The Use and Limitations of Filter-paper Electrophoresis

Abstract

In 1935 Tiselius, using the apparatus he had devised in 1930, demonstrated that human serum globulin fell into three reasonably defined groups based on their electrophoretic mobilities. For the next 15 years a steady flood of papers reported the fluctuations of albumin and the globulins in young and old, in health and disease, as disclosed by analyses carried out using Tiselius apparatus with and without the numerous minor modifications which were introduced.In spite of brilliant efforts at simplification it was evident that the method, because of its relative complexity, could never have widespread use in routine clinical chemistry. In 1950 three groups of workers, among them Tiselius himself, described methods which indicated that, using filter paper as a matrix, a relatively cheap and rapid method could be evolved of obtaining a rough analysis of the serum proteins akin to the analysis obtained by the classical technique.The method required no more than 0.5 ml. of serum, and though estimates of the total protein concentration would still have to be made by other means, it was conceivable that this simple technique would dispense with the tedious salt fractionation and subsequent analyses required heretofore to obtain the albumin/globulin ratio.The last four years have produced a spate of papers devoted to the description of modifications in methods of separation and analysis with numerous technical and mathematical tricks for improving the correlation of the results with the substantial body of data already obtained with the classical technique. With few notable exceptions scant attention was paid to the fundamental principles underlying the technique or to the points in which their application to it differed from the classical procedure.As paper succeeded paper it became increasingly evident that except within fairly wide limits the two methods were not quantitatively comparable.Even worse, because of lack of time spent on a consideration of the basic principles involved, G it seemed probable that differing techniques for the production of " electrophoretograms " were not comparable among themselves.One was faced with the prospect of scores of laboratories turning out hundreds of results year by year; each group, because of deviations in technique unappreciated and therefore unrecorded, differing to an unknown extent one from the other. The clinical chemical chaos that could arise demanded a reassessment of the situation in the hope that workers in the field would stop, think, and start again.Electrokinetic phenomena, as the derivation of the word implies, are concerned with the

Fibrinogen Estimation by Paper Electrophoresis

“…Because of the ease with which fibrinogen becomes denatured (17,19,20) results may be fallaciously low as part of it may be left at the starting line. Furthermore, spuriously high results may be obtained because of the formation of an abnormally dense band, as shown in figure lb.…”

“…Finally, recognition of the numerous difficulties in the estimation of individual serum protein fractions (17,21,22,23,24) which would apply equally to the estimation of fibrinogen, has resulted in the general abandonment of the expression of results as absolute amounts, and they are now usually expressed as a percentage of the total dye absorbed under standard conditions. Although the standard methods of estimating fibrinogen are by no means free from error (25) they appear to be no more time-consuming and considerably more accurate than techniques using paper electrophoresis.…”

“…With the advent of paper electrophoresis, serum was used in the investigation of blood proteins to the almost complete exclusion of plasma because of the unsatisfactory results obtained with the latter (17). Antweiler (18) stated that fibrinogen adhered to the paper and did not move on electrophoresis : however, the present study confirms the work of Berkes and Karas (1) and Antonini and Piva (2) who produced evidence that fibrinogen does move on paper and does produce a definite band.…”

Electrophoretic Analysis of Serous Pleural Effusions

“…Changes occurring in other diseases have been reported by Seibert and others (1947), Flynn (1954), Hardwicke (1954), and many others. Further, Martin and Franglen (1954) have shown that paper-strip electrophoresis cannot properly be used for quantitative analysis. Gilliland and others (1956) found that the albumin/a2 globulin ratio could be correlated with the activity of pulmonary tuberculosis, varying from 3.45 for patients with extensive active tuberculosis to 5.69 for those with arrested disease.…”

Electrophoretic Partition of -Globulins in Human Sera by a Non-ionic Detergent

“…These workers attribute this effect to the formation of complexes between borate ions and a-glycol groups in the lipoid components of the a-globulins (Martin and Franglen, 1954 (1) paper-strip electrophoresis. It seems likely, however, that 83-globulin is the main source, with occasional and variable increments from the a-globulins depending upon the concentration and distribution of the associated lipids which are transferable to the new position.…”