Preparation of Antibodies

00:01 Antibodies are incredibly useful in a number of different diagnostic assays and actually throughout the whole of biology, there are uses for monoclonal antibodies because their really high degree of specificity makes them excellent probes to detect the presence of particular molecules.

00:23 One can produce polyclonal antibodies, that is antibodies produced from a number of different B-cells by immunizing an animal with an antigen.

00:33 So here we have an example, taking a protein and immunizing a sheep with this particular protein.

00:40 One can then collect blood from the sheep a period of time later, one gives the sheep a chance to make some antibodies, the immune response gets going.

00:50 And then a few weeks later, after a couple of boosters, one can take some blood from the sheep.

00:56 And there will be polyclonal antibodies that are specific for this antigen.

01:01 And the proportion of antibodies because you’ve immunized the sheep, the proportion of antibodies against this particular antigen will be increased.

01:10 So they won’t all be against this antigen, there’ll be antibodies against many other antigens as well.

01:14 But the percentage of antibodies against the immunizing antigen will have been increased by the immunization procedure. So you’ll have different antibodies against different parts of the particular antigen of interest. So for example, immunizing with human IgG, you can develop sheep anti-human IgG as a reagent to use in immunodiagnostic tests, where you want to see if a patient has IgG antibodies that bind to a particular antigen. For example, an autoantigen.

01:51 And using these kind of sheep antibodies and then labeling them with a fluorescent dye or with an enzyme will allow their use in immunodiagnostic assays.

02:05 So those are polyclonal antibodies, a mixture of antibodies.

02:09 And very often it’s useful to have a mixture of antibodies rather than a single specificity.

02:15 But sometimes you want to have all of the antibody absolutely identical.

02:20 And specific for perhaps one single epitope on an antigen.

02:26 It’s possible to do this using the hybridoma approach to produce monoclonal antibodies.

02:36 In this approach, an animal is immunized with the antigen of choice.

02:40 The spleen is removed from the mouse in this example, and it’s making-- within the spleen will be B-cells that are making antibody against the antigen that the mouse was immunized with.

02:54 B-cells don’t survive for very long outside of the body.

03:00 However, B-cells can become malignant, become tumor cells.

03:07 And these tumor cells will have long term survival, that’s a characteristic of a tumor cell.

03:13 It divides very rapidly and it survives.

03:17 By fusing together, the normal B-cells from the spleen of the immunized mouse, together with a malignant B-cell in the form of a myeloma, one can produce what are called hybridomas.

03:35 And these hybridomas which is a hybrid of the normal B-cell and the tumor B-cell, inherit two properties.

03:45 They inherit the antigen specificity of the normal B-cell but they also inherit the immortality of the tumor cell, in other words the myeloma cell.

03:58 So in this methodology, there is fusion between the immune spleen cells and the myeloma tumor cells.

04:07 These cells, hybrid cells are then cultured in a selective medium that kills off the unfused tumor cells, because otherwise they’d keep growing, they’re immortal.

04:18 They’d keep growing and they’d outgrow the hybrids.

04:21 So you want to just have the hybrids, the normal spleen cells die off naturally.

04:27 The myeloma cells that have not fused will be killed by culture in a selective medium.

04:33 So only the few cells survive after a few days.

04:37 And the cells are then diluted into a microtitre plate, so that there is on average one cell per well.

04:47 The cells are then grown in the individual culture plate wells.

04:52 And the culture supernatant, what’s being secreted by these hybridomas, can be assayed to see whether they are producing the antibody of interest.

05:04 So the supernatants from the wells containing the growing hybrid cells are screened for the presence of the desired antibody using the enzyme-linked immunosorbent assay, ELISA.

05:18 Then the positive wells are grown up to a very large volume and there is a clone of antibody producing fused cells.

05:29 This clone, the hybridoma is an immortal producer of the desired monoclonal antibody.

05:35 So these can be kept growing for years and years, and years, producing antibody that is all of identical specificity, monoclonal antibody.

05:49 There is another approach to making monoclonal antibodies and this is called phage display.

05:56 For example, maybe one wants to develop a monoclonal antibody against a tumor antigen as a therapeutic agent to treat patients with a particular tumor.

06:10 One can immunize a mouse with this tumor-derived antigen, take the spleen from these mice and isolate the B-cells.

06:22 Messenger RNA is then taken from these B-cells and converted into cDNA.

06:34 PCR, the polymerase chain reaction, is then used to amplify the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region using specific primers.

06:49 These two sequences, the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region are then linked together using a flexible linker.

07:00 And this gene sequence is inserted into bacteriophages which are viruses that infect bacteria.

07:09 Now what you have is a mixture of phages on the cell surface, or on the surface of these phages rather, you will have the antibody present.

07:25 And inside the phage, you will have the gene encoding that particular specificity of antibody.

07:33 You can then use a method that is often referred to as panning; it’s a bit like panning for gold.

07:39 But here you’re panning for a phage that has on its surface an antibody fragment that recognizes the antigen of interest.

07:48 So by incubating a mixture of different phages with different antibodies on their surface, you’re after one particular antibody with specificity for a particular tumor antigen, by panning on a plate where there is immobilized tumor antigen.

08:02 You can select phages that have high affinity binding.

08:06 They will have inside them the gene sequence that you want.

08:10 And then you can express that in a particular expression system.