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Immunochemical Assays for Determination of Organophosphorus Pesticides in Milk
Yavor Ivanov and Tzonka Godjevargova*
Prof. dr A. Zlatarov University, Bulgaria
Submission: April 10, 2019; Published: April 22, 2019
*Corresponding author: Tzonka Godjevargova, Department of Biotechnology, Prof. dr A. Zlatarov University, Burgas, Bulgaria
How to cite this article: Yavor Ivanov, Tzonka Godjevargova. Immunochemical Assays for Determination of Organophosphorus Pesticides in Milk. Dairy
and Vet Sci J. 2019; 11(3): 555811. DOI: 10.19080/JDVS.2019.11.555811
The detection of pesticide residues is an important task in ensuring the safety of milk. In the last decade, organophosphorus insecticides have been used, but they also pose a serious risk because of their high toxicity. For fast screening of milk, immunochemical methods of analysis are appropriate. Enzyme-linked immunosorbent assay is the most common assay mode and base of many commercialized assay kits. Residues of some organophosphorus compounds in milk and dairy products determined by ELISA are shown. For fast screening of pesticides, the lateral flow strips are also described. The advantages of a new developed method MNPs-based immunoassay for determination of phosphorus pesticides in milk was described and some publications were represented. In recent years, simultaneous identification of more than one pesticide is increasingly perceptual, that way the examples for multi-immunoassays for determination of organophosphorus pesticides was presented.
Milk and dairy products are widely used food products which are being consumed by people of different age groups. In this regard, the quality and safety of raw milk is an important issue [1,2]. The detection of pesticide residues is an important task in ensuring the safety of milk. The use of organochloride pesticide like dichloro diphenyl trichloroethane (DDT) and hexachlorocyclohexane (HCH) was banned. However, the residual effects are still causing problems [3-5]. The residues of DDT persist in the environment for long periods and enter the food chain. In the last decade, organophosphorus insecticides (OPP) and carbamyl derivatives have been used that are relatively easy to degrade in the environment but also pose a serious risk because of their high toxicity. There are variety publications reporting organic compounds exceeding established maximum residue limits (MRL), for different OP pesticides, like fenthion, dichlorvos, methyl parathion, chlorpyrifos, and malathion in milk [6-8]. Melgar et al.  founded that the frequency of total samples containing detectable levels of OPP residues was 6.73 % in total milk and 8.67 % in raw milk. The highest percentage incidence measured was for dichlorvos (5.78 %), followed by coumaphos (2.06 %), and parathion methyl (0.83 %). Salas et al.  described that
approximately 39.6% of the pasteurized Mexican milk samples contained detectable levels of OP pesticide residues. Eight samples contained residues exceeding established maximum residue limits (MRL). They found that the OP pesticides present in these samples were dichlorvos, phorate, chlorpyrifos, and chlorfenvinphos). OP pesticides are strong cholinesterase inhibitors and they are like neurotransmitters for acetylcholinesterase, butyrylcholinesterase and pseudocholinesterase. When these enzymes are inhibited, acetylcholine accumulates in the neurons of the muscle nodes or synapses and changes the natural path of the nervous impulse. Acetylcholine accumulation causes a number of diseases like Alzheimer’s disease and Parkinsonian disorders, and also many other degenerative diseases [9,10].
Pesticide analysis is performed by liquid, gas and high performance liquid chromatography (HPLC), and HPLC along with Mass Spectrometry [11,12]. These methods are very suitable for monitoring toxic pesticides but have a number of disadvantages: expensive, time-consuming to prepare samples, require well-trained personnel, and are not adapted to conduct on-site and (on time) real-time analysis. For fast screening of milk, immunochemical methods of analysis  are appropriate. They are simple, fast, cheap, sensitive methods, provide real-time and
time-based analysis, and do not require costly instrumentation.
In addition, the immunoassay is also suitable for the analysis of
milk, since milk samples can be analyzed after simple pretreatment
such as dilution or protein precipitation.
Immunochemical methods are based on the reaction between
the antigen (the pesticides studied) and the respectively antibody
specific, which is very selective and rapid . Since pesticides
are small molecules, they cannot be detected by sandwich
immunoassay. A competitive method should be used whereby
the antigen in the sample competes with the labeled pesticide to
bind to the antibody . From immunochemical methods, the
largest application for the determination of organophosphorus
pesticides is the enzyme-linked immunosorbent assay (ELISA)
and the immunofluorescent assay (IFA).
A significant number of ELISA assays were developed to
identify different organophosphorus pesticides . Analyzes
are sensitive and detect very low concentrations of pesticides.
Enzyme-linked immunosorbent assay (ELISA) is the most
common assay mode and base of many commercialized assay
kits. The preparation of monoclonal and polyclonal antibody
against organophosphorus pesticides, and the antigen-enzyme
conjugate synthesis, is the very important task [17-20]. The
most common markers for those conjugates are the enzymes
horseradish peroxidase and alkaline phosphatase. Quantification
of the target analytes is based on corresponding substrate color
change. Residues of some organophosphorus compounds in milk
and dairy products determined by ELISA are shown in Table 1.
In recent years, a number of analyzes for pesticides in milk
have been done based on fluorescent-linked immunosorbent
assay (FLISA) [21-24]. This method is highly sensitive, because
combined selectivity of antibody and sensitivity of fluorescent
marker [25,26]. Different fluorescent markers are used: organic
fluorescent dyes, fluorescent marker proteins, green fluorescent
proteins, quantum dots . ELISA and FLISA are simple and
low cost methods, but analysis is multi-stage, requires multiple
washing and takes a long time (Figure 1).
For fast screening of pesticides, the lateral flow strips are
also used, but they are less sensitive and inaccurate. Lateral flow
tests are also known as lateral flow immunochromatographic
assays. The lateral flow strips are based on a competitive
analysis. Unlabeled antigen and competitive antigen-marker
are transported by the flow onto a nitrocellulose membrane,
and they are competing for coupling with antibody immobilized
onto the membrane . The label most commonly used in
immunochromatographic assay is colloidal gold, enzyme,
magnetic nanoparticles . The lateral flow strips are able to
detect low pesticide concentrations. Тheir low detection limit is
in the range of ng/mL, but often have false results. Residues of
some organophosphorus compounds determined by lateral flow
tests are shown in Table 2 [29-32].
The IFA for pesticide determining does not draw back from
the sensitivity of the ELISA method but does not require repeated
washing and is a faster method . Fluorescence polarization
immuno-analysis (FPIA) is quick and applicable method. There
many publications about OP pesticide detection. Anna Yu.
Kolosova, et al.  was developed and optimized monoclonal
antibody for the detection of parathion-methyl (PM). PM
concentration determinable by the FPIA ranged from 25 to 10000
ppb. The detection limit was 15 ppb. Xu et al.  make the FPIA
which detect 5 OPs simultaneously with a limit of detection below
10ng/mL. Heterogeneous IFA is preferable to homogeneous
as it provides a more stable test, due to the immobilization of
antibodies onto solid supports . Application of nanoparticles
as carriers increases the degree of immobilization of the antibody
enhances the sensitivity of the immunoassay and preserves the
activity of the immobilized antibody over time . Particularly
suitable for this purpose are magnetic nanoparticles (MNPs),
since due to the applied magnetics separation accelerates and
facilitates the conduct of the assay, reducing the interference of
the interfering components in the milk sample and preserves the
activity of the immobilized antibody over time . MNPs have a
high specific surface area and provide good contact between the
immobilized antibody and the antigen and correspondingly high
rate of immunoreaction. The principle of the method is based on
competitive analysis. The target antigen and antigen-fluorescent
dye conjugate compete for coupling to antibody immobilized
onto MNPs (Table 3).
At the first step, OPP containing sample is added to MNPs
with immobilized antibody. Then, fluorescent conjugate (OPPfluorescent
dye) is added and the conjugate is coupled to
the non-occupied binding sites of the antibody. The MNP are
collected with a permanent magnet. After that, the fluorescence
intensity of the uncoupled conjugate (OPP-fluorescent dye)
in the supernatant is measured. The excess of the conjugate is
directly proportional to the OPP concentration in the sample.
On this principle have been developed assays for the detection
of ochratoxin, aflatoxin M1, enterotoxin A, and antibiotics in
milk [38-41]. There are few publications about MNP-based
immunoassay for the determination of phosphorus pesticides in
Testing several pesticides simultaneously in one sample
is a very perceptual analysis. In recent years, simultaneous
identification of more than one pesticide  is increasingly
perceptual. To achieve this, it is possible to increase the number
of antibodies that recognize individual antigens in one sample.
A more economical alternative, however, is to create a single
antibody that is able to identify several analytes in a single test
and which is termed a broad-specific antibody. First, multihapten
antigen was prepared by sequentially conjugating
haptens of four different pesticides (chlorpyrifos, triazophos,
carbofuran and parathion methyl) to the carrier protein bovine
serum albumin (BSA) . An antibody with wide cross reactivity
is then obtained.
For example, Liang et al. [47,48] synthesized a common
hapten of several O, O-dimethyl organophosphorus pesticides to
produce a broad-spectrum antibody. The hapten is conjugated
to bovine albumin (BSA) to produce an immunogen. Rabbits
were immunized with the resulting immunogen and polyclonal
antisera was produced against each of the antigens.
A common, broadly specific enzyme-linked immunosorbent
assay for malathion, dimethoate, phenate, phosmet, methidathion,
fenitrothion, methyl parathion and fenthion has been developed.
The following IC50 values were determined under optimal
conditions: 30.μg / L for malathion, 28.9μg / L for dimethoate,
88.3μg / L for fentoate, 159.7μg / L for phosmet, 191.7μg / L for methidathion, 324.0μg / L for fenitrothion, 483.9μg / L of methyl
parathion and 788.9μg / L for fenthion. Antibodies produced
using the multi-hapten strategy have much lower affinities to
analytes compared to antibodies derived from a single hapten.
Probably this is due to a lower hapten-protein molecular ratio
for each individual hapten in the multi-hapten immunogen
compared with those immunogens that contained only a single
hapten . Mixing several single antigens together based on
a certain proportion to form a multi-hapten immunogen was
also used for the preparation of mixed antibodies [49,50]. Each
hapten is coupled to a carrier protein individually and then the
mixture is used as immunogen. In this case, a lower titer of multivariate
was established as compared to the obtained antibodies
with only one individual hapten.
The immunochemical assays for determination of organophosphorus
pesticides are simple, fast, cheap, sensitive methods,
provide real-time and time-based analysis, and do not require
costly instrumentation. In addition, the immunoassay is
also suitable for the analysis of milk, since milk samples can be
analyzed after simple pretreatment such as dilution or protein
precipitation. The developed MNPs based IFA and multi-assays
are very perspective assays, suitable for determination of organophosphorus
pesticides in milk.