An Enzyme lmmunoassay for Human Transferrin Salvatore F. Russo and Judy Utter Dahlberg Western Washington University. Bellingham, WA 98225 Proteins carry out several types of biological functions including transport and storage, muscle contraction, mechanical support, immune protection, generation and transmission of nerve impulses, and the control of growth and differentiation. When a protein performs a catalytic function, i t can be assayed by a suitable choice of substrate coupled with a method of following the change in substrate or product concentration with time. However, this cannot be done when the protein is not an enzvme. and the assav of noncatalytic prckeins may be difficult:~ortunatel~, the field a powerful method for detectine of immunoloay -. provides . specifir noncatalytic proteins. In an enzyme immunoassay (EIA) utilizina dor-immunohinding', the antigen is first bound to a nitro~ellulosemembrane. The antigen is the protein that is to be detected. Following hinding of antigen, the remaining unhound sites on the nitrocellulose are blocked with gelatin, a protein that will not hind to antigen. Membrane-hound antigen is then incubated with an antihody specific for the antigen to he detected, and the membrane is washed to remove unhound antihody. This washing is followed by the addition of a second antihody which combines with the first. The second antibody contains a covalently attached enzyme which can he detected by a n appropriate assay. In this experiment, the antigen is human transferrin, a protein of 90,000 molecular weight found in the 0 electrophoretic fraction of human blood plasma. The human transferrin was injected into rabbits to produce antibodies to human transferrin. There are five classes of immunoelobin. Immunoglobulin G (IgG) molecules have two light chains and two heavy chains connected by several disulfide bonds. T h e combination of human antigen (Ag) with rabbit IgG (Ah) molecules can be depicted as follows since this tvpe of antihody molecule contaihs two hinding sites:
human transferrin (Ad
rabbit IgG (lOAh)
(Agz . loAb)
The second antihody was produced by injecting rabbit IgG into goats and is called goat antirabbit (GAR). The second antihody has the enzyme horseradish peroxidase (HRP) covalently attached and will detect any antigenrabbit IgG complex. The goat antirabbit antihody binds to the antigenic determinants on both heavy and light chains of the rabbit antihody.
Horseradish peroxidase catalyzes the following oxidationreduction reaction between 4-chloro-l-nanhthol and -~ hvdrogen peroxide: ~
The l,4-naphthoquinone produced in this reaction is purple, which allow* detection of the Dresence of the initial antieen. High-grade reagents must be used in this experimeni to avoid problems with impurities that miaht interfere with the analy&. Azide ion is 'specially trouhiesome since it is an inhibitor of horseradish peroxidase. Experimental Equipment Sheets of 15- X 15-cmnitrocellulose may he purchased from BioRad (Cat. No. 162-0116).Aplatformshakeris needed. Cover a 10- X 10-in. niece of Stvrofoam with aluminum foil since this ~rovidesa clean aid resilient surface for the nitrocellulose membrane. Continuously adjustable digital micropipets such as P20 and P200 Gilson Pipetman are needed. Reagents Distilled deionized water (ddH20)should be used throughout this experiment. Aqueous solutions of bovine serum albumin (BSA) and lysazyme (Lyso) are needed, each at a concentration of 2 mg/mL. Human transferrin should he prepared at a concentration of 10 mg/ 100 FL using a 0.75-mL Eppendorf tube and high-resolutionbuffer (HRB) as the solvent. Normal human blood plasma (Cat. No. IL 200020) may he purchased from Fisher Scientific. This product is derived from material that has tested negative for the hepatitis B surface antigen and the AIDS virus antibody. The buffer (Gelman Cat. No. 51104) contains tris(hydroxymethyl)aminomethane,barbital, and sodium barbital at pH 8.8 and ionic strength 0.06 M. The first antibody, rabbit antiserum to human transferrin, may be purchasedfrom Accurate Chemical and Scientific (Cat. No. A061). The second antibody, goat antirabbit IgG (H + L) horseradish peroxidase conjugate, is available from Bio-Rad (Cat. No. 170-6515). Blocking solution contains 3%w/v gelatin (EM grade, Bio-Rad Cat. No. 170-6537).Tris-buffered saline (TBS) contains 20 mM tridhydroxymethyl)aminomethane (EIA grade, Bio-Rad Cat. No. 1610716) and 500 mM NaCl at pH 7.5. To prepare TBS add 4.84 g of tris(hydroxymethyl)aminomethane to 58.45 g NaC1, and bring to approximately 1800 mL with ddHnO.Adjust to pH 7.5 with HC1, and then dilute to a total volume of 2.00 L. Tween tris-buffered saline (TTBS) is prepared by adding 0.5 mL tween-20 (EIA grade, Bio-Rad (Cat. No. 170-6531)to 1.00 L of TBS. Horseradish peroxidase (HRP) color development reagent can be purchased from BiaRad (Cat. No. 170-6534). To nrenare solution A. add 10 uL of stock transferrin to 90 uL HRB tuhk. - in aa0.75-m~ ~. and follow hv - ~nnendorf ., , thoroueh mixi&. T o prepare solution H, add 10 rl. of A to 9.) pL HRR in a 0.75-ml. Eppendorftuhe, and rollw by thorough mixing. C'mrinu?rhis serial dilution procedure to produce C, D, E, and F. ~~~
(Ag, . loAh)
z0 Ab
~~
~
~
' Hawkes, R.; Niday, E.:Gordon, J. Anal. BIocbem. W82, 119, 142-
147.
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Procedure Cover a 50-mL Erlenmeyer flask with aluminum foil, and place this in a freezer for later use in color development. Place the hlockingsolution (3%aqueousgelatin)in a 37 "C bath until liquefied.Cut out acireular piece of nitrocellulose filter that is approximately8 cm in diameter. Wear disposable gloves to avoid contaminating the filter. Put your initials on it using a hall-point pen (not felt-tip). Then write letters on the filter to identify the locations of each sample using the following code: S (stock), A, B, C, D, E, F,BSA, Lyso, and BL (blood). Sample Application. In this and all subsequent steps, it is important to submerge the filter rather than having it float on the surface of the solution. Immerse it at a 459 angle into ddHnOin a 9-emdiameter Petri dish. The water should be evenly adsorbed. Using forceps,take the filter out, and carefully wave it in the air to remove excess liquid. Then suspend it for 5 min over straight pins stuck into the aluminum-foil-covered Styrofoam. After the nitrocellulose is completely dry, move the filter onto the aluminum foil surface, and pin it in place. Using a P20 Pipetman, spot on 1 of stock transferrin and each dilution below the code Letters (A, B, C, D, E, F); repeat for BSA, lyso, and BL. Avoid pressing too hard on the nitrocellulose with the tip of the pipet. Air dry for 5-10 min by again suspending the filter on pins. Fill a Petri dish with 20 mL of blocking solution. Immerse the membrane into the blocking solution, and swirlon a platform shaker for 30 min. First AntOody. Place 7 mL blocking solution plus 14mL TBS in a Petri dish. Immediatelyprior to use, add 21 pL of the first antihody. Then transfer the filter from the blocking solution into the first antihody solution, and swirl for 1 h. After that time, rinse in ddHzO in another Petri dish for 1min. Washin 20mLTTBS for 5min with swirling. Repeat with fresh 20 mL TTBS for another 5 min with swirling. Second Antibody. Just prior to use, add 7 pL of second antihody to a Petri dish containing 7 mL blocking solution and 14 mL TBS. Transfer the filter to thesecond antibody solution, andswirl for 1h. After that time, rinse in ddHnO in aPetri dish for 1 min. Then wash in 20 mL TTBS for 5 min with swirling. Wash again in 20 mL TTBS for another 5 min with swirling. Wash in 20 mL TBS for 5 min with swirling. Color Deuelopment. Prepare the following solutions: I . Caution: Weorgioues to avoid contact uith thr HRPrcagent. Didaolve 12 mg HRI' reagent in 4 ml. ire-cdd methanol in n 10. ml. beaker. Covrr with aluminum foil rrnce the rrngenr is light
sensitive. 2. Remove the cold Erlenmeyer flask from the freezer. Add 20 mL TBS followed by 12 pL ice-cold 30%H202with mixing. Mix solutions 1and 2 immediately before use, and then transfer to a Petri dish. Transfer the filter to the Petri dish, and allow to incubate at room tem~eraturefor 15-30 min. Stoo the develooment by imhrraina the iilter in ddH>Ofor 10 min, ;hanging thewtrr atleast onw. Dry the mrmhrane suspended on pins.
Discussion The EIA procedure will not detect BSA or lysozyme since they do not contain antigenic determinants in common with human transferrin. The most intense spot is observed for L lo5 ng) followed by A (lo4 stock (1 FL of 1.0 X 1 0 h g l ~ or ng), B (10" ng), C (10%ng), D (10 ng), and E (1.0 ng). The spot observed for E is only slightly different from background so this defines the sensitivity of the assay. The BL (blood plasma) may be compared to the standards to obtain an approximate value for transferrin concentration. Normal adult hlood serum is reported to have a transferrin concentration in the ranee 220400 me1dL (2200-4000 n -d u L ) as measured hy nephelometry: The entire procedure requires about 4 h. In addition to the qualitative examination of spots, t h e r e sults may he tpaniitated using the reflectanck mode of a densitometer.' This assay has two advantages over enzyme immunoassays that employ microtiter plates. First, the amount of the antigen needed is ereatlv reduced because of the small s ~ o t &."second, the uie of &rocellulose permits the color ti be viewed against a white background which enhances the discriminatory power of the assay. ~ the use of rabbit antiA recent ~ u b l i c a t i o ndescribes transferrin bound to microtiter plates followed by human transferrin and then mouse antitransferrin attached to HRP for the analysis of transferrin in human hlood serum. This is an example of a method that relies on two different sites on the transferrin molecule. Another use for the assay described involves the electronhoretic seoaration of oroteins followed bv electro~horetic transfer to ;litrocellulode (Western blotting). Since proteins are alreadv on the solid matrix. samole has been . a~olication .. accomplished,and one would begin with blocking solution in the exoerimental ororedure. This provides a powerl'ul tool for identifying specific proteins in complex mixture since only proteins recognized by the first antihody will lead to ~ ~ o r in the Western eventual color d e ~ e l o p m e n t . ~ example, blot test for individuals carrying antibodies to the human immunodeficiency virus (HIV) that causes AIDS, viral proteins are first separated by electrophoresis followed by transfer to nitrocellulose. A positive test will depend on the binding of specific antibodies in blood serum to these proteins. The final step is accomplished with HRP attached to antibody that recognizes the previously used antiviral antibody.
a
Tietz, N. W. Texibmk of hinical Chemistry: Saunders: Philadelphia, 1986; p 1847. Guindi. M. E.; Skikne, 8. S.; Covell. A. M.; Cook. J. D. Am. J. Clin. Nuh: 108447.37-41. 'Towbin. H.: Staehlin. T.: Gordon, J . Proc. Nat. Acad. Sci.. USA 1979, 76.4350-4354. Reiser, J.; Wardale, J. Eur. J. Biochem. 1981, 114, 569-575.
Second Edition of Modern Experiments for Introductory College Chemistry Available A new edition of Modern Experiments for Introductary Chemistry, containing experiments from the Journal of Chemical Education that were selected because they reflect exciting innovations in introductory laboratory work, has just been published. Compiled by H. Anthony Neidig and Wilmer J. Strattan, the experiments are written for professors to use, not as prestyled, specific directions, but as inspiration and guide for adoption and adaptation in their individual courses. The 144-pagepaperback is $16.50 ($17.50 foreign) and can he purchased by sending a prepaid order to: Subscription and Book Order Department, Journal of Chemical Education, 20th and Northamptan Streets, Easton, PA 18042. All orden must he prepaid and the price includes shipping.
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