Rapid Screening of Immunoreagents for Carbaryl Immunosensor

Chapter 9

Rapid Screening of Immunoreagents for Carbaryl Immunosensor Development 1

1

2

Downloaded by NORTH CAROLINA STATE UNIV on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch009

1

S. Morais , M. A. González-Martinez , R. Puchades , Angel Montoya , A. Abad , and A. Maquieira 1

2

1

2

Departamento de Quimica andLaboratorioIntegrado de Bioingenieria, Universidad Politécnica de Valencia, Apdo. de Correos 22012, Camino de Vera s/n, 46071 Valencia, Spain

A fast, accurate selection of antibodies, solid supports, assay format and dissociating agents for immunosensor systems may be achieved by means of a screening tool based on the Enzyme Linked Immuno Filtration Assay principles. This approach is demonstrated in this paper using the immunological reaction between the pesticide carbaryl as a model analyte and a set of monoclonal antibodies raised against carbaryl. Future development of immunosensors may be simplified using this method.

Environmental contamination is partly caused by the pesticides applied to crops to protect them against pests. Consequently, the presence of pesticide residues needs to be monitored to protect both human health and environment. To make constant monitoring feasible, a greater effort should be made to develop sensitive, rapid, costeffective and automated methods for routine analyses (i). Suitable immunoassays are being tested as alternative techniques to traditional methods (2) when high sample throughput or on-site screening analyses are required. For example, there has been an increasing interest in the development of immunosensors because of its great promise for on-site analysis. An immunosensor is an analytical device composed by an immobilized immunoreagent in intimate contact with a physical transducer that converts the product of the immunoreaction into a quantificable signal. Flow Injection Immuno Analysis provide attractive approaches to immunosensor development (3). In this type of immunosensor the sample is incorporated in a carrier stream which enters a reaction chamber containing the immobilized immunoreagent, where the immunological reaction takes place. In immunosensor development, the following aspects have to be optimized: appropriate immobilization support material, suitable immunoreagents, and feasible dissociating agents. In this report, the rapid optimization of these parameters using the principles of the Enzyme Linked ImmunoFiltration Assay (ELIFA) (4) is described.

© 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

9. MORAIS ET AL.

Rapid Screening of Immunoreagents

107

Experimental section


Reagents. Carbaryl analytical standard was purchased from Dr. Ehrenstorfer (Augsburg, Germany). Haptens 6-[[(l-naphthyioxy)carbonyl] amino] hexanoic acid (CNH), 3-[[(l-naphthyloxy)carbonyl] amino] propanoic acid (CNA) (Figure 1), and monoclonal antibodies (MAbs) were obtained, purified, and characterized as previously described (5). Horseradish peroxidase (HRP) was from Boehringer (Mannheim, Germany) and rabbit anti-mouse peroxidase-conjugated immunoglobulin from Dako (Glostrup, Denmark). All other reagents were of analytical grade.

CARBARYL

n

* n -5

~

W

A

CNH

Figure 1. Structures of Carbaryl and the haptens CNA and CNH. Solid supports. Support selection criteria were: good dynamic properties, ease of reagent immobilization (antibodies or haptens), high stability to regeneration conditions and the variety of geometries available, i.e. spectroscopic cells, fiber optics, membranes, and electrodes. Beaded supports included controlled pore glass (CPG) and derivatized agarose gels. CPG (PG240-200) was purchasedfromSigmaAldrich Quimica (Madrid, Spain). Affi-gel hydrazide (Affi-gel Hz) and Affi-gel 15 werefromBio-Rad laboratories (Richmond, CA). Several membranes of different chemical compositions were tested: Immobilon-P transfer membrane was from Millipore Corporation, (Bedford, MA), glass microfibre filters from Whatman International Ltd. (Maidstone, Kent, U.K), Loprodyne (Pall, Madrid, Spain), and homemade polyetherimide membranes (6). Apparatus. The Easy Titer ELIFA device was acquired from Pierce Chemical Company (Rockford, IL). A Gilson Minipuls-3 peristaltic pump (Villiers, Le Bel, France) was attached to the ELIFA device to control the flow rate. Polystyrene 96well microtiter plates (Nunc, Roskilde, Denmark) were used, and absorbance was read and recorded with a Dynatech MR-700 microplate reader (Sussex, U.K.). Procedures Preparation of hapten-protein conjugates. To visualize the antigen-MAb reaction in direct immunoassays, a CNH-HRP conjugate was prepared by a variation of the

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


108

IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

anhydride mixed method (7). Briefly, to 4 mg of C N H (13.3 pmol) in 197 p L of N N'dimethylformamide (DMF) was added 2.9 pL of tri-n-butylamine (12.2 umol) followed by 1.6 pL of isobutyi chloroformate (12.2 umol). The mixture was stirred for 1 hour at room temperature, and 200 pL of the resulting activated hapten was diluted in 1.8 mL DMF. A volume of 136 pL of this mixture was added to 3 mg of HRP diluted in 1.4 mL of 50 mM carbonate buffer, pH 9.6. The coupling reaction was incubated at room temperature for 5 h with stirring, and the resulting conjugate was purified by gel filtration on Sephadex G-25 using 100 m M sodium phosphate buffer, pH 7.4, as eluant. For indirect immunoassay, C N H was covalently attached to bovine serum albumin (BSA) using the modified active ester method (8). A mixture of 30.2 mg of C N H (0.1 mmol), 12.5 mg of N-hydroxysuccinimide (0.1 mmol), and 20.6 mg of N,N'dicyclohexylcarbodiimide (0.1 mmol) in 1 mL of D M F was stirred at room temperature for 2 h to convert the carboxyl group into the succinimide ester function. After centrifuging, (12,000 g; 15 min), 600 pL of the clear supernatant containing the active ester was added dropwise to 3 mL of a 15 mg/mL B S A solution in 50 m M carbonate buffer, pH 9.6. The mixture was allowed to react at room temperature for 4 hours with stirring. After centrifuging as above, futher purification of the C N H B S A conjugate was carried out using the gel filtration technique described for the C N H - H R P conjugate. Although both C N H and proteins show absorbance peaks at almost the same wavelength (280 nm), the C N H spectrum displays little characteristic shoulders in this spectral zone, so when conjugation occurred, the modification of the protein spectra was very evident (5). Then, the extent of coupling of C N H to proteins was determined by U V absorbance spectrophotometry at 280 nm by assuming additive absorbance values of C N H and proteins in the conjugates. The estimated hapten to protein molar ratios were 17 and 2 for the B S A - C N H and H R P - C N H conjugates, respectively. MAbs immobilization for direct immunoassays. MAbs to carbaryl raised from C N H (5) and C N A (9) were immobilized on both alkylamined CPG and Affi-gel Hz beads. Periodate oxidation of vicinal hydroxy 1 groups of the carbohydrate results in the formation of aldehydes for specific coupling to both supports, resulting in an oriented and covalent immobilization. Finally, the immunosorbent suspension was prepared by the addition of storage buffer, 20 mM sodium phosphate, pH 7.4, 0.02% NaN , and stored at 4°C till use. 3

Conjugate immobilization for indirect immunoassays. Affi-gel 15 was used to immobilize C N H - B S A conjugate through free amino groups of BSA, following the manufacturer's protocol. In addition, CPG was prepared (10) by glutaraldehyde activation prior to immobilization of the C N H - B S A conjugate. After coupling, the remaining active binding sites were blocked with 1 M ethanolamine, pH 8.0/HC1 and left for 1 hour at room temperature for the blocking reaction to be completed. Both coupled blocked gel and CPG were washed with deionized water.


9. MORAIS ET AL.

109



Finally, the antigen-coated immunosorbent suspensions were prepared and stored as described above for the Mab-coated immunosorbents. Screening of Immunoreagents. A n inert membrane sheet wetted with 2.5% B S A solution was sealed over two gaskets covering the 96 wells, and the sample application plate clamped on top of the membrane (Figure 2). First, 200 pL/well of 2.5% B S A solution was pipetted and left to flow through the membrane for 5 min. Then, 50 pL of immunosorbent suspension which contained aproximately 1 mg of immunosorbent were added and the following solutions sequentially pipetted: For the direct competitive ELIFA assays, 200 p L of a mixture solution in phosphatebuffered saline (PBST; 10 m M phosphate, 137 m M NaCl, 2.7 m M KC1, p H 7.4 containing 0.05% Tween 20) of Carbaryl:CNH-HRP (1:1 v/v), and wash solution (5 times with 200 pL PBST). For the indirect competitive ELIFA assays, 200 p L of a mixture solution of CarbaryhMAbs (1:1 v/v), wash solution (5 times with 200 pL PBST), peroxidase conjugated rabbit anti-mouse immunoglobulins solution (200 pL) and, wash solution (6 times with 200 pL PBST). A peristaltic pump controlled the flow rate as well as the vacuum required to ensure that all wells were emptied simultaneously (in 5 min for immunoreagent solutions and 2 min for the wash solution). Then, a microtiter plate was hold in the lower chamber of the device in order to collect the product of the enzymatic reaction. The enzyme substrate solution consisting in 75 pL, 2 mg/mL OPD and 0.012% H 0 in 25 m M citrate, 62 m M phosphate buffer, pH 5.4 was added. After 5 min the reaction was stopped by adding 75 pL of 2.5 M sulfuric acid. Finally, the plate was removed from the device and the absorbance at 490 nm was read in the microplate reader. Both direct and indirect competitive formats were studied. The concentration of carbaryl which inhibited 50% of the maximum signal (I ) was used to estimate the strength of the apparent antibody affinity in each format. The lower the I value the greater is the binding between the M A b and antigen. Negative controls (zero analyte) were included to estimate the maximum signal for 1^ determination. Competition curves were obtained by plotting absorbances vs the logarithm of competitor concentration and were fitted to four-parameter logistic equations (11). 2

2

50

50

Results and Discussion Optimization of the system parameters. The nature of the filtration membrane was the first parameter optimized. A n inert membrane without nonspecific physical adsorption of the immunoreagents was required to ensure that the immunological reaction ocurred exclusively on the immobilization support. Several membranes such as Immobilon-P, Loprodyne, glass fiber membrane and homemade polyetherimide membranes were tested by ELIFA. First, homemade membranes with available pore size were eliminated because of sporadic flow impediment when the wells are filled. For the rest of the membranes, the nonspecific adsorption was measured by means of adding enzymatic conjugates to the membrane which had been previously treated with B S A solutions. After washing the membrane with PBST, the presence of



110


Figure 2. Schematic view of the Easy Titer ELIFA device. (1: Sample application plate, 2: Sample well, 3: Membrane, 4: Gaskets, 5: Transfer canula, 6: Collection chamber).



9. MORAIS ET AL.

111


nonspecifically adsorbed enzyme conjugates was determined enzymatically as described above. Immobilon-P, a nitrocellulose membrane designed for high protein binding was tested. BSA solutions of 1-5% were used as blocking agents. In spite of using high BSA solution (5%) to block active sites, high levels of the immunoreagents (CNH-HRP conjugate as well as rabbit anti-mouse peroxidase-conjugated immunoglobulin) were adsorbed in both direct and indirect formats, respectively. The Loprodyne membrane, designed for low protein adsorption, precoated with the above blocking agent also showed high nonspecific adsorption capacity for both labeled immunoreagents. These high nonspecific signals prevented the use of both Immobilon-P and Loprodyne membranes. The glassfibermembrane blocked with 2.5% BSA gave the lowest nonspecific signals, i.e. 0.03 and 0.11 optical density (O.D) units under the best conditions of the method in direct and indirect formats, respectively, and was therefore used throughout this work. Flow rate parameters ranging from 1-10 mL/min were investigated. A flow rate of 4 mL/min was chosen as a trade-off between speed and sensitivity of the assay. In these conditions, wells were emptied simultaneously in approximately 5 min. Direct format. The I of eight anti-carbaryl MAbs, immobilized on both CPG and Affi-gel Hz supports, were measured. Different solvents were tested to determine the regeneration of the antibodies after disrupting the MAb-antigen complex. Optimal concentrations of immobilized MAbs (0.03-1 mg/g bead) and HRP-conjugates ranging from 0.1-0.5 pg/mL were selected by non-competitive ELIFA to give absorbances in the range of 0.8-1.2 O.D. Table I shows the carbaryl I obtained by direct competitive ELIFA for the MAbs immobilized on both Affi-gel Hz and CPG supports. The activity of immobilized MAbs was dependent on the type of support used. The enzyme conjugate concentration required to achieve optimal absorbance was lower on Affigel Hz than on CPG. I values were 3-6 times higher on CPG, probably due to the fact that physical adsorption dominates the chemical orientation of immobilized MAbs (72). The MAbs LIB-CNH 3.2, LIB-CNH 3.6, LIB-CNH 4.5, LIB-CNH 10.3, and LIB-CNA 3.6 displayed the lowest I to carbaryl, corresponding to the highest apparent affinity in this assay format. 50

50

50

50

Table I. Carbaryl 1^ values (nM) determined by direct competitive ELIFA format for MAbs immobilized on Affi-gel Hz and CPG MAb (LIB-) Support

CNH 3.2

CNH 3.6

CNH 3.7

CNH 4.5

CNH 8.9

CNH 10.1

CNH 10.3

CNA 3.6

Affi-gel Hz

10

25

29

25

74

34

25

23

CPG

60

85

150

115

200

150

125

130



112


Figure 3. Inhibition curves of monoclonal antibody L I B - C N A 3.6 which was covalently immobilized on Affi-gel Hz (•) and CPG (•) (direct format). Each point represents the average of four wells in the same application plate.



9. MORAIS ET AL.

113


For immunosensor to become a practical quantitative analytical tool, regeneration of the immunoreagents must be accomplished after disrupting the antigen-MAb complex. One drawback of using dissociating agents is the eventual decrease in antibody activity. Different reagents for antibody regeneration, such as highly concentrated NaSCN (3M), NaCl (0.5M-3M), methanol in water (50:50), and buffers with low pH (glycine, acetate, phosphate) were studied with regard to the fast and complete regeneration of immobilized MAbs. 0.1 M glycine/HCl, pH 2.0 turned out to be the best dissociating reagent, since it allowed the effective immunocomplex disruption after the application of few (1-3) short-time (5 min) regeneration cycles. Most of the MAbs tested lost their native abilility (>50%) to recognize the enzyme conjugate after 4 or 5 runs on both supports (Table II). Only LIB-CNA 3.6 was able to maintain its native activity (100%), estimated as the maximum signal in non-competitive conditions (zero analyte), after at least 15 regenerations without signal decline. Figure 3 shows the inhibition curves displayed by LIB-CNA 3.6 on Affi-gel Hz and CPG. The 1^ values for both immunosorbents in the competitive direct format were 5 and 13 ng/mL, respectively. As indicated by the manufacturer, the binding of antibodies to Affi-gel Hz is highly stable under low pH conditions. Therefore, at least for this immunosorbent, the MAb activity decrease should not be attributed to dissociation of the immunoreagent (MAb) from the solid support, but to MAb denaturation.

Table H . Remaining activity (%) of MAbs immobilized on both Affi-gel Hz* and CPG supports, after dissociating steps in the direct format b

MAb (LIB-) Run

CNH 3.2

1

CNH 3.6

CNH 3.7

CNH 4.5

CNH 8.9

CNH 10.1

CNH 10.3

CNA 3.6

80 -60

75-70

72-65

45-70

70-80

30-30

45-55

100-100

2

80-55

70-65

45-60

35-70

60-60

30-20

40-50

100-100

3

40-50

60-50

45-50

25-55

50-50

30-20

40-50

100-100

4

35-50

48-50

35-25

25-50

50-50

25-15

25-50

100-100

5

25-45

45-50

20-20

20-45

50-45

25-15

20-30

100-100

15

Rapid Screening of Immunoreagents for Carbaryl Immunosensor

Recommend Documents