Single Radical Immuno Diffusion technique and Laurell’s Rocket Immuno electrophoresis technique are two most commonly used techniques employed for quantification of Human serum albumin. The underlying principle of both the techniques employ the precipitation of the antigen making use of a mono specific antibody (raised in rabbits) in semi-solid agarose gel plate (Bodle et al.2013). The difference between both the techniques lies in the size of the precipitates formed which is radial in case of SRID technique whereas rocket shaped in case of Rocket Immuno electrophoresis.
Advantages and Disadvantages of the techniques:
The advantages of using SRID technique includes a highly sensitive reaction outcome using a lesser amount of antigen and the disadvantages include long duration of reaction process ranging from 18 to almost 48 hours. On the other hand, the advantages of Rocket Immuno electrophoresis technique includes a faster reaction rate with quantitative accuracy, whereas disadvantages include maintenance of antibody to antigen ratio with precision and detection of only one antigen per plate.
Calculation of the diameter of wells employed in SRID Plates:
The diameter of the precipitation ring formed can be calculated by the formula:
Diameter of precipitation ring = Diameter of whole circle-Diameter of well
According to the formula,
Diameter of well= Diameter of precipitation ring-Diameter of whole circle
If the rockets would have been run at 90V overnight, instead of 320V in two hours,
The Rocket Immuno electrophoresis works on the principle of loading antigen samples of varying concentration on the wells along the edge of the agarose gel that contains the specific antibody (Kricka and Park 2014). On application of an electric field, the sample moves towards the anode and forms an antigen-antibody complex (Westermeier 2016). As the antigen present in the sample migrates towards the anode it encounters more and more antibody molecules till it reaches the ‘equivalence point’. Therefore, depending on the concentration of antigen present in the sample, the increase in peak of the rockets are seen. On running the gel at 90V overnight, the antigen present in the sample would have migrated out of the gel and no alteration in the rocket height would have been found. Further, it should be noted that running the gel overnight would lead to complete saturation of the antigen-antibody complex.
Normal range for Human Serum albumin levels,
The normal range for Human Serum Albumin level is documented to be 3.5 to 5.5g/dL or around 35-55g/L (Anguizola et al.2013). However, the serum albumin levels differ slightly from laboratories to laboratories depending on the optimal conditions observed while the test is being performed.
The gel is prepared in 1X PBS Buffer followed by the addition of antibody and then allowed to spread uniformly in a plate. After the gel solidifies uniformly, wells are cut and the known antigen solution along with the unknown antigen sample is loaded and the gel is run for a period of 2 hours at 320V. A graph is then plotted on the basis of the observed height pattern of the rocket appearance in the gel.
Rocket electrophoresis is one of the most convenient molecular biology tool in order to determine the concentration of unknown antigen present in a sample. It should be kept in mind that higher the concentration of antigen present in the sample, farther the antigen would travel in the electrophoresis field and an increase in the height of the rocket series would be seen in the gel. Higher the rocket height, more would be the concentration of antigen in the sample. The highest precipitate value recorded in the experiment was 31.0mm and the lowest was 21.5mm.
Anguizola, J., Matsuda, R., Barnaby, O.S., Hoy, K.S., Wa, C., DeBolt, E., Koke, M. and Hage, D.S., 2013. Glycation of human serum albumin. Clinica Chimica Acta, 425, pp.64-76.
Bodle, J., Verity, E.E., Ong, C., Vandenberg, K., Shaw, R., Barr, I.G. and Rockman, S., 2013. Development of an enzyme?linked immunoassay for the quantitation of influenza haemagglutinin: an alternative method to single radial immunodiffusion. Influenza and other respiratory viruses, 7(2), pp.191-200.
Kricka, L.J. and Park, J.Y., 2014. 15 Immunochemical Techniques. Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics-E-Book, p.236.
Westermeier, R., 2016. Electrophoresis in practice: a guide to methods and applications of DNA and protein separations. John Wiley & Sons.