Scandinavian Journal of Clinical and Laboratory Investigation

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A sensitive enzyme immunoassay for the quantitation of human IgM B. Dinesen & O. Saxtrup Nielsen To cite this article: B. Dinesen & O. Saxtrup Nielsen (1979) A sensitive enzyme immunoassay for the quantitation of human IgM, Scandinavian Journal of Clinical and Laboratory Investigation, 39:5, 449-454, DOI: 10.3109/00365517909106130 To link to this article:

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Date: 16 April 2016, At: 09:10

Scand. J. clin. Lab. Invest. 39, 449-454, 1979.

A sensitive enzyme immunoassay for the quantitation of human IgM

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B . D I N E S E N & 0. S A X T R U P N I E L S E N Department of Clinical Chemistry CL, Rigshospitalet, University Hospital, Blegdamsvej 9, DK-2100, Copenhagen, Denmark

Dinesen, B. & Saxtrup Nielsen, 0. A sensitive enzyme immunoassay for the quantitation of human IgM. Scand. J. clin. Lab. Invest. 39, 449-454, 1979. A highly sensitive and specific two-site solid-phase enzyme immunoassay for the quantitation of human immunoglobulin M (IgM) in biological fluids is presented. Detection limit is 20 pg/l (12 ngltube). Routine working range is 30-1000 pg/l. Between-assay coefficient of variation is 10%.The assay allows quantitation of IgM in human cerebrospinal fluid. Key-words: cerebrospinal fluid; enzyme immunoassay; IgM; serum B. Dinesen, M.Sc., Department of Clinical Chemistry, Rigshospitalet, University Hospital, Blegdamsvej 9 , DK-2100, Copenhagen, Denmark

The primary antibody response in an immunization process consists mainly of production of immunoglobulin M (IgM), later followed by immunoglobulin G (IgG), which perhaps is suppressing the IgM production [3, 141. I n cerebrospinal fluid (CSF), the concentration of IgM is so low, that assays for determining IgM until recently have been too insensitive to measure the concentration in the healthy state. Several attempts to measure human IgM using different immunological principles have been described recently [6,9,11-13, 161. Among these, only radioimmunoassay (RIA) [9] and enzyme immunoassay (EIA) [6] were sensitive enough to measure the IgM concentration in CSF in the healthy state. The purpose of the present work was to establish a method for human IgM estimation with a sensitivity sufficient to quantitate IgM in normal adult 0036-5513/79/0900-0449$02.00 8 1979 Medisinsk Fysiologisk Forenings Forlag

CSF. EIA was chosen to gain experience with this analytical principle and because EIA does not require a laboratory specialized for isotopic work. A sandwich (two-site immunoenzymometric) technique and not a competitive procedure has been applied because of the difficulties involved in the preparation of pure human IgM with a high yield. In the present method a solid-phase technique was used for the separation of bound and free marker. Disposable polystyrene cuvettes, made for the LKB 8600 Reaction Rate Analyzer, were used as solid phase. The assay procedure is schematically shown in Fig. 1 and consists of the following steps: coating of cuvettes with rabbit anti human IgM, antigen incubation, incubation with peroxidaselabelled goat anti human IgM and finally measurement of the cuvette-bound enzyme activity-the individual steps separated by a careful washing procedure. Descriptions of problems related to these individual steps are


B. Dinesen & 0 .Saxtrup Nielsen


Human albumin. ‘Trocken, reinst’, Behringwerke, West Germany. Reference material. WHO Immunoglobulin reference preparation 67/97. (IgM concentration 0.82 g/l.)


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Working standard. Serum poot from 1000 healthy blood donors. The pool was frozen (-20°C) and thawed (10°C) twice before the addition of 0.1 % (w/v) sodiumazide, filtered through a series of membrane filters ending with pore size of 0.2 pm, and finally stored in 1 ml aliquots at -80°C. All other reagents and chemicals applied were analytical grade. Polystyrene cuvettes were no. 750 (51/12) from Sarstedt, West Germany. Coating bufer. 100 mmol/l sodium carbonate buffer, pH 9.8. Washing solution. 150 mmol/l sodium chloride containing 0.05 % Tween-20.

Wash Enzyme activity rneosurement


= polystyrene surface

z- =


human I g M

rabbit anti human I g M peroxidase conjugated goal anti humon I g M

FIG. 1 . Schematic representation of the solid-phase sandwich enzyme immunoassay. 1 = Coating procedure. 2 = Antigen incubation. 3 = Labelled antibody incubation.

set out below, together with performance data obtained using the developed method.

MATERIAL A N D METHODS Material Coating antibody. Anti human IgM (p-chain specific) from rabbit, code no. 10-091, lot nos. 027, 037 and 107, DAKO-immunoglobulins, Denmark. Labelled antibody. Peroxidase-conjugated IgG fraction of goat anti human IgM (heavy chain specific), product no. 66-2136, lot nos, 9781 and 10679, Cappel Laboratories, USA.

Antigen-incubation buffer. 20 mmolil sodium phosphate buffer, pH 7.2 containing 150 mmol/l sodium chloride. 0.05 % Tween-20, 0.02% sodium azide and 1 % human serum albumin. Labelled antibody diluent. As antigen-incubation buffer, but without human serum albumin. Methods The washing procedure used to separate the individual steps of the assay was: 3 times 3.5 ml of washing solution as described by Engvall et al. (5). Measurement ofenzyme activity. Quantitation of the resultant cuvette bound peroxidase activity was performed by a kinetic method using the LKB 8600 Reaction Rate Analyzer (LKB Sweden), as described by Brock [4]. In order to get a larger response the concentration of 2-methoxy-phenol was doubled to 2.6 mmolll in the final substrate solution, Agarose gel precipitation methods. Single radial immunodiffusion and double diffusion were performed as described by Mancini et al. [8] and

Enzyme Immunoassay for IgM

Ouchterlony [lo], respectively. Rocket immunoelectrophoresis and crossed immunoelectrophoresis were performed according to the methods described by Weeke [151.




in 0

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Assay procedure Coating. 1 ml aliquots of rabbit anti human IgM, diluted 1:500 in coating buffer, were pipetted into polystyrene cuvettes, incubated at 37°C overnight and stored at 4°C until used, still containing the coating solution. (Stable for at least 1 month.) Washing. A sufficient number of coated cuvettes were washed three times with washing solution. Antigen incubation. 600 jil of blank (antigenincubation buffer), working standard (diluted 500 to 4OOOO times with antigen-incubation buffer) or samples were pipettedinto the washed, empty antibody-coated cuvettes. Samples were incubated in duplicate and standards in triplicate. Incubation took place overnight at room temperature (21°C) with constant gentle stirring on a rotating table inclined 60" from the horizontal plane. The washing procedure was repeated followed by : Labelled antibody incubation. To the washed cuvettes, to which IgM was now bound, through rabbit anti human IgM, 600 p1 of peroxidase labelled goat anti human IgM (diluted 1:75 in labelled antibody diluent) was added. Incubation, lasting 90 rnin and was under the same conditions as the antigen incubation. The washing procedure was repeated again followed by: Measurement of enzyme activity. To the washed cuvettes,coated with the entire sandwich, 1000jil of 100 mmol/l potassium-phosphate buffer (pH 7.0) was added. The cuvettes were vortex-mixed for 30 sec and left at room temperature for 30 min. 50 ji1 of 60 mmol/l2-methoxy-phenol was added followed by vortex-mixing for 30 sec. After preincubation for 10 min at 37"C, 100 p1 of 1.2 mmol/l H Z 0 2was added to start the reaction. The increase in absorbance (AA) at

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FIG.2. Influence of the coating time and buffer upon the dose-response curve using dilutions of the working standard as antigen. Other parameters of the assay were unchanged. Each point and bar indicates mean+ SEM, n = 4. fl-tl: 100 mmol/l sodium-carbonate buffer, pH 9.8 [ 5 ] . A - - - A : 50 mmol/l sodiumcarbonate buffer, pH 9.6 [I]. 0----0: 20 mmol/l sodium-phosphate buffer, pH 7.2 containing 1SO mmol/l sodium-chloride and 0.02% sodium-azide [2, 51.

405 nm was followed for 2 rnin on a LKB 8600 Reaction Rate Analyzer.

Conditions of the assay Coating procedure. The procedures for coating polystyrene surfaces with antibodies described in the literature [l, 2, 51 vary widely (neutral pH, high salt concentration versus basic pH, low salt concentration). Therefore experiments were performed to establish optimal conditions for the coating with the present rabbit anti human IgM, concerning the type of buffer, coating time and concentration of coating antibody. The influence of different coating times and various buffers in the antibody coating procedure upon the dose-response curve is shown in Fig. 2. It is seen, that an overnight coating procedure was superior to a 3 h procedure. 100 mmol/l sodium carbonate buffer (pH 9.8) gave rise to the steepest doseresponse curve in the concentration range 1061060 jig IgM/I, and was chosen for the final assay. The influence of the concentration of antibody in the coating procedure upon the cuvette-bound enzyme activity was examined by using coating antibody dilutions from 1:SO to 1:2OOO and


B. Dinesen & 0.Saxtrup Nielsen



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FIG. 3. Influence of the amount of labelled antibody upon the cuvette-bound enzyme activity. Other parameters of the assay were unchanged. Each point and bar indicates rnean+SEM, n = 4. ( : Working standard diluted I :1000. - - - : Working standard diluted 1 :3500. . . . : Working standard diluted 1 :10000. ' ' ' . . : Blank.




sample IgM concentrations of 0, 106, 303 and 1060 pg/l. Acceptable standard curves could be obtained using a wide range of antibody concentrations in the coating procedure (1 :loo1 :2000). The antibody dilutions 1 : 500, used in the final assay procedure, was selected as a balance between economy and maximal response. The incubation volume (600 pl) is the volume sufficient to wet the entire coated surface of the cuvettes during the incubation. The overnight incubation time (18 h) was chosen paying regard to an eight-hour working day. Antigen incubation.

Labelled antibody incubation. The influence of

incubation time with labelled antibody upon the level of enzyme activity bound to the cuvettes was examined in the range 0.5-23 h, using sample IgM concentrations of 106, 303 and 1060 pg/l. There is a plateau of maximal response throughout the examined IgM concentration range (106--1060pg/l) between 90 min and 3 h of incubation. Beyond 3 h of incubation the response in the higher IgM concentration range (303-1060 pg/l) declined, probably because of desorption of the cuvette-bound anti IgM antibodies. An incubation time of 90 min was chosen for the assay procedure as the

shortest incubation time, leading to maximal response. The influence of the concentration of labelled antibody upon the level of enzyme activity bound to the cuvettes is shown in Fig. 3. With increasing amount of labelled antibody the cuvette-bound enzyme activity increased. Unfortunately, this increase calls for a more intensive washing procedure -at a labelled antibody dilution of 1 :50 the selected washing procedure became insufficient. For these reasons the labelled antibody dilution of 1:75 was adopted as the best balance between optimal response and acceptable washing efforts. Measurement of enzyme activity. The vortexmixing and 30 min delay time before enzyme

activity measurement reduced the analytical variation. The enzyme activity was stable for 3 h, which corresponds to the processing time of 100 cuvettes. Sensitivity and precision of the assay The sensitivity, expressed as the lower limit of detection, was 12 ng IgM per tube, corresponding to an IgM concentration of 20 pg/l.

The unulyticul variation between days was determined by calculating the standard deviation (SD) on paired results performed between days on thirty-five sera. The SD was 0.07 g/l. With a mean IgM concentration of 0.85 g/I the corresponding coefficient of variation was 8.2%. Calculated from the same data, a withinassay SD of 0.06 g/l was found, corresponding to a within-assay coefficient of variation of 7.1 %. The concentrations of IgM in the diluted sera were evenly distributed within the range 110-325 pg/l. Further, a pooled cerebrospinal fluid was analysed in eleven consecutive assays over a 2 month period. The pool was analysed undiluted, the concentration of IgM was 470k 50 pg/l (mean f SD), corresponding to a between-assay coefficient of variation of 10.6%. Standardization and specificity of the assay

WHO immunoglobulin reference preparation 67/97 was used as standard. As a substandard a pool of serum from lo00 blood-donors was used. These two preparations behaved identically in the assay as shown in Fig. 4.

Enzyme Immunoassay for IgM



double diffusion against five sera and the substandard without the appearance of non-specific reactions. Furthermore, the specificity was tested by the ability of the antibody to bind to monoclonal bone marrow cells from patients with IgA- or IgG-myeloma or IgM-macroglobulinemia, according to methods described by Hijmans et al. [7].This test was performed by Dr A.Wiik using a peroxidase staining technique with 3-amino-9-ethylcarbazole as the chromogen. Binding could only be demonstrated on IgM-producing cells.


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FIG.4. Comparison of the dose-response curves obtained using substandard (C,) and WHO immunoglobulin reference preparation 67/97 (IgM concentration 0.82 g/l) ( 0 ) . Each point and bar represents the mean value and SD of six separate triplicate estimations. The insert shows the preparations diluted 1 :20000 to 1 :80000with expanded ordinate scale.

The specificity of the first antibody (rabbit anti human IgM) was evaluated by using it in single radial immunodiffusion, double diffusion and crossed immunoelectrophoresis. As antigens served five serum samples and the substandard, non-specific reactions with the antibody could not be detected. The specificity of the second antibody (peroxidase labelled goat anti human IgM) was tested by






Rochet-ImmunoelectrophoresIs IgM ( g / I )

FIG. 5. Serum IgM concentration as measured by enzyme immunoassay and by rocket immunoelectrophoresis. (0): sixty-six serum samples from our normal ten serum samples from patients with routine. (0): myeloma of heavy chain class a or y.

Comparison with rocket-immunoelectrophoresis Sixty-six sera from the normal routine of the laboratory and ten sera from patients with myeloma of heavy chain class ry and y were compared by the two methods. The results are shown in Fig. 5. The coefficient of correlation was r = 0.96. The best line to fit the data, calculated by the least squares method, is described by the equation: ‘EIA’ = 0.15+ 1.02 x ‘Rocket’, (n = 76). The EIA-axis intercept is not significantly different from zero ( t = 0.15, d.f. = 74). The statistics do not indicate any significant difference between results obtained by the two methods.


The present enzyme immunoassay for the quantitation of human IgM is specific in the sense, that other serum proteins including IgA and IgG do not interfere. Its results correlate highly with results obtained with the established rocket immunoelectrophoresis, and are therefore equally useful. The sensitivity obtained by the present method (12 ngltube or 20 pg/l) is compaiable to what has been obtained using RIA (10 ngitube or 100 pg/l) 191. In its present form (with manual washings), the assay cannot replace rocket immunoelectrophoresis or single radial immunodiffusion when serum samples are to be assayed, because it is too time-consuming. But its sensitivity and specificity makes it a good tool for the quantitation of IgM in samples with very low IgM-concentration as cerebrospinal fluid and cell culture supernatants. Work is in progress examining cerebrospinal fluid concentrations of IgM in patients with acute infections within the central nervous system.


B. Dinesen & 0.Saxtrup Nielsen

ACKNOWLEDGMENT We express our gratitude to D r Allan Wiik, Immunofluorescence Laboratory, Rigshospitalet, Copenhagen, for his performance of the test for specificity concerning our second antibody-peroxidase labelled goat anti human IgM.

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REFERENCES 1 Bartlett, A., Dormandy, K.M., Hawkey. C.M..







Stableforth, P. & Voller, A. Factor-V111-related antigen : measurement by enzyme immunoassay. Brir. med. J. i, 994, 1976. Belanger, L., Sylvestre, C. & Dufour, D. Enzymelinked immunoassay for alpha-fetoprotein by competitive and sandwich procedures. Clinica chim. acta 48, 15, 1973. Bennich, H . Imrnunoglobuliner. pp. 148-149 i n Blomback, B. & Hanson, L.A. (eds) Plasmaproteiner. Kabi, Stockholm, 1976. Brock, A . Peroxidase activity in human granulocytes, inter- and intra-individual variations. Scand. J . d i n . Lab. Inaesr. 37, 163, 1977. Engvall, E., Jonsson, K . & Perlmann, P. Enzymelinked immunosorbent assay. I I . Quantitative assay of protein antigen, Immunoglobulin C, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim. biophys. Acta ( A m s t . ) 251, 427, 1971. Guesdon, S.-L. & Avrameas, S. Magnetic solid phase enzyme-immunoassay. Immunochemistry 14, 443, 1977. Hijmans, W., Schuit, H.R.E. & Klein, F. An immunofluorescence procedure for the detection of

intracellular immunoglobulins. Clin. expl. Imnurri. 4,457, 1969. 8 Mancini, C., Carbonara, A.O. & Heremans, J.F. lrnmunochemical quantitation of antigens by single radial irnmunodiffusion. Immunochemistry, 2 , 235, 1965.

9 Nerenberg, S.T. & Prasad, R. Radioimmunoassays for Ig classes G , A, M, D, and E in spinal fluids: normal values of different age groups. J. Lab. d i n . M e d . 86, 887, 1975. 10 Ouchterlony, 6. Immunodiffusion and immunoelectrophoresis. pp. 678-688 in Weir, D.M. (ed.) Handbook of Experimental Immunology. Blackwell Scientific Publications, 1967. 1 I Schmidt, W.A.K., Klein, M. & Brade, H. Ein empfindlicher Nachweis von Immunoglobulin G und M mit Hilfe sensibilisierter Latexpartikeln. Immun. Infekt. 5, 30, 1977. 12 Sieber, A. Plasmaprotein-Bestimmung durch LaserNephelometrie: Laborpraxis. Loboratoriumshlutter 21, 109, 1977. I 3 Sternberg, S.C. A Rate Nephelometer for Measuring specific proteins by lmmunoprecipitin Reactions. Clin. Chem. 23, 1456, 1977. 14 Wang, A.-C. The structure of Immunoglobulins. Caldwel1,S.L. p. 18 in Fudenberg, H.H..Stites, D.P., & Wells, S.V. (eds) Basic and clinical immunology. Lange Medical Publications, California, 1976. I5 Weeke, 8. Rocket irnrnunoelectrophoresis and Crossed immunoelectrophoresis. In Axelsen, N.H., K r d l , S. & Weeke, B.(eds) A Manualof Quantitaticr Immunoelertrophoresis. Universitetsforlaget, Oslo, 1973. 16 Weisner, 9.. Schnedler, R. & Bernhardt, W. Immunoglobulin A, G und M in lumbal entnommenem Liquor cerebrospinalis. Neruenarrt 46, 532, 1975.

Received 7 October 1978 Accepted 17 December 1978

A sensitive enzyme immunoassay for the quantitation of human IgM.

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