Quantitative filter paper electrophoresis by means of reflectance densitometry

CLINICA

538

QUANTITATIVE REFLECTANCE

FILTER

PAPER

ELECTROPHORESIS

BY

CHIMICA

MEANS

ACTA

OF

DENSITOMETKY

SUMMAKY

A method of quantitative paper electrophoresis is described which is suitable for clinical application and most research purposes. The method depends upon reflectance densitometry and the assumption is made that the various globulins are laid down on a carpet formed by the albumin tail. Within the range 0.125-3.0 g albumin per IOO ml tailing has been found to be independent of concentration. The validity of the method has been demonstrated

by analysis of protein mixtures

of known composition.

INTRODUCTION

Direct scanning of the stained strip is by far the most convenient means of evaluating mixtures separated by paper electrophoresis. Photometric analysis may be achieved by either transmittance or reflectance measurements. Scanning by transmission has so far proved to be the more generally used procedure, but reflectance densitometry has many advantages to commend it. This technique has been used by Latnerl and by Rottger2 and a critical evaluation which provided favourable support was undertaken by 0wen3. The subject of quantitative zone electrophoresis on paper has an enormous literature in which the inherent difficulties have been discussed and explored. Many of the problems which arise can be overcome by preliminary experiments and careful standardisation of technique. It is possible to select a dye for which the different protein fractions have approximately the same affinity and to calibrate a densitometer to correct for the non-linear relationship between the densitometer response and the concentration of the dye on paper. One outstanding problem, however, is that of albumin tailing and this represents a very serious limitation. Albumin, being the fastest moving component, precedes the other fractions and may be said to “lay down a carpet” for them. Several workers have measured the degree of albumin tailing and sought to apply corrections31s. \\?th transmission densitometry, tailing causes low measurements of the albumin fraction and high

QUANTITATIVE

values surface

ELECTROPHORESIS

for the globulins. layer,

BY DENSITOMETRY

Since the major

reflectance

scanning

539

part of reflection

should

is likely to be from the

prove satisfactory

for the globulin

com-

ponents which in the drying process are deposited over the albumin and so form the surface layer from which reflection occurs. In order to obtain true albumin values, however, it would be necessary to add the amount in the “tail” to the determined albumin peak. It was therefore decided to undertake the present work in order to test this hypothesis

and to attempt

to determine

the degree of albumin

tailing.

EXPERIMENT.tL

Method

of electrophoresis

The electrophoresis apparatus used was that designed by LatnerG. The filter paper strips were Whatman IOO fat-free paper 46 x 2 cm and the buffer a conventional barbitone buffer, pH 8.6, ,u 0.05. A 120 V dry battery served to supply the current which was 0.2 mA per cm width of paper. The sample, either serum or CSF, was diluted with buffer or concentrated by ultrafiltration to a total protein content of approximately 2 g/Ioo ml and applied by means of a paint brush in the form of a narrow band across the dry strip. Using a technique previously described6 the paper was moistened without allowing the buffer to displace the applied protein. The volume of sample used was of the order 0.02 ml. On close examination the two surfaces of the brand of filter paper used are different: the sample was always applied to the upper machined side and it was always this surface which was scanned. This was adhered to strictly, especially when scanning paper step wedges. Up to ten such strips at a time could be placed in the electrophoresis apparatus. Electrophoresis was allowed to proceed overnight for 16 h. The strips were dried in a horizontal position in a hot air oven at IIO’ for 20 min and then immersed in a solution of L&amine Green S.F. 150 (zoo mg “,/oin 3% sulphosalicylic acid ‘). After 30 min they were washed in several changes of 2% acetic acid, rinsed twice in tap water and hung to dry at room temperature. reflectance

densitometer

The strips were scanned in an automatic

(“Chromograph”

Joyce

Loebl Co. Ltd., Gateshead

recording on Tyne).

Investigation of the factors which influence quantitative electrophoresis (a) The dye uptake of normal whole semm on$lter paper In order to investigate the dye uptake on whole serum protein on filter paper, step wedges were prepared after the manner of Latner et a1.8. Using Whatman IOO fat-free papers 2 cm wide, a series of strips was prepared by saturating each strip with a different dilution of normal serum in barbitone buffer. A blank step was prepared in the same way by saturating the strip of paper with diluent only. The total protein of the stock serum was estimated in duplicate by a biuret method9. The middle 2.5 cm of each strip was cut out and stained with Lissamine Green. The pieces of paper, each representing a different protein concentration, together with the blank segment were arranged in ascending order on a strip of white paper and the whole mounted in the densitometer strip holder and scanned. The diagram obtained consisted of a series of plateaux. The height of each of these minus that of the blank step gave the height of pen displacement for each protein concenClin.

Chim.

Acta,

II

(1965) 538-546

A. L. LATNER,

540

D. C. PARK

t&ion. On plotting such displacement against the concentration, a curvilinear relationship was obtained, as illustrated in Fig. I. The figure shows the results on two occasions. Although there is an appreciable difference between them in regard to the height of pen deflection for a given protein concentration, the shapes of the curves are virtually identical. In other words the proportionality for given protein concentrations is the same on each curve.

kc+

60 80 100 Serum protein

&

120

140

160

concentration

of pen deflection

180 (mg/lOOml)

Fig. I. The relationship between pen deflection and serum protein concentration. Each point represents the mean of duplicate estimations. The different symbols represent the results on two separate occasions.

(b) The dye uptake of the diflerent sewm protein fractions To investigate the dye affinity of the different protein fractions, filter paper step wedges were prepared for each fraction. Solutions of human albumin-, c(~-, /G, and y-globulin were obtained by preparative cellulose column electrophoresis and the protein content estimated by weighing (see later-Analysis of semwn protein mixtures). Step wedges previously described

were then prepared for whole serum.

for each protein

In each case the relationship between displacement was found to be a curvilinear

fraction

in the manner

protein concentration and height of pen one. The curves obtained are shown in

Fig. 2. The close agreement between the four curves and that of whole serum indicates that the different protein fractions and whole serum had closely similar dye affinities. 60-

0, I

I

20

40

60 Protein

80 100 120 concentration

160 140 (mg/lOOml)

180

Fig. 2. The relationship between pen deflection and protein Each point represents the mean of duplicate estimations. Clin.

Chim.

Acta,

II

(1965)

538-546

for each of the protein

fractions.

QUANTITATIVE

ELECTROPHORESIS

BY DE~SITO~~ETRY

541

(c) The relationshifi between the amount of dye present and the densitomctev yesponse The optical wedge in the densitometer used is an annular grey wedge which is linear with respect to density. It is known, however, that beyond a small concentration of dye neither reflection or transmission densitometry yields a linear relationship between dye concentration on paper and density. It has been suggested by Jencks et al.1° that this is due to the nature of the dye on paper. Step wedges of filter paper stained with different dilutions of Lissamine Green were prepared in the following manner. Serial dilutions were prepared from a stock solution of dye. Each “step” was prepared from a strip of filter paper (~~lhatrnan IOO, 2 cm wide) which was dropped into a large Buchner tube containing dilute dye solution. Negative pressure was applied for a few minutes to remove any air bubbles at the surface of the paper. The strip was then lifted out carefully and hung from a polythene peg. Excess dye solution was removed by gently blotting the free end of the strip. The strips were left to dry at room temperature. A piece about 2.5 cm in length was cut from the middle of each strip and a series of these pieces, each representing a different dye concentration, together with a piece of untreated filter paper as a blank, was arranged in ascending order of concentration and scanned. As was expected, the relationship between dye concentration and pen displacement was a curvilinear one. With these data it was then possible to compare the stained-protein pen deflection curve and the dye pen deflection curvre. A point on each curve showing the same pen deflection was selected. By adjusting the abscissa scale of the dye curve, these two points could be made to coincide. The dye curve replotted on this new abscissa scale was found to coincide exactly with that of the stained protein curve. This was interpreted as evidence that the actual relationship between protein concentration and dye uptake was a linear one and that the curvilinear response was an optical property of the dye on paper. (d) Method of correcting scan diagrams Having determined the shape of the curvilinear relationship between dyed protein concentration and pen deflection, it was then necessary to convert the curve to a straight line in order to be able to correct paper electrophoresis scan diagrams. This was done according to the method described by Latner*l which is a general procedure applicable wherever it is required to convert a curve into an ideal straight line. The resulting right-angled triangle and its contained curve lying partly along and partly just under the hypotenuse were then reproduced in transparent Perspex, the c.urve being engraved on the triangle on the under-surface to avoid inaccuracies due to parallax. Scan diagrams could then be corrected quite simply with the aid of this Perspex triangle. The side of the triangle representing the original abscissa was aligned along the base line of the electrophoresis diagram and wherever the curve engraved on the triangle cut the electrophoresis tracing, the point was replotted on the hypotenuse vertically above the point of intersection. Since the electrophoresis scan diagrams were recorded on graph paper, the points to be corrected could be replotted quickly. When there was a sufficient number of points, a free hand curve was drawn connecting them, and a corrected electrophoresis diagram obtained. Clin. Chirn. Acta, IT

(1965) 538-546

542

A. L. LATNER,

D. C. PARK

(e) Iwestigation of the nature and extczt of albun& tailing These experiments were carried out first with bovine albumin and then repeated with human albumin. Whatman IOO fat-free paper was used throughout and experimental conditions (buffer composition, drying, staining, scanning) were as described

above. (I) The

adsorption

of albumin

protein to be carried through applying an albumin solution

by paper was demonstrated

by allowing

the

the paper strip by liquid flow. This was arranged by to some paper strips and placing these in the electro-

phoresis tank as for an electrophoresis run but without applying a current. The tank was tilted towards the anode and the protein was carried through the paper by the slow syphoning of buffer from the cathode to the anode side. When the strips were stained and scanned, each showed a single protein peak of albumin followed by a “tail”

of adsorbed albumin over that part of the paper traversed by the albumin. (2) An albumin solution was applied to each of five paper strips and subjected

to electrophoresis in the manner already described and then the current reversed until the albumin peak had returned about half-way. Rromphenol blue was previously added to the protein solution applied to one of the strips in order to indicate the position of the main albumin peak. When the other strips were stained and scanned, a peak was obtained with tails on either side of equal depth to each other, showing that albumin

tailing was no greater in the region where the albumin

versed the paper twice. saturation phenomenon.

This

experiment

demonstrated

that

band had trans-

albumin

tailing

is a

(3) Albumin solutions were subjected to electrophoresis and then the strips taken from the electrophoresis tank, hung albumin peak downwards in a descending chromatography tank and washed chromatographically for 24 h with the barbitone buffer used for electrophoresis. When the strips were stained and scanned it was seen that the albumin peak had been washed off the paper but that the tail still remained. This confirmed that tailing is an irreversible phenomenon. (4) The effect of protein concentration upon the degree of tailing was studied. First serial dilutions of bovine albumin solution were subjected to electrophoresis and the strips scanned. Solutions containing 1.0 and 0.5 g bovine albumin per IOO ml showed an albumin peak with a tail 3 mm above the base line. Solutions containing 0.25 and 0.125 g per IOO ml showed no peak since all the albumin was consumed in the path of migration: the tail began 3 mm above the base line and eventually petered out.

These experiments were repeated with serial dilutions of a stock solution of 3 g human albumin per IOO ml. The same effect was observed, that is, where the concentration was sufficient to show an albumin peak after electrophoresis, the tail approximated closely to 3 mm above the base line. It was therefore concluded that within the range of albumin concentrations likely to be encountered in practice, tailing was independent of concentration and in evaluating scan diagrams could be assumed to be 3 mm above the base line. Other investigators have also found that .tailing is independent of albumin concentration 4s12, I3 but some disagree with this finding59 ll. The analysis of scau diagYam.s Since the relationship between pen deflection and dyed protein concentration had been shown to be non-linear above pen deflections of 2 cm, it was necessary to

QUANTITATIVE

ELECTROPHORESIS

BY DENSITOMETRY

543

correct these points on the scan diagram. This was done by means of the Perspex correction triangle in the manner already described. In practice it was usually only the albumin peak which had to be corrected. The whole area under the corrected curve was then divided into areas corresponding to the different protein fractions. The albumin peak was drawn in symmetrically about a perpendicular line dropped from the maximum to the base line. The other peaks were then fitted in a corresponding fashion, so that their points of intersection represented half the total pen deflection at that point (see Fig. 3). With a little practice this procedure was found to be reproducible and not unduly time consuming. The extent of albumin tailing during the run was assessed by drawing a line parallel to the base line and 3 mm above it from the point of application to the foot of the albumin peak (see Fig. 3). The area underneath this line represented the

;A,

i

Fig. 3. Scan diagram of an electrophoresis strip showing the method of analysing applying corrections. The corrected albumin peak is shown as a dotted line. The represents the area corresponding to the albumin tail.

the cur\ e and shaded portion

albumin tail, and was added to the area under the albumin peak to give the albumin value. Since we believe that the globulin fractions are deposited over the albumin tail, we consider that the amount of light reflected from the surface indicates the amount of each globulin present and therefore the globulin peaks were drawn down to the true base line (see Fig. 3). The areas corresponding to the various protein fractions were then traced on to tracing paper, cut out and weighed on an automatic balance. Iioulet et al.15 also consider that the gravimetric method of determining areas is preferable to the more time consuming and more exacting use of a planimeter. In considering the area of the albumin tail it may be argued on theoretical grounds that protein adsorption occurs over the whole width of the albumin band. If one makes the probably false assumption that there is no adsorption under the albumin peak it is somewhat simpler to assess the area of peak and tail since both may be cut out together on the same piece of tracing paper. In actual fact the error introduced is so small as to be of no practical significance. qf serum protein nuitures In order to test the validity of this method of evaluating scan diagrams, solutions were prepared containing known amounts of each protein fraction. The protein fractions were obtained from normal human serum by preparative electrophoresis on ethanolysed cellulose after the manner of Porath16.

Analysis

Clin.

Chim.

A&,

II

(1965)

538-546

A.

544

L. LATNER,

D. C. PARK

Each of the major protein fractions was concentrated to a final volume of approximately I ml by ultrafiltration using wide Visking tubing (inflated diameter 24/32”) supported by sacs of nylon mesh. The concentrated protein obtained was now diluted while still in the Visking sac with approximately IO ml 0.1% sodium chloride and ultrafiltration repeated down to a volume of I ml. The protein solution was “washed” in this way several times with 0.1% sodium chloride until all the barbitone had been removed. This was checked by demonstrating analytically that the Na and Cl concentrations were equivalent. The identities of the protein fractions were confirmed by comparing their behaviour on paper electrophoresis with that of normal whole serum. In preparing the first two protein mixtures and the step wedges referred to earlier, protein concentration was determined by weighing. The Na and Cl contents of each protein solution were estimated by flame photometry and mercuric nitrate titration respectively. 1.0 ml of the protein solution was taken to dryness in a vacuum desiccator and the weight of the dry residue minus the weight of sodium chloride per ml gave the concentration of protein per ml in the original solution. In preparing the third protein mixture, protein concentrations were determined more rapidly by U.V. spectrophotometry reading at ZIO rnp and comparing with bovine albumin standard’?. These

solutions

of determined

protein

composition

and concentration

were

mixed together in known proportions to prepare appropriate protein mixtures which were then subjected to paper electrophoresis and the strips analysed according to the method

of reflectance

densitometry

described

above. The results obtained

in this

way were compared with the known composition of the mixture. Table I shows the good agreement between the actual percentage composition and that found. Evidence of reproducibility is given by the duplicate analyses shown in mixture 3 and received further confirmation by the results shown in Table II of the duplicate analyses of human sera and cerebrospinal fluids. DISCUSSION

cedure,

There has been much criticism of paper electrophoresis as a quantitative probut we consider that our results lend justification to it as a reliable direct

TABLE

I

COMPARISON OF PAPER STRIP PERCENTAGE ____

ANALYSIS

OF

Albumin + cc-globulin

Mixture

Mixture

Mixture

PROTEIN

MIXTURES

,VITH

ACTUAL

COMPOSITION

or,-Globulin

@-Globulw

yGlobztli$~

0 ,”

22

!” 20

‘5

19 48 4s

96

ra

Found

55 60

3.0 4.8

2 Actual Found

37 38

-

14 ‘5

7.4 7.2 6.0

TT

1-

‘3 12

1; lb

I Actual

3 Actual Found

Clin. Chim.

Acta,

65 6.3 67 II (1965) 538-546

0,

QUANTITATIVE TABLE DUPLICATE

ELECTROPHORESIS

BY DENSITOMETRY

54.5

II ANALYSES

REPRODUCIBILITY

OF

OF THE

SOME

SERA

AND

CEREBROSPINAL

FLUIDS

TO

ILLUSTRATE

THE

METHOD

method of analysis. For most clinical investigations a quantitative analysis is not required but it can sometimes be necessary, for example, in estimating CSF y-globulin as a diagnostic test for multiple sclerosis r8. It may also be helpful on occasion in following a patient’s progress and for specific research projects a quantitative procedure is usually required. The satisfactory results obtained for the analysis of protein mixtures support our original concept that the globulins are deposited on an albumin carpet, which does not itself interfere with reflectance scanning. It is interesting that over a fairly wide range of concentrations we have found that albumin tailing is constant, that is, that the density of the tail is independent of concentration. In this respect we agree with Hardwicke” and Henry ct al.l”, and disagree with Yeoman5. Afthough there is some possibility that the a,-globulin fraction has not quite the same dye affinity as the other fractions (see Fig. z), we consider that this defect is of no real practical significance. Gorringe19 also showed that albumin, y-globulin and the mixed proteins of whole serum have the same dye affinity for L&amine Green:

he did not study the LX-and j’$globuIins.

We use a paint-brush for applying the protein because this must produce minimal damage to the paper surface. It might be argued that the taking up of a protein solution in this manner would result in denaturation by virtue of surface effects. Although this argument is by no means entirely sound, it is obvious that surface denaturation cannot be avoided whilst electrophoresis in filter paper is being carried out. There is, however, no reason why denaturation at this stage should interfere with the electrophoresis pattern from the point of view of proportionality of the various protein sub-groups, since in any case all the strips are heated prior to staining. MThilst our experiments have been carried out solely with Whatman IOO fat-free paper, we see no reason why the technique we describe should not be used with other grades.

Clin. Chim.

Ada,

II (1965) 538-546

546

A. L. LATNER,

I).C. PakRK

REFERENCES 1 2 3 4 5 b ; N g 10 1I 12 ~3 14 15 10

A. I,.I.ATNER,&~G~~WZ. J.,5r (1952) XXIXP. H. ROTTGER, Fx@vienfia, 9 (1953) ISO. J. h. OWEN, .4nal?~sf, 81 ('956) 26. J. H.~RDWWE, Bwchem. ,I.,57 (1954) 166. w. I3.~EOh%AN,C~i~.C~ta‘~.‘4&z, 4 (1959) 246. A. L. LATNER, Riochsm. J., 51 (1952) XIII-'. G. DISCOMRE, R. I;.JONES A~'D 11.I'.\VINSTANLEY, J. Cli?f. Paihol., 7 (1954) 106. A. I,.LATNER, L. MOLYNEVS ?IND J. DUDFIELD KUSE, J. Lab. CEztz. ,Xed., 4.1 (1954) 157. J. FINE, Biochent.J,, 29 (1935) 799. \I:.P. JENWS, M. R. JETTON AXD E. 1~.DL!RRUM,B~&WX. J., 60 (1955) 205. A. 1”. L~TN~SR, I. Lnb. Cl&. Med., 45 (1955) 147, S. CHR. SOM~~ERFELDT, Stand. J. Chin. Lab. Invest., 5 (1953) 299. R. J. HENRY, 0. J. GOLUB AND C. SOBEL, CZzn.Chew., 3 (1957) 49. C. RI. KAPLAN, H. I;.WEISBIIRG A~YD L. Dow, J. Lab. Clw?. Med., 50 (1957) 657. H. ROULET, J. A. C)\VEN AXI) C. I'.STEWART, Clin.Chive.Acta, I (1956) 417. J. PORATH, ‘4rk.I