CHAPTER 9 Immunochemical Techniques

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.. Co

CHAPTER 9

1 Introduction The scope of this book does not allow a complete description of the many techniques available for improving and facilitating immunoassays

in general There is a large amount of literature covering techniques, and these can be consulted for specific problems 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

first desired in starting ELISA The book recommended (I) should be con- sidered definitive in the laboratory since it is extremely “digestable” and covers a large field of methods, all of which are relevant to ELISA

2 Labeling Antibodies with Enzymes

Antibodies can be readily labeled by covalent coupling to enzymes (2-7) 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, alkaline phosphatase, and p- 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 products and insoluble prod- ucts (for immunoblotting, immunocytochemical techniques)

The purchasing of enzyme-linked reagents from commercial sources

is recommended, but for laboratory-produced specific reagents, such as monoclonal antibodies (MAbs) or affinity-purified antibodies, conju- gates will need to be prepared in the laboratory

207

2.1 Coupling Antibodies to Horseradish Peroxidase

Two general methods are used for the preparation of antibody peroxi- dase conjugates, the twstep glutaraldehyde method and the periodate method Good batches of horseradish peroxidase (HRP) can be deter- mined 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

2.1.1 Gluteraldehyde 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 glutaraldehyde 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 HRP-antibody conjugates need to

be separated from unconjugated material for optimum sensitivity The HRP must be pure to minimize crosslinking of enzyme molecules to con- taminating proteins during the first step of the following procedure

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

2 After overnight incubation at room temperature; remove excess free glut- araldehyde by gel filtration.*

3 Concentrate the enzyme solution to 10 mg/rnL (1 rnL 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 rnM 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/rnL in 0.15M NaCl) to the enzyme solution and check that the pH > 9.0

5 Incubate at 4OC for 24 h

*Use a gel matrix with an exclusion hmit of 20,~50,000 for globular protems Use medmm- sized beads (approx 100 pm 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 pL of glycerol and 20 pL of 1% xylene cylanol The column should be prerun with a minimum of 10 column volumes of 0.15M NaCl Allow the column to run until the buffer level drops just below the top of the bed resm Stop the flow of the column Carefully load the column with the glutaralde- hyde-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 0.15M NaCl Run the column with 0.15M NaCI Pool the fractions that look brown, These contain the active enzyme

Labeling Antibodies with Enzymes 209

6 Add 0.1 mL of 0.2M ethanolamine, pH 7.0 Incubate at 4°C for 2 h At this stage, there will be present in the solution the uncoupled HRP, the uncoupled antibody, and the HRI-antibody conjugate For some assays, no further puri- fication 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 between 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 as described later 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 0.2M glucose or methylmannoside) Alternatively, the whole separation can be achieved on the basis of size by gel filtration Column eluates can be assayed by enzyme activity, absor- bance at 403 nm, or absorbance at 280 nm

2.1.2 Periodate Coupling Periodate treatment of carbohydrates opens the ring structure and allows them to bind to free amino groups Coupling antibodies and horseradish peroxidase with periodate linkage are an efficient method This method

is based on refs (4) and (5)

1 Resuspend 5 mg of HRP in 1.2 mL of water Add 0.3 mL of freshly pre- pared O.lM sodium periodate in 10 mM sodium phosphate, pH 7.0

2 Incubate at room temperature for 20 mm

3 Dialyze the HRP solution versus 1 mM sodium acetate, pH 4.0, at 4OC with several changes overnight

4 Prepare an antibody solution of 10 mg/mL in 20 mit4 carbonate

5 Remove the HRP from the dialysis tubingm and add to 0.5 mL of the anti- body solution

6 Incubate at room temperature for 2 h

7 The Schiff’s bases that have formed must be reduced by adding 100 pL of sodium borohydride (4 mg/mL in water) Incubate at 4OC for 2 h

2.1.2.1 NAKANE AND KAWAOI METHOD OF ENZYME ACTIVATION

1 Dissolve the horseradish peroxidase (HRPOSigma Type VI, RZ = 3) in 1 O

mL of freshly prepared 0.3M sodium bicarbonate, pH 8.1 (should be this

pH on making up) Note the mg/mL on bottle of HRPO

2 Add 0.1 mL of a 1% solution (v/v) fluorodinitrobenzidine in absolute etha- nol Mix for 1 h (leave on bench and gently swirl every 10 mm)

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

4 Add 1 O mL of 0.16M ethylene glycol (ethanediol) in distilled water Mix gently (as above) for 1 h

5 Dialyze against O.OlM sodium carbonate/bicarbonate buffer, pH 9.5, at 4°C (three changes vs 500-1000 mL)

Conjugation is as follows:

1 Add the IgG (or other protein) dialyzed against O.OlM carbonate/bicarbon- ate buffer, pH 9.5) at a ratio of 5 mg IgG (protein) to 1.33 mg of activated enzyme Note: You know the volume of your activated enzyme and know the original mg/mL 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 ~3 h (overnight is suitable)

2 Add 1 mg of sodium borohydride (NaBH,)/mg of enzyme used Make the sodium borohydride up fresh to about 200 mg/mL, and add relevant vol- ume containing the correct number of milligrams

3 Dialyze against PBS

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

3 Coupling Antibodies to Alkaline Phosphatase

Conjugatation of antibodies to alkaline phosphatase can be made using

a one-step procedure with glutaraldehyde The conjugates retain good immunological and enzymatic activity, but can be large and heteroge- neous 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 alkaline phosphatase in a final vol- ume of 1 mL Alkaline phosphatase is usually supplied as a suspension in 65% saturated ammonium sulfate, which should be centrifuged at 4OOOg for 30 min (5 min in microfuge) The antibody solution can then be added

to resuspend the enzyme pellet

2 Dialyze the mixture against four changes of O.lM 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 mag- netic stirrer Slowly, with gently stirring, add 0.05 mL of a 1% solution of

EM 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 1M ethanolamine, pH 7

5 After 2 h of further incubation at room temperature, dialyze overnight at 4°C against three changes of PBS

Coupling Antibodies to Alkaline Phosphatase 211

6 Centrifuge at 40,OOOg for 20 min

7 Store the supematant at 4°C in the presence of 50% glycerol, 1 mM ZnCl,,

1 mM MgC12, 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

3.1 AvidbBiotin 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 3M) 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 will deal 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 can

be outlined as shown in Fig 1

3.1.1 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 mol- ecule(s) Again after washing, the complex is detected by the addition of avidin, which is linked to enzyme followed by addition of the relevant substrate

3.1.2 BRAB System This is essentially the same as the LAB system, except that the avidin

is not conjugated to an enzyme Here the avidin acts as a bridge to con- nect the biotinylated secondary antibody and biotinylated enzyme Since the avidin has multiple biotin binding sites, this system allows more biotinylated enzyme to be complexed with a resulting amplification of sig- nal, thus making the system potentially more sensitive than the LAB system

Labelled Avidin-Biotin System (LAB)

Substrate

c

Primary antibody

ElUple Secondary detecting

antibody antibody Coloar development

Fig 1 Different systems for use of avidin and biotin in ELISA

Preparation of Immunoglobulins 213

Table 1 Compounds Available for Introduction of Biotin

NHS-LC-Biotin NHS-Biotin SULFO-NHS-Biotin NHS-LC-Biotin NHS-SS-Biotin Photo Activatable Biotm-HPDP Iodoacetyl-LC-Biotin Biotm hydrazide Biotinylated Protein A

Primary amines Primary amines Primary ammes Primary amines Primary amines Nucleic acids Thiols Thiols Carbohydrates

3.1.3 ABC System

This is almost identical to the BRAB system except that it requires preincubation of biotinylated enzyme with avidin to form large complexes that are then incubated with the secondary antibody In this way there is a large increase in signal owing to the increase in enzyme molecules

3.2 Methods for Labeling with Biotin

There are many biotinylated commercial reagents designed for use in ELISA Reference 7 illustrates the various methods for the introduction

of biotin onto reagents for use in ELISA using a variety of chemicals A brief survey is shown in Table 1 to illustrate the versatility of labeling methods for proteins, carbohydrates, and nucleic acids

4 Preparation of Immunoglobulins

About 10% of serum proteins are immunoglobulins After immuniza- tion, the specific antibodies produced are about l-25% of this fraction,

so that the required immunoglobulin (in ELISA) may be from O l-2.5%

of the total protein in a serum Some assays are favored by the relatively crude fractionation of serum to obtain immunoglobulins, e.g., for use in binding to plates in trapping (sandwich assays) to avoid competition for plastic binding sites by other serum proteins There are several methods for separation of immunoglobulins for use in ELISA These procedures are suitable for polyclonal antibodies, but not necessarily for MAbs The isolation of total immunoglobulins (Ig) as compared to the purification

of specific immunoglobulins is relatively simple

4.1 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 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 (5x less at +4”C) The isolation of mammalian IgG and IgA by ammonium sulfate precipi- tation 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 concentrated 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 here is given as 10 mL Adjust the volumes accordingly to suit the starting vol- ume of your serum

1 To 10 mL serum add 2.7 g of (NH&S04 Add a small quantity in steps Stir constantly at room temeperature

2 Incubate at room temperature for 1 h while stirring

3 Centrifuge at approx 5OOOg at 4°C for 10-15 mm

4 Discard the supernatant fluid

5 Dissolve the pellet in 2-3 mL of distilled water

6 Add 0.5 g (NH&SO+ Stir constantly at room temperature

7 Centrifuge as before

8 Dissolve pellet in 10 mL distilled water or PBS

9 Dialyze against appropriate buffer for use in ELISA, and so on, or dialyze against distilled water and then freeze-dry

4.2 Ion-Exchange Chromatography

After salt fractionation, IgG can be purified further on DEAE-cellu- lose, DEAE-Sephadex A-50, or DEAE-Sephacel Such methods are not described in this volume, but a large amount of literature is available

4.3 Protein A and Protein G Protein A has some affinity for the Fc of most mammalian IgGs and

can be used for their isolation Although protein A as used in immunoas- says has little practical use in detecting sheep, bovine, and goat IgG, 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, Richmond, VA) or glass beads Such reagents are

Immunosorbents 215

very 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 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 using a 0.9% sodium chloride solution containing 0.6% acetic acid

or by addition of a solution of sodium thiocyanate (2-W) Such meth- ods are particularly useful in the purification of mouse IgGs from MAb ascites preparations

5 Immunosorbents

A breakthrough in the easy use of immunosorbents has been made with the availability of reagents, such as n-Hydroxysuccinimide-deri- vatized agarose (Bio-Rad) This gel is can be washed three times in cold distilled water and then be used to covalently attach any protein, merely

by incubation of that protein(s) in a wide variety of buffers (such as 0.1 sodium carbonate buffer) Blocking of unreacted active sites on the gel is achieved by the addition of ethanolamine or by merely leaving the gel overnight Such gels are thus very easy to prepare Antisera can then be added in neutral buffers, and addition of some detergents (e.g., 0.5% Tween 80) minimizes nonspecific adsorption of serum proteins Desorp- tion of bound antibodies is then achieved by addition of chaotropic ions (sodium thiocyanate), or organic acids with low surface tension or pH extremes Thus, such affinity techniques can be used to get rid of unwanted cross reactions from sera, e.g., if a serum has antibovine IgG activity, this can be adsorped out by passing that serum over an affinity column with bound bovine serum or IgG In this case, the antibodies passing through the column will be free from antibovine activity

Other immunosorbents are available commercially based on beaded agarose or glass A wide variety of proteins, such as whole serum or IgG, can be purchased attached covalently to beads and are extremely conve- nient (but expensive) for the removal (as an example) of unwanted crossreactive antibodies from small volumes of antisera The beads are simply added to an antiserum and, after a short incubation, are separated

by centrifugation (microfuge, 12,000 rpm for lo-30 s) The agarose beads thus capture any unwanted antibodies on the solid-phase, leaving the antiserum free of that “contaminant.” This method has the advantage over blocking by addition of high levels of specific protein (against which

the unwanted antibodies react) in that there is complete separation of immune complexes, which may interfere with ELISAs Such reagents can be reused by eluting the immunologically bound protein using simi- lar methods (low pH, and so on) as described above, followed by exten- sive washing The section on immunoaffinity purification in ref I should

be consulted for extensive practical details of methods

6 Production of Antisera The raising of antisera in laboratory animals would fill a manual in itself The variability in immune response within and between species and the various antigens used mean that no brief rules can be given, and reading of the relevant scientific literature is essential Generally, the administration of a nonreplicating agent requires the addition of an adju- vant, whose effect is to stimulate the immune system so that efficient presentation of the antigen takes place

6.1 Immunization The purpose of immunization is to obtain high-titered antisera that binds strongly to antigen (high avidity) The properties of antisera are determined by the genetic composition of the animal injected (particu- larly the Ir genes) This means that there can be great variation in the quantitative and qualitative aspects of antisera from between species and even between individuals of the same species This should be borne in mind when considering the use to which the serum is being made In preparing sera one should: (1) always obtain a preimmunization serum and (2) never automatically pool sera

Point (2) is particularly important if a defined property of an antiserum

is required, e.g., in discrimination of antigens Up to a certain degree, an increase in the dose (weight) of antigen will increase antibody titer, how- ever this may also increase crossreactivity Adjuvants also increase the immunogenicity of proteins Haptens should also be labeled with carrier proteins to elicit an immune response The carrier protein should also be foreign to the host to be recognized by the T-cells For most immunogens the interaction of T- and B-cells is essential for antibody production The animal species chosen can be important, The animal species most often used in laboratories are rabbits, goats, guinea pigs, pigs, sheep, goats, and rats Commercial companies may favor horses and donkeys for large-scale preparations Many animals contain crossreactive anti- bodies in their serum before immunization This could complicate their