al. investigated the effect of difference between
The emergence of MAb has helped in overcoming the issues the conjugates prepared via the N­ aIO4 oxidation method and
with PAb. Since then, advances in DNA technology have the Mannich reaction on the specificity of the resultant PAb
enabled the production of recombinant antibodies, which against miroestrol, which is a strong estrogenic compound
include single-chain variable fragment (scFv) antibody, produced in Pueraria candollei [40]. The PAb obtained from
bispecific Bis-scFv, fragment antigen-binding (Fab) anti- the hapten conjugate derived from the Mannich reaction
body, bispecific ­Fab2, trispecific ­Fab3, bivalent minibody, exhibits higher specificity to miroesterol than that of the PAb
and multibody (diabody, triabody, and tetrabody) [29]. All of obtained via the ­NaIO4 oxidation method, suggesting that
the aforementioned recombinant antibodies can be applied the Mannich reaction is an important reaction for obtaining
to ELISA, although the corresponding secondary antibodies specific anti-hapten antibodies.
need to be prepared. In addition to the method to prepare hapten-carrier pro-
tein conjugates, the number of hapten molecules bound to

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Table 3  Chemical structures of representative isoflavonoids and

cross-reactivities (CRs) of PAb [32], MAb [33] produced from DZ–
BSA conjugates prepared by N ­ aIO4 oxidation method, and MAb [39]
produced from DZ–cBSA conjugates obtained by Mannich reac-
tion

Isoflavonoids R1 R2 R3

Daidzin (DZ) H Glc– H

Daidzein H H H
Genistin H Glc– OH
Genistein H H OH
Puerarin Glc– H H
Compound CRs of PAb [32] ­(NaIO4 oxidation) CRs of MAb [33] ­(NaIO4 oxidation) CRs of MAb [39]
(Mannich reac-
tion)

Diadzin (DZ) 100 100 100

Daidzein 93.4 16.2 1.6
Genistin 49.0 82.4 0.044
Genistein 45.1 24.4 < 0.015
Puerarin 0.1 3.4 < 0.015

carrier proteins affects the specificity of antibodies. The Utilization of antibody in icELISA depending
hapten numbers are typically evaluated via matrix-assisted on specificity
laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF–MS) using sinapinic acid as the matrix Antibodies exhibiting broad CR sometimes act as a useful
[41]. The relationship between the number of hapten mol- and effective tool for recognizing a bioactive skeleton or a
ecules and antibody specificity has been investigated using group of bioactive compounds because of the simultaneous
a mercaptopropionic acid derivative of atrazine: high anti- determination by icELISA using the antibodies. Ginseno-
body titers with moderate antibody specificity are induced sides are the major compounds produced in ginseng and
from 15–30 hapten molecules per carrier protein, while are classified into two groups according to their structure:
a lower number of hapten molecules exhibits a slower 20(S)-protopanaxadiol and 20(S)-protopanaxatriol [45]. As
immune response with higher specificity [42]. This obser- they are considered as active compounds that exert various
vation was also supported by the PAb against miroesterol pharmacological activities of ginseng, such as tonic, immu-
reported by Kitisripanya et al. [40]. Recently, MAbs against nomodulatory, antimutagenic, and anti-aging activities, they
the Cephalotaxus alkaloid harringtonine and pyrrolizidine are focused as a target for quantitative/qualitative analysis
alkaloid monocrotaline have been independently produced in ELISA. With respect to 20(S)-protopanaxadiol, MAbs
from their BSA conjugates using the ­NaIO4- and N,N′- against G-Rb1 [46] and Rg3 [47] have been produced, while
carbonyldiimidazole-mediated methods, respectively, for those against G-Re [48], G-Rg1 [49], and Rh1 and Rg2 [50]
their determination in plants. Both of the resultant MAbs have been produced as representatives of 20(S)-protopanaxa-
exhibit extremely high specificity to both targets with high triol for their specific determination. Interestingly, Morinaga
sensitivity, although only two hapten molecules are bound et al. have produced MAb against G-Re exhibiting broad CR
to BSA [43, 44]. with G-Rd (76.2%) and G-Rg1 (70.9%) in addition to G-Re
(100%) itself, enabling the development of icELISA for the

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simultaneous determination of the total ginsenosides in plant specificity to Nar in sandwich ELISA using two MAbs dra-
samples (Table 4) [51, 52]. matically increases as compared with that in icELISA using
a single MAb.
Sandwich ELISA
ELISA using a recombinant antibody
Sandwich ELISA has been widely accepted to exhibit higher
specificity and wider working range as compared to the other DNA recombinant technology has enabled the production
types of ELISA. However, it is difficult to prepare two anti- of antibodies in Escherichia coli [56] and other organisms
bodies possessing different epitopes, especially for haptens [57–60]; currently, recombinant antibodies (rAbs) have been
because steric hindrance may disturb the antigen–antibody reported to exhibit several advantages over conventional
reaction because of the small size of the haptens. Therefore, MAb and PAb in terms of production speed, the ability to
true sandwich assays for hapten can be rarely developed, modify properties through mutagenesis, and information on
except for tacrolimus [53], angiotensin II [54], and naringin antibody–target interaction. Among rAbs, scFv and antigen-
(Nar; which is a major flavonoid glycoside found in citrus binding fragment (Fab) of an antibody are structurally inde-
fruits) [55]. Among these haptens, Nar is the smallest hap- pendent units containing antigen-binding sites (Fig. 6). scFv
ten with a molecular weight of 580 Da and the only plant consists of ­VH and ­VL chains with a flexible peptide linker
secondary metabolite. Two hybridoma cell lines secreting of Gly and Ser, where the C-terminus of ­VH is linked to the
different antibodies have been carefully screened, which ena- N-terminus of ­VL and vice versa. Thus, their size decreases
bled construction of a sandwich for Nar [55]. Interestingly, approximately to one-sixth of the original parental IgG

Table 4  Chemical structures of representative ginsenosides and cross-reactivities (CRs) of MAb 4G10 and scFv used for simultaneous determi-
nation of total ginsenosides in plant samples by icELISA [51, 52, 62]

Ginsenosides R1 R2 R3

Protopanaxatriol
 G-Re H Rha1–2Glc–O– Glc–
 G-Rg1 H Glc–O– Glc–
Protopanaxadiol
 G-Rb1 Glc1–2Glc– H Glc1–6Glc–
 G-Rc Glc1–2Glc– H Ara(f)1–6Glc–
 G-Rd Glc1–2Glc– H Glc–
Compound CRs of MAb 4G10 [51] CRs of
GRe-scFv
[62]

Protopanaxatriol
 G-Re 100 100
 G-Rg1 70.9 67.2
Protopanaxadiol
 G-Rb1 < 0.009 < 0.009
 G-Rc < 0.009 < 0.009
 G-Rd 76.2 73.5

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Fig. 6  Schematic diagram of representative antibodies, IgG molecule (a), single chain variable fragment (scFv) antibody (b), and antigen-bind-
ing fragment (Fab) (c)

molecule. Fab consists of a two-binding arm containing V ­ H were found to exhibit better affinity and sensitivity than those
and ­VL chains, in addition to the constant regions of heavy fusing at the N-terminus of GFP.
­(CH1) and light (­ CL) chains. They have become popular as a
probe for ELISA because the original affinity and specificity
of the original IgG molecule are maintained (Table 4). Conclusion
A secondary antibody is required to detect rAbs in
icELISA. The Fc region of immunoglobulin (MAb/PAb) To date, various methods for the quantitative or qualitative
is typically used as the epitope of secondary antibody for analysis of plant secondary metabolites have been developed
high versatility, while tags such as poly His-tag, T7-tag, and because a lot of marketed drugs are generated from plant
E-tag are commonly used as epitopes of secondary antibod- secondary metabolites, such as morphine (analgesic drug),
ies for rAb because they can be genetically incorporated into vinblastine (antineoplastic drug), paclitaxel (antineoplastic
genes without disturbing the tertiary structure and activity drug), quinine (antimalarial drug), digitoxin (cardiotonic
of the rAb. Thus far, various scFvs against plant secondary drug), and so on, and the accurate, sensitive, and selective
metabolites have been constructed and expressed in E. coli evaluation of these drugs leads to safe clinical and general
to develop icELISA, including plumbagin [61], G-Re [62], usages.
DZ [63], wogonin glucuronide [64], and paclitaxel [65]. Simi- In this review, ELISA has been discussed in detail; it is
larly, Fab-based icELISA has been reported for artemisinin, representative of various analytical methods because of its
which is produced from traditional Chinese herbal medicines, several advantages over other analytical methods in terms
e.g., Artemisia annua L. and wogonin glucuronide, for their of simplicity, cost efficiency, and selectivity. However, all
determination [66, 67]. They can be genetically engineered; types of ELISA exhibit more or less advantages and disad-
therefore, fluorescent single-domain antibodies (fluobodies), vantages. A barrier for further development of ELISA is the
chimera proteins of a green fluorescent protein (GFP), and an preparation of specific antibodies against the target hapten.
scFv also have been utilized in immunoassays. This combina- Even in this advanced era, there are many important plant
tion always results in a 1:1 ratio between the fluorochrome and secondary metabolites for which antibodies are not avail-
scFv, which overcomes the disadvantage of direct methods in able. ELISA would be more familiar to us if the antibody
immunoassays, i.e., deactivation of the antibodies with labe- or antibody-mimicking probes that are alternatively used in
ling enzymes. Furthermore, immunoassays using fluobodies ELISA could be obtained more easily.
enabled skipping of the time-consuming secondary antibody
step with high sensitivity. Some studies have focused on these Acknowledgement  This work was funded by a Grant-in-Aid for Young
Scientists (B) [17K15466] of the Japan Society for the Promotion of
useful fluobodies to develop rapid and sensitive fluorescent- Science (JSPS). This work was also supported by the Chulalongkorn
linked immunosorbent assays (FLISA) for plant secondary visiting professor grants.
metabolites, including plumbagin [68] and G-Re [69]. In these
reports, the fluobodies fusing scFv at the C-terminus of GFP

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Journal of Natural Medicines (2018) 72:32–42 41

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