Antibodies in molecular biology

4.13 Antibodies in molecular biology

The majority of techniques in molecular biology are beyond the scope of this book, but some of the contributions of immunology to this important discipline deserve mention.

If monoclonal antibodies had not already existed, then they would certainly have had to be invented for molecular biology. Similarly, the ability of polyclonal sera to ‘view’ a polypeptide as

a series of discrete epitopes has been crucially important for the detection of incompletely expressed protein sequences fused in the middle of completely unrelated proteins. Antibodies provide a means for defining and testing the function of previously unknown pro- tein molecules. They are an essential tool for the characterization of the protein via recombinant DNA technology.

4.13.1 Analysis of in vitro translation products

Routes from the first definition of an antigen, for example a novel, functionally important cell- surface glycoprotein, to a full determination of its amino acid sequence, are now well established. Often the first step in characterization is to determine its apparent molecular weight by immuno- precipitation (see Section 3.13) and SDS-PAGE analysis (see Appendix B).

Some of the questions to be resolved before attempting the full cycle of cloning, expression and sequencing are: (a) Does the cell really make the antigen or has it been acquired exogenously? (b) Is the antigenic determinant protein- or carbohydrate-based? (c) Is the epitope sequence- or conformation-based? If the latter is the case then it is unlikely that

the antibody would react with the unglycosylated, incomplete segment of the original molecule expressed in the middle of a bacterial protein. Immunoprecipitation of in vitro translation products from cell-derived mRNA not only

definitively resolves these questions but also provides confirmation that the mRNA is acceptable for the preparation of cDNA.

A typical result is shown in Fig. 4.9. The mRNA was extracted from the cell expressing the antigen of interest and mixed with 35 S-methionine and micrococcal nuclease-treated rabbit retic- ulocyte lysate (the latter is a rich source of all the molecules to make protein, but devoid of the

156

C H A P T E R 4: Antibodies as probes

Fig. 4.9 Immunoprecipitation of in vitro translation products. Messenger RNA, extracted from three different stages of the life-cycle of a protozoan parasite, was translated in vitro in the presence of 35 S- methionine. The resulting polypeptides were fractionated on an SDS-PAGE gel, either immediately (tracks 2–4), or after immunoprecipitation with either a monoclonal antibody (tracks 5–9) which reacted with a polypeptide in a single life-cycle stage (track 4), or an irrelevant antibody (results not shown). Track 1 contains radiolabelled molecular weight standards, molecular weight is in thousands and migration position is indicated by arrowheads. Tracks 2–4 show polypeptides in the starting material with a molecular weight of up to 100 000, indicating good translation. Tracks 5–7 show the effect of increasing the amount of monoclonal antibody added as ascitic fluid. Although the 85-kDa band increases in intensity, so does the non-specific binding. Tracks 8 and 9 show the lack of binding of the antibody to other life-cycle stages (represented in tracks 2 and 3).

‘message’). The polypeptides synthesized in vitro have been visualized by fluorography after immunoprecipitation and SDS-PAGE analysis. Significantly, the photograph shows discrete polypeptides some of which are in excess of 90 kDa apparent molecular weight, indicating that the mRNA is in good condition. The single, large polypeptide band precipitated by the mono- clonal antibody, which is slightly smaller than that synthesized by intact cells (not shown), confirms that the epitope is not carbohydrate dependent, either directly or for its conformation. (The techniques for in vitro translation are explained in full in Hames & Higgins 1999.)

4.13.2 Screening of expression libraries

Antibody can provide a powerful tool by which a mature molecule, for example a cell-surface glycoprotein expressed by a eukaryotic cell, can be related to a set of partial sequences expressed in bacterial fusion proteins.

The method described was used to screen a cDNA library from a protozoan parasite expressed in the bacteriophage λgt11. The methods in recombinant DNA technology referred to below are fully described in Glover (1996) or Sambrook and Russel (2000).

4.13ANTIBODIES IN MOLECULAR BIOLOGY

Preparation in advance

1 Calculate the titre of the recombinant phage stock and dilute to produce about 300 discrete plaques per plate (adjust the total number per plate according to the size of the plaques).

2 Grow up a stock of the Escherichia coli (Y1090) plating cells and mix an aliquot with the appro- priate dilution of phage and plate out.

3 Incubate the culture plates at 42°C until plaques appear (between 3 and 4 h, but longer if the

E. coli has been cooled for storage).

4 Transfer the cultures to a 37°C incubator, overlay each plate with a sterile, isopropyl-β,d- thiogalactopyranoside (IPTG)-impregnated nitrocellulose filter (previously soaked in 10 mm IPTG and dried before use) and incubate overnight to induce fusion protein expression.

5 Before removing the filters, use a syringe loaded with dye and fitted with a large gauge needle to pierce each filter at three places round its periphery to provide orientation marks in the filter and agar. (By this means it will ultimately be possible to match up the black spots on the auto- radiograph or coloured spots on the filter with phage plaques on the original culture plate.)

6 Unless you are using a monoclonal antibody produced in vitro, it will be necessary to ex- haustively absorb all antisera or ascitic fluids to remove the naturally occurring anti-E. coli and antibacteriophage antibodies. Commercial preparations are now available which will allow this to be accomplished conveniently, according to the manufacturer’s instructions.

MATERIALS AND EQUIPMENT Nitrocellulose filters bearing replicates of recombinant phage plaques Antibody, poly- or monoclonal, specifying antigen of interest

10 m M tris(hydroxymethyl)-aminomethane (Tris), pH 9.6, containing 150 m M sodium chloride and 0.05% v/v Tween 20 (wash buffer)

Bovine serum albumin (BSA), RIA grade

3 MM paper Clear acetate sheets Pen with water-insoluble ink Radioactive ink Orbital shaker Bag sealer Role of continuous plastic tube In addition: Materials for autoradiography or enzyme labels

METHOD

1 Remove the nitrocellulose filters from each plate and immerse them in a large volume of

10 m M Tris, pH 9.6, containing 150 m M sodium chloride and 0.05% v/v Tween 20 (wash buffer). Store the agar plates face down at 4°C until immunoscreening is complete.

2 Leave the filters in the wash buffer on the orbital shaker for 10 min at room temperature. Use sufficient buffer to ensure that the filters are moving freely.

3 Transfer the filters to wash buffer containing 3% w/v BSA (RIA grade) and leave rocking for

30 min at room temperature. (The BSA will block the unoccupied protein-binding sites on the filters and so prevent non-specific uptake of antibody protein.)

Continued

C H A P T E R 4: Antibodies as probes

4 Seal the filters into a plastic sac, excluding as much air as possible.

5 Dilute the pre-absorbed antibody (see step 6, Preparation in advance) in wash buffer containing 3% w/v BSA, allowing 5 ml of solution for every 20 filters, and load it into the sac using a syringe and needle. As a guide, dilute the antibody 1 : 100 or use neat tissue culture supernatant from hybridoma cells.

6 Reseal the plastic sac and leave it rocking for 1 h at room temperature.

7 Cut open one edge of the sac and recover the antibody solution. So little is consumed during this procedure that it may be stored at –20°C for further use.

8 Transfer the filters to a tank of wash buffer and rock gently for 10 min at room temperature. For efficient washing, use sufficient buffer to allow the filters to move freely.

9 Wash twice more under the same conditions.

10 Remove the filters from the final wash, gently blot each one dry on 3 MM paper and seal all together into a plastic sac, excluding as much air as possible.

11 Dilute the anti-immunoglobulin antibody or protein A in wash buffer containing 3% BSA. See Technical notes for selection of label.

12 Add the labelled second reagent to the plastic sac, using a syringe and needle, and reseal the sac.

13 After incubating on the rocking platform for 1 h at room temperature, open the sac, recover and store the labelled reagent, and wash the filters three times in wash buffer as in step 8 above.

A For immunoscreening with enzyme labels

14 After the final wash, blot each filter dry on 3 MM paper, and process with chromogenic

substrate to reveal the binding sites of the enzyme-conjugated second reagent, as described in Section 4.11.4.

B For immunoscreening with radioactive labels

14 Remove each filter from the final wash solution, blot dry on 3 MM paper and mount onto a sheet of card covered with Saran wrap. (Saran wrap is best but cling film may be used if necessary.) Finally, cover the filter and card tightly with Saran wrap.

15 Cover each orientation hole on the filters with a small sticky label and mark the position of the hole with radioactive ink (this aids orientation of the autoradiograph when preparing the template for isolation of the phage plaques).

16 Expose to X-ray film using tungsten intensifying screens and process photographically.

17 Prepare a duplicate of the coloured dots on the filter (enzyme labels) or the black dots on the autoradiograph (radioactive labels) using a clear acetate sheet and a fine pen. Remember to transfer the orientation marks.

18 Mark the regions of the positive plaques on the acetate sheet and use this to guide a Pasteur pipette to remove a plug of agar from the corresponding region in the original plate.

The plugs of agar are each dispersed in individual tubes containing a storage buffer. If the original phage plaques were discrete and optimally spaced, each plug should contain one, or a limited number, of types of recombinant phage. It is necessary to repeat the ‘screening and picking’ pro- cess until the phage plaques are uniformly positive on immunoscreening (Fig. 4.10).

You should now have a series of recombinant phages containing segments of the DNA coding for the protein defined by the original antibody.

4.13ANTIBODIES IN MOLECULAR BIOLOGY

(a)

(b)

Fig. 4.10 Screening of a λ λgt11 expression library with antibodies. A λgt11 genomic library of the protozoan parasite Trypanosoma cruzi was screened with a mixture of antisera from infected patients. (a) The first round of screening identified a single-phage colony in this plate (corresponding to the black dot on the photograph of the nitrocellulose filter) producing a fusion protein which contained one or more parasite- derived epitopes. Once the agar plug containing the recombinant phage had been replated onto a fresh culture of Escherichia coli (b) the proportion of positive colonies increased dramatically.

TECHNICAL NOTES • It is necessary to remember that polyclonal sera or ascitic fluids are biological materials and

not chemical reagents. The pre-absorption step, to remove the anti-E. coli and antiphage antibodies, must be carried out to completion.

• Binding of the first antibody can be visualized with an anti-immunoglobulin antibody or protein

A, labelled with either a radioactive or enzyme label. • It is good practice to keep the filters moist during autoradiographic exposure as it is possible to continue the washing procedure if the radioactive background is unacceptably high, or to repeat the immunoscreening if no signal is detected after 7 days’ exposure. This is not possible with enzyme labels.

• All materials and equipment should be sterile and aseptic technique should be used through- out. The screening can take between 7 and 9 days; therefore, even at 4°C, microbial contamin- ants can overgrow and make plaque identification difficult.

• Monoclonal antibodies raised against short peptides, especially C-terminal, are likely to be better reagents than antibodies which recognize conformational determinants.

4.13.3 From partial sequence to mature protein

The rapid progress seen in the characterization of important proteins in infectious organisms has been made possible by the application of the techniques of molecular biology, rather than those of protein chemistry. Consequently, there is a frequent need to relate an incomplete, and often unknown, sequence of DNA expressed in a prokaryote to the mature molecule expressed in the eukaryotic cell. The multiperspective specificity of polyclonal antibodies combined with the discriminatory power of immunoblotting allow this link to be established with relative ease.

C H A P T E R 4: Antibodies as probes

Once an unknown sequence has been expressed, e.g. as a plasmid-derived fusion protein in β -galactosidase, then the whole bacterial lysate may be used to immunize a mouse and so pro- duce an antiserum suitable for immunoblotting.

An important note: adequate specificity controls are needed when using the antiserum for immunoblotting. It is essential to blot against not only the organism or cell of interest but also the E. coli host strain (not infected with the recombinant plasmid). Only then is it possible to

be reasonably confident that the mature polypeptide identified on the blot of the SDS-PAGE- fractionated organism really does relate to the fusion protein.

It is possible to adsorb the fusion protein onto nitrocellulose and use it for affinity purification of antibodies from polyclonal sera. This has been particularly useful in clinical situations, where immunization with fusion protein is not possible.

The technique • A small square of nitrocellulose filter is placed in contact with a single clone of recombinant

phage growing in E. coli. After induction of the fusion protein, the filter is ‘blocked’ with BSA and pre-eluted with 0.1 m glycine–HCl buffer, pH 2.5, containing 150 mm sodium chloride.

• Antibody is absorbed from the patient’s serum onto the filter, which is then extensively washed and eluted by immersion in a small volume of glycine–HCl buffer. • After neutralization with a few crystals of solid Tris, the affinity-purified antibody can be used for a miniature immunoblot (Section 4.11 and Miniblotter apparatus). This technique both relates the fusion protein to the mature protein (identified by size in the blot) and establishes its importance to the patient’s response to infection.

It has been possible to relate a DNA sequence of unknown function to a gene product using an antiserum raised against the synthetic oligopeptide inferred from the triplet code. Oligopeptides of < 10 amino acids are relatively poor immunogens; however, once their chain length exceeds 10–15 the chance of obtaining an antiserum is high. This approach tends to favour detection of epitopes in accessible regions of high hydrophilicity and N- or C-terminal regions, even when the resulting antisera are used in immunoblotting.