The Elisa guidebook

Fig. 29.

Situations 4¨C6 on titration of reagents.

7. The methods described here should be used to routinely examine reagents throughout their lifetime of use as a kit for testing. When there is an identified drift to lower OD values for Cm, then the reasons can be investigated using the

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methods. The danger signals are when the mean OD of the Cm approaches the allowable mean ¡À2 ¡Á SD limit. Remember to observe the lower limit of error bars (2 ¡Á SD error bar of Cm mean per plate). If this falls inside the is within the mean Cm value ¨C3 SD limits, then the system should be examined.

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10¡ª

Immunochemical Techniques

The scope of this book does not allow a complete description of the many techniques available for purification and treatment of reagents for facilitating immunoassays in general. There is a large amount of literature covering techniques, and these can be consulted for specific problems. The examination of many of the catalogs produced by commercial companies is useful since they often include good technical sections describing methods using their products. This chapter contains the practical basics of conjugation (a large field in itself), and details other immediately useful techniques that might be first desired in starting an ELISA. The book Antibodies: A Laboratory Manual (1) should be regarded as definitive in the laboratory because it is extremely ''digestible" and covers a large field of methods, all of which are relevant to ELISA.

1¡ª

Labeling Antibodies with Enzymes

Antibodies can be readily labeled by covalent coupling to enzymes (2¨C7). The ideal product for any coupling reaction should have a 1:1 ratio of antibody to enzyme with no loss of specific activity of either reactant, but this is technically unachievable. However, owing to the amplification of the signal by the enzyme action, even relatively poor conjugates have required sensitivities. A large number of enzymes have been used to label antibodies. The most commonly used are horseradish peroxidase (HRP), alkaline phosphatase (AP) and β-galactosidase. The ideal enzyme considerations are cost, stability, size, and ease of conjugation. The enzyme should have a high catalytic activity and a range of substrates that yield both soluble and insoluble products (for immunoblotting and immunocytochemical techniques). The purchasing of enzyme-linked reagents from commercial sources is recommended but for laboratoryproduced specific reagents, such as monoclonal antibodies (mAbs) or affinity-purified antibodies, conjugates will need to be prepared in the laboratory.

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1.1¡ª

Coupling Antibodies to HRP

Two general methods are used for the preparation of antibody peroxidase conjugates: the two-step glutaraldehyde method and the periodate method. Good batches of HRP can be determined by measuring the ratio of the HRP absorbance at 403 and 280 nm (RZ = OD 403 nm/OD 280 nm). This ratio should be at least 3.0. Good reagents designed for coupling are available commercially.

1.1.1¡ª

Glutaraldehyde Coupling

In the two-step glutaraldehyde method, glutaraldehyde is first coupled to pure HRP via the relatively few reactive amino groups available on the enzyme. By performing this step in high gluaraldehyde concentrations, very few HRP-HRP conjugates are formed. The HRP-glutaraldehyde mixture is then purified and added to antibody in solution. This method has a low coupling efficiency, so the HRPantibody conjugates need to be separated from unconjugated material for optimum sensitivity. The HRP must be pure to minimize crosslinking of enzyme molecules to contaminating proteins during the first step of the procedure.

1.Dissolve 10 mg of HRP in 0.2 mL of 1.25% glutaraldehyde (electron microscopic grade) in 100 mM sodium phosphate (pH 6.8). Caution: glutaraldehyde is hazardous. Work in a fume hood.

2.After overnight incubation at room temperature, remove excess free glutaraldehyde by gel filtration. To do this, use a gel matrix with an exclusion limit of 20,000¨C50,000 for globular proteins. Use medium-sized beads (approx 100 µm in diameter). Prepare a column with 5 mL of bead volume according to the manufacturer's instructions. To make the column easier to load and run, first add 20 µL of glycerol and 20 µL of 1% xylene cylanol. The column should be prerun with a minimum of 10 column vol of 0.15 M NaCl. Allow the column to run until the buffer level drops just below the top of the bed resin. Stop the flow of the column. Carefully load the column with the glutaraldehyde-treated HRP. Release the flow and allow the HRP to run into the column. Just as the level of the HRP solution drops below the top of the column, carefully add 0.15 M NaCl. Run the column with 0.15 M NaCl. Pool the fractions that look brown. These contain the active enzyme.

3.Concentrate the enzyme solution to 10 mg/mL (1 mL final volume) by ultrafiltration or by dialysis against 100 mM sodium carbonate/sodium bicarbonate buffer (pH 9.5) containing 30% sucrose. Change the buffer to 100 mM sodium carbonate-bicarbonate (pH 9.5) either by dialysis or by washing on the ultrafiltration membrane.

4.Add 0.1 mL of antibody (5 mg/mL in 0.15 M NaCl) to the enzyme solution and check that the pH is >9.0.

5.Incubate at 4¡ãC for 24 h.

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6. Add 0.1 mL of 0.2 M ethanolamine (pH 7.0). Incubate at 4¡ãC for 2 h. At this stage there will be present in the solution, the uncoupled HRP, the uncoupled antibody, and the HRP-antibody conjugate. For some assays, no further purification is necessary. In these cases, the uncoupled HRP will not bind to any antigen and will be lost during any washes prior to enzyme detection. Further purification will require separation based on the differences among the three species. The easiest separation will be between the uncoupled HRP and the two antibody-containing fractions. If the antibody binds to protein A, the antibodies can be removed simply, by low pH treatment. Separation between the two antibody fractions can be achieved by gel filtration (a 50-mL S300, or equivalent) or affinity chromatography on a concanavalin A column (eluted with 0.2 M glucose or methyl-mannoside). Alternatively, the whole separation can be achieved on the basis of size by gel filtration. Column eluates can be assayed by enzyme activity, absorbance at 403 nm, or absorbance at 280 nm.

1.1.2¡ª

Periodate Coupling

Periodate treatment of carbohydrates opens the ring structure and allows them to bind to free amino groups. Coupling antibodies and HRP with periodate linkage is an efficient method. This method is based on ref. 4 and 5.

1.Resuspend 5 mg of HRP in 1.2 mL of water. Add 0.3 mL of freshly prepared 0.1 M sodium periodate in 10 mM sodium phosphate (pH 7.0).

2.Incubate at room temperature for 20 min.

3.Dialyze the HRP solution against i mM sodium acetate (pH 4.0) at 4¡ãC with several changes overnight.

4.Prepare an antibody solution of 10 mg/mL in 20 mM carbonate.

5.Remove the HRP from the dialysis tubing and add to 0.5 mL of the antibody solution.

6.Incubate at room temperature for 2 h.

7.The Schiff's bases that have formed must be reduced by adding 100 µL of sodium borohydride (4 mg/mL in water). Incubate at 4¡ãC for 2 h.

1.1.2.1¡ª

Nakani and Kawaoi (4) Method of Enzyme Activation

1.Dissolve the HRP (HRPO, Sigma Type VI, RZ = 3) in 1.0 mL of freshly prepared 0.3 M sodium bicarbonate, pH 8.1 (should be this pH on making up). Note the milligrams/milliliter on the bottle of HRPO.

2.Add 0.1 mL of a 1% solution (v/v) of fluorodinitrobenzidine in absolute ethanol. Mix for 1 h (leave on bench and gently swirl every 10 min).

3.Add 1.0 mL of 0.08 M sodium periodate (NaIO4) in distilled water. Mix gently for 30 min at room temperature (swirl every 5 min).

4.Add 1.0 mL of 0.16 M ethylene glycol (ethanediol) in distilled water. Mix gently (as in step 3) for 1

h.

5.Dialyze against 0.01 M sodium carbonate/bicarbonate buffer, pH 9.5, at 4¡ãC (three changes using 500¨C1000 mL each change).

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1.1.2.2¡ª Conjugation

1.Add the IgG (or other protein) dialyzed against 0.01 M carbonate/bicarbonate buffer, pH 9.5) at a ratio of 5 mg of IgG (protein) to 1.33 mg of activated enzyme. (Note: You know the volume of your activated enzyme and know the original milligrams/milliliter, and, therefore you know the effective concentration of the activated enzyme and can add so many milligrams in a certain volume. Mix and stand at room temperature for not less than 3 h (overnight is suitable).

2.Add 1 mg of sodium borohydride (NaBH4)/mg of enzyme used. Make the NaBH4 up fresh to about

200mg/mL and add a relevant volume containing the correct number of milligrams.

3.Dialyze against phosphate-buffered saline (PBS).

4.You may wish to separate the free enzyme by methods already described, but in most ELISAs this is not necessary.

1.2¡ª

Coupling Antibodies to AP

Conjugation of antibodies to AP can be made using a one-step procedure with glutaraldehyde. The conjugates retain good immunological and enzymatic activity but can be large and heterogeneous in nature. The major drawbacks are the high cost of the enzyme and the need to use very concentrated solutions of enzyme and antibody.

1.Mix 10 mg of antibody with 5 mg of AP in a final volume of 1 mL. AP is usually supplied as a suspension in 65% saturated ammonium sulfate, which should be centrifuged at 4000g for 30 min (5 min in a microfuge). The antibody solution can then be added to resuspend the enzyme pellet.

2.Dialyze the mixture against four changes of 0.1 M sodium phosphate buffer (pH 6.8) overnight. This is essential to remove free amino groups present in the ammonium sulfate precipitate.

3.Transfer the enzyme-antibody mixture to a container suitable for stirring small volumes. In a fume hood add a small stir bar and place on a magnetic stirrer. Slowly, with gentle stirring add 0.05 mL of a 1% solution of electron microscopy, grade glutaraldehyde. Caution: Glutaraldehyde is hazardous.

4.After 5 min, switch off the stirrer and leave for 3 h at room temperature. Add 0.1 mL of 1 M ethanolamine (pH 7.0).

5.After an additional 2 h of incubation at room temperature, dialyze overnight at 4¡ãC against three changes of PBS.

6.Centrifuge at 40,000g for 20 min.

7.Store the supernatant at 4¡ãC in the presence of 50% glycerol, 1 mM ZnCl2, 1 mM MgCl2, and 0.02% sodium azide.

The procedure may be scaled down to the 1-mg antibody level if the antibody and enzyme concentration is reduced by a factor of 10. Here, the time allowed for coupling should be increased to at least 24 h. The yield of conjugate may be reduced.

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1.3¡ª

Avidin/Biotin Systems in ELISA

The specific binding between avidin (an egg white protein) and biotin (a water-soluble vitamin) has been exploited in ELISA. Avidin is a tetramer containing four identical subunits, each of which contains a very high-affinity binding site for biotin. The binding is not disturbed by extremes of salt, pH, or chaotropic agents such as guanidine hydrochloride (up to 3 M). The avidin/ biotin system is well suited for use as a bridging or sandwich system in association with antigen/antibody reactions. The biotin molecule can be easily coupled to either antigens or antibodies, and avidin can be conjugated to enzymes (and other immunological markers such as fluorochromes, colloidal markers, and ferritin). This section deals briefly with applications of the biotin/ avidin system to ELISA. An excellent outline of reagents and biotin/ protein-labeling methods (biotinylation) can be found in (ref. 8). Three basic systems are outlined.

1.3.1¡ª

LAB System

An antigen immobilized on a microtiter well is detected by incubation with a primary antibody. After washing, this is detected by incubation with an antispecies antibody that is biotinylated (linked to biotin molecule[s]). Again, after washing, the complex is detected by the addition of avidin that is linked to enzyme followed by the addition of the relevant substrate.

1.3.2¡ª

BRAB System

2¡ª

Preparation of Immunoglobulins

About 10% of serum proteins are immunoglobulins (Igs). After immunization, the specific antibodies produced are about 1¨C5% of this fraction, so the required Ig (in ELISA) may be from 0.1 to 2.5% of the total protein in a serum. Some assays are favored by the relatively crude fractionation of serum to obtain Igs, e.g., for use in binding to plates in trapping (sandwich assays) to avoid competition for plastic binding sites by other serum proteins. Several methods for separation of Igs are available for use in ELISA. These procedures are suitable for polyclonal antibodies but not necessarily for mAbs. The isolation of total Igs as compared to the purification of specific Igs, is relatively simple.

2.1¡ª

Salt Fractionation

Two salts are used for selective Ig precipitation: ammonium sulfate and sodium sulfate. The concentration of ammonium sulfate is expressed as a percentage of saturation whereas the concentration of sodium sulfate is expressed as percentage (w/v). The concentration of salt at saturation depends on temperature, particularly for sodium sulfate (five times less at +4¡ãC). The isolation of mammalian IgG and IgA by ammonium sulfate precipitation depends on the volume of the serum being processed. For large volumes, the salt is added directly, whereas for small volumes, the salt is added as a con-

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centrated solution. As already indicated, proteins are precipitated by different amounts of ammonium sulfate. This is a method that can be used to obtain samples of sufficient purity for most ELISAs. The initial volume of serum given here is 10 mL. Adjust the volumes accordingly to suit the starting volume of your serum.

1.To 10 mL of serum add 2.7 g of (NH4)2SO4. Add a small quantity in steps. Stir constantly at room temperature.

2.Incubate at room temperature for 1 h while stirring.

3.Centrifuge at approx 5000g at 4¡ãC for 10¨C15 min.

4.Discard the supernatant fluid.

5.Dissolve the pellet in 2¨C3 mL of distilled water.

6.Add 0.5 g of (NH4)2SO4, and stir constantly at room temperature.

7.Centrifuge as in step 3.

8.Dissolve the pellet in 10 mL of distilled water or PBS.

9. Dialyze against the appropriate buffer for use in ELISA or dialyze against distilled water and then freeze-dry.

2.2¡ª

Ion-Exchange Chromatography

After salt fractionation, IgG can be purified further on DEAE-cellulose, DEAE-Sephadex A-50, or DEAE-Sephacel. Such methods are not described in this book, but much literature is available.

2.3¡ª Protein A

Protein A (SpA) is isolated from the cell walls of Cowan 1 or other strains of Staphylococcus aureus. It consists of a single polypeptide chain (mol wt of approx 42,000). Protein A has a high affinity (K = 108 L/mol) for the Fc of most mammalian IgGs and can be used for their isolation. A genetically engineered recombinant form of protein A (mol wt 32,000) is marketed, in which most of the nonessential regions have been, removed leaving four IgG binding domains intact.

Although protein A as used in immunoassays has little practical use in detecting sheep, bovine, and goat IgGs, they can be purified when the protein A concentrations are high, as in the commercially available protein A-Sepharose or protein A conjugated to Affi-gels (Bio-Rad) or glass beads. Such reagents are quite useful in rapid separation of most mammalian IgGs. Briefly, 5 mL Protein A columns are equilibrated with PBS. Serum or crude IgG is then added and elution with PBS is maintained. The IgG attaches to the column (via reaction to the protein A bound to the inert matrix), and the other serum proteins pass through the column. The bound IgG is then eluted by using a 0.9% sodium chloride solution containing 0.6% acetic acid or by adding a solution of sodium

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thiocyanate (2¨C5 M). Such methods are particularly useful in the purification of mouse IgGs from mAb ascites preparations.

2.4¡ª Protein G

Protein G is isolated form group G Streptococcus sp. The protein is similar to protein A in that it binds to a variety of mammalian Igs through their Fc region, but generally with a higher affinity. Unlike protein A, protein G binds strongly with bovine, ovine, and caprine IgGs.

2.5¡ª Protein A/G

Protein A/G is a genetically engineered protein produced by a gene fusion product from a nonpathogenic Bacillus strain. The protein is engineered to have four Fc binding domains of protein A and two of protein G per molecule. The product binds to all classes of mouse IgG, but not with IgA or IgM.