Western Blotting as a Diagnostic Method Pirkko Soundy and Bronwen Harvey

1. Introduction

Protein blotting, the transfer of proteins from a separating gel onto a thin uniform support matrix, first appeared in 1979. Continuing the geographic theme following Southern's publication of his method for the identification of specific DNA fragments (1) in 1975 and the introduction of Northern blotting (2) not long after, the technique became known as Western blotting. Today, the original article by Towbin et al. (3) is cited many thousands of times a year. The technique itself has been modified and extended over the years (4). Once on a solid support, procedures that would otherwise proved difficult or impossible in the gel can be undertaken. A blot allows for rapid staining and destaining protocols of the separated proteins. Low concentrations of sample are more easily detected because they are not spread throughout the thickness of the gel but are "concentrated" on the surface; also, membranes are easier to handle and manipulate.

2. Protein Blotting Techniques

In a classic Western blotting (3), protein samples are separated in an electrophoretic gel (5,6) and then electroblotted onto a support matrix. Once retained, proteins can be probed with almost anything that can selectively bind peptide motifs in order to identify the presence or absence of particular entities. Commonly, detection is achieved via an enzyme suitable for use with a variety of colorimetric, fluorescent, or chemiluminescent substrates. Not unexpectedly, there is substantial commercial interest in the technique.

2.1. Transfer Techniques

Electroblotting is by far the most widely used Western blotting technique. Capillary blotting and diffusion blotting provide alternative techniques; however, these are relatively slow procedures. The basic equipment for the separation and transfer of proteins using polyacrylamide gel electrophoresis is commercially available from several companies. There are two basic types of transfer systems: (1) semidry and (2) tank buffer apparatus. By far the most widely used are the latter tank buffer systems (for reviews, see refs. 7-9). The transfer of protein from the gel is achieved by applying an electric potential across the gel and the membrane, which are in contact. The membrane, nitrocellulose, polyvinylidene difluoride (PVDF), or Nylon, provides a more stable matrix than the gel, making subsequent manipulations much easier. In tank systems, an arrangement of sponge pad, filter paper, gel, membrane, filter paper, and sponge pad soaked in transfer buffer are held together, in that order, by a ridge plastic cassette. This cassette is place into a vertical tank filled with transfer buffer between platinum electrodes (see Fig. 1). The arrangement of these electrodes ensures a uniform voltage gradient over the whole

From: Medical Biomethods Handbook Edited by: J. M. Walker and R. Rapley © Humana Press, Inc., Totowa, NJ

Polyacrylamide Gel Anode
Fig. 1. Diagrammatic representation of electroblotting.

gel. The cassette must be placed in such a way to ensure that the membrane is between the gel and the anode. With semidry blotting apparatus, a similar arrangement of buffer-soaked filter paper, gel, membrane, and filter paper is placed between two large graphite electrodes. However, in this case, no additional liquid is present; hence the term semidry. Again, the membrane layer should be between the gel and the anode. In both cases, transfer times are short, varying from 30 min to a few hours. Modifications to the composition of the transfer buffer (3,10,11) and the use of cooling and circulation devices in the case of tank systems improve the effectiveness and reproducibility of the process. The transfer conditions required depend on the nature of the experiment and are dependent on factors such as the type of gel matrix, the nature of the proteins to be transferred, the type of membrane used, and so forth. A review of these criteria influencing this key step is provided in Table 1.

A critical factor in achieving successful protein blotting is the quality of the sample. Pro-teolysis leads to the appearance of smears, producing erroneous molecular-weight determination and/or false negatives. Proteolysis is the fragmentation of intact protein and can commonly be caused by proteolytic enzymes (proteasease) and/or physical forces such as shearing to which the protein is exposed during the extraction process. Key factors to consider in sample preparation include temperature, avoidance of freeze/thaw cycles (which subject the protein to excessive physical forces), the use of nonionic detergents (which can disrupt the protein conformation), use of protease inhibitors (which prevent proteolysis), and the use of subcellu-lar fractionation of the sample. The latter assists in a more effective separation of the protein mixture by reducing its complexity.

Traditionally, proteins were transferred onto nitrocellulose sheets. In 1986, Millipore Corporation introduced PVDF (12), which is now widely used. This membrane offers the advantages of a higher mechanical strength than unsupported nitrocellulose, a high protein-binding

Table 1

Factors Influencing Protein Transfer

Parameter

Comments

Gel composition

Membrane selection Ionic strength Buffer type pH

Methanol SDS

Physical parameters Temperature

Transfer time Power

The larger the pore size (low concentrations of monomer), the faster the transfer (10). The larger the protein the slower the transfer. Gradient gels are more effective at resolving protein mixtures, containing a wide range of molecular weights.

The properties of the membrane must be considered when determining transfer conditions.

Dilute buffers allow the use of high transfer voltages, minimizing heating. Reuse of transfer buffers is not advised.

Different buffers lead to different transfer efficiency. Tris buffers are more efficient that phosphate or acetate buffers. Proteins near their isoelectric point will transfer poorly.

Effects buffer conductivity; alternation can lead to initial current drain and decreased resistance.

Methanol is essential to improve binding of proteins to nitrocellulose. Its presence can reduce protein mobility because of fixation of the protein within the gel.

Sodium dodecyl sulfate (SDS) improves transfer by increasing protein mobility; however, resulting denaturation can reduce recognition via antibodies. SDS can lead to reduced binding to the nitrocellulose membrane. Its use depends on the nature of the proteins requiring detection.

Poor contact between gel and membrane reduces the efficacy of transfer. Uniform conductivity and temperature within the transfer tank improve transfer.

Temperature can alter buffer resistance and, subsequently, the power delivered, as well as the state of denaturation of the protein being transferred.

Heating should be minimized during long transfer times.

Use of high currents achieves rapid elution of the proteins out of the gel.

capacity (125-160 |g/cm2), and chemical stability. Nylon membranes have found favor with some users; there are examples indicating that groups of proteins only bind to Nylon (13) or more strongly to Nylon than nitrocellulose (14). The high background associated with this type of membrane in this application is probably responsible for its intermittent use. Nitrocellulose with its good binding capacity and low background is probably the most popular membrane and is available as a pure ester in both supported and unsupported forms. Unsupported membranes are produced when the active substrate is cast as a pure sheet. Because of their fragile nature, unsupported membranes should be handled with care. In supported membranes, the active substrate is cast on either side of an inert "web" or support, usually a polyester. Membranes with this type of internal support have a greater tensile strength, improving the handling characteristics of the membrane. Mixed membranes containing cellulose acetate and other cellulose derivatives as well as nitrocellulose are also available, but these types of membrane have a lower binding capacity than pure nitrocellulose. Most methods utilize a 0.45-|im pore size membrane. However not all proteins/peptides with a molecular weight less than 20 kDa are retained by nitrocellulose of this pore size (15,16). Membranes with 0.2 or even 0.1 |im are available for this application. PVDF is a Teflon-type polymer composed of repeating (-CH2-CF2-)«; it offers high mechanical strength and a high protein-binding capacity. Most PVDF membranes on the market are hydrophobic and unsupported. The membrane needs to be prewet in methanol prior to use. Proteins interact noncovalently through dipolar and hydrophobic interactions. The high binding capacity is the result of its open, porous polymeric structure and large surface area. The membrane is stable in many solvents, making it a more suitable membrane than nitrocellulose in some applications. For example, this characteristic has led to the use of PVDF in the harsh conditions employed in protein and peptide sequencing (17-19). Separated proteins are blotted onto PVDF, their positions are identified using a total protein stain such as Coomassie blue or Ponceau red, and the portion of the blot carrying the protein of interest is excised and further analyzed to determine its sequence. The protein is subjected to N-terminal sequencing in an appropriate instrument or, alternatively, subjected to internal sequencing. In this latter case, the protein immobilized on the membrane is first digested with specific proteases (such as trypsin, thermolysin, endoproteinase Lys-C, endoproteinase Glu-C, endoproteinase Asp-N clostripain). Hydrophilic peptides are released from the membrane into the digestion mixture and is then be separated by reverse-phase high-performance liquid chro-matography (HPLC). Each peptide can then be individually sequenced. In both of these examples, sequencing is achieved using Edman chemistry, which removes amino acid residues from the N-terminus of the protein or peptide, one at a time in sequence. Each cycle consists of the following steps:

1. Coupling with phenyl isothiocynate (PITC) under mild alkaline conditions, forming a phenylthiocarbamyl peptide.

2. Cleavage of the residue as a anilinothiazolinone (ATZ), an amino acid derivative.

3. Conversion of the cleaved ATZ derivative to a stable phenylthiohydantoin (PTH) derivative.

This stabilized PTH amino acid derivative is then identified by reverse-phase HPLC. With direct sequencing, the membrane on which the target protein is retained is subjected directly to Edman chemistry within the instrument. The methodology is limited to the identification of approx 30 residues. In addition, gaps in the sequence often result, particularly when the level of target protein is low. Often, the proteins are also blocked at the N-termini in the course of the process, leading to a reduction in the target available for sequencing. The frequency of blockage increase with time, suggesting that other processes are at work (20,21).

2.2. Detection of Proteins/Peptides

In Western blotting following transfer, detection of target proteins is general achieved using a specific antibody. The attachment of specific antibodies to their targets can be readily visualized by using methods based on direct or indirect immunoassays. These methods involve the use of various labels conjugated to an antibody (see Fig. 2). Some of these labels can directly produce a detectable signal (e.g., fluorescent molecules, such as fluorescein, or an radioisotope such as iodine-125), but more commonly, today, indirect immunoassay systems based on enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are used. In this case, the detectable signal is generated by the action of the enzyme on a particular substrate; hence, the systems are described as indirect. Chromogenic, chemiluminescent, or fluorescent enzyme substrates are available. Some of the more commonly used substrates with some useful websites are given in Table 2. Chromogenic substrates result in the deposition of a colored product at the site of the conjugated enzyme and, hence, the protein of interest. Chemilumines-cent and fluorescent substrates both produce light that can be detected as appropriate via X-ray film, charge-coupled device (CCD) cameras, or scanners. At present, the most widely used signal generation system in Western blotting is chemiluminescence. This has several advantages, including the ability to achieve high levels of sensitivity and fast processing times. Chemiluminescence is the generation of electromagnetic radiation, as light through the release

Outline Western Blot Procedure
Fig. 2. Examples of Western blotting detection strategies.

Table 2

Commonly Available Enzyme Substrates for Use in Western Blotting Applications

Enzyme Alkaline phosphatase

Substrate

Peroxidase

Colormetric (color produced follows each substrate listed)

Fluorescent

Chemiluminescent

3,3',5,5'-Tetramethylbenzidine (blue) 4 Chloro- 1-naphthol (blue/black) Diaminobenzidine ± cobalt chloride (produces a red/brown color in the absence of cobalt chloride, black when present) AmplexGold™

('www. molecularprobes. com) FluorBlot™ (www.piercenet.com)

Luminol/peroxide

(www.amershambiosciences.com) Acridans (e.g., 2',3',6'-trifluorophenyl 10 methylacridan-9-carboxylate) (www.lumigen.com)

5-Bromo-4-chloro-3-indolylphosphate/nitroblue-tetrazolium (black/purple) Naphthol phosphate/Fast Red (red)

2'-[2-Benzothiazoyl]-6'-hydroxybenzothiazole phosphate (www.jblsci.com) 2-Hydroxy-3-naphtoic acis-2'-phenylaniide phosphate (www.roche-applied-science.com) DDAO phosphate (www.molecularprobes.com) Dioxetanes (e.g., disodium 3-[4

methoxyspiro {1,2 dioxetane-3,2'-tricyclo [3.3.1.1] decan}-4-yl phenyl] phosphate [www.tropix.com])

Note: Full details of the substrate mode of action and recommended applications and condition can be found at the website cited. In general, additional information about all enzyme substrate for Western blotting can be found at these sites.

Hrp Detection With Luminol
3-aminophthalate Fig. 3. Oxidation of luminol.

of energy from a chemical reaction. Usually, these reactions involve the cleavage of a relatively weak O-O bond, which liberates a large amount of energy. HRP systems generally are based on the oxidation of the cyclic diacylhydrazide luminol in the presence of hydrogen peroxide (see Fig. 3) (22). Immediately following oxidation the luminol is an excited state, which decays back to the ground state via a light-emitting pathway. The presence of phenolic enhancers cause the light emission to be increased 1000-fold (23). Often, the process is referred to as enhanced chemiluminescence. AP systems are almost exclusively based on the use of 1,2-dioxetane substrates (24) (see Fig. 4).

Key to any methodology is efficient blocking of the membrane. This will minimize background or nonspecific binding of the antibodies and reagents used for detection and, therefore, maximize the specific signal-to-noise ratio. A number of protein and detergent solutions are routinely used for this purpose. These include nonfat dried milk, Tween-20®, bovine serum albumin (BSA), whole serum, and gelatine. Care must be taken to ensure that the blocking agent is compatible with the detection reagents used. The most common in use today are nonfat dried milk and BSA (3,25) Figure 5 outlines a few of the available detection methodologies in Western blotting; many variations on these themes exist in the reagents and kits that are available from suppliers. In a single-layer system, the primary (or recognition) antibody is conjugated either to an enzyme or a direct label. A double-layer system involves the use of an anti-immunogobulin capable of recognizing the primary antibody. This anti-immunogobulin is conjugated to an enzyme or a direct label. Another method utilizes a biotin-tagged anti-immunogobulin to introduce a third layer. The biotin molecule binds a streptavidin or avidin molecule which is conjugated to a suitable label, usually an enzyme. Increasing the number of layers within the detection procedure increases the sensitivity of the system. However, the label itself and the type of substrate employed also contribute the sensitivity of the system.

3. Western Blot Assays in the Diagnosis of Viral Infections

Western blotting has mostly found use in detecting immunogenic responses elicited by infectious agents, such as bacteria, viruses, and parasites, where the presence of these agents is difficult to detect with methods that aim to isolate and culture the infectious agent from patient samples. Other diagnostic uses for Western blot assays include detection of the presence of abnormal cellular proteins such as the prion protein. In general, Western blot assays do not provide truly quantitative information. In these applications, the technique is usually used as a secondary method to confirm initial results obtained with enzyme immunoassays (EIAs) or enzyme-linked immunosorbent assays (ELISAs) or provides additional information not obtainable with these procedures. Often, many different antigens can be assayed together, thus providing a wider picture of the antigenic response being studied.

Fig. 4. Example of 1,2 dioxetane hydrolysis by alkaline phosphate.

In addition to many in-house-developed diagnostic Western blot systems, several companies are manufacturing commercial kits for use in serological diagnostics of different infectious diseases. Confirmation of human immunodeficiency virus (HIV) infections is an important application area in viral diagnosis. Detection of immunological responses in Lyme disease is a major application for bacterial diagnosis. Cysticercosis represents a parasitic disease where the Western blot technique demonstrates the presence of the parasite Taelia cysticercosis, which is difficult to prove by other means. Western blotting methods have proved useful for showing the presence of disease-related human proteins in autoimmune diseases. How Western blot assays are currently applied to these clinical areas will now be discussed.

3.1. Western Blot Assays in the Diagnosis of Viral Infection

3.1.1. HIV-I Diagnostics

HIV-1 and HIV-2 are the two causative agents of autoimmune deficiency disease (AIDS). Whereas HIV-2 infections are mostly restricted to Africa, HIV-1 has spread worldwide. Following infection by either virus, the patient develops antibodies against viral proteins. Detection of these antibodies with EIAs is used in the routine detection of HIV infection in patients and screening of blood donors. Western blotting is recommended for use as a secondary confirmatory test to further analyze samples that have repeatedly produced positive EIA results (26, and references therein). Anti-HIV antibodies can be found in most body fluids, including serum, saliva, and urea. All of these can be used as samples for testing for the disease. These Western blot kits contain specific HIV proteins fractionated according to their molecular weight by electrophoresis on a polyacrylamide gel in the presence of sodium dodecylsufate (SDS). These separated proteins are electrotransferred from the gel to a nitrocellulose membrane, which is then washed, blocked, and packaged. In the clinical laboratory, these nitrocellulose strips are incubated with HIV antibodies present in the patients serum (or other body fluid). These are the primary antibodies of our description in Fig. 5. The strips are washed to remove unbound material. Visualization of the human immunoglobulins specifically bound to the HIV proteins is achieved using a series of incubations in, for example, goat anti-human IgG conjugated with biotin, followed by streptavidin conjugated with HRP and then the HRP substrate 4-chloro-1-naphthol (see Fig. 5C). If antibodies to any of the target HIV proteins or antigens are present, bands corresponding to the positions of one or more of the HIV proteins result. The kit will contain suitable control material to ensure that the nitrocellulose strip and other reagents are performing correctly.

The structure and protein composition of HIV-1 and 2 viruses are well characterized, allowing identification of antigens acting in the immunological response. Following HIV infection, the earliest antibodies are against group-specific antigen (GAG) protein p24 and its precursor p55, but with later progression of the disease, these antibodies can decrease in quantity and be difficult to detect. Antibodies against envelope protein (ENV) precursor protein gp160 and its precursors gp120 and gp41 are usually found in most HIV-infected patients. Multimeric forms of gp41 can give rise to bands of 120 and/or 160 kDa (27). Antibodies against HIV polymerase (POL) enzyme proteins p31, p51, and p66 can also be detected in patient samples (27-31).

Immunodetection Western Blot
Fig. 5. Diagrammatic representation of some immunodetection regimes used in Western blotting.

In addition to the complexities in antigen patterns arising from the HIV viruses, the interpretation of blotting results is complicated by the different combinations of antibodies that may be found in patients. Antibody patterns may change during the progression of the infection (2731). This biological variation has resulted in different guidelines being put forward for scoring Western blot results as positive for detecting HIV-1/2 virus infection. The requirement for detecting antibodies against all three major protein groups (ENV, GAG, and POL) for a positive score results in a relatively high number of indeterminate results, as only 72-79% of positive samples type contained antibodies against all three different groups of HIV proteins (32). The World Health Organisation (WHO) has recommended using the presence of at least two bands, including those corresponding to gp41, gp120, or gp160 (33), as a criteria for a positive finding. A negative test result is the absence of any reactive bands on Western blot. Although detection of anti-HIV antibodies can indicate the presence of viral infection, final diagnosis should only be based on clinical examination.

Saliva and urine have been established as alternatives to serum samples for testing for HIV antibodies. These fluids offer the advantage of easy collection. Although the antibody concentration is lower than in serum, it is still suitable for detection with sensitive immunological methods such as Western blotting. Factors such as increase in urine volume following consumption of large quantities of liquids or the use of diuretics (34) further reduce antibody concentrations. Saliva contains secretions from salivary glands, oral microorganisms, cells, and gingival-crevicular transudate, which has been shown to contain immonoglobulin G against HIV proteins in infected individuals (34).

The Federal Drug Administration (FDA) has approved three different kits for HIV testing. The Western Blot HIV-1 kits from Cambridge Biotech and Genetic Systems (Bio-Rad Laboratories) can be used with serum/plasma samples for confirmatory testing of blood donors. The Cambridge Biotech kit can also be used with urine samples and the Genetic Systems kit with dried blood spots. Organon Teknika's OraSure HIV-1Western blot kit is exclusive for use with oral fluid samples. Many other diagnostic kits exist for detecting HIV antibodies using the technique; see Table 3 for details.

All of the kits for HIV diagnosis are based on the same principal design. The kit contains several identical test nitrocellulose strips to which size-separated HIV proteins have been transferred and immobilized. These strips are stable for several months if stored at cold room temperatures. The viral proteins have been obtained from virus purified from infected H-9/ HTLV-IIB, T-lymphocyte cell line with ultracentrifugation (OraSure), or partially purified and inactivated by treatment with psoralen and ultraviolet (UV) light (Cambridge Biotech). In addition to HIV antigens, the blotted membrane can also contain control bands such as the staphy-lococcal protein (JN HIV-1/2 Kit). These controls are for detecting the presence of general IgG proteins in a sample and act as positive confirmation for correct functioning of the kit.

Before the individual membrane strips are incubated with a patient samples, they are blocked to inhibit nonspecific binding. The patient sample is diluted in sample buffer. The extent of dilution required depends on the test kit used and the sample. For example, the OraSure kit requires 150 |L of saliva diluted 1 : 7 for use; the Genelabs HIV-1 blot 1.3 kit requires 20 |L of serum diluted 1:100 for use. If anti-HIV antibodies are present in the patient sample, they bind to their cognate antigen on the test strip. After washing, the bound antibodies are detected with a secondary anti-human IgG antibody-alkaline phosphatase conjugate (OraSure), which catalyzes the conversion of nitroblue tetrazolium (NBT) into a colored deposited product in the presence of 5-bromo-4-chloro-2-indolyl-phosphate (BCIP). Alternatively, the Cambridge Biotech HIV-1 kit uses a biotin-labeled goat anti-human IgG and an avidin conjugated to HRP. Color reaction is achieved using the substrate 4-chloro-1-naphthol. All of the HIV-test kits contain negative control serum, as well as low- and high-positive control serums, which help in assessing the patient test results.

The actual protocol for scoring the bands present on membranes reacted with patient sera vary from one manufacturer to other. With the OraSure kit, the intensity of bands on the patient test strip are compared to the intensity of the gp41 band on the low-positive test strip. If bands of equal or higher intensity are detected, the band is scored as present. In the Cambridge Biotech kit, the intensity of the p24 band, seen with a weakly reactive serum, is used as reference for scoring. The overall test with both kits is considered positive only if at least two of the major bands (gp160, gp41, gp120, or p24) are present, according to the recommendations by WHO.

Antibodies against HIV-2 proteins can give positive reaction on Western blot containing HIV-1 antibodies, however, often the results are indeterminate. This is why some of the HIV-1 Western blot kits contain additional antigens derived from HIV-2. The JN HIV-1/2 Kit contains a recombinant HIV-2 membrane protein, Genelabs HIV-1 blot 2.4 contains a line of gp46

Table 3

Examples of Commercially Available HIV Western Blot Kits

Table 3

Examples of Commercially Available HIV Western Blot Kits

Test kit

Manufacturer/supplier

Sample type

FDA approval for diagnostic puiposes

Genetic Systems HIV-1 Western Blot

Bio-Rad Laboratories

Human serum,

Yes

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