Rocket Immunoelectrophoresis

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Abstract

Rocket electrophoresis (also referred to as electroimmunoassay or electroimmunodiffusion) is a simple, quick, and reproducible method for determining the concentration of a specific protein in a protein mixture. The method, originally introduced by Laurell (1), involves a comparison of the sample of unknown concentration with a series of dilutions of a known concentration of the protein, and requires a monospecific antiserum against the protein under investigation. The samples to be compared are loaded side-by-side in small circular wells along the edge of an agarose gel that contains the monospecific antibody. These samples (antigen) are then electrophoresed into the agarose gel, where interaction between antigen and antibody takes place. As the protein antigen starts to leave the well and enter the gel, antigen molecules will start to interact, and bind with, antibody molecules. However, at this early stage, there is considerable antigen excess over antibody and no precipitation occurs. However, as the antigens sample electrophoreses further through the gel, more antibody molecules are encountered that interact with the antigen, until eventually there is sufficient antibody-antigen crosslinking such that “equivalence” is reached and the antigen-antibody complex precipitates. One might therefore expect to see some sort of precipitation line appear in the gel. In practice, a “rocket” shape is seen (Fig. 1). The majority of the antibody-antigen precipitate is indeed at the head of this rocket, but the fine precipitation lines up the side of the rockets are formed by a small amount of antigen diffusing sideways as the antigen passes through the gel. This small amount of antigen very quickly meets sufficient antibody to reach equivalence and precipitate. The greater the amount of antigen loaded in a well, the further the antigen will have to travel through the gel before it can interact with sufficient antibody to form a precipitate. Therefore, if a series of wells are loaded with increasing antigen concentration, then a series of rockets of increasing height should be produced, with the area under the curve (rocket) being proportional to the amount of antigen in the well. However, since the rockets are nearly perfect isosceles triangles, the height of the rocket is also proportional to the area under the curve (and hence the antigen concentration), and it is this easier-to-measure parameter that is normally recorded. Rocket electrophoresis is therefore carried out by loading a series of samples of different antigen concentrations, with one sample being the unknown. The unknown sample can be a highly complex mixture of proteins (e.g., serum sample, tissue extract, urine sample, cerebrospinal fluid, and so forth), but only one rocket will be produced from this sample owing to the interaction of the antibody in the gel with the antigen of unknown concentration. A calibration graph of protein concentrations vs peak height is then constructed and, knowing the peak height of the unknown sample, the concentration can be read off from the graph. Concentrations of proteins as little as 1 µg/mL can be measured in this manner (requiring as little as 20 ng of protein to be loaded in a well). An excellent detailed review of this subject has been published (2). References (38) give some idea of the range of applications of this technique. This chapter will describe the construction of a simple calibration curve using bovine serum albumin (BSA) as antigen and anti-BSA. This is an ideal trial system for the first-time user and will give the user confidence to use the technique with his or her own, often more precious, antigen and antibody.