Hermanson G. Bioconjugate Techniques, Second Edition

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930 24. Preparation of Colloidal Gold-Labeled Proteins

ﬁ lter. Centrifugation also may be done. Each protein–gold complexation should be optimized

for the proper amount of protein to add to maintain stability of the colloid. This can be done

according to the method described in Section 1, this chapter.

3. Preparation of Protein A–Gold Complexes

Protein A–gold probes (as well as other immunoglobulin binding proteins adsorbed to gold)

have been used to visualize antibody binding to antigenic sites in tissue sections, cells, and blots

(Hearn, 1987; Jemmerson and Agre, 1987; Lethias et al., 1987; Yokota, 1988; Bendayan and

Garzon, 1988; Bendayan, 1989; Herbener, 1989; Roth et al., 1989; Stump et al., 1989). Gold

labeling of immunoglobulin binding proteins provides “universal” probes for detection of any

antibody–antigen interaction ( Figure 24.2 ). Thus, only one gold-labeled reagent need be pre-

pared to visualize many different immunochemical targeting procedures. This avoids having

to make antibody–gold probes for each speciﬁ c immunoglobulin used. The downside of this

approach, however, is the potential nonspeciﬁ city of protein A in binding other antibodies that

may be present within the sample.

Figure 24.2 Antigens may be detected in cells or tissue sections through the use of protein A-coated gold par-

ticles. The binding of a speciﬁ c primary antibody to its target antigen can be localized by the immunoglobulin

binding capability of protein A, which occurs in the Fc region of the antibody.

Protocol

1. Determine the minimum amount of protein A required to stabilize the colloidal gold sol

being used. The colloidal suspension should be adjusted, if needed, with 0.1 M K

pH 6–7. Measure the pH of the sol using a gel-ﬁ lled electrode. Determining the stabiliza-

tion amount of protein A can be done according to the method described in Section 1,

this chapter.

2. Mix a stabilizing amount of protein A plus an additional 10 percent with the appropri-

ate volume of colloidal gold. For example, Herbener (1989) mixed 10 ml of a 14 nm gold

particle sol at pH 6.9 with 0.3 mg of protein A dissolved in 0.2 ml water. Mix well.

3. After 1 minute, add 250 l of 1 percent polyethylene glycol (MW 20,000) per 10 ml of

gold sol used. The PEG helps to stabilize further the sol against aggregation.

4. Stir for an additional 5 minutes.

5. To remove excess protein A, centrifuge the preparation at a minimum of 50,000 g for

30 minutes to several hours (4°C), depending on the size of the particles and the amount

of solution. Discard the supernatant, and resuspend the protein A–gold pellet in 0.01 M

sodium phosphate, pH 7.4, containing 1 percent PEG.

4. Preparation of Antibody–Gold Complexes

Immunocytochemical staining with antibody–gold probes is a powerful way to detect, localize,

and quantify antigen molecules in tissue sections and cells ( Figure 24.3 ). Metabolic processes

can be followed, epitope mapping of the structural characteristics of macromolecules can be

Figure 24.3 Antibodies coated on colloidal gold particles can be used to detect speciﬁ c antigens in cells.

4. Preparation of Antibody–Gold Complexes 931

932 24. Preparation of Colloidal Gold-Labeled Proteins

done, and detection of pathogens or other foreign substances within cells can be accomplished

using gold-labeled antibodies (Ellis et al., 1988; Albrecht et al., 1989; Cramer et al., 1989;

De Waele et al., 1989; Nielsen et al., 1989; Martinez-Ramon et al., 1990; van den Brink et al .,

1990).

The optimal coupling pH for an antibody should be determined by measurement of the rela-

tive pI range of the immunoglobulin. Many antibodies, however, adsorb best at a pH of 8–9.

The optimal level of protein addition to the gold sol to prevent aggregation should be deter-

mined according to the method of Section 1, this chapter. In addition, bovine serum albumin

(BSA) is often added instead of PEG (see the protein A coupling procedure, described previ-

ously) to further stabilize the antibody–gold suspension.

Protocol

1. Determine the minimum amount of antibody required to stabilize the colloidal gold sol

being used. The colloidal suspension should be adjusted, if needed, with 0.1 M K

or NaOH to pH 8–9. Measure the pH of the sol using a gel-ﬁ lled electrode. Determining

the stabilization amount of antibody can be done according to the method described in

Section 1, this chapter.

2. Mix a stabilizing amount of antibody plus an additional 10 percent with the appropri-

ate volume of colloidal gold. For example, Geoghegan (1988) found that an addition

of 10–14 g of antibody per ml of gold colloid resulted in stable preparations. Mix well

after addition of antibody to the gold suspension.

3. After 1 minute, add a quantity of 10 percent BSA to bring the concentration to 0.25 per-

cent in the antibody–gold suspension. The BSA helps to stabilize further the sol against

aggregation and also blocks nonspeciﬁ c binding sites. Alternatively, PEG may be added

according to step 3 of Section 3, this chapter.

4. Stir for an additional 5 minutes.

5. To remove excess IgG, centrifuge the preparation at a minimum of 50,000 g for 30 min-

utes to several hours (4°C), depending on the size of the particles and the amount of solu-

tion. Discard the supernatant, and resuspend the antibody–gold pellet in 0.01 M sodium

phosphate, pH 7.4, containing 0.25 percent BSA (or 1 percent PEG, as desired).

5. Preparation of Lectin–Gold Complexes

Lectins, or proteins with speciﬁ c binding sites for carbohydrates, can be used as targeting mole-

cules to localize particular glycoconjugates such as glycoproteins or glycolipids on cell surfaces

(Figure 24.4 ). Labeled with gold particles, lectins are important probes for detection of cell-

surface components, intracellular receptors, and in immunological or biochemical assay procedures

(Bog-Hansen et al., 1978; Kimura et al., 1979; Nicolson, 1978; Roth, 1983; Benhamou et al .,

1988; Nakajima et al ., 1988).

The following generalized protocol is an adaptation for the labeling of 15 nm gold particles

with Aplysia gonad lectin, as described by Benhamou et al., 1988. Each lectin–gold preparation

will have its own unique pH optimum and ratio of lectin-to-gold for the absorption process.

Protocol

1. Determine the minimum amount of lectin required to stabilize the colloidal gold sol

being used. The colloidal suspension should be adjusted, if needed, with 0.1 M K

or NaOH to a pH equal to or slightly above the pI of the lectin being used. For Aplysia

gonad lectin, the optimal pH for adsorption was determined to be 9.5. Nakajima et al.

(1988) include pI conditions for a number of different lectins. Measure the pH of the sol

using a gel-ﬁ lled electrode. Determining the stabilization amount of lectin can be done

according to the method described in Section 1, this chapter.

2. Mix a stabilizing amount of lectin plus an additional 10 percent with the appropriate

volume of colloidal gold. For example, Benhamou et al., 1988, found that an addition of

5 g of lectin per ml of gold colloid resulted in stable preparations. However, in their ﬁ nal

lectin–gold conjugate preparation a 5-fold increase in this ratio (25 g lectin/ml gold) was

used to stabilize fully the sol. Mix well after addition of lectin to the gold suspension.

3. After 1 minute, add 250 l of 1 percent polyethylene glycol (MW 20,000) per 10 ml of

gold sol used. The PEG helps to stabilize further the sol against aggregation.

4. Stir for an additional 5 minutes.

5. To remove excess lectin (particularly important if the 5-fold excess ratio is used), centri-

fuge the preparation at a minimum of 50,000 g for 30 minutes to several hours (4°C),

depending on the size of the particles and the amount of solution. Discard the superna-

tant, and resuspend the lectin–gold pellet in 0.01 M sodium phosphate, pH 7.4, contain-

ing 1 percent PEG.

Figure 24.4 Lectins coated on gold particles can be used to detect speciﬁ c carbohydrate sequences in cell-

surface glycoconjugates.

5. Preparation of Lectin–Gold Complexes 933

934 24. Preparation of Colloidal Gold-Labeled Proteins

6. Preparation of (Strept)avidin–Gold Complexes

Avidin– or streptavidin–gold conjugates can be used to detect, localize, or quantify the binding

of biotinylated molecules in cells, tissue sections, or blots (Morris and Saelinger, 1984; Bonnard

et al., 1984; Gillitzer et al., 1990; Bronckers et al., 1987) ( Figure 24.5 ). These reagents are similar

to the use of protein A–gold complexes in detecting immunoglobulins (Section 3, this chapter) in

that they are “universal” for detecting any biotin-labeled molecules. Thus, targeting molecules

need not be directly modiﬁ ed with gold, only biotinylated so that they are able to interact with

avidin– or streptavidin–gold conjugates. See Chapter 11 and Chapter 18, Section 3 for biotinyla-

tion reagents and protocols that can be used to add a biotin tag to macromolecules. Also, see

Chapter 23 for additional information on (strept)avidin–biotin techniques, including conjuga-

tion protocols.

The following protocol is based on the method of Morris and Saelinger (1984) for the labeling

of succinylated avidin with gold particles of 5.2 nm diameter. Succinylated avidin was used to

reduce the pI of the protein, thus eliminating nonspeciﬁ c binding due to the strong positive

Figure 24.5 Streptavidin-coated gold particles can be used to detect biotinylated antibodies that are bound to

speciﬁ c antigenic determinants.

charge of the native tetramer. Alternatively, streptavidin or NeutrAvidin (Thermo Fisher) can

be used to coat gold particles without the need for succinylation.

Protocol

1. Prepare a 200 ml gold sol by using white phosphorus reduction as described in Section 2,

this chapter.

2. Prepare 5 ml of a 1 mg/ml succinylated avidin solution by dissolving the protein in 50 mM

sodium phosphate, pH 7.5.

3. With stirring, add the succinylated avidin solution to the colloidal gold suspension at

room temperature.

4. React for 30 minutes with constant mixing.

5. Remove excess protein by centrifugation at a minimum of 50,000 g for several hours.

6. Suspend the succinylated avidin–gold pellet in 50 mM Tris, 0.15 M NaCl, pH 7.5, con-

taining 0.5 mg/ml PEG (MW 20,000).

7. Centrifuge again under the same conditions to assure complete removal of non-adsorbed

protein.

8. Resuspend the pellet in Tris buffer containing PEG and store at 4°C.

A similar protocol has been used by Bonnard et al. (1984) in the preparation of streptavidin–

gold probes.

Protocol

1. Prepare 20 ml of a gold sol by the white phosphorus method described in Section 2, this

chapter.

2. Dissolve streptavidin in 0.1 M sodium phosphate buffer, pH 7.4, at a concentration of

1 mg/ml.

3. With stirring, add the streptavidin solution to the gold suspension. Immediately add

200 l of 1

M sodium bicarbonate.

4. React for 10 minutes at room temperature.

5. To stabilize further the colloid, add 200  l of 2 percent PEG 6000.

6. Centrifuge the streptavidin–gold suspension at a minimum of 50,000 g for several hours

to remove excess protein solution.

7. Resuspend the pellet in 0.1 M sodium phosphate, 0.02 percent PEG, pH 7.4, and store

at 4°C.

6. Preparation of (Strept)avidin–Gold Complexes 935

936

Modiﬁ cation or attachment of proteins or other molecules with synthetic polymers can pro-

vide many beneﬁ ts for both in vivo and in vitro applications. Covalent coupling of polymers to

large macromolecules can alter their surface and solubility properties, creating increased water

solubility or even organic solvent solubility for molecules normally sparingly miscible in such

environments. Polymer modiﬁ cation of foreign molecules can provide increased biocompatibility,

reducing the immune response, increasing in vivo stability, and delaying clearance by the retic-

uloendothelial system. Modiﬁ cation of enzymes with polymers can dramatically enhance their

stability in solution. Polymer attachment can provide cryoprotection for proteins sensitive to

freezing. Polymers with multivalent reactive sites can be used to couple numerous small mol-

ecules for creating pharmacologically active agents that possess long half-lives in biological

systems. Similar complexes can be formed to create highly potent immunogens consisting of

hapten–polymer conjugates for induction of an antibody response toward the hapten. Polymer

modiﬁ cation of surfaces can effectively mask the intrinsic character of the surface and thus

prevent nonspeciﬁ c protein adsorption. Finally, multifunctional polymers can serve as extended

crosslinking agents for the conjugation of more than one molecule of one protein to multiple

numbers of a second molecule, creating large complexes with increased sensitivity or activity in

detecting or acting upon target analytes.

Many polymers have been studied for their usefulness in producing pharmacologically active

complexes with proteins or drugs. Synthetic and natural polymers such as polysaccharides,

poly(

L-lysine) and other poly(amino acids), poly(vinyl alcohols), polyvinylpyrrolidinones,

poly(acrylic acid) derivatives, various polyurethanes, and polyphosphazenes have been coupled to

with a diversity of substances to explore their properties (Duncan and Kopecek, 1984; Braatz

et al., 1993). Copolymer preparations of two monomers also have been tried (Nathan et al .,

1993).

The two polymers most often used in these applications are dextran and polyethylene glycol

(PEG). Both polymers consist of repeating units of a single monomer—glucose in the case of

dextran and an ethylene oxide basic unit in the case of PEG. The polymers may be composed

of linear strands or branched constructs. An additional similarity is that both of them pos-

sess hydroxyl and ether linkages, lending hydrophilicity and water-solubility to the molecules.

Dextran and PEG can be activated through their hydroxyl groups by a number of chemical

Modification with Synthetic Polymers

methods to allow efﬁ cient coupling of other molecules. Dextran can be activated at multiple

sites throughout its chain, since each monomer contains hydroxyl resides. PEG, by contrast,

only has hydroxyls at the termini of each polymer strand. Derivatives of both polymers are

commercially available.

The following sections discuss the major properties and conjugation chemistries associated

with the use of these polymers in modifying or conjugating proteins and other molecules.

1. Protein Modiﬁ cation with Activated Polyethylene Glycols

Since the ﬁ rst report by Abuchowski et al. (1977a, b) concerning the alteration of immuno-

logical properties toward bovine serum albumin (BSA) that had been modiﬁ ed with PEG, the

interest in polymer modiﬁ cation of biological molecules has grown incessantly. PEG coupled

to other molecules can be used for altering solubility characteristics in aqueous or organic sol-

vents (Inada et al., 1986), for modulation of the immune response (Delgado et al., 1992), to

increase the stability of proteins in solution (Berger and Pizzo, 1988), to enhance the half-life

of substances in vivo (Knauf et al., 1988), to aid in penetrating cell membranes, to alter phar-

macological properties (Dunn and Ottenbrite, 1991), to increase biocompatibility, especially

toward implanted foreign substances, and to prevent protein adsorption to surfaces.

PEG consists of repeating units of ethylene oxide which terminate in hydroxyl groups on

either end of a linear chain. Some constructs have branches that have multiple linear strands

emanating from these points. PEG is made from the anionic polymerization of ethylene oxide,

resulting in the formation of polymer strands of various potential molecular weights, depend-

ing on the polymerization conditions. Thus, all polymeric PEGs are polydisperse and exist as

distribution of multiple lengths and molecular weights. Most forms of PEG useful in biocon-

jugate applications have molecular weights less than 20,000 and are soluble both in aqueous

solution and in many organic solvents.

Unlike the PEG molecules formed from anionic polymerization techniques, there now exist

highly discrete forms of the polymer made by controlled addition of small PEG units to create

chains of exacting molecular size. These discrete PEGs have a single molecular weight and do

not display the polydispersity of the traditional PEG polymers. See Chapter 18 for a complete

discussion of discrete PEG-based reagents and their applications.

Since the polymer backbone of PEG is not of biological origin, it is not readily degraded

by mammalian enzymes (although some bacterial enzymes will break it down). This property

results in only slow degradation of the polymer when used in vivo, thus extending the half-life

of modiﬁ ed substances. PEG modiﬁ cation serves to mask any molecule that it is coupled to—the

“ PEGylated ” molecule being protected from immediate breakdown or from being complexed

and inactivated by immunoglobulins in the bloodstream.

1. Protein Modiﬁ cation with Activated Polyethylene Glycols 937

938 25. Modiﬁ cation with Synthetic Polymers

PEG ’s properties in solution are especially unusual, frequently displaying amphiphilic ten-

dencies, having the ability to both solubilize in aqueous layers and in hydrophobic membranes

or organic phases. The partitioning quality of PEG across membranes is important in aiding the

formation of hybridomas in the production monoclonal antibodies (Goding, 1986b). The par-

titioning characteristics of PEG also create the ability to use it in aqueous two-phase systems

for the puriﬁ cation of biological molecules (Johansson, 1992).

PEG in solution is a highly mobile molecule that creates a large exclusion volume for its

molecular weight, much larger in fact than proteins of comparable size. Whether in solution or

attached to other insoluble supports or surfaces, PEG has a tendency to exclude other polymers.

This property forms a protein-rejecting region that is effective in preventing nonspeciﬁ c protein

binding (Bergstrom et al., 1992). Conjugation with PEG can create the same exclusion effects

surrounding a macromolecule, even preventing interaction between a ligand and its target

(Klibanov et al., 1991), an enzyme and its substrate (Berger and Pizzo, 1988), or the immune

system and a foreign substance (Davis et al., 1979). Thus, PEG-modiﬁ ed molecules display low

immunogenicity, have good resistance to proteolytic digestion, and survive in the bloodstream

for extended periods (Abuchowski et al ., 1977a; Dreborg and Akerblom, 1990).

PEG can be conjugated to other molecules through its two hydroxyl groups at the ends of each

linear chain. This process is typically done by the creation of a reactive electrophilic intermediate

that is capable of spontaneously coupling to nucleophilic residues on a second molecule. To

prevent the potential for crosslinking when using a bifunctional polymer, monofunctional PEG

polymers can be used which contain one end of each chain blocked with a methyl ether group.

Monomethoxypolyethylene glycol (mPEG) contains only one hydroxyl group per linear chain,

thus limiting activation and coupling to one site and preventing the crosslinking and polymeri-

zation of modiﬁ ed molecules. The mPEG derivative also stabilizes the blocked end to degradation

in solution.

1.1. Trichloro- s -triazine Activation and Coupling

The most common activation methods for PEG create amine-reactive derivatives that can form

amide or secondary amine linkages with proteins and other amine-containing molecules. The

oldest method of PEG activation is through the use of trichloro- s-triazine (TsT; cyanuric chlo-

ride) (Abuchowski et al., 1977). TsT is a symmetrical heterocyclic compound containing three

reactive acyl-like chlorines. This reagent and its derivatives are extensively used in industrial

applications to form strong covalent bonds between dye molecules and fabrics. The compound

also has been used to activate afﬁ nity chromatography supports for the coupling of amine-

containing ligands (Finlay et al., 1978). Reaction of the TsT with PEG results in the formation

of an activated derivative with an ether bond to the hydroxyl group of the polymer. If mPEG

is used, TsT activation will be restricted to the one free hydroxyl, thus forming a monovalent

intermediate that can be coupled to proteins without polymerization ( Figure 25.1 ).

The three reactive chlorines on TsT have dramatically different reactivities toward nucle-

ophiles in aqueous solution. The ﬁ rst chlorine is reactive toward hydroxyls as well as pri-

mary and secondary amine groups at 4°C and a pH of 9.0 (Abuchowski, 1977a; Mumtaz and

Bachhawat, 1991). Once the ﬁ rst chlorine is coupled, the second one requires at least room

temperature conditions at the same pH to react efﬁ ciently. If two chlorines are conjugated to

nucleophilic groups, the third is even more difﬁ cult to couple, requiring at least 80°C at alkaline

Figure 25.1 mPEG polymers may be activated by trichloro- s-triazine for the modiﬁ cation of amine-containing

molecules.

pH. After activation of mPEG with TsT, it is therefore, for all practical purposes, only possible

to couple one additional component to the triazine ring.

TsT activation provides a simple route to an amine-reactive PEG derivative and it has been

used extensively as an activation method for modifying proteins (Wieder et al., 1979; Zalipsky

and Lee, 1992; Gotoh et al., 1993). The modiﬁ cation of primary amine-containing molecules

such as proteins is pH dependent. At physiological pH values, the reaction will proceed slower

than in a more alkaline pH environment. Optimal derivatization efﬁ ciency is reached at con-

ditions equal to or above pH 9.0. However, TsT reactivity is not exclusive toward amines.

TsT–mPEG modiﬁ cation of proteins can result in modifying other nucleophilic groups such as

sulfhydryls and the phenolate ring of tyrosine. In addition, there is potential for toxicity associ-

ated with TsT and its derivatives—an especially important consideration for in vivo use.

The following protocol for mPEG activation using TsT and its coupling to proteins is based

on the protocols of Abuchowski et al . (1977b) and Gotoh et al . (1993).

Protocol for the Activation of mPEG with TsT

Note: All operations should be done in a fume hood. Dispose of hazardous waste according to

EPA guidelines.

1. Dissolve 5.5 g of TsT in 400 ml of anhydrous benzene which contains 10 g of anhydrous

sodium carbonate.

2. Add to the TsT solution, 50 g of mPEG-5000 (monomethoxypolyethylene glycol having

a molecular weight of 5000). Mix well to dissolve.

1. Protein Modiﬁ cation with Activated Polyethylene Glycols 939