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The Dot Blot ELISA: A Rapid & Simple Experiment to Demonstrate Antibody-Antigen Interactions Author(s): Donald G. Gerbig, Jr., Christopher J. Fenk and Amy S. Goodhart Source: The American Biology Teacher, Vol. 62, No. 8 (Oct., 2000), pp. 583-587Published by: on behalf of the University of California Press National Association of Biology

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S S be

The Dot Blot ELISA A Rapid c& Simple Experiment To Demonstrate Antibody-Antigen Interactions

Donald G. Gerbig, Jr. Christopher J. Fenk Amy S. Goodhart

The understanding of antibody-antigen interactions is of fundamental importance in introductory science courses that include immunology as an essential component. Differentiat- ing the terms antigen and antibody are major accomplishments in any immunology discussion. Unfortu- nately, many students never have the opportunity to "see" the specificity of these interactions. Ironically, these same students may have already encountered rapid diagnostic tests based on the specificity of antibody- antigen interactions.

Rapid diagnostic tests are now available in kit form and are used to iden- tify infectious agents or body metabo- lites. Two commonplace examples are the rapid streptococcal identification kit and the home pregnancy test kit. The rapid streptococcal identification kit is a fixture in physicians' offices and has superceded the traditional approach of isolating Streptococcus pyo- genes on blood agar plates.

Two popular laboratory techniques that demonstrate antibody-antigen binding concepts are the Enzyme Linked Immunosorbent Assay (ELISA) and Western blot (Ausbel et al. 1997; Harlow & Lane 1998). Rapid diagnostic tests, similar to those referred to above, were initially developed using ELISA or Western blot assays. In both techniques, antigen is detected using an antiserum that contains reactive antibodies. Students, particularly those in the allied health fields, should recog-

nize that ELISA and Western blot are used for detection and/or confirma- tion of Lyme disease and HIV infection (Ziska et al. 1996; Burke 1989).

Both the ELISA and Western techniques have been introduced successfully in the undergraduate laboratory (Fenk et al. 2000; Farrell & Farrell 1995; Grimes et al. 1998). The reported Western blot experiments, although powerful, use more expensive equipment, include the use of primate- derived reagents (Farrell & Farrell 1995), and require a minimum of two laboratory periods to complete. The reported ELISA technique, while useful, is in fact a laboratorv simulation based on biotin-streptavidin interactions and does not involve actual antibody-antigen binding.

A complementary technique that serves as an effective introduction to antibody-antigen interactions is the dot immunobinding assay or dot blot ELISA (Hawkes 1986). The dot blot ELISA demonstrates fundamental features of both the ELISA and Western blot techniques, yet is simpler to perform. Unlike a conventional ELISA that is quantitative, the dot blot ELISA is generally qualitative with a single dot observed and viewed against a white background. Also, the dot blot ELISA does not require PAGE, as in a Western blot.

The dot blot ELISA is well suited for the undergraduate laboratory, as evidenced by the recent availability of commercial kits and a published experiment based on human transferrin (Russo & Dahlberg 1990). However, commercial kits are relatively expensive and contain proprietary compo- nents, compromising the instructor's ability to fully explain the kits' actual mechanisms. In addition, the use of primate-derived reagents, such as human transferrin, is a serious deter- rent for incorporation into the introductory laboratory setting due to con- cerns regarding potential exposure to

HIV and hepatitis B. We have devised a simple and fun dot blot immunobinding experiment for introductory science laboratories to illustrate the specificity of antibody-antigen interactions. This dot blot experiment demonstrates the key features of both the conventional ELISA and Western blot techniques. The procedure is versatile, inexpensive to perform (after initial investment in supplies, cost is esti- mated at less than $1.00 per strip), and uses nonprimate-derived proteins.

The design and implementation of this dot blot experiment is simple and straightforward. Bovine serum albumin (BSA) and ovalbumin (OVA) are "dotted" onto a support membrane and probed with rabbit anti-albumin serum. Horseradish peroxidase conjugated recombinant protein G and substrate are used to selectively detect bound antibodies. This introductory experiment can be performed in less than three hours and uses readily available reagents. In addition, it involves actual antibody-antigen interactions; thus, the basic principles of the experiment are the same as those incorporated into a rapid diagnostic kit. The procedure is simple enough to be used in high school as well as introductory college laboratories. Mod- ifications in the procedure can adapt it toward a variety of student skill levels. The reagents can be prepared in advance for students with little laboratory experience. Advanced students can be challenged by preparing the necessary reagents. This experiment has been successfully incorporated into a number of introductory courses including anatomy, microbiology, and physiological chemistry.

Materials The following list of materials is

needed for this experiment. All equipment and supplies are available through a distributor such as Fisher

Donald G. Gerbig Jr., Ph.D. is an Assis- tant Professor of Biology and Christo- pher J. Fenk, Ph.D., is an Assistant Pro- fessor of Chemistry at Kent State Uni- versity-East Liverpool, East Liverpool, OH 43920. Amy S. Goodhart is an undergradute student in the Depart- ment of Biology, Kent State University- East Liverpool. Professor Gerbig can be reached by e-mail at dgerbig@ kenteliv.kent.edu.

DOT BLOT ELISA 583



Scientific, Pittsburgh, PA. Immunologi- cal reagents are available through a number of vendors such as Sigma- Aldrich, St. Louis, MO; Zymed, San Francisco, CA; and AMRESCO, Solon, OH.

* Bovine Serum Albumin (BSA) pow- der; Fraction V

* Chicken Egg Albumin (Ovalbumin, OVA); Grade V

* Primary Antiserum (Rabbit anti- bovine albumin whole serum); Sigma catalog number B-1520

* Recombinant protein G-Horserad- ish Peroxidase (HRP) conjugate; Zymed catalog number 10-1223

* HRP substrate (Diaminobenzidine, DAB tablets); AMRESCO catalog number E-733

* Tween 20 * 20X Tris Buffered Saline (TBS) * Pelikan' India ink * Poly(vinylidene difluoride) mem-

brane (PVDF) * Methanol * 3% Hydrogen peroxide * Adjustable digital pipettors (0-10 pI)

or Hamilton' syringe * 15-ml Conical centrifuge tubes or

similar capped tubes * Latex gloves * Orbital or rocking shaker * Glass or plastic pipets * Vacuum pump or water aspirator

Methods: Pre-Lab Preparation

These methods may vary according to student number and participation level. The following protocol assumes the instructor will make dilutions and provide pre-made reagents to students. All formulations assume volume will be lost due to student and instructor error and are generous volumes for a class of 12. More advanced students can follow the pre-lab preparation in carrying out this exercise. However, the instructor must keep in mind that this will require the exercise to be performed in two laboratory periods as opposed to one.

1. Prepare a 20X solution of TBS for subsequent dilutions. Add 163.6 grams of sodium chloride (NaCl), 4.4 g of potassium chloride (KCI) and 60.6 g of Tris base to 700 ml of distilled water. Stir until dissolved, adjust pH to 7.4 using 1M or concentrated HCl and adjust volume to 1000 ml with distilled water. Note: We found it convenient and economical to purchase the 20X TBS

solution commercially (AMR- ESCO; Solon, Ohio).

2. Prepare a liter of TBS-0.05% Tween (wash buffer) for diluent and washing the membranes. Add 50 ml of 20X TBS buffer to 950 ml of distilled water. Then add 0.5 ml of Tween 20 to the solution and mix thoroughly.

3. Prepare 100 ml of blocking buffer for the PVDF strips. Add 5 ml of 20X TBS buffer to 95 ml of distilled water. Then add 0.2 ml of Tween 20 to the solution and mix thoroughly.

4. Prepare 100 ml of India ink staining buffer. Add 5 ml of 20X TBS buffer to 95 ml of distilled water. Then add 0.3 ml of Tween 20 and 100 pl of India ink to the solution and mix thoroughly.

5. Prepare a 1/4000 dilution of primary antiserum in TBS-0.05% Tween (wash buffer). Add 10 ,ul primary antiserum to 40 ml of diluent and mix thoroughly.

6. Prepare a 1/2000 dilution of recombinant protein G-HRP in TBS-0.05% Tween (wash buffer). Add 20 ,ul recombinant protein G-HRP to 40 ml of diluent and mix thoroughly.

7. Prepare a 1.0 mg/ml solution of BSA to be used for dotting. Weigh 1.0 g of BSA and dissolve into 10 ml of TBS. Dilute 10 ,ud of this BSA solution into 1 ml of TBS solution and mix thoroughly.

8. Prepare a 1.0 mg/ml solution of OVA to be used for dotting. Weigh 1.0 g of OVA and dissolve into 10 ml of TBS. Dilute 10 pl of this OVA solution into 1 ml of TBS solution and mix thoroughly.

9. Cut the PVDF membrane into strips that will fit into 10-ml conical tubes. Cut the bottom of the strip asymmetrically to distinguish front from back. One-by-four centimeter strips work well. Approximately 16 strips are needed for the experiment (12 students + 1 reference standard + 3 extra). Take care to use latex or other suitable gloves to avoid touching the PVDF.

10. Dotting procedure: Wet a PVDF strip with -1 ml methanol for 15 seconds. Discard the methanol, then wet the membrane with -3 ml distilled water for 15 seconds. Discard the water wash. Using a vacuum device, apply 5 ,lL of BSA solution onto the strip of PVDF. Do not move the membrane until all the liquid has

aspirated through the PVDF. Apply 5 ,l of OVA solution about 5 mm below the BSA dot. Again, do not move the membrane until all the liquid has aspirated through the PVDF. For verification purposes, indicate the location of each protein dot by inscribing the edge of the membrane with a razor blade. Allow the membrane to dry completely. Note: Some laboratories may not have vacuum lines readily available. A water aspirator is useful to create the vacuum. Attach a 10-ml volumetric pipet to rubber tubing connected to a water aspirator. If available, a 96- or 48-well dot blot plate system is useful in place of the pipet and rubber hose. These devices allow for the easy preparation of multiple samples.

11. Before class starts, mix diaminobenzidine (DAB) as described by the manufacturer. Briefly, 1 DAB tablet (48.0 mg) is dissolved in 10 ml of distilled water followed by addition of 50 pl of 3% H202.

12. One PVDF strip is stained with India ink (1 jil/ml) prior to class to show the dotted proteins. Place a PVDF strip in a 15-ml conical tube. Wet the PVDF strip with -1 ml methanol for 15 seconds. Discard the methanol, then wet the membrane with -3 ml distilled water for 15 seconds. Discard the water wash. Stain the PVDF membrane with the India ink buffer solution by agi- tating on an orbital shaker for 1 to 3 hours.

13. (Optional) A negative control strip may be prepared with one of the extra PVDF strips. After wetting and blocking, this strip is incubated directly with recombinant protein G-HRP, omitting the anti-albumin serum. This demonstrates the specificity of recombinant protein G-HRP for antibody.

Methods: Student Procedure

Volumes listed are for individual tubes. Each student is supplied a PVDF strip in a 15-ml conical tube, pre- diluted blocking buffer, primary antibody, recombinant protein G-HRP and DAB substrate. Wash buffer may be provided in a polypropylene squeeze bottle to facilitate washing the PVDF strips. A water aspirator similar to

584 THE AMERICAN BIOLOGY TEACHER, VOLUME 62, NO. 8, OCTOBER 2000



the one described for applying the proteins to PVDF is useful for the washing steps. A Pasteur pipet is sub- stituted for the volumetric pipet. With this setup, students can carefully aspi- rate the fluid from the tubes without disturbing the membrane. This elimi- nates potential loss of the membrane encountered by pouring fluid from the tubes.

Although an orbiting shaker or rocker is desirable for this exercise, it is not required. If a shaker or rocker is not available, students can agitate the tubes manually every 30 seconds during the incubation times and achieve satisfactory results.

IMPORTANT: During the procedure, the PVDF should never be allowed to dry. If this occurs the membrane must be wetted as described in Step 1 below.

1. Wet the PVDF membrane with -1 ml of methanol for 15 seconds. Discard the methanol, then wet the membrane with -3 ml distilled water for 15 seconds. Discard the wash water.

2. Block the nonspecific binding sites with 3 ml of blocking buffer for 20 minutes using an orbital shaker, then discard the blocking buffer solution.

3. Wash the membrane for 1 minute with distilled water followed by two 5-minute washes in 3 ml of wash buffer solution. Each wash should be done at room temperature on the rotary shaker. Imiiportanit: Discard wash solutions tipon1 completion of each wash step.

4. Add 3 ml of primary antibody solution to the PVDF strip and incubate for 20 minutes at room temperature on the rotary shaker. Discard the antibody solution.

5. Wash as described in Step 3. 6. Add 3 ml of recombinant protein

G-HRP solution to the PVDF strip and incubate for 20 minutes at room temperature on the rotary shaker. Discard the recombinant protein G-HRP solution.

7. Wash as described in Step 3. 8. Add 3 ml of the pre-made DAB

substrate solution to the blots and watch for the appearance of a dark colored dot. When dots appear, discard substrate solution and wash blot with distilled water to stop the reaction.

9. Air dry the strips and compare to India ink reference standard.

10. Record observations.

Results & Discussion Analysis of the experimental results

is simple and direct. After completing

the procedure, students will observe a dark brown dot on the PVDF membrane probed with anti-albumin serum (Figure 1). This "dot" is the result of a specific interaction between anti- albumin serum and albumin. HRP- conjugated recombinant protein G is used to recognize the antibodies binding to the deposited protein. Diamino- benzidine (DAB) is oxidized and deposits a brown precipitate where the antibody-antigen complex is found on the membrane. (Figure 2). The specificity of this interaction is demonstrated by comparing the developed PVDF membrane with protein dotted PVDF stained with India ink. India ink stains both the albumin and ovalbumin and shows the student where proteins are deposited on the PVDF membrane (Hancock & Tsang 1983). This compar- ison is important in demonstrating a number of concepts. First, antibody- antigen specificity is stressed. The student visualizes two dots on the India ink strip and only one dot on the antibody probed strip. The anti-albumin serum recognizes albumin and not ovalbumin. Second, the concept of controls can be introduced. The instructor can discuss the consequences of omitting primary antibody, secondary binding proteins (recombinant protein G-HRP), albumin or ovalbumin from the membrane. We found it convenient to use a "negative" control as part of the experiment. This strip is incubated

directly with recombinant protein G- HRP, omitting the anti-albumin serum and demonstrating the specificity of the recombinant protein G-HRP for antibody (Figure 1).

Several features of this laboratory procedure are unique and make it convenient to use in introductory science courses. All of the immunological reagents are nonprimate-derived (bovine albumin, chicken ovalbumin, rabbit anti-albumin serum and HRP conjugated protein G) and can be purchased commercially. The instructor can ac- complish the procedure without exten- sive pre-laboratory preparation. In most immunological procedures, antisera concentration must be titrated in order to optimize signal response. With this procedure, titration is not necessary. A wide range of antibody dilutions (1/ 1000 to 1/20,000) work effectively. Since the detectable levels of antibody are so small, consumption of valuable immunological reagents is minimized, making the procedure more economical.

The entire procedure can be com- pleted in a single three-hour laboratory period at ambient temperature. Con- ventional ELISA and Western blots contain multiple incubation steps at 370 C and require many hours to complete. Incubation times have been reduced to 20 minutes each at room temperature. To accommodate this minimal time frame, unoccupied

OVA

A B C

Figure 1. Photograph of Dot Blot ELISA. Strip A was probed with the anti-albumin serum, followed by recombinant protein G-HRP and substrate. Strip B was stained with India ink to visualize both the dotted albumin and ovalbumin. Strip C was probed with recombinant protein G-HRP only.

DOT BLOT ELISA 585



binding sites are blocked with a TBS solution containing 0.2% Tween 20 (Harlow & Lane 1988) instead of skim milk, albumin (a problem with this exercise) or gelatin. The use of Tween 20 as a blocking agent avoids solubility issues associated with protein-based blocking reagents. These modifications reduce the time required for the exercise and permit completion in a three- hour laboratory.

The more traditional nitrocellulose membrane used in most dot blot immunoassays is replaced by PVDF membrane. Two advantages to using PVDF membrane are that it is more resistant to tearing in routine handling and it is nonflammable. These are important considerations in high school and undergraduate laboratories.

The use of recombinant protein G- HRP as a secondary binding protein is also of considerable significance. Protein G is an immunoglobulin G (IgG) binding protein isolated from groups C and G streptococci. This protein binds human serum albumin and two portions of IgG, the Fc (fragment crystalline), and Fab (fragment antigen binding) regions (Erntell et al. 1988; Kato et al. 1995). Recombinant protein G is produced in Escherichia coli and lacks the albumin binding site found in

the natural protein. The recombinant product only binds to the F, and Fab regions of IgG (Figure 3). The use of recombinant protein G avoids the possibility of "false" positive dots that may occur with secondary animal antisera binding to albumin and ovalbumin. The use of a recombinant protein is an excellent introduction to the world of molecular biology.

Other important features of this laboratory exercise include the minimum of specialized laboratory equipment and use of commercially prepared

reagents. Unlike the ELISA or Western blot, which call for expensive supplies and materials, this exercise uses equipment commonly found in most biology laboratories. The reagents for this experiment are relatively inexpensive and may be purchased pre-mixed, adding extra convenience for use in the undergraduate or high school laboratory.

Classroom Applications There are a number of variations on

this exercise that make it appropriate not only for introductory classes, but more advanced science courses as well. The procedure can be modified to demonstrate disease transmission as published by Dickey (1989) and Grimes et al. (1998). Antibody solutions could serve as simulated body fluids that are exchanged from person to person. If the exchanges are coordinated prop- erly, the individual who infected the others can be determined upon completion of the dot blot procedure. Anti- body detection in the described procedure is extremely sensitive, allowing for multiple dilutions without signal loss. This allows a single person to serve as the reservoir of infection.

Another suggestion for more advanced classes is the use of both anti- albumin and anti-ovalbumin sera. The anti-ovalbumin serum can be used at the same dilution as described for anti- albumin antiserum. The specificity of the anti-ovalbumin serum is such that cross reactivity between albumin and ovalbumin is not observed. Using both antisera in a class could serve as an exercise involving immunological unknowns and reinforce the concept of antibody-antigen specificity. Albumin, ovalbumin or both proteins could be visualized, depending upon the instruc- tors' planned outcomes. If simple protein detection doesn't sound exciting, students could be assigned a "blood" sample (diluted anti-albumin and anti- ovalbumin serum) and sent to the laboratory to screen for a fictitious infectious agent (albumin and ovalbumin).

If time is a limiting factor in conduct- ing this exercise, the procedure may be interrupted after incubation with primary antiserum. Store the tubes containing the PVDF strips and primary antiserum horizontally over- night. The experiment may then be continued at that point the following day. Alternatively, incubation with primary antiserum may be continued on an orbital or rocking shaker for 24 hours.

A useful pre-laboratory exercise is to require students to find literature

b::1FHRP

Il/ Substrate (colorless)

ABSA Q OVAHR

Anti-albumin Recombinant Protein G-HRP

Figure 2. Diagram of the Dot Blot ELISA. Proteins are dotted on PVDF membranes, probed with primary antiserum followed by recombinant protein G-HRP and substrate.

IgG molecule

> ~~~~~region

0Fab region _

Figure 3. Typical immunoglobulin (IgG) illustrating the Fc and Fab portions.

586 THE AMERICAN BIOLOGY TEACHER, VOLUME 62, NO. 8, OCTOBER 2000



that uses the dot blot ELISA in screen- ing procedures. A great deal of useful information is available via the Internet through sources such as MED- LINE available through www.ncbi. nlm.nih.gov/PubMed/. If the papers are not readily available, the published abstracts should provide enough information to initiate discussion of the application. It is important to note, however, that great care must be exer- cised when using other Internet resources which may not be directly associated with the primary literature.

Conclusions The dot blot experiment described

is a simple and versatile laboratory exercise that effectively introduces the immunological concepts of antibody- antigen interactions to introductory science classes. The procedure is adaptable to a broad range of student skill levels. Pre-made solutions can be supplied to students with little laboratory experience. These students will observe antibody-antigen interactions without being overwhelmed by detailed laboratory techniques. More advanced students can prepare the PVDF membranes and make the necessary reagents for the exercise. These students gain an appreciation of the preparation necessary for performing an ELISA or a Western blot procedure. In addition, if the Western blot technique is part of the curriculum, this is an effective introduction to its principles. This dot blot ELISA has been used successfully to provide students with the background necessary to exe- cute a Western blot laboratory exercise (Fenk et al. 2000).

If laboratory exercises are to fully engage students in learning, it is important that they be relevant to their experiences and not merely cookbook demonstrations. Immunological techniques, widely used for clinical testing, fulfill this criterion. The simplicity and flexibility of the exercise detailed here allows for a variety of students to experience scientific principles in a meaningful context.

Acknowledgments We would like to thank Dr. Roxanne

Burns for her insightful and helpful comments in critically reviewing this manuscript. We would also like to thank Sharon Campbell for her techni- cal assistance with the laboratory procedures.

References Ausbel, F., Brent R., Kingston, R.E.,

Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. (Eds.). (1997). Short Protocols in Molecular Biology. New York: Wiley.

Burke, D.S. (1989). Laboratory diagnosis of human immunodeficiency virus infection. Clinics in Laboratory Medicine, 9(3), 369-392.

Dickey, J.L. (1989). A quick and easy simulation of disease transmission. The American Biology Teacher, 51(6), 364-365.

Erntell, M., Myhre, E.B., Stobring, U. & Bjork, L. (1988). Streptococcal protein G has affinity for both Fab and Fc fragments of human IgG. Molecular Immunology, 25(2), 121-126.

Farrell, S.O. & Farrell, L.E. (1995). A fast and inexpensive Western blot experiment for the undergraduate laboratory. Journal of Chemical Educa- tion. 72(8), 740-742.

Fenk, C.J., Grooms, S.Y. & Gerbig, D.G., Jr. (2000). A convenient and highly specific Western blot experiment for introductory biochemistry. Journal of Chemical Education, 77(3), 373-374.

Grimes, W.J., Chambers, L., Kubo,

K.M. & Narro, M.L. (1998). Trans- mission of a viral disease (AIDS) detected by a modified ELISA reaction: A laboratory simulation. The American Biology Teacher, 60(5), 362-367.

Hancock, K. & Tsang, V.C. (1983). India ink staining of proteins on nitrocellulose paper. Analytical Bio- chemistry, 133(1), 157-62.

Harlow, E. & Lane, H. (1988). Antibod- ies: A Laboratory Manual. New York: Cold Spring Harbor.

Hawkes, R. (1986). The dot immunobinding assay. Methods in Enzymology, 121, 484-491.

Kato, K., Lian, L., Barsukov, I.L., Der- rick, J.P., Kim, H., Tnaka, R., Yos- hino, A., Shiraishi, M., Shimada, I., Arata, Y. & Roberts, G.C.K. (1995). Model for the complex between protein G and an antibody F, fragment in solution. Structure, 3, 79-85.

Russo, S. F & Dahlberg, J. U. (1990). An enzyme immunoassay for human transferrin. Journal of Chemical Educa- tion, 67(2), 175-176.

Ziska, M. H., Donta, S. T. & Demarest, F. C. (1996). Physician preferences in the diagnosis and treatment of Lyme disease in the United States. Infection, 24(2), 182-186.

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DOT BLOT ELISA 587



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