1275
Methods and Devices NEW ULTRAMICROMETHOD FOR CONCENTRATION OF CEREBROSPINAL FLUID SIDNEY N. KAHN
E.
J.
THOMPSON
Department of Chemical Pathology, Institute of Neurology, National Hospital, Queen Square, London WC1N 3BG ROUTINE
hospital laboratory electrophoresis of cerebrospinal
fluid (c.s.F.) is normally carried
out on support media such as cellulose acetate,3 and even paper.4 This requires concentration of the c.s.F. by 20-100 times for sufficient protein to be applied in the small (1-5 .1) application volume demanded by these media. Present methods of concentration involve the use of positive or negative pressures and membranes of different pore sizes and require rather large volumes which, in the case of c.s.F., are often not available. The most efficient way of using the limited volumes of c.s.F. available would be to concentrate about 100 .1 down to 1-5 ,1. We describe such a method which requires no apparatus and permits multiple simultaneous sample processing. Conventional ultrafiltration invaribly requires large pressures in order to initiate flow through the membrane. There is one commercially available product for the concentration of urine and large volumes of C.S.F. which does not depend on such pressure (’Minicon’, Amicon Limited, High Wycombe, Bucks). This is achieved by pre-treatment of the membrane with a wetting agent.5 We have used the same technique for the preparation of the ultramicrofilter. A disc of ’Diaflo’ PM 10 ultrafiltration membrane (nominal cut-off 104 daltons), 62 mm diameter (Amicon Ltd.) is soaked for 2 hours in 50% (v/v) aqueous glycerol containing 1% (v/v) ’Triton X-100’ (British Drug Houses Ltd., Rohm & Haas Co.) and allowed to dry thoroughly. The membrane is then placed on an absorbent pad of blotting-paper on a rigid, flat base-e.g., a glass slide. The membrane is held in contact with the absorbent pad by transverse ribs (wooden applicators, 2 mm diameter, have been found suitable) kept in place by
agar,’ agarose,2
spring clips (fig. 1). The sample to be concentrated is then applied to the surface
Fig. 2--Agarose gel electrophoresis, in 13% lactate buffer2 0.075 mol/1, pH 87.
agarose,
barbitone/calcium-
A single C.S.F. of total protein 38 mg/dl, concentrated 26x. Application volume 5 1. Left to right: the first two electrophoretograms represent concentrated c.s.F., the third the native c.s.F.
of the membrane, which must be horizontal. The volume to be applied will depend on the total volume of fluid to be concentrated and the final volume desired. To concentrate 100 1, the circular area of application should be about 3-4 mm diameter-i.e., 5-10 jjd is applied initially. When the volume of fluid remaining on the surface of membrane is about 10-20% of the initial volume, in 2-3 minutes, a second aliquot is applied and the process is repeated until the desired concentration is reached and the sample removed. It is crucial to avoid damage to the membrane surface and consequent loss of concentrating ability. We have found both microlitre syringes and adjustable micropipettes (e.g., ’Finnpipette’) with disposable plastic tips suitable. We have used this technique for the concentration of single C.S.F.S to a uniform protein concentration of 10 g/1 before applying 5 pj to an agarose slide for electrophoresis; representative electrophoretograms are shown in fig. 2. CONCLUSIONS
This new method of ultramicroconcentration applicable to and other biological fluids has the advantages of speed (20-fold concentration in about 30 minutes), ease of manipulation, economy (up to 20 samples can be concentrated on one 62 mm diameter disc), simultaneous multiple sample processing, and the facility to vary the molecular-weight cut-off by selecting different filters. In addition no special apparatus is required. The technique is extremely simple, but reasonable care must be taken to avoid damage to the membrane. Continual attention is also needed to avoid complete drying of the sample with possible secondary loss of protein by precipitation. c.s.F.
We are grateful to Dr W. F. Blatt, (N.B.), Holland, for technical advice.
of Amicon B.V., Oosterhout
REFERENCES
Link, H. Acta neurol. scand. 1967, 43, suppl. 28, p. 7. Laurell, C-B. Scand. J. clin. Lab. Invest. 1972, 29, suppl. 124, p. 79. 3. Kohn, J. in Chromatographic and Electrophoretic Techniques (edited by Smith); vol II, p. 114. London, 1968. 4. Mies, H-J. Klin. Wschr. 1953, 31, 159. 5. Blatt, W. F. Personal communication. 1. 2.
Fig. 1---CoLuplete absorbent pad.
apparatus, showing membrane held in apposition
to
I.
Methods and Devices NEW ULTRAMICROMETHOD FOR CONCENTRATION OF CEREBROSPINAL FLUID SIDNEY N. KAHN
E.
J.
THOMPSON
Department of Chemical Pathology, Institute of Neurology, National Hospital, Queen Square, London WC1N 3BG ROUTINE
hospital laboratory electrophoresis of cerebrospinal
fluid (c.s.F.) is normally carried
out on support media such as cellulose acetate,3 and even paper.4 This requires concentration of the c.s.F. by 20-100 times for sufficient protein to be applied in the small (1-5 .1) application volume demanded by these media. Present methods of concentration involve the use of positive or negative pressures and membranes of different pore sizes and require rather large volumes which, in the case of c.s.F., are often not available. The most efficient way of using the limited volumes of c.s.F. available would be to concentrate about 100 .1 down to 1-5 ,1. We describe such a method which requires no apparatus and permits multiple simultaneous sample processing. Conventional ultrafiltration invaribly requires large pressures in order to initiate flow through the membrane. There is one commercially available product for the concentration of urine and large volumes of C.S.F. which does not depend on such pressure (’Minicon’, Amicon Limited, High Wycombe, Bucks). This is achieved by pre-treatment of the membrane with a wetting agent.5 We have used the same technique for the preparation of the ultramicrofilter. A disc of ’Diaflo’ PM 10 ultrafiltration membrane (nominal cut-off 104 daltons), 62 mm diameter (Amicon Ltd.) is soaked for 2 hours in 50% (v/v) aqueous glycerol containing 1% (v/v) ’Triton X-100’ (British Drug Houses Ltd., Rohm & Haas Co.) and allowed to dry thoroughly. The membrane is then placed on an absorbent pad of blotting-paper on a rigid, flat base-e.g., a glass slide. The membrane is held in contact with the absorbent pad by transverse ribs (wooden applicators, 2 mm diameter, have been found suitable) kept in place by
agar,’ agarose,2
spring clips (fig. 1). The sample to be concentrated is then applied to the surface
Fig. 2--Agarose gel electrophoresis, in 13% lactate buffer2 0.075 mol/1, pH 87.
agarose,
barbitone/calcium-
A single C.S.F. of total protein 38 mg/dl, concentrated 26x. Application volume 5 1. Left to right: the first two electrophoretograms represent concentrated c.s.F., the third the native c.s.F.
of the membrane, which must be horizontal. The volume to be applied will depend on the total volume of fluid to be concentrated and the final volume desired. To concentrate 100 1, the circular area of application should be about 3-4 mm diameter-i.e., 5-10 jjd is applied initially. When the volume of fluid remaining on the surface of membrane is about 10-20% of the initial volume, in 2-3 minutes, a second aliquot is applied and the process is repeated until the desired concentration is reached and the sample removed. It is crucial to avoid damage to the membrane surface and consequent loss of concentrating ability. We have found both microlitre syringes and adjustable micropipettes (e.g., ’Finnpipette’) with disposable plastic tips suitable. We have used this technique for the concentration of single C.S.F.S to a uniform protein concentration of 10 g/1 before applying 5 pj to an agarose slide for electrophoresis; representative electrophoretograms are shown in fig. 2. CONCLUSIONS
This new method of ultramicroconcentration applicable to and other biological fluids has the advantages of speed (20-fold concentration in about 30 minutes), ease of manipulation, economy (up to 20 samples can be concentrated on one 62 mm diameter disc), simultaneous multiple sample processing, and the facility to vary the molecular-weight cut-off by selecting different filters. In addition no special apparatus is required. The technique is extremely simple, but reasonable care must be taken to avoid damage to the membrane. Continual attention is also needed to avoid complete drying of the sample with possible secondary loss of protein by precipitation. c.s.F.
We are grateful to Dr W. F. Blatt, (N.B.), Holland, for technical advice.
of Amicon B.V., Oosterhout
REFERENCES
Link, H. Acta neurol. scand. 1967, 43, suppl. 28, p. 7. Laurell, C-B. Scand. J. clin. Lab. Invest. 1972, 29, suppl. 124, p. 79. 3. Kohn, J. in Chromatographic and Electrophoretic Techniques (edited by Smith); vol II, p. 114. London, 1968. 4. Mies, H-J. Klin. Wschr. 1953, 31, 159. 5. Blatt, W. F. Personal communication. 1. 2.
Fig. 1---CoLuplete absorbent pad.
apparatus, showing membrane held in apposition
to
I.
