Mohammad Reza Khakzad1, Farzin Hadizadeh2*, Abdolreza Varasteh1, Maliheh Moghaddam2, Mohammad Saadatmandzadeh3 and Mojtaba Sankian2*
1 Department of Immunology, Islamic Azad University, Iran
2Mashhad University of Medical sciences, Iran
3 Ferdowsi University of Mashhad, Iran
Submission: August 20, 2018; Published: November 15, 2018
*Corresponding author: Mojtaba Sankian & Farzin Hadizadeh, Bu-Ali Research Institute, Ferdossi Square, Bu-Ali Square, Mashhad, Iran.
How to cite this article: Mohammad Reza Khakzad, Farzin Hadizadeh, Mojtaba Sankian, et al. An Improved Method to Conjugation of KLH-Diazinon.
Open Acc J of Toxicol. 2018; 3(5): 555621. DOI: 10.19080/OAJT.2018.03.555621.
Monoclonal antibodies are now widely used for hapten detection by immunoassay kits. The first and critical step in development of an immunoassay kit is selection and synthesis of a suitable hapten-carrier. Diazinon as a non-immunogenic hapten should be attached in an appropriate way to a carrier molecule. The suitable design of active hapten is an important stage and challengeable problem for monoclonal antibody production. This study indicates that our procedure for making an active hapten is time saving, easier and less expensive in comparison with previous study findings and, furthermore, resulting in production of a high affinity anti-diazinon monoclonal antibody
The immunoassay techniques are recently used in the assay and detection of low molecular weight poisons, like the residue of organophosphate pesticides in agricultural and food products. The first and critical step in development of an immunoassay kit is selection and synthesis of a suitable hapten.Traditionally, gas chromatography, high performance liquid chromatography and mass spectroscopy are validated techniques for the determination of small molecules, however, these methods are not cost effective and user-friendly.The immunoassay techniques such as enzyme-linked immunosorbent assay (ELISA) have been developed as a simple, rapid, and cost-effective alternative to above mentioned techniques. Diazinon is one of the organophosphate pesticides with wide application in agriculture that are toxic to humans.
Monoclonal antibodies are now widely used for hapten detection by immunoassay kits. The application of immunization is essential as an adjunct to monoclonal antibody production to do so, diazinon as a hapten, should first be attached correctly to a carrier molecule. The suitable design of hapten is an important stage for immunization in animal models to monoclonal antibody production against small molecules. A precise attachment of the hapten to a carrier is very challenging and requires high skilled researchers. The chemical cross-linkers strongly influence a hapten structure and limit availability of its antigenic active sites leading to a loss of effective interaction between hapten and antibodies in immunoassay techniques.
Small organic molecules such as diazinon are non-immunogenic. Therefore, design and synthesis of suitable carrier conjugate is a high fundamental parameter for its immunoassays. Unfortunately, in most cases the complexity of hapten synthetic process, leads to the masking of its functional groups and so lowers titer and affinity of monoclonal antibodies against hapten structure. Another problem is selection of a suitable spacer between hapten and carrier. Conformational effects of the spacer arm on hapten most probably cause invalidity of some immunoassays. Furthermore, type and length of spacer arms and most noteworthy, attachment site is very critical and affect the affinity of the resulting antibodies.
Few cases have been reported regarding the correct attachments of a diazinon to carrier molecules. Unfortunately, the developed assays had low sensitivity and specificity for diazinon detection. Furthermore, one of the strong problems is selection of spacer with suitable length (n = number of atoms) between hapten and carrier protein to prevent possible changes in electrostatic effects of hapten-protein conjugates and, hence, perturbation in the three-dimensional conformation of the hapten. In previous studies for diazinon hapten synthesis, spacer arms such as 3-aminopropanol (n=3) or 6-aminocaproic acid (n=6) have been used.
Commonly, two strategies are discussed for the design of diazinon hapten synthesis. One approach is linker attachment directly to the its aromatic ring. In this strategy, the attachment of the hydrocarbon spacer arm to the oxygen atom
of the diazinon aromatic ring has been used. This approach is
an easy synthesis pathway; however, it is suitable for production
of polyclonal antibodies against diazinon. The other approach
is the connection of a spacer to the aromatic ring through
thiophosphate moiety that is suitable for maximizing exposure
of aromatic ring. At first, this method was introduced by
Heldman et al and was developed later by McAdam et al.& Tae Lee
[5,9,10]. Although, this strategy has difficult synthesis pathway,
but it can be used for monoclonal antibody production against
diazinon. We encountered some difficulties in synthetizing
the reactive hapten diazinon by McAdam [9,11] and Tae Lee 
methods. So, we improved their method in order to optimize
reaction yields. Herein, we described an effective synthesis of
diazinon-linker (6-aminocaproic acid) conjugate, attached to
the diazinon thiophosphate group (Figure 1). Finally, we have
prepared conjugated diazinon (hapten)-KLH which could be
useful as an immunogen.
Sodium hydride (NaH), ethyl dichlorothio phosphate, 6-
aminocaproic acid, N-hydroxy succinimide (NHS), dichloro
methane, 4-dimethyl aminopyri dine, N, N-dicy clohexyl
carbodiimide (DCC), Keyhole limpet hemocyanin (KLH), were
purchased from Sigma Aldrich (USA). Technical diazinon was
gifted by Farad Company, Iran.
All the other reagents, including KOH, anhydrous
dimethylformamide (DMF), Toluene, MeOH, EtOH, silica gel/
hexane/ethyl acetic acid (200:65:7), Sephadex G-25, MgSO4,
Fatty acid free bovine serum albumin, were of the highest quality
available from commercial sources. Dialysis membrane (cut off
12000 Da) was obtained from Himedia Laboratories (India).
The Chemical reactions were monitored and purified by thinlayer
chromatography (TLC) plates (Silica Gel 60 F 254, Merck Germany) eluting by chloroform-petroleum ether, methanol
(30:70:1) solution and detected with a UV-transilluminator
at wavelength of 365nm. H NMR spectra were run on Bruker
spectrometer (300MHz) using TMS as an internal standard.
UV spectra were taken on a CE9500 Cecil Spectrophotometer
Preparation of pyrimidone : To prepare Pyrimidone
intermediate , Technical diazinon 1 (23g) dissolved in 4ml of
EtOH70%, containing 50mg KOH, mixed and refluxed for 6 hours
in 100°C under stirring.The reaction mixture was neutralized
with 2N HCl. Afterwards, pyrimidone  was extracted from the
reaction mixture by addition of an equal volume of, chloroform
in petroleum ether (1:9) (×2). The extract was vacuum-dried for
1h at 40-45 °C to give 6.4280g (yield of 55.6 %) of pyrimidone
2. The 1H-NMR spectrum was then recorded. 1H NMR (CDCl3): δ
6.74 (1H, s, H-py), 2.93 (1H, m, J = 6.9, CH), 2. 30(3H, s, py-CH3),
1.33(6H, d, C H3).
Preparation of O-Ethyl O- [6-methyl-2-(1-methylethyl)-
pyrimidinyl] phosphorochloridothioate : Next, Pyrimidone
 (150mg, 0.98mmol) dissolved in 1 mL anhydrous DMF. Sodium
hydride (NaH, 60mg, 2.5mmol) was dissolved into anhydrous
toluene (5mL) and added spontaneously to pyrimidone 
solution. The mixtures were stirred for 15min at RT. Salt sodium
of 2 was prepared and ethyl dichlorothiophosphate was added
(140μL) and stirred (rpm= 100) at RT for 3 days. Then, the
product was isolated by a TLC method with chloroform-ethyl
acetate-methanol (30:70:1) to give oily 164.64mg, 0.56mmol
(yield of 57%) of 3. The 1H-NMR spectrum was then recorded.1H
NMR (CDCl3): δ 6.73 (1H, s, H- py), 4. 47 (2H, q, CH2O), 3.15 (1H,
m, CH), 2.53(3H, s, py-CH3), 1.37 (3H, t, CH3), 1.32(6H, d, CH3).
Preparation of O-Ethyl O-[6-methyl-2-(1-methylethyl)-
pyrimidinyl] N-(4-carboxypentyl) phosphorami
dothioate: A stirred solution of 100mg (0.34 mmol) of 3 in
0.3mL of MeOH cooled in an ice-water bath was added dropwise
to a solution of 50mg (0.9 mmol) of KOH. 53mg (0.41mmol) of
6-aminocaproic acid in 1 mL MeOH was added and stirred for 15
min at RT. After stirring, to transfer the product  from aqueous
phase to organic phase, the mixture was filtered and acidified
with adding 1N HCl (2ml) extracted with 2ml of chloroform
The organic extract was dried over MgSO4, and concentrated
in a rotary evaporator at 40-45°C to about 500μl and then
purified by chromatography on silica-gel column eluting with
hexane-ethyl acetate-acetic acid (200:65:7). A yellow oil
(95mg,0.24mmol, 71.7% yield)and stored at -20°C until used.
The 1H-NMR spectrum was then recorded.1H NMR (CDCl3): δ
11.5(1H, bs, COOH), 6.31 (1H, s, py), 4.07 (2H, m, CH2O), 3.72
(3H, m, NHCH2), 3.14(1H, m, CH) , 2.54(3H, s, py-CH3), 2.25
(2H,m, CH2CO2), 1.55 – 1.20(9H, m, CH2, CH3).
Preparation of Hapten active ester : For immunization
purposes, the carboxylic acid group of the above prepared Hapten
 was covalently attached to amino groups of carrier protein
(KLH or OVA), using NHS and DCC as an activating ester reagent.
Briefly, NHS, N-hydroxysuccinimide (11mg, 0.0095mmol)
dissolved in 1.5mL dichloromethane and Hapten (40mg,
0.10mmol), 4-dimethylaminopyridine (1.1mg, 0.0087mmol),
and DCC, N, N-dicyclohexylcarbodiimide (20mg, 0.095mmol)
were added to it. The reaction mixture was incubated for 4h at
RT with stirring. Then filtered to remove the dicyclohexylurea,
and the solvent was evaporated under reduced pressure at 40-
50°C to give crude yellow oily active ester . This crude product
was used in the next step without any further purification.
Preparation of Hapten KLH conjugate : Crude activated
hapten  was dissolved in anhydrous DMF (1ml). A solution
of carrier protein (KLH) in 800μL, 0.1M Borate buffer (pH 9),
was added dropwise to 5 solution. Immediately, 2μL of 2N NaOH
was added. The mixture was stirred overnight at 4°Cand then
centrifuged for 5min at 10000rpm to remove any precipitate.
Finally, KLH-hapten conjugate 6 were separated from uncoupled
haptens by dialysis.The reaction solution was dialyzed using
dialysis membrane (cut-off 12 KDa, Himedia, India) against PBS
(10mM phosphate buffer, 137mM NaCl, 2.7 mM KCl, PH 7.4) at
4°C with 3 changes per day for 2 days. The maximum absorption
of native carrier was at 280nm and it was found to be 414nm in
This study is an effort to modify the synthetic process in
preparation of diazinon hapten (Figure 1). Mc Adam and then
Yang Tae Lee have previously reported method for preparation
of diazinon hapten. In their method diazinon  is converted
to pyrimidone intermediate  which is later is converted
to chlorinated intermediate . The latter is subsequently
linked to 6-aminocaproic acid to produce . Acid derivative is
activated by NHS to give active ester  of hapten.Purification
of each product was performed by recrystallization and / or
column chromatography. And finally, active ester is conjugated
to suitable carrier protein such as KLH to give . We repeated
the method exactly as described previously. The major problem
we encountered during our work was insufficient product yield
for in each step and so we could not get enough active ester
as our finished product. So, we have used an improved and
modified approach in developing diazinon hapten conjugates. At
the first stage in preparation of 2 we omitted crystallization step
and after evaporation of organic phase we used the oily residue
2 without any further purification. In the preparation of 3 and 4
we did not change the procedure. In the next step in preparation
of active ester 5 we deleted the final purification process of 5 by
column chromatography and used crude 5 directly in the next
step of carrier protein conjugate 6 preparation. This approach
was significantly successful in an increased production. After
conjugation of 5 to carrier protein, the purification process
by dialysis bag was performed to delete any small molecule
impurity, including unreacted active ester 5 and acid derivative
4 from reaction mixture. The overall yield of our process was
significantly increased, and we isolated carrier protein conjugate
6 in enough quantity.
Due to deleting final time-consuming crystallization and
column chromatography procedure the overall reaction time in
our study was relatively shorter than that of McAdam and others.
The setting of column chromatography usually takes a lot of time
and it is a relatively expensive technique. The yield reported by
Lee for column chromatography of active ester 5 was relatively
low (28%). As it was mentioned above, we repeated the method
exactly as described previously and had low yields. Therefore,
we attempted to replace it by purification of carrier protein
conjugate 6 through dialysis. Of course, this did not affect our
results and we got the final product 6 with a less expensive and
This study involved an improvement in immunoassay method
by using monoclonal antibody against haptenic structure. The
preparation of specific antibodies has a lot of complexity using an
immunizing hapten. The synthesis of the immunizing hapten has
been the most important key in monoclonal antibody production.
Developing immunoassay technology is often expensive, timeconsuming
and challengeable for researchers. Nevertheless, we
well know that we are not allowed to do any major modifications
in the hapten synthesis procedure. Some attempts have been
reported regarding production of monoclonal antibody against
diazinon but none of them were successful.
The structure of carrier conjugate was confirmed through
comparing UV maximum absorption of native KLH and
conjugate and a shifting from 280 to 414nm was observed.
Although, there are many factors that influence on making a
monoclonal antibody, one of the most important of these factors
is correct hapten synthesis. The sensitivity of our monoclonal
antibody detected was found to be 10-100pg/ml (data patented
139650140003001715, Iran).This was significantly lower than
that reported by Lee et al. (4.0ng/ml).
In conclusion, we can say that our reported procedure for
hapten synthesis was time saving, easy and less expensive,
resulting in production of diazinon monoclonal antibody with