In the Laboratory
Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk A Modern Laboratory Project Luis A. Inda, Pedro Razquín, Fermín Lampreave, and María A. Alava* Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza 50009-Zaragoza, Spain Miguel Calvo Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza 50013-Zaragoza, Spain
Background In response to the interest of the students of organic chemistry enrolled in biochemistry laboratory and to the relatively new biotechnology industry, we have developed a new enzyme immunodotting assay to be included as a laboratory exercise. This assay, which is based on a previously described method (1) with some modifications, allows the rapid detection of milk adulterations. The experiment was developed to accomplish two general goals: first, to stimulate students to apply the general principles learned in theoretical courses (such as the antigen–antibody interaction and the enzyme reaction for signal amplification), emphasizing their critical thinking; and second, to give them the opportunity to solve an applied science problem. In addition, we use this experiment to analyze with the students other situations where immunoenzymatic techniques can be applied. The practical course enrolls approximately 60 students, most of whom are taking organic chemistry. They are divided into three sections of about 20 students each. Each section has daily 4-hour laboratory sessions for a period of two weeks. This enzyme-immunodotting-assay project is ideally performed by students working in groups of two during one laboratory session at the end of the biochemistry course. It requires only the specific antibodies, the nitrocellulose membrane, and common equipment found in a chemistry laboratory. It has been favorably accepted by the students because their results are clear and reproducible. Overview of Experiment Students are expected to read the experiment before coming to the laboratory. The laboratory period begins with a 20-min briefing by the professor, in which the fundamental concepts learned in lecture are reviewed. A brief overview of the experimental objectives and the detailed protocol and related literature are presented. The students are also given the milk samples to analyze and the particular supplies required for the experiment. Thus the professor can explain the basis of the immunoenzymatic assay, which is summarized in Figure 1. *Corresponding author.
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The assay starts when the first antibody is immobilized on a nitrocellulose membrane (Fig. 1a). To eliminate nonspecific binding, the remaining free sites on the membrane are blocked with a foreign protein that will not bind to the antibody. Then, the samples containing the antigen to be detected are incubated with the antibody-containing membrane (Fig. 1b). The antigen is captured by the antibody and the membrane is washed to remove the unbound proteins. Finally, the treated membrane is incubated with the second enzyme-labeled antibody that captures the antigen. This assay is called a sandwich assay because the immunocomplex is formed by antibody–antigen–antibody interactions. The detection of the immunocomplexes is achieved by performing the appropriate enzymatic reaction to develop colored products (Fig. 1c). If there is no antigen in the sample, there will be no binding to the immobilized first antibody. When the second antibody is added, it will not find the antigen and, therefore, it will also be eliminated during the washing step. No color reaction will be observed in this case. The enzyme attached to the second antibody is horseradish peroxidase (HRP). HRP catalyzes the oxidation–reduction reaction between 4-chloro-1naphthol and hydrogen peroxide. The 1,4-naphthoquinone produced is a purple precipitate and it allows the detection of the initial antigen. Azide ion, routinely used in the stock buffers to prevent biological contamination, has to be avoided because it is an inhibitor of HRP (2). In general, when either the first antibody or the antigen is bound to a solid phase (e.g., nitrocellulose membrane), as in this experiment, the technique is referred to as an enzyme-linked immunosorbent assay (ELISA) (3, 4). When the sample is applied to the membrane as a dot, the procedure is called immunodotting (2, 3). Students should understand the economic importance of the large annual world production of sheep-milk cheese, especially the production of cheeses with appellation d’origine (such as the Roquefort in France, the Manchego in Spain). Each country has developed specific legislation to regulate the manufacture of these cheeses. One important concern is the use of milk from other species. The adulteration is usually made with cow and goat milk, which are generally cheaper. Besides the economic problem, these adulterations are a public health concern, as some consumers may be allergic to milk proteins from these animals. An important conclusion from these facts is that the quality control tests on each milk shipment should
Journal of Chemical Education • Vol. 75 No. 12 December 1998 • JChemEd.chem.wisc.edu
In the Laboratory
be performed before it is used in cheese manufacture. Many methods for detection of goat and cow milk in sheep milk have been proposed (5, 6 ). These methods (gas– liquid chromatography, electrophoresis, etc.) were based on the specific composition of the milk fat or proteins. However, since most of these methods involve one or more timeconsuming steps, several immunological approaches have been developed, including immunodotting (1) and ELISA, which use monoclonal antibodies (7, 8 ). Students use the enzymeimmunodotting assay described below to analyze several samples of sheeps milk for the presence of cows milk adulteration. Experimental Protocol
Materials At the beginning of the session, each group of students will have the following materials. Equipment. Nitrocellulose membrane is obtained from Amersham (Hybond-C extra, Cat. No. RPN203E). Latex gloves
Figure 1. Representation of the enzyme immunodotting assay for the detection of cow milk in milk of other species. Step a: the first antibody is added to the nitrocellulose membrane (1) to immobilize it on a solid support (2). To avoid nonspecific binding, the remaining free sites of the nitrocellulose membrane are blocked with ovalbumin (3). The assay can be stopped here and the wet membrane containing the antibody can be stored at 4 °C until further use. Step b: the unkown milk samples are incubated with the immobilized antibody (4) to form the specific antigen–antibody immunocomplexes (5). The second enzyme-labeled antibody is added to form the antibody– antigen–antibody sandwich complex (6). Unbound proteins from the samples and the excess second antibody are removed by washing. Step c: this is the development of the enzymatic reaction using a colorless enzyme substrate (6). The formation of colored enzyme product indicates the presence of the antigen of interest (7).
are worn when handling the membrane. The membrane is cut into 2 × 10-cm sheets. Adjustable digital micropipets such as Gilson’s P20 and P100 Pipetman are needed. A glass plate is used as a surface on which to place the nitrocellulose. Small plastic boxes are used in the incubation steps of the immunoassay. Reagents. Pure sheep- and cow-milk standards are obtained from local stores or farms, or are provided by the Faculty of Veterinary of the University of Zaragoza (Spain) or by Z.E.U. Inmunotech S.L. (Zaragoza, Spain). Each group will have one test tube containing the corresponding standards (0.5 mL/ tube). Both antibodies—the sheep antibody against bovine immunoglobulins (IgG), used in the first step, and the horseradish peroxidase (HRP)-labeled antibody against bovine IgG, used in the second step—are provided by Z.E.U. Inmunotech S.L. Alternatively, antibodies can be purchased from other sources (Sigma, Madrid, Spain). The blocking solution contains 5% w/v ovalbumin (Sigma, Cat. No. A-5253) prepared in phosphate-buffered saline solution (PBS). PBS contains 0.15 M NaCl, 0.01 M K2HPO4/KH2PO4, pH 7.4, and is prepared using distilled deionized water (d-water). The HRP color development reagent, 4-chloro-1-naphthol, can be purchased from Sigma (Cat. No. C-8890). This reagent is light-sensitive and should be prepared immediately before using, by mixing 12 mg of 4-chloro-1-naphthol in 4 mL of cold methanol in a beaker covered with aluminium foil. It is important to wear latex gloves to avoid contact with the HRP reagent. H2O2 (Perhydrol, 30%), used in the HRP reaction, is obtained from Merck (Spain). For this procedure, second antibodies labeled with HRP have been chosen; however, second antibodies labeled with other enzymes such as alkaline phosphatase (AP) can also be used. In this case, the immunocomplexes are obtained using 0.2 µg/mL of goat anti-rabbit IgG coupled to alkaline phosphatase (Sigma) and then detected with bromo-4-chloro-3-indolylphosphate (BCIP, Sigma) and nitro blue tetrazolium (NBT, Sigma).
Procedure Preparation of samples. The assay is optimized to analyze crude milk samples. It is advisable to use milk that has not been heated. As and alternative to crude milk, skim milk or whey proteins can be used. Only analysis of sheep-milk cheese requires sample preparation. To get a homogeneous solution, 3 g of cheese is mixed with 5 mL of 0.15 M NaCl solution using a glass potter or mortar. The homogenate is centrifuged at 300 × g for 10 min or is filtered through a paper filter (Whatman No. 1). The collected supernatant or the filtrate is then ready to be used in the immunoassay. Pure sheepand cow-milk standards are used in the assay as negative and positive controls, respectively. A 1:3 (v/v) dilution of the complete milk with d-water is sufficient for the assay. The students’ samples can be prepared by the professor by mixing pure sheep milk or cheese homogenates with various amounts of cow milk. In this work, we prepare three samples of sheep milk containing 1, 5, and 20% (v/v) cow milk. These samples are stored at 4 °C until the analysis is performed. First antibody immobilization. The nitrocellulose is placed on a clean glass plate used as a support. Latex gloves should be worn when handling the membrane. Forceps are used to remove membranes from their box. Using a waterresistant pen, the positions of the antibody applications are
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In the Laboratory
labeled on the membrane for sample identification. The applications should be at least 1.5 cm apart to avoid crosscontamination among samples during the assay. Five microliters of the first antibody anti-bovine IgG solution is applied as a circular drop on the membrane. Then the membrane is air-dried for 5 min. Blocking of the remaining free sites of the nitrocellulose membrane. The antibody-containing membrane is immersed into 5 mL of the blocking solution (5% ovalbumin prepared in PBS) contained in a small plastic box (3 × 12 cm) and incubated for 30 min at room temperature. If the membrane floats, submerge it carefully using forceps. Finally, the blocking solution is removed and the membrane is washed extensively with d-water. Sample application. The problem sample to be analyzed (40 µ L/dot) is carefully applied to the membrane in the position where the first antibody was immobilized. Pure cow milk and pure sheep milk are also applied as positive and negative sample controls, respectively. To allow the formation of the specific immunocomplexes, incubate for 5 min. The samples are removed independently with a micropipet to avoid cross-contamination. Then, the membrane is washed to eliminate unbound proteins. To reduce the risk of mixing samples, the membrane is removed with forceps and the dwater is applied separately to each application dot. Second Antibody. The HRP-labeled anti-bovine IgG antibodies are applied (40 µL/dot) to the membrane in the same position where the samples were added, and the treated membrane is incubated for 10 min to form the double antibody sandwich. The excess of second antibody is removed by washing with copious amounts of d-water. The treated membrane is placed in a small plastic box for performance the enzymatic reaction. Color development. Mix the HPR reagent solution with PBS in a 1:5 ratio (v/v). Immediately before use, mix this solution with 5 µL of 30% H2O2 and transfer it to the plastic incubation box containing the treated membrane. Incubate at room temperature until a purple color is observed in those application dots where the antigen is present (10 min, or until a color background is observed in the membrane). To stop the development, remove the solution and fill the box with d-water, changing the water at least once. Finally, air-dry the membrane and make a picture of the results. Results and Discussion The assay described here allows the detection of cow milk adulteration in milk of other species, mainly in sheep milk. In general, if the milk is not pure, the antibodies coated on the membrane will capture specifically any bovine IgG present in the samples. If the sheep milk is pure, no milk proteins will bind to the membrane. The second HRP-conjugated antibody (anti-bovine IgG) will bind specifically bovine proteins captured in the first step. Finally, a substrate–chromogen solution for the HRP reaction results in the formation of an insoluble colored product. If the result is positive a purple dot is observed in the membrane, indicating that the sheep-milk sample contains the cow-milk antigen. If the result is negative, the membrane will remain white because all the HRPconjugated antibody was removed during the washes. At the end of the assay, students will have a nitrocellulose membrane showing several purple dots of different intensities 1620
Figure 2. Immunodot analysis of pure sheep milk (1) and adulterated sheep milk containing cow milk: 1% (2), 5% (3), and 20% (4). Pure cow milk is used as positive control (5). Purple precipitates form in all samples containing cow milk. The formation of the colored precipitates correlates well with the degree of adulteration in the samples.
corresponding to the samples analyzed. Figure 2 shows the results of the analysis of three fraudulent sheep-milk samples prepared in the laboratory. These samples contained 1, 5, and 20% of cow milk (v/v) (Fig. 2, dots 2–4, respectively). Pure sheep milk (Fig. 2, dot 1) and pure cow milk (Fig. 2, dot 5) were analyzed as negative and positive controls, respectively. The presence of the purple dots correlates with degree of cow milk adulteration of the samples. No color was observed when pure sheep milk was analyzed. This result indicates that the antibodies used are highly specific for bovine IgG. However, in some experiments a slight colored background might appear in the control sample. This artifact is likely due the blocking or the washing steps not having been properly performed. It could also be observed if the antibodies used are not completely specific. The presence of high levels of fatty acids in the milk, and especially in the cheese, could affect the formation of the antigen–antibody complex and give false-negative results. In the case of cheese, we have described the procedure to prepare a homogenate for the assay. For milk samples, the removal of the milk fat is not necessary. Student Response The most important benefit is that students usually appreciate the opportunity to deal with real-life problems. They see that they have already learned the principles necessary to perform the laboratory work on time and successfully. Most students also enjoy the practical course, and sometimes they try to design alternative procedures to solve the same problem. All students have been fascinated by the potential for application of the antibody–antigen interaction. Figure 3 shows a very personal interpretation by one of our students of the antibody–antigen interaction. Conclusions The most relevant characteristics of this enzyme immunodotting assay are (i) the short time needed to develop the test (around 30 min if the assay starts with the first antibody already immobilized in the nitrocellulose); (ii) the high sensitivity (it detects cow milk adulterations as low as 1%); (iii) the high specificity (no cross-reactivity with other milk proteins of other species, such as goat milk, is observed) (data not shown). In addition, the assay is easy to perform. It consists of three steps and the milk sample is analyzed directly with no preprocessing. The results of several analyses can be stored in the laboratory diary without loss of the color dots. The immunodot method described here was designed as a qualitative method. It has the advantage that it requires no special equipment or trained operators, making it suit-
Journal of Chemical Education • Vol. 75 No. 12 December 1998 • JChemEd.chem.wisc.edu
In the Laboratory
Another enzyme immunodotting assay method has been developed to detect transferrin in normal human blood plasma (2). However, the use of blood-related material has serious safety implications. Because the aim of the assay described here is mainly pedagogic, we have developed this assay so that students can understand the fundamentals of the method while using materials that represent little risk. Other immunochemical methods, such as double immunodiffusion and radial immunodiffusion (3) could be also used but they usually require overnight incubations. The ELISA performed with microtiter quantities is also specific and sensitive, but it requires special equipment to measure the results (3, 4). The students will know that in addition to the application described here, these techniques are used in many laboratories dedicated to quality control, especially in the detection of food adulterations, in the analysis of food contaminants, and in clinical assays to detect various antigens (3, 4). Acknowledgments Figure 3. A funny interpretation of the specific interaction between an antibody and the corresponding antigen. This drawing was kindly provided by D. Gabriel Mainer, Fellow associated with the Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza.
able as a “field method” in dairy factories and laboratories for quality control before using milk in cheese manufacture. It is an interesting assay for inclusion in laboratory practical courses at colleges and universities, because of its low cost and ready availability of laboratory supplies required. Thus students in different specialities, such as chemistry, clinical biochemistry, immunochemistry, food quality control research, or veterinary medicine, can learn through this assay to solve a problem of quality control.
We wish to thank Andrés Piñeiro, Full Professor in the Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain, for his experience and helpful suggestions during the development of this assay; and D. Gabriel Mainer, Associated Fellow in the Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, for providing Figure 3, an artistic interpretation of the assay. Literature Cited 1. Aranda, P.; Sánchez, L.; Pérez, M.D.; Ena, J. M.; Puyol, P.; Oria, R.; Calvo, M. Food Control 1993, 4, 101–104. 2. Russo, S. F.; Dahlberg, J. U. J. Chem. Educ. 1990, 67, 175–176. 3. Principles and Techniques of Practical Biochemistry, 4th ed.; Wilson, K.; Walker, J., Eds.; Cambridge University Press: New York, 1994. 4. O’Kennedy, R.; Byrne, M.; O’Fagain, C.; Berns, G. Biochem. Educ. 1990, 18, 136–140. 5. Ramos, M.; Juarez, M. Int. Dairy Fed. Bull. 1984, 181, 3–9. 6. Rodriguez, M. A. Rev. Española Lechería 1995, 10, 26–30. 7. Levieux, D.; Venien, A. J. Diary Res. 1994, 61, 91–99. 8. Immunology. A Short Course, 3rd ed.; Benjamini, E.; Sunshine, G.; Leskowitz, S., Eds.; Wiley-Liss: New York, 1996.
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