Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide

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1 Chapter 15 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide Shuo Wang, Can Zhang, and Yan Zhang Summary In recent years, immunochromatographic lateral flow test strips are used as a popular diagnostic tool. There are two formats (noncompetitive and competitive) in gold-based immunoassay. Noncompetitive gold-based immunoassay also called sandwich assay is applied for the detection of large molecular mass. For small molecular mass such as pesticide, competitive format of lateral flow colloidal gold-based immunoassay is described in this chapter. The preparation of gold colloidal and the conjugation between antibody and gold colloidal are described. Hi-flow plus nitrocellulose membranes are separately coated with goat anti-rabbit IgG (control line) and hapten-ova conjugate (test line). Thus, the degree of intensity of color of the test line is the reverse of the concentration of pesticide in the sample and the visual result is immediately observable. Colloidal gold-based immunoassay can also be applied for multianalysis in one test strip if the detected targets show different physico-chemical properties and their haptens show great differences in chemical structure. Key words: Lateral flow, Gold-based immunoassay, Non-competitive, Competitive, Multi-analysis. 1. Introduction Lateral flow tests are also called immunochromatographic strip (ICS) tests. They have been a popular platform for rapid tests since their introduction in the late 1980s. ICS tests are used for the specific qualitative or semiquantitative detection of many analyses including antigens, antibodies, and even the products of nucleic acid amplification tests. Urine, saliva, serum, plasma, or whole blood can be used as specimens. Test specificity can also be very high. The tests use colloidal gold, dye, or latex bead conjugates to generate signal. Early rapid tests used colored latex to form the visual signal, and some current versions continue to Avraham Rasooly and Keith E. Herold (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol. 504 Humana Press, a part of Springer Science + Business Media, LLC 2009 DOI: / _15 237

2 238 Wang, Zhang, and Zhang use this method. Latex is originally, and still is, the prime labeling method used in agglutination tests. This is because of its predisposition to agglutinate in the presence of binding components. For rapid tests, in which stability of the conjugate is critical for avoiding false positives, this predisposition to agglutinate can become a major problem. The use of colloidal particles as versatile and efficient templates for the immobilization of biomolecules has been recognized since early 1980s. Because of its superior stability, sensitivity, and precision and reproducibility of manufacture, colloidal gold is more suitable for use in rapid tests (1 5). Colloidal gold immunoassay has been developed and applied increasingly in various research field such as for the detection of hormone (pregnancy, fertility), virus (HIV, hepatitis B and C), and bacteria (streptococcus A and B) (6, 7). The nanocolloidal gold particles could replace the enzyme to be labeled to antibody in pesticide immunoassay since it has very large surface area and good biocompatibility and stability. Compared with enzyme immunoassay (EIA), colloidal gold-based immunoassay can be completed rapidly in one-step. When antibody labeled with colloidal gold particles is combined with corresponding antigen, the colored immuno-reactant can be visually detected. There are two formats in immunochromatographic strip tests: noncompetitive and competitive reaction (8). They can be explained graphically in Notes 1 and 2. The format of noncompetitive immunochromatographic test is also called sandwich assay, which is applicable for target analyte with more than one epitope (high molecular mass analyte). For the smaller molecular mass analyses, the competitive format assay can be used (9 11). The major steps of strip preparation and analysis of competitive format assay were shown in the Scheme 1. In this type of assays, the detector reagent is typically colloidal gold-labeled antibodies against the analyte. The capture line is normally analytes conjugated to a carrier protein immobilized on the membrane. Analytes in samples will compete with analytes immobilized on the membrane for binding to the detector antibody. The more analytes present in the sample, the more effectively it will be able to block the capture of colloidal gold-labeled antibodies. Hence, an increase in the amount of analytes in samples will result in a decrease in signal in the readout zone (test line). So far, the competitive strip tests have been mainly used for the detection of low molecular mass (hapten), especially for drugs of abuse or pesticide in agricultural products. The detection limits of this competitive assay can achieve to ppb level (nanograms of analyte per gram of sample). Here colloidal gold for conjugation is used as available detection system. The signal reagent is solubilized and binds to the antigen or antibody in the sample and moves through the membrane by capillary migration. The tests can be run individually or

3 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide 239 Scheme 1. The major steps of strip preparation and analysis of competitive format assay. in limited-size batches. Results can usually be read in 5 15 min. All tests include an internal procedural control line that is used to validate the test result. The benefits of immunochromatographic tests include (a) user-friendly format, (b) very short time to get test result, (c) long-term stability over a wide range of climates, and (d) relatively inexpensive to make. These features make strip tests ideal for applications such as home testing, rapid point of care testing, and testing in the field for various environmental and agricultural analytes. The assembled strips are dried and packaged, making them stable for months when properly protected from moisture and excessive heat. 2. Materials 1. HAuCl 4 (0.01%): 0.01 g HAuCl 4 dissolved in 100 ml deionized water. 2. Trisodium citrate (1%): 1 g trisodium citrate dissolved in 100 ml de-ionized water. 3. ph 7.2, 10 mm phosphorate buffer (PBS) and 0.05% Tween- PBS. 4. Conjugate storage buffer (ph 7.2): 2 mm sodium borate containing 1% BSA and 0.1% sodium azide (1% BSA is added in order to stabilize the gold nanoparticles).

4 240 Wang, Zhang, and Zhang 5. ph 9.0, 5 mm borate buffer is used as dilution buffer in optimal condition studies for conjugation between colloidal gold and antibody. 6. Species-specific anti-immunoglobulin antibodies (protein A or goat anti-rabbit IgG, coated as control line) and hapten conjugated with protein (coated as test line). All the reagents should filter with 0.45 μm membrane Nitro-cellulose Hi-Flow plus membrane (HF ), Nylon + membrane (INYC 00010), Polyvinylidene fluoride membrane (IPVH 10100), semi-rigid polyethylene sheets, and filter paper were purchased from Millipore (Bedford, MA). Hydrogen tetrachloroaurate trihydrate (CAS Number: ) was purchased from Sigma (St. Louis. MO). Protein A-Sepharose 4B was purchased from Amersham Biosciences (Uppsala, Sweden). Camag Linomat 5 automatic sampler (CAMAG, Switzerland, Fig. 1) and Milli-Q purified water system were also used. The synthesis of carbaryl haptens (shown in Fig. 2) was performed as previously described by Wang et al. (12). The synthesis of endosulfan haptens (shown in Fig. 3) reported by Lee et al. Fig. 1. The photo of Camag Linomat 5 sample application.

5 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide 241 Fig. 2. Chemical structures of carbaryl and the haptens used for immunogen (haptena) and used for coating on the membrane as test line (haptenb). Fig. 3. Chemical structure of endosulfan and the haptens used for immunogen (haptena) and used for coating on the membrane as test line (haptenb). was accomplished according to their procedures (13). The haptens were coupled to keyhole limpet hemocyanin (KLH) for used as immunogens or coupled to OVA for used as coating on the membrane. Antibodies were produced in rabbits as described by Wang et al. (12). Female white rabbits were immunized by intradermal

6 242 Wang, Zhang, and Zhang and intramuscular injections of haptens conjugated with KLH. IgG from antiserum was purified by protein A-Sepharose 4B affinity chromatography (14). 3. Methods 3.1. Membrane Selection 3.2. Preparation and Selection of Colloidal Gold For immunochromatographic strip (ICS) tests, membrane with high protein-binding capacity is used as support body. Different membranes and some binding properties are presented in Table 1. Nitrocellulose Hi-Flow plus membrane is often used widely. Nitrocellulose membranes are completely neutral, and their binding properties are independent of the ph of the immobilization solution (although ph can have an effect on both the solubility and immobilization efficiency of a particular protein). The immobilization buffer with ph is chosen. Sometimes the surfactants and detergents such as Tween-20 and Triton-X100 in very low concentration are added in the buffer to reduce the background and nonspecific binding. Gold colloids were prepared by controlled reduction of gold chloride with sodium citrate using the procedure described by Frens (15). The strength of color showing was closely related to the size and quality of the colloidal gold particles. The size of the colloidal gold particles was directly dependent on the amount of trisodium citrate used in its preparation process. The results were summarized in Table 2. It was found that if the diameter of gold particles were small (<40 nm), it was hard to indicate a clear and bright color (low reactant concentration results in low rates). It was also found that gold particles with large diameter (>40 nm) were unstable, self-coagulation occurred. For small molecular like pesticide, the optimize size selection of diameter for colloidal gold particle was 40 nm. Table 1 Binding propertities of different membrane polymers Membrane polymer Nitrocellulose Polyvinylidene fluoride (PVDF) (Charged modified) Nylon Mechanism of binding Electrostatic Hydrophobic (Ionic) Electrostatic

7 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide 243 Table 2 Changes of diameter size and color of formed colloidal gold with different amount of trisodium citrate Amount of 1% trisodium citrate added in a 100 ml 0.01% gold chloride solution (ml) Color of colloidal gold particles Diameter size of colloidal gold particles (nm) 2 Reddish Salmon pink 25 1 Dark red Purple-red Purple Purple-gray Blue 150 The procedures for preparing colloidal gold with 40 nm diameter were as follows: Step 1: First, 100 ml of 0.01% HAuCl 4 was boiled throughly for 5 min. Step 2: Then1 ml solution of 1% trisodium citrate was added under constant stirring. Step 3: It was observed that the color of the solution had changed in less than 2 min, then it was boiled for another 5 min. Step 4: After cooling, de-ionized water was added to the initial volume. The diameter of the particle was checked with a transmission electron microscope (Fig. 4) Optimal Condition Study for Conjugation Between Colloidal Gold and Antibody The ph of colloidal gold solution for labeling antibody was adjusted with 0.1 M K 2 CO 3 or 0.1 M HCL (ph 9.0 for polyclonal antibody and ph 8.2 for monoclonal antibody). Here, polyclonal antibody was used. First, antibodies were purified with protein A sepharose-4b, then dialysis in ph mm PBS. For conjugation, antibody was directly absorbed on colloidal gold particle surface, mediated mainly by London-van der Waals force and hydrophobic interaction (16). To have a strong adsorption between the gold and antibody conjugate, a preliminary titration was performed. The colloidal gold was formed in solution by virtue of a balance between electrostatic repulsion and Londonvan der Waals attraction among the particles. However, on addition of ionic substance, the attracting force became greater than the counteraction, which led to an aggregation accompanying a color change from red to blue. Coating the colloidal surfaces

8 244 Wang, Zhang, and Zhang Fig. 4. Colloidal gold particle with 40 nm diameter observed by transmission electron microscope. with protein molecules, such as SPA, antibody, and BSA, could prevent this instability. Optimal conditions of antibody concentration for the coating could be determined by comparing the adsorption between 520 and 580 nm. Anti-carbaryl antibody conjugated with colloidal gold was used as example, the procedures for conjugation between colloidal gold and anti-carbaryl antibody were as follows: Step 1: Gold colloidal suspension adjusted to ph 9.0 was pipetted into a series of tubes at 1 ml per each tube. Step 2: 100 μl antibody solution diluted in a series of concentration ( mg/ml) was added to each colloidal gold solution. Step 3: After being incubated for 5 min, each tube received 0.1 ml of 10% NaCl and was shaken for 10 min. Step 4: Absorption of each tube at 520 and 580 (A 520 A 580 ) was determined. As shown in Fig. 5, the minimal antibody to stabilize 1 ml of gold colloidal suspension was approximately 8 μg anti-carbaryl antibody. The antibody was determined to be 120% of the minimum concentration to ensure complete reaction with the colloidal gold particles. Under gentle stirring in ice bath, anti-carbaryl antibody was added drop by drop to the gold colloidal suspension within 20 min. Step 5: After overnight incubation at 4 C, the mixture was centrifuged at 10,621 g at 4 C for 30 min, and the pellet was resuspended in conjugate storage buffer (2 mm sodium borate containing 1% BSA and 0.1% sodium azide) and diluted for use.

9 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide 245 Fig. 5. Optimization of conjugation between anti-carbaryl antibody and colloidal gold Preparation of Membrane Strip for Lateral-Flow 3.5. Procedure and Optimization of Lateral-Flow Gold- Based Immunoassay Nitrocellulose Hi-flow plus membrane was cut into sections ( cm 2 ), for the detection of small molecular (such as pesticide). The procedures of preparation of membrane strip were shown: Step 1: The membrane was coated with hapten-ova conjugates in a volume of 1 μl containing 1 μg hapten conjugate as test line with Camag Linomat 5 automatic TLC sampler. The control line was coated with 0.5 μl of anti-rabbit IgG from goat diluted 1/100 in PBS buffer (ph 7.2). The distance between the two lines was 0.5 cm. Step 2: The coated test strips were dried at 37 C for 15 min in constant temperature desiccator. Step 3: The remaining protein-binding sites of the membrane were blocked by immersing the strips in PBS containing 1% BSA at 37 C for 30 min. Step 4: The test strips were washed with PBST and dried. The coated test strips were stored in a desiccator at 4 C. Lateral-flow device used in this study was shown in Fig. 6. In lateral-flow device, the dried filter acted as an absorbent actively. The main purpose of the assay was to allow visual evaluation, thus it was only used as a qualitative assay to detect contamination at a threshold level. For this, the color intensity of the test strip must be high enough to be seen and enable observation of difference in color intensity between negative control and samples. For this purpose, optimization experiments were used to determine the optimal immobilization concentration of hapten conjugate, optimal ratio of gold antibody conjugate mixed with antigen and incubation time. Optimal immunoreagent concentration was selected as a clear appearing in the negative control with the shortest time, and the comparison of the intensity of color among samples and control could be easily distinguished by eye.

10 246 Wang, Zhang, and Zhang Fig. 6. The analytical device for lateral-flow immuno-gold assay. C: control line, T: test line. The procedures were shown in the following: series dilutions of standard solutions in 5% MeOH (prepared in PBS-0.05% Tween buffer) were mixed with corresponded PAb colloidal gold conjugate at a certain ratio (9:1 or 8:1). After incubation for 5 min, a certain volume mixture ( μl) was added to sample application site of the test strip. The liquid reagent migrated by capillary toward the test line and control line. After the liquid reagent passed through the reaction zone (test line and control line), different intensity of color on the test line were separately compared with negative control. The intensity of the color of the test line was the reverse of the concentration of detected target. As shown in Fig. 7, under optimized condition different concentrations of carbaryl were detected by strip test Validation of the Test Strip Immunoassay by Comparing with Instrumental Analysis The reliability of the test strip immunoassay was determined by carrying out the test with the uncontaminated samples spiked with detected target at three levels and analyzed by strip test and instrumental analysis. The results obtained by strip test should be consistent with the results obtained by instrument. The correlation between the two methods should be good. An example of validation of gold-based lateral flow immunoassay and analysis of food samples for the detection of carbaryl was shown in the following.

11 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide 247 Fig. 7. Lateral-flow immunogold assay for the detection of carbaryl (C, control line; T, test line; carbaryl concentration A: control; B: 100 μg/l; C: 500 μg/l; D: 1000 μg/l). Food samples were used for carbaryl screening and determination. Carbaryl was spiked at three levels in samples and extracted using methanol, which was earlier found to be an efficient extractant for this compound in food and environmental matrices. The samples were screening by lateral-flow gold-based immunoassay and confirming by HPLC (shown in Table 3). 3.7 Colloidal Gold- Based Immunoassay for Multianalysis Colloidal gold-based immunoassay could also be applied for multianalysis. It was known that if the detected targets show different physico-chemical properties and their haptens show great differences in chemical structure, multianalysis in one test strip is feasible. The different corresponding coating antigen for each pesticide could be fixed at different sites as respective test lines on the strip. As shown in Fig. 8, using lateral-flow gold-based assay for the detection of carbaryl and endosulfan, when the mixture of two gold antibody conjugates was added to the reaction zone, both test lines (carbaryl line and endosulfan line) and control line had color development. When gold antibody (specific to endosulfan) conjugate was added to the test lines, only the

12 248 Wang, Zhang, and Zhang Table 3 Comparison of results obtained by lateral-flow gold-based immunoassay and HPLC Matrix Spiked level (mg/kg) Results obtained by gold-based lateralflow immunoassay (n = 3) a Results obtained by HPLC (mg/ kg) b Chinese cabbage 5,, ±, ±, ± , +, ± ,, 3.57 Rice (grain) 10 ±, +, ± , +, Rice (Powder) 5,, ±, ±, ± , +, Barley (grain) 5,, ±, +, ± , +, Barley (powder) 5,, , ±, , +, a (+) positive: carbaryl concentration was more than 10 mg/kg; (±) positive/ negative: carbaryl concentration was around 10 mg/kg; ( ) negative: carbaryl concentration was less than 10 mg/kg b HPLC confirmation for all samples was carried out according to the method described in the previous paper (17) color of endosulfan test line appeared, and no color developed in carbaryl test line. It was also found that there was no color development in endosulfan test line when gold antibody conjugate (specific to carbaryl) was added. For multianalyte colloidal gold immunoassay and single pesticide (carbaryl or endosulfan) colloidal gold immunoassay, the visual detection limits of carbaryl and endosulfan were the same for both formats. The existence of endosulfan (or carbaryl) did not affect the binding between carbaryl (or endosulfan) and anti-carbaryl (or endosulfan) antibody gold conjugate by comparing the visual results of single pesticide gold-based immunoassay and multianalysis gold-based

13 Lateral Flow Colloidal Gold-Based Immunoassay for Pesticide 249 Fig. 8. Multianalysis flow-through gold-based immunoassay. Up line (C): control line (goat anti-rabbit IgG coated); middle line (ET): endosulfan test line (endosulfan hapten-ova coated); down line (CT): carbaryl test line (carbaryl hapten-ova coated). (A) The mixture of two gold antibody conjugates was added; (B) Anti-carbaryl antibody colloidal gold conjugate was added; (C) Anti-endosulfan antibody colloidal gold conjugate was added. immunoassay. It should be feasible to detect multiple pesticides by combining different conjugates in one NC membrane. 4. Notes 1. Noncompetitive formats This format is used when testing for larger analyte with multiple antigenic sites, such as LH, hcg, and HIV. As shown in Scheme 2, in this case, less than an excess of sample analyte is desired, so that some of the antibody gold conjugates will not be captured at the first line (test line, antibody coated), and will continue to flow toward the second line of immobilized antibodies (the control line). These are species-specific anti-immunoglobulin antibodies (usually protein A or goat anti-rabbit IgG), specific for the conjugate antibodies on the colloidal gold particles. 2. Competitive format for small molecular mass (pesticide) This is used most often when testing for small molecules (shown in Scheme 3) with single antigenic determinants, which cannot bind to two antibodies simultaneously. The assay is designed such that if there is no pesticide present in the test, the gold antibody will accumulate on the nitrocellulose membrane where they are trapped by the immobilized pesticide hapten conjugate while liquid migrate towards the test strip. For samples containing pesticide, the binding sites on the specific antibody molecules will be occupied first by pesticide, leaving fewer binding sites for hapten-ovalbumin (OVA) conjugate on the membrane. Consequently, less colloidal gold-labeled antibody will remain at the hapten-ova location on the nitrocellulose

14 250 Wang, Zhang, and Zhang Scheme 2. Double antibody sandwich reaction (noncompetitive). membrane. Thus, the degree of intensity of gold-color of the test line is the reverse of the concentration of pesticide in the sample and the visual result is immediately observable. If this format is chosen, it is important to pay close attention to the amount of antibody bound to the gold particles, in relation to the amount of free antigen in the sample. Acknowledgments The authors are grateful for financial supports from the Ministry of Science and Technology of the People s Republic of China (project No. 2006BAD05A06), the New Century Talent Program of Ministry of Education of the People s Republic of China (project No. NECT ).

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