93

Clinica Chimica Acta, 97 (1979) 93-95 0 ElsevierjNorth-Holland Biomedical Press

BRIEF TECHNICAL

NOTE

CCA 1096

EST~ATION OF Ighiz IN CEREBROSPINAL FLUORO~MUNOASSAY

FLUID BY

E.A.H. HISCHE a, H.J. VAN DER HELM a-* and T. OUT b a Department of Neurology, Academic Hospital “Wilhelmina Gasthuis”, le Helmerstraat 104, 1054 EG Amsterdam (The Netherlands) and’b Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (The Netherlands) (Received

March 9th, 1979)

Introduction For the estimation of IgM in concentrations that are normally present in cerebrospinal fluid (CSF) only radioimmuno~say is sensitive enough. The BioRad Immuno-Fluor procedure for IgM was modified to obtain the required low detection limit and compared with a radioimmunoassay. Materials and methods Fluoroimmunoassay Reagents Immune-FluorTM kit for IgM, cat. no. 171-1300 (Bio-Rad Laboratories, 2200 Wright Avenue, Richmond, CA 94804) consisting of: standard buffer (200 ml concentrate to make a buffer pH 7.5, containing 150 mmolfl NaCl and 10 mmol/l phosphate). Lyophilized Immunobead reagent consisting of a rabbit anti human-IgM, covalently bound to derivatized polyacrylamide beads. Lyophilized fluorescein-conjugated rabbit anti human-IgM. Lyophilized immunoglobulin standard prepared from clarified human serum with assayed values for IgG, IgA and IgM. Sodium chloride (Merck 6404), di-sodium hydrogenorthophosphate dibydrate (Merck 6580), potassium dihydrogen orthophosphate (Merck 4873). Procedure To lower the detection realize the same reaction * To whom coneepondence

limit 250 ~1 of sample was used instead of 100 ~1. To conditions as the original protocol prescribed, the fol-

should be addressed.

94

lowing modifications were introduced in the preparation of the reagents: Immunobead reagent was suspended in 30 ml buffer pH 7.5 containing 150 mmol/l NaCl and 20 mmol/l phosphate (instead of 51 ml standard buffer), the fluorescein labeled anti human-IgM antibodies were dissolved in 3.25 ml of standard buffer (instead of 2.75 ml). As blank a solution of 150 mmol/l NaCl, and for standardization IgM standards with concentrations ranging from 14 I.U./l up to 170 I.U./I, were used. The following procedure was found to be satisfactory: pipette 250 ~1 sample into a glass tube (size 100 X 15 mm), add 250 ~1 Immunobead reagent, mix by Vortex and incubate for 1.5 h at 37°C. Add 25 ~1 fluorescein labelled anti human-IgM reagent, mix by Vortex and incubate for 1 h at 37°C. Add 1 ml standard buffer, mix by Vortex and centrifuge for 15 min at 1700 X g. Immediately after centrifugation, decant the supernatant and blot the inside of the tube to just above the sediment with absorbent paper that leaves no fibres. Add 1.5 ml of standard buffer and repeat the procedure of mixing, centrifugation, decanting and blotting. Finally add 1.0 ml standard buffer, mix and measure the fluorescence at 495 nm excitation and 525 nm emission wavelength. The fluorescence was measured with a standard Aminco Bowman spectrophotofluorometer (American Instrument Company, Silver Springs, U.S.A.) supplied with an off-axis ellipsoidal mirror condensing system and a xenon lamp. Slits of 3 mm before the excitation monochromator and 5 mm before the emission monochromator were used. The resolution was improved by placing a filter of 525 nm wavelength (maximal transmittance 0.59, bandwidth 6 nm) between the sample and the second monochromator. Radioimmunoassay

The radioimmunoassay

of IgM was performed

as described

for IgE [ 11.

Results and conclusions The detection limit of the proposed procedure standard deviation of the blank, n = 8).

Fig. 1. Relationship

between

fluoroimmunoassay

is about 5 I.U./l IgM (3X the

and radioimmunoassay

(RIA) of IgM.

95

The precision on a day-to-day basis was assessed by repeated analysis of a pool of CSF. Small portions were stored at -20°C until the analysis was performed. A coefficient of variation of 6.6% (n = 27, X = 97.2 I.U./l) was found. The proposed procedure was compared with the radioimmunoassay. In 46 samples of CSF, duplicate estimations were performed by both methods. Fig. 1 shows a scatter diagram of the results. Linear regression analysis gave a correlation coefficient T = 0.949. Conclusion: This fluoroimmunoassay procedure is suitable for determination of IgM in CSF. Acknowledgements We thank Mrs. L.R. Bastiaans-Tjahjadi, Miss L.K.E. Tjian for their skilful technical

Mrs. F.J.Th. assistance.

Reference 1 Stallman. P.J. and Aalberse. R.C. (1977)

Int. Arch. Allergy 54.9-18

Gorter-Louwerier

and