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Dimethyl Suberimidate asan Effective Crosslinker forAntibody-Enzyme ConjugationM. C. Sekhar a , G. L. Sharma a , S. Gangal b , A. P.Joshi a & P. U. Sarma aa Centre for Biochemicals , Delhi Univ. , Campus MallRoad, Delhi, 110 007, Indiab Immunology Division , Cancer Research Institute ,T.M.R.C., Parel, Bombay, 400 007, IndiaPublished online: 24 Sep 2006.
To cite this article: M. C. Sekhar , G. L. Sharma , S. Gangal , A. P. Joshi &P. U. Sarma (1991) Dimethyl Suberimidate as an Effective Crosslinker forAntibody-Enzyme Conjugation, Preparative Biochemistry, 21:4, 215-227, DOI:10.1080/10826069108018574
To link to this article: http://dx.doi.org/10.1080/10826069108018574
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PREPARATIVE BIOCHEMISTRY, 21(4), 215-227 (1991)
DIMETHn SUBERIMIDATE AS AN EFFECTIVE CROSSLINKER FOR ANTIBODY-ENZYME CONJUGATION
1 1 1 M.C. Sekhar G. L. Sharma , 'S .Gangal 2A.P. Joshi and P .U. Sarma 'C'entre forBiochemicals , Delhi Univ. Campus Mall Road,
Delhi-110 007, India.
21mmunology Division, Cancer Research Institute, T.M.R.C., Parel, Bombay-400 007, India.
ABSTRACT
Eimethyl suberimidate (DMS), a bifunctional reagent was used for the first time to crosslink the -feto protein monoclonal antibodies (AFPMAb) to horse raddish peroxidase (HRP). Three batches of conjugates were prepared, purified by Sephadex gel chromatography and evaluated for their immunological reactivity. The Rz values obtained for AFPMAb-HRP conjugate were 0.39 to 1.36. Under optimised conditions the ELISA results showed the optical density of 1.9. The iso-electric focusing for the conjugate revealed different degrees of crosslinking between antibodies and HRP. It was evident that isoperoxidase-C was involved in the crosslinking process. From the dot ELISA,as low as 25 pg of AFP in the test samples could be detected with AFPMab-HRP conjugate. The conjugate prepared by DMS was stable at O°C for more than 10 months.
INTRODUCTION Bifunctional reagents are often used for crosslinking
and determining the interresidual distances in biologically active proteins .1-6 A wide variety of bifunctional reagents such as glutaraldehyde, bis succinic acid N-hydroxysuccinimic ester (BSNHS), N,N-0-phenylenedi maleimide (OPDM)r N-succi- nimidyl-3-(2-pyridyldithio) ,Jropionate (SPDP) and imidoesters are commonly used for preparing various types of conjugates. However, these reagents are reported to
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Copyright 0 1991 by Marcel Dekker, Inc.
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216 SEKHAR ET AL.
have advantages and disadvantages in relation to the reactivity, specificity, solubility and stability of the antibody-enzyme conjugates.
Glutaraldehyde conjugation decreases significantly the immunological activity of IgG-alkaline phosphatase
and the enzyme can be coupled only in very low yields. '-11 It may also cause intramolecular crosslinking, if the excess reagent is not removed. The bifunctional ester BSNHS is useful for the conjugation of heme-octapeptide- microperoxidase to antibodies. l2 But, excess BSNHS is required for complete conversion of microperoxidase to an intermediate monoactive ester without significant crosslinking. The OPDM reacts through the thiol group of the protein and subsequently maleimide derivatized protein is conjugated with the enzyme.13 Hamaguchi & & observed that the maleimide decomposes significantly from the derivatized protein at a high rate and causes problems in conjugation.14 Although the enzyme is coupled almost completely, only 15% of the IgG and 40% of the Fab' are recovered in the conjugate. The conjugates prepared by the OPDM are quite heterogeneous and about one third undergo aggregation. Such aggregates are harmful in immunological assays. Further, OPDM may not be useful in case of several enzymes like p-glucosidase and alkaline phosphatase since these enzymes lose their activity by thiolation. l5 The use of SPDP results in homo polymerization.
Intermolecular crosslinking reaction in proteins, such as hemoglobin, bovine pancreatic ribonuclease .,I6 RNA polymerase from E.coli, etc. with the help of imidoesters have been studied. The imidoesters form imidine structure with the proteins and form stable conjugates in the two proteins .17 The binfunctional reagent dimethyl suberimidate (DMS) cross-links the -amino groups of lysine in proteins and imidoester groups of the crosslinker (Fig-1). Monoclonal antibodies to -feto protein (AFPMAb) was coupled with horse raddish peroxidase (HRP) using dimethyl suberimidate
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DMS AS AN EFFECTIVE CROSSLINKER 217
c1- c1-
HN-C-(CH ) -C-NH / I t 2 6 1 1 \
(Protein A ) NH; NH; (Protein B)
FIGURE 1. Structure of Dimethyl suberimidate (DMS) and crosslinking of DMS to protein A (AFPMAb) and protein B (HRP). The reaction between & -amino groups of lysine groups in protein and imidoester groups of the reagent produce the crosslinking.
(DMS) as a crosslinker for the preparation of a stable antibody-enzyme conjugate.
MATERIALS Monoclonal antibodies to AFP (1H 7H4) were prepared
at the Cancer Research Institute, Bombay, using standard
2-mercaptoethanol, 0-phenylenediamine (OPD) and DMS were obtained from Sigma Chemical Co., USA and other reagents were purchased from local companies. Standard AFP, polyclonal antibodies to AFP and conjugate were purchased from Dakopatts.
procedure. Peroxidase, lauryl sulphate (SDS)
METHODS
Preparation of AFPMAb-HRP Conjugate Conjugate was prepared according to the method
described by Carpenter and Harrington with slight
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218 SEKHAR ET AL.
modifications. Two hundred micrograms of antibodies was dissolved in 0.2 ml of 0.2 M triethanolamine bufferlTEA), pH 8.5 and 0.3 mg of solid DMS. To this, 2 mg of HRP type VI was added and incubated at 37OC for 30 min. Fifty microliters of TEA buffer containing 2% SDS and 2% 2-mercaptoethanol was added to the tube. The sample was frozen in dry ice and kept at -1OOC for 16 h. A glass column (40 x 0.5 cm) was packed with Sephadex G-25 and equilibrated with 0.2 M TEA buffer, pH 8.5. The conjugate sample was loaded and the fractions were collected with the same buffer at a flow rate of 0.25 ml/min. The conjugate was recovered in the void volume and both protein concentrations and RZ values were monitored for each fraction. Activity was checked for each fraction with substrate OPD and hydrogen peroxide. Upto 1 mg concentrations of AFPMAb were used for preparing the antibody-enzyme conjugate. The fractions were stored at O°C with 0.1% BSA and immunological reactivity was evaluated by double antibody sandwich ELISA and dot blot techniques.
Isoelectric focusinq (IEF)
Isoelectric point of the conjugate was determined by using 3.5-9.5 pH gradient isoelectric focusing gels. Aproximately 150 pg of purified conjugate was loaded on the gel and electrophoresis was performed at 500 v for 4 h at 4OC. The gel was stained with Coomassie Brilliant Blue.
ELISA
Antiserum to AFP was diluted (1:lOOO) in coating buffer [2.5 mM sodium dihydrogen phosphate, 7.5 mM disodium hydrogen phosphate and 0.145 M sodium chloride, pH 7.2 (PBS)] and 100 pl was used to coat the wells. The plate was left overnight at 4OC. The plate was washed with washing buffer (PBS with 3% sodium chloride and 0.1% Tween-20) and blocked with 3% BSA in PBS for 2 h. One hundred microliters of AFP (conc. 10 to 300 ng/ml)was added to each well and incubated for 2 h at 37O C. Commercial and AFPMAb-HRP conjugates were
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DMS AS AN EFFECTIVE CROSSLINKER 219
diluted in washing buffer and 100 p1 was added to each well. The plate was kept at 37OC for 1 h. After washing the plate, the chromogenic substrate OPD (0 .0666% in 0.1 M phosphate-citrate buffer, pH 5.0 containing 5 1-11 of 30% hydrogen peroxide) was added to develop color. The reaction was stopped after 20 min by adding 50 pl of 5 N H2S04. The plate was read at 490 nm using ELISA reader model Immuno Reader NJ 2000, Nunc GMBH.
Dot ELISA
Nitrocellulose (NC) membranes with a pore size 0.45 pm were marked for the loading positions with a soft pencil and left for 10 min in Petri dish containing PBS (0.01 M phosphate buffer containing 0.15 M sodium chloride), pH 7.2.
After air drying, different concentrations of AFP were spotted and air dried. The NC membranes were blocked with 3% BSA (PBS containing 3% BSA and 0.5% Tween-20) for 30 min. After washing with washing buffer (0.3% Tween-20 in PBS), the NC membranes were treated with 1:20 diluted AFPMAb-HRP conjugate for 30 min at 37OC. Chromogenic substrate diaminobenzidine (DAB) and hydrogen peroxide (for SO ml of PBS, 25 mg of DAB and 20 p1 of 30% hydrogen peroxide) were used for the detection of inmuno reaction.
RESULTS
An effective antibody-enzyme conjugation could be brought about by the use of DMS. The AFPMAb-HRP conjugate was prepared by DMS as a crosslinker and the conjugate was purified by Sephadex G-25 column chromatography. The proteins of the reaction mixture were resolved into two peaks. The peak-I containing conjugate was eluted in void volume (Fig.2). The enzyme activity and the Rz values of various fractions are shown in Table 1. The Rz values of peak-I fractions of AFPMAb-HRP conjugate ranges from 0.39 to 1.36. Peak-I1 did not show any enzyme activity with substrate OPD or immunological reactivity in ELISA test. Peak-I fractions showing enzyme activity were pooled and used
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1.01
0 1 1 1 1 1 1 1 1 1 1
4 8 12 16 20 0 Fraction no
FIGURE 2. Elution profiles of AFP conjugate reaction mixture on Sepha- dex G-25 column. 200 pl of conjugate was loaded to the column and eluted with TEA buffer pH 8.5, flow rate of 0.25 ml/min. Optical densities were read at 280 nm ( 0 - 0 ) and 405 nm ((-0 respectively.
Table 1. Rz values of AFPMAb-HRP conjugate and the HRP activity with substrate O-phenylene- diamine (OPD).
AFPMAb-HRP Rz value Activity Fraction (OD at 405nm/275nm) with OPD number’ -
4 0 .83 +++ 5 0.97 +++ 6 1.36 +++ 7 0.86 ++ 8 0.39 +
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DMS AS AN EFFECTIVE CROSSLINKER 221
0
AFP Log CONCENTRATION
FIGURE 3. Dose response curve with 1:20 diluted AFPMAb conjugate ( 0 - 0 )
and 1:SOO diluted commercial conjugate ( 0 - 0 ) .
in immuno assays. The conjugated protein obtained in the immunologically active fractions were found to be 46.64%. The dose response curve (Fig.3) followed the sigmoid path with increase in optical density as antigen concentration increased. The ELISA values for the conjugate with different concentrations of AFP are shown in Table 2. With 100 ng/ml of antigen, an optical density of 0.98 was recorded with 1:20 diluted AFPMAb-HRP conjugate.
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222 SEKHAR ET A L .
Table 2. ELISA values of AFPMAb-HRP conjugate at 1 : 2 0 dilution with diffevent antigen concentrations. ( * ) Each value is an ave- rage of six readings.
AFPMAb-HRP AFP Conc. ELISA readings con jugate nglml at 490 nm*
Peak-I (1:20 dil.) 10 30
100 300
Peak-I1 (undil.) 100
0.43
0.59
0.98
1.19 0.02
Results obtained using dot ELISA, indicated that the detection limits for AFP was below 25 pg (Fig.4). Figure 5
shows the protein profiles of the conjugate on 3.5-9.5 pH gradient isoelectric focusing gel. The schematic diagram of iso-electric focusing gel (Fig.6) indicates the antibody- peroxidase conjugate with an isoelectric point (PI) around 4 . 4 (Lane 2) and it is comparable with that of commercial conjugate. Peroxidase C which has PI around 8.9
(Lane 3 ) is absent in lane 2 indicating thereby that isoperoxidase C was utilized in the coupling reaction. Faint bands may be the unreacted immunoglobulin or aggregates of peroxidase. It was observed that the isoperoxidase-C was involved in the conjugation process.
DISCUSSION
The results of present investigation show that the bifunctional imidoester DMS can be used as a crosslinker for the conjugation of AFPMAb with HRP. The glutaraldehyde moved unsuccessful in initiating the crosslinking reaction
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FIGURE 4. Detection of AFP using AFPMAb-HRP conjugate by dot ELISA. The detection limit was found to be less than 25 pg of AFP.
FIGURE 5. Isoelectric focusing of antibody enzyme conjugate on 3.5-9.5 pH gradient ampholine PAGE plate. Lane 1) commercial conjugate, 2) AFPMAb-HRP conjugate and 3) peroxidase Type VI (Sigma) .
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2 24 SEKHAR ET AL.
-< -C 3.5-
FIGURE 6. Schematic diagram of isoelectric -focusing gel showing commercial conjugate and AFPMAb conjugated to isoperoxidase-C in lane 1 and 2 respectively ( * I . Lane 3 shows isoperoxidase-C at pH 8.9 ( 0 ) and anodic isoenzyme in lane 2 and 3 ( > I .
between HRP and AFPMAb. This might be because the HRP was, poorly activated even at high concentrations of glutaral- dehyde. It has been reported that in isoperoxidase-C, there is a shielding of 6 lysine groups surrounding the carbohydrate molecules. Therefore, activation by glutaraldehyde was restricted to minor isoenzymes leading only to low levels of HRP activation. l8 Adams and Wisdome used one step glutaraldehyde mediated peroxidase conjugation and obtained only 1% of the enzyme activity and 5 % of immunoreactivity in the conjugate. Clyne et a1 (1979) recovered 32.06% of the protein in the conjugated form using glutaraldehyde. 2o Conjugates prepared by SPDP have been
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Dns AS AN EFFECTIVE CROSSLINKER 225
shown to yield upto 60% of the immunoglobulin in the conjugated form.21 In the present investigation an average recovery of 46.64% of protein was obtained in the immuno- logically active fraction.
The conjugate was purified by Sephadex G-25 column chromatography, which separated the high molecular conjugated protein from low molecular weight components. Better resolution was obtained with Sephadex G-75 and G-100 as compared to G-25 (data not shown here). The conjugate recovered in the void volume fractions (peak-11) strongly reacted with the OPD substrate (Fig.2). Peak-I1 fractions failed to give any reaction with substrate or in the ELISA test. When peak-I1 fractions were monitored at 405 nm for its heme content, the optical densities were near zero suggesting that these fractions did not contain heme.
The double antibody sandwich assay was carried out using polyclonal anti-AFP antibodies as capturing antibodies. Three to 300 ng/ml AFP were used as standard. Results showed that the conjugate prepared by DMS was specific to AFP. With AFP-MAb-HRP conjugate the dose response curve was similar to the sigmoid curve obtained by MacDonald and Kelly?' Five hundred times diluted commercial conjugate gave an optical density of 1.23 with 2.5 ng/ml of AFP, whereas 1:20 dilution of AFP-MAb-HRP conjugate was needed to give an optical density of 1.27 with an equal concentration of AFP.
The HRP conjugates have been reported to be stable with Rz values between 0.3-0.4. In our preparation of AFPMAb conjugate the Rz values were obtained between 0.39-1.36. When the conjugate was stored with 0.1% BSA at OOC, even after repeated freezing and thawing immunological activity was retained up to 10 months. Dot ELISA results showed that the detection limit on NC was less than 25 pg AEP. isoelectric focusing showed that in our preparation isoperoxi- dase-C, which was dominant in all peroxidases, was involved in the crosslinking process.
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226 SEKHAR ET AL.
CONCLUSIONS
Dimethyl suberimidate, an imidoester crosslinker, was successfully attempted to conjugate q-feto protein monoclonal antibodies to peroxidase for the first time. Unlike the diffi- culty that exists in elimination of glutaraldehyde polymers from conjugation medium, DMS could be eliminated easily during the purification process and thus did not interfere i n further evaluation of the conjugate. It was found that the conjugate prepared by using DMS was quite stable and showed good immuno- reactivity even after 10 months.
ACKNOWLFDGMENTS
The f i m t a u t h o r is gx.ateful t o t h e C o u n c i l f o v S c i e n t i f i c and I n d u s t r i a l R e s e a r c h (CSIR). New D e l h i . f o r f i n a n c i a l s u p p o r t . Thanks are due t o Mr. Sui.esh S a r o h a f o r t e c h n i c a l a s s i s t a n c e and f o r t y p i n g t h e manusc i - ip t .
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