~~
Original Article
Ann CIin Biochem 1991; 28: 461-466
Determination of kappa and lambda light chains in serum immunoglobulins G , A and M A Haraldsson, M J H Kock-Jansenl, M Jaminon, P B J M v Eck-Arts1 T de Boo2, C M R Weemaes and J A J M Bakkeren From the Department of Paediatrics, the Central Clinical Chemistry Laboratory' and the Department of Medical Statistid, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands
An enzyme-linked immunosorbent assay is described for measuring x and X light chains within each of the serum immunoglobulin classes G, A and M. The detection limit was 0.06 U/L for total IgG, IgG x and IgG X, 0 . 2 U/L for total IgA, IgA x and IgA X and O.SU/L for total IgM, IgM x and IgM X. The concentrations of x plus X light chains from the three different immunoglobulins correlated well within those of total immunoglobulin G, A and M as measured by enzyme-linked immunosorbent assay or by immunonephelometry. Adult values for the x/X light chain ratio were found to be 2 - 0 for IgG x/X, 1 1 for IgA x/X and 1 - 7 for IgM x/X. SUMMARY.
-
Additional key phrases: immunoglobulin light chains, kappa/lamba light chain ratio
Immunoglobulin x and A light chains have been studied on various occasions. In an earlier study,' a method for measuring the total kappa/lambda light chain (x/X) ratio in serum of healthy and diseased children was described. Definite variation was noted between different age-groups. Other investigators have studied the total x/X ratio in children and a d ~ l t s . ~ The - ~ x/A ratios within the various immunoglobulin classes have also been de~cribed.~J However, such studies have usually determined ratios within monoclonal immunoglobulins, established from myeloma patients or diseased individuals. These results cannot be used to provide reference values for normal immunoglobulins. Abnormal total x/X ratios have been found in various diseases. Patients with hypogammaglobulinaemia have been found with abnormal total x/A ratios2 and children with IgA deficiency had a significantly wider spread of x concentrations.' Patients suffering from I g A nephropathy also had abnormal IgA x/X ratios.6 In some autoimmune disorders such as systemic lupus erythematosus, Sjflgren syndrome and juvenile rheumatoid arthritis an abnormal total x/X ratio has been d e ~ c r i b e d . ~ - ~ Correspondence: Dr
A
Haraldsson.
The total x/X ratio represents essentially the light chain ratio of the most abundant serum immunoglobulin, usually IgG. The total IgG x/X ratio therefore overshadows possible abnormalities in the ratio of the separate immunoglobulins. We have now developed a method which allows the separate estimation of the x and X light chains in human polyclonal immunoglobulins G, A and M and we have measured the x/X ratio of IgG. I g A and IgM separately in healthy adults. This method provides a means by which further investigation of the x/X ratio in health and disease is possible.
MATERIALS AND METHODS Reagents Affinity-purified goat antisera directed against human y, Q or p heavy chains (Cappel, WestChester, PA, USA) were used as capture antibodies. To obtain a coating solution these antisera were diluted 1:lOOO in 0-05 mol/L NaHCO, and the pH adjusted to 9 - 5 with 1 mol/L NaOH. As second antibody goat antisera (IgG fraction) conjugated with horseradish peroxidase (HRP) and directed against either x or X human light chains (anti-x-Po and anti-X-Po, respectively; Cappel) were applied. These antisera were diluted with incubation buffer for the 46 1
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462
Haraldsson et al.
measurement of x and X light chains. The dilutions varied with the antiserum used. For measurements of total immunoglobulins G, A and M by the ELISA the second antibody was a peroxidase-labelled anti-y, a or p (anti-?-Po, anti-ol-Po, anti-p-Po, respectively).
The ranges of the standard curves used were; for total IgG, IgG x and IgG X 0-1 U/L, for total IgA, IgA x and IgA X 0-9 U/L and for total IgM, IgM x and IgM X 0-15 U/L. For a control serum another batch of pooled serum from 500 healthy Dutch blood donors was measured at three different dilutions which were expected to lie at different points on the standard curve. The same standard and control sera were used for all measurements, i.e. x and X in the different immunoglobulins and total IgG, IgA and IgM in the ELISA method, and when measuring total IgG, IgA and IgM and total x/X ratio using immunonephelometry .
Buffers The incubation buffer contained 50 mmol/L KH2P04 (Merck, Miinchen, Germany), 154 mmol/L NaCl 0-1 mL/L Tween 20 (Sigma. St Louis, MO, USA) and 10 drops/L merthiolate with a minute amount of phenol red, adjusted to pH 7 - 4 by addition of 1 mol/L NaOH. The incubation medium was made by adding 2 g of Serum samples bovine serum albumin (Sigma) to each litre of the A total of 114 serum samples were examined. In incubation buffer shortly before use. order to obtain low as well as high concentrations The substrate solution was freshly prepared by adding 2 g/L o-phenylene-diamine-dihydro- of the immunoglobulins, serum samples were collected from 86 healthy children aged 1 month chloride (Sigma) and 1 pL of 300 g/L hydrogen to 15 years and 20 healthy adult Dutch blood peroxide (Merck) per mL of a substrate buffer, containing 34 7 mrnol/L citrate and 66 7 mmol/L donors. In addition, eight individuals known to have low values of immunoglobulin G were Na,HPO,, adjusted to pH 5.0 with 12 mol/L NaOH. As stop solution, 2 mol/L sulphuric acid enrolled to provide more samples with x/X ratios in the low immunoglobulin G range. The x/X was used. ratio of the different immunoglobulin classes was measured by our assay and total immunoglobulin Standard and control pools concentrations were measured by the ELISA A pooled serum from 500 healthy Dutch blood method and by immunonephelometry for donors was used for the preparation of a comparison of the different methods. standard. It was stored for 2 weeks at 4"C, centrifuged at 60 OOO x g for 1 h and the precipitate For the estimation of adult values, serum and lipids were removed. The serum was filtered, samples were measured from 20 healthy indivifirst through a 0.45 p n and subsequently through duals. In addition, pooled serum from 500 healthy Dutch blood donors was measured to obtain a 0 - 2 2 pm filter (Millipore Corp., Bedford, MA, mean values. All serum samples were diluted in USA), quickly frozen in dry CO, and acetone the incubation medium and measured in three and stored at -70 "C. The immunoglobulin concentrations of the standard serum were different concentrations as described for the standard and control sera. In order to get an compared to a WHO standard 67-86'O and a standard serum from the Central Laboratory of insight into the values to be expected for the light The Netherlands Red Cross Blood Transfusion chains, before diluting the serum samples the total Service (CLB H00-02). For the standardization values of IgG, IgA and IgM as well as total x and of the x/X ratios we absorbed x or X light chains X values were measured by nephelometry. from the pooled serum with rabbit anti-x or anti-X Equipment antiserum, respectively, and subsequently measured with separate immunoglobulins by immunoPolystyrene 96-well, flat-bottomed microtitre nephelometry using pig antisera, according to an plates (Nunc- Immuno Plate Maxi Sorpz; Roskilde, earlier described method.' We were thus able to Denmark) were used for the assay. Washing was establish a standard for the immunoglobulin G , A performed with incubation buffer using an autoand M x/X ratios in the pooled serum. This was matic plate washer. The absorbance reading was further confirmed by correlating x plus X in the performed by using an automated reader (Titertek different immunoglobulins with the total immunoMultiskan MC, Flow Labs, Maclean, VA, USA) globulins as measured by ELISA or immunopheland calculated on a P C using Titersoft ETA ometry. A standard curve was prepared by Software (version 2.OA, Flow Labs). Total diluting the standard serum with incubation immunoglobulin concentrations were measured medium to give 10 different concentrations, by laser nephelometry, using a DISC 120
-
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Kappa and lambda light chains Nephelometer (Hyland, Nivelles, Belgium) and rabbit antisera against human y, a and p chains and x and X light chains (Dako, Glostrup, Denmark).
Procedure The microtitre plates were coated with 200pL coating solution, containing either anti-y, anti-a or anti-p antibody as the first antibody, and incubated for at least 18 h at 4°C. The plates were thereafter washed four times with incubation buffer. Subsequently, l00pL of the serum samples in three different dilutions, standard serum in 10 different dilutions and control serum in three different dilutions were added, in duplicate, to the wells. To avoid edge effects, incubation buffer alone was added to the edge wells. The plate was incubated again for at least 18 h at 4°C and washed four times. The second antiserum (anti-x-Po, anti-A-Po, anti-y-Po, anti-a-Po or anti-p-Po) was then added, 100pL to each well in the different dilutions according to the antisera used and incubated for 90min at 37°C. The plates were then washed four times with incubation buffer. Subsequently, 100 pL of fresh substrate solution was added and incubation was carried out at room temperature for 5 to 7 min,dependent on the colour development. Finally, the reaction was stopped with 15OpL of 2mol/L sulphuric acid and the absorbance was measured at 492 nm. Statistical procedures To compare our results to earlier methods the x plus X values of immunoglobulin G , A and M were compared to the total IgG, IgA or IgM. respectively, as measured by ELISA and by immunoephelometry. For the comparison of the two different measurement techniques we followed the procedure of Bland and Altman'l in which the 'limits of agreement' are the mean difference ? 2SD of the differences. To visualize the degree of agreement, plots are made of the differences versus the average value of the two methods after log (natural logarithm, In) transformation. RESULTS
The standard curves were made over the range 0-1 U/L for total IgG, IgG x and IgG X, 0-9 U/L for total IgA, IgA x and IgA X and 0- 15 U/L for total IgM, IgM x and IgM X, the actual range for each measurement depending on the most accurate part of the curve. The detection limit for total IgG, IgG x and IgG X, was 0.06 U/L, for total
463
TABLE1. Between-run precision of the ELISA Mean
(KUIL) 78 39 118 64 57 122 72 44
118
SD (KU/L)
cv (Yo)
n
7-0 3.7 10.9 3.2 3.1 5.6 2.6 2.1 4.5
8.9 9.6 9.3 4.9 5.5 4.6 3.6 4.7 3.8
32 33 33 29 30 30 28 29 29
IgA, IgA x and IgA X 0.2 U/L and for total IgM, IgM x and IgM X O.SU/L. The within-run precision of the method was calculated using the differences of the duplicates for all measurements. The coefficient of variation ranged from 2.0 to 4.5% (n = 50). The between-run precision is shown in Table 1. When measuring serum samples from patients with IgG deficiency, no IgG x or X light chains were detected. The same negative result was found when measuring IgA x and IgA X from IgA deficient patients. Measurement of the pooled serum revealed X A ratios of 2 - 0 for IgG, 1.1 for IgA and 1.7 for IgM. The measurements of 20 healthy individuals revealed comparable results with an acceptable standard deviation. The results of the measurements in serum of healthy adults and in pooled serum of 500 healthy Dutch blood donors are shown in Table 2. One hundred and fourteen serum samples were used when comparing our method to earlier techniques. A high degree of agreement was found between the x plus X for IgG, IgA or IgM and the total IgG, IgA and IgM, respectively, as measured by the ELISA. The mean differences of the two methods after log (natural logarithm, In) transformation within the limits of agreement expressed as k 2SD were 0.00& 0 - 14 for IgG, 0.01&0-17 for IgA and 0-OlkO.10 for IgM TABLE 2. Zmmunoglobulin x A ratio in ZgG, IgA and ZgM in pooled serum of SW healthy Dutch blood donors and mean x A ratio in 20 healthy adults. 10 men and 10 women (statistics are calculated from 10 healthy individuals)
Pooled serum IgG IgA
IgM
Total
2.0 1.1 1.7 2.0
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20 individuals (SD) Men (n = 10)
Women (n = 10)
2.05 1.01 1.65 2.01
2.07 1.12 1.76 2.02
(0.36) (0.20) (0.26) (0.32)
(0.34) (0.24) (0.39) (0.37)
Haraldsson et al.
464
I
-0 3 4 4
and - 1.4 and for IgM 0.99, 1.01 and 6.3, respectively. The agreement between the x plus X for IgG, IgA and IgM as measured by ELISA and immunonephelometry was lower revealing a mean difference after log (In) transformation of - 0 - 0 3 + 0 - 2 3 for IgG, 0*14?0-28 for IgA and 0-08+_0-28 for Igh4 (Fig. 2). This implies that the quotient of IgG ELISA divided by IgG x plus IgG X lies between 0.77 and 1-22. For IgA the
6
5
Averoge In of the twomethods
.
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.
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-03
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4 5 Averoge Inof the t w o methods
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two methods
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00
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Averoge In of thetwo methods
3 Averoge In of the t w o methods
FIGURE 1 . Agreement between measurements of total immunoglobulin as measured by ELISA and x plus X of that particular immunoglobulin as measured by our method, expressed after log (natural logarithm) transformation as mean difference -t 2SD. (a) Total IgC versus IgC x plus IgG A. (b) Total IgA versus IgA x plus 1gA X. (c) Total IgM versus IgM x plus IgM X.
(Fig. 1). This implies that the quotient of IgG ELISA divided by IgG x plus IgG X lies between 0.87 and 1.15. For IgA the quotient lies between 0-85 and 1.20 and for IgM between 0.91 and 1.12. The difference between these two methods was not statistically significant (P>0-05). When comparing the x plus X for IgG, I g A or IgM to the total IgG, IgA and IgM, respectively, all measured by ELISA, the correlation coefficient, slope and intercept for IgG was 0-98,0-98and 2.3, for IgA 0-99, 1-07
..
0 4 ............................................................................
-0-4
Averoge In of the t w o methods
FIGURE 2. Agreement between measurements of total immunoglobulin as measured by imrnunonephelometry and x plus X of that particular immunoglobulin as measured by our method, expressed after log (natural logarithm) transformation as mean difference 250. (a) Total IgC versus IgC x plus IgG X. (b) Total IgA versus IgA x plus IgA A. (c) Total ISM versus IgM x plus IgM X.
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Kappa and lambda light chains quotient lies between 0.87 and 1.52 and for IgM between 0.82 and 1 .43. The correlation coefficient, slope and intercept for the comparison of the measured x plus X values of the separate immunoglobulins and the immunonephelometry was 0-95 and 3.0 for IgG, 0.96,1-06 and 6.1 for IgA and 0.97, 1-12and - 1.9 for IgM, respectively. The serum samples showing the lowest agreement between the different methods could not be correlated with any age or patients' groups. O m % ,
DISCUSSION
465
Recently, light chain ratios of IgG, IgA and IgM measured by an ELISA method using rabbit antisera have been de~cribed.'~The results obtained in that study reveal definitely higher x/X ratios of IgG, IgA and IgM than ours. We question the way of calculating absorbance ratios by simply adding the absorbances of the different immunoglobulin x and X values. We clearly prefer using different standards for each determination. However, both studies show the X Aratio for I g A to be the lowest and the ratio for IgG the highest. In certain diseases and in autoimmune disorders x or X preference has been implicated. Some infections have been found to carry with them a high total x/X ratioI2 and a high total x/X ratio has also been found in systemic lupus erythematosus, juvenile rheumatoid arthritis and some other autoimmune disorders. 1~3-7*8,12 In addition, an imbalanced total x/X ratio has been described in hypogammaglobulinaemia,2 agammaglobulinaemia and in ataxia telangiectasia.12An abnormal x concentration has also been detected in IgA deficiency.l Imbalanced x or X deposits have also been demonstrated in various tissues in certain diseases such as IgA nephropathy6 and Sjdgren d i ~ e a s e . ~ The bulk of the total immunoglobulin concentration in serum of healthy individuals derives from IgG. Therefore, IgG x and X accounts for the majority of the light chains in serum. The ratio of x and X in other immunoglobulins is therefore partly overshadowed by the IgG light chains. Measuring the x/X ratios separately in IgG, IgA and IgM may provide a means for understanding better the function of the immunoglobulin light chains in health and disease.
Concentrations of x plus X of the separate immunoglobulin classes IgG, I g A or IgM, measured by our method, compared well with the total immunoglobulins measured by ELISA. The difference between the two methods was not statistically significant and a good agreement was consistent. Agreement was poorer when comparison was made with immunonephelometry. This was to be expected. The sensitivity of the nephelometric method causes greater deviation for the measurement of IgA and IgM than for IgG. Measuring IgM by nephelometry requires a particularly high precision and sensitivity. Furthermore, since many of the serum samples obtained from children contained low values of IgA, high precision was needed for IgA as well. This possibly explains a certain bias in these measurements. Additionally, as goat antisera were used in the ELISA and rabbit antisera in the immunonephelometry a slight difference in results due to these antisera may be expected. Our values show a distinct difference between the x/X ratios of the different immunoglobulins. Interestingly, IgA shows a lower x/X ratio (1.1) Acknowledgements than IgG (2.0) and IgM (1.7). The reason for this The work was supported in part by a research difference is not known. The total x/X ratio has earlier been reported to be 1.7 to 2.0.1-4*9*'2*13grant from the Icelandic Council of Science. As IgG comprises the majority of total immunoglobulins it was expected that the IgG x/X ratio REFERENCES would resemble that of the total immunoglobulins. 1 Renckens ALJM, Jansen MJH, Van Munster PJJ, Studies of the x/X ratio in different immunoWeemaes CMR, Bakkeren JAJM. Nephelometry of the globulin classes have been carried out5 in patients kappa/lambda light chain ratio in serum of normal with monoclonal production of immunoglobulins. and diseased children. CIin Chem 1986; 32: 2147 These results were clearly different from ours 2 Skvaril F, Barandun S, Morel1 A, Probst M. showing a IgM x/x ratio of 3.2 and a IgA x/x ratio Imbalances of k/l immunoglobulin light chain ratios of 1.4-1.6. This variation underlines the differin normal individuals and in immunodeficient patients. In: Peters H, ed. Twenty-second Colloquium ence between light chain ratios of monoclonal and Bruges: Protides of Biological Fluids. New York: polyclonal immunoglobulins. The results gained Pergamon Press, 1975; 415 from monoclonal immunoglobulins can hardly be 3 Normansell DE. Use of kappa and lambda chain compared to those obtained in healthy individuals. quantitation for the detection of immunoglobulin Hitherto, healthy individuals have scarcely been abnormalities in serum. Diagn CIin Imrnunol 1987; 5: loo investigated.
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4 Ford RP, Mitchell PEG, Fraser CG. Desirable performance characteristics and clinical utility of immunoglobulin and light-chain assays derived from data on biological variation. CIin Chem 1988; 34: 1733 5 Eisen HN. Antibody structure: The immunoglobulins. In: Davis BD, Dulbecco RO, Eisen HN, Ginsberg HS, Wood WB Jr, eds. Microbiology 2nd ed. Maryland: Harper International Edition, 1988; 446 6 Lai KN, Chan KW, Mac-Moune Lai F, et al. The immunochemical characterization of the light chains in the mesangial IgA deposits in IgA nephropathy. Am J Clin Pathol 1986; 85: 548 7 Knight J, Laing P, Knight A, Adams D, Ling N. Thyroid-stimulation autoantibodies usually contain only L-light chains: evidence for the ‘forbidden clone’ theory. J CIin Endocrinol Metab 1986; 62: 342 8 Knight JG, Laing P , Adams DD, Bray JJ, Ling NR. Autoantibodies to acetylcholine receptor in myasthenia gravis: light chains. Neurology 1986; 36: 1531 9 Jasani B. Immunohistologically definable light chain
restriction in autoimmune disease. J Pathol 1988; 154: 1 10 Reimer CB, Smith SJ, Hannon WH, et al. Progress towards international reference standards for human serum proteins. J Biol Stand 1978; 6 : 133 11 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurements. Lancet 1986; i: 307 12 McKelvey EM, Fahey JL. Immunoglobulin changes in disease: quantitation on the basis of heavy polypeptide chains, IgG (gG), IgA (gA)and IgM (gM), and of light polypeptide chains, type K(I) and type L(I1). J CIin Invest 1965; 44: 1778 13 Barandun S, Skvaril F, Morel1 A. Imbalances of the k/l ratio of human immunoglobulins. CIin Immunol Biol 1976; 3: 57 14 Chui SH. Lam CWK, Lai KN. Light-chain ratios of immunoglobulins G, A and M determined by Enzyme immunoassay. CIin Chem 1990; 36: 501 Accepted for publication 2 April 1991
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Original Article
Ann CIin Biochem 1991; 28: 461-466
Determination of kappa and lambda light chains in serum immunoglobulins G , A and M A Haraldsson, M J H Kock-Jansenl, M Jaminon, P B J M v Eck-Arts1 T de Boo2, C M R Weemaes and J A J M Bakkeren From the Department of Paediatrics, the Central Clinical Chemistry Laboratory' and the Department of Medical Statistid, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands
An enzyme-linked immunosorbent assay is described for measuring x and X light chains within each of the serum immunoglobulin classes G, A and M. The detection limit was 0.06 U/L for total IgG, IgG x and IgG X, 0 . 2 U/L for total IgA, IgA x and IgA X and O.SU/L for total IgM, IgM x and IgM X. The concentrations of x plus X light chains from the three different immunoglobulins correlated well within those of total immunoglobulin G, A and M as measured by enzyme-linked immunosorbent assay or by immunonephelometry. Adult values for the x/X light chain ratio were found to be 2 - 0 for IgG x/X, 1 1 for IgA x/X and 1 - 7 for IgM x/X. SUMMARY.
-
Additional key phrases: immunoglobulin light chains, kappa/lamba light chain ratio
Immunoglobulin x and A light chains have been studied on various occasions. In an earlier study,' a method for measuring the total kappa/lambda light chain (x/X) ratio in serum of healthy and diseased children was described. Definite variation was noted between different age-groups. Other investigators have studied the total x/X ratio in children and a d ~ l t s . ~ The - ~ x/A ratios within the various immunoglobulin classes have also been de~cribed.~J However, such studies have usually determined ratios within monoclonal immunoglobulins, established from myeloma patients or diseased individuals. These results cannot be used to provide reference values for normal immunoglobulins. Abnormal total x/X ratios have been found in various diseases. Patients with hypogammaglobulinaemia have been found with abnormal total x/A ratios2 and children with IgA deficiency had a significantly wider spread of x concentrations.' Patients suffering from I g A nephropathy also had abnormal IgA x/X ratios.6 In some autoimmune disorders such as systemic lupus erythematosus, Sjflgren syndrome and juvenile rheumatoid arthritis an abnormal total x/X ratio has been d e ~ c r i b e d . ~ - ~ Correspondence: Dr
A
Haraldsson.
The total x/X ratio represents essentially the light chain ratio of the most abundant serum immunoglobulin, usually IgG. The total IgG x/X ratio therefore overshadows possible abnormalities in the ratio of the separate immunoglobulins. We have now developed a method which allows the separate estimation of the x and X light chains in human polyclonal immunoglobulins G, A and M and we have measured the x/X ratio of IgG. I g A and IgM separately in healthy adults. This method provides a means by which further investigation of the x/X ratio in health and disease is possible.
MATERIALS AND METHODS Reagents Affinity-purified goat antisera directed against human y, Q or p heavy chains (Cappel, WestChester, PA, USA) were used as capture antibodies. To obtain a coating solution these antisera were diluted 1:lOOO in 0-05 mol/L NaHCO, and the pH adjusted to 9 - 5 with 1 mol/L NaOH. As second antibody goat antisera (IgG fraction) conjugated with horseradish peroxidase (HRP) and directed against either x or X human light chains (anti-x-Po and anti-X-Po, respectively; Cappel) were applied. These antisera were diluted with incubation buffer for the 46 1
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462
Haraldsson et al.
measurement of x and X light chains. The dilutions varied with the antiserum used. For measurements of total immunoglobulins G, A and M by the ELISA the second antibody was a peroxidase-labelled anti-y, a or p (anti-?-Po, anti-ol-Po, anti-p-Po, respectively).
The ranges of the standard curves used were; for total IgG, IgG x and IgG X 0-1 U/L, for total IgA, IgA x and IgA X 0-9 U/L and for total IgM, IgM x and IgM X 0-15 U/L. For a control serum another batch of pooled serum from 500 healthy Dutch blood donors was measured at three different dilutions which were expected to lie at different points on the standard curve. The same standard and control sera were used for all measurements, i.e. x and X in the different immunoglobulins and total IgG, IgA and IgM in the ELISA method, and when measuring total IgG, IgA and IgM and total x/X ratio using immunonephelometry .
Buffers The incubation buffer contained 50 mmol/L KH2P04 (Merck, Miinchen, Germany), 154 mmol/L NaCl 0-1 mL/L Tween 20 (Sigma. St Louis, MO, USA) and 10 drops/L merthiolate with a minute amount of phenol red, adjusted to pH 7 - 4 by addition of 1 mol/L NaOH. The incubation medium was made by adding 2 g of Serum samples bovine serum albumin (Sigma) to each litre of the A total of 114 serum samples were examined. In incubation buffer shortly before use. order to obtain low as well as high concentrations The substrate solution was freshly prepared by adding 2 g/L o-phenylene-diamine-dihydro- of the immunoglobulins, serum samples were collected from 86 healthy children aged 1 month chloride (Sigma) and 1 pL of 300 g/L hydrogen to 15 years and 20 healthy adult Dutch blood peroxide (Merck) per mL of a substrate buffer, containing 34 7 mrnol/L citrate and 66 7 mmol/L donors. In addition, eight individuals known to have low values of immunoglobulin G were Na,HPO,, adjusted to pH 5.0 with 12 mol/L NaOH. As stop solution, 2 mol/L sulphuric acid enrolled to provide more samples with x/X ratios in the low immunoglobulin G range. The x/X was used. ratio of the different immunoglobulin classes was measured by our assay and total immunoglobulin Standard and control pools concentrations were measured by the ELISA A pooled serum from 500 healthy Dutch blood method and by immunonephelometry for donors was used for the preparation of a comparison of the different methods. standard. It was stored for 2 weeks at 4"C, centrifuged at 60 OOO x g for 1 h and the precipitate For the estimation of adult values, serum and lipids were removed. The serum was filtered, samples were measured from 20 healthy indivifirst through a 0.45 p n and subsequently through duals. In addition, pooled serum from 500 healthy Dutch blood donors was measured to obtain a 0 - 2 2 pm filter (Millipore Corp., Bedford, MA, mean values. All serum samples were diluted in USA), quickly frozen in dry CO, and acetone the incubation medium and measured in three and stored at -70 "C. The immunoglobulin concentrations of the standard serum were different concentrations as described for the standard and control sera. In order to get an compared to a WHO standard 67-86'O and a standard serum from the Central Laboratory of insight into the values to be expected for the light The Netherlands Red Cross Blood Transfusion chains, before diluting the serum samples the total Service (CLB H00-02). For the standardization values of IgG, IgA and IgM as well as total x and of the x/X ratios we absorbed x or X light chains X values were measured by nephelometry. from the pooled serum with rabbit anti-x or anti-X Equipment antiserum, respectively, and subsequently measured with separate immunoglobulins by immunoPolystyrene 96-well, flat-bottomed microtitre nephelometry using pig antisera, according to an plates (Nunc- Immuno Plate Maxi Sorpz; Roskilde, earlier described method.' We were thus able to Denmark) were used for the assay. Washing was establish a standard for the immunoglobulin G , A performed with incubation buffer using an autoand M x/X ratios in the pooled serum. This was matic plate washer. The absorbance reading was further confirmed by correlating x plus X in the performed by using an automated reader (Titertek different immunoglobulins with the total immunoMultiskan MC, Flow Labs, Maclean, VA, USA) globulins as measured by ELISA or immunopheland calculated on a P C using Titersoft ETA ometry. A standard curve was prepared by Software (version 2.OA, Flow Labs). Total diluting the standard serum with incubation immunoglobulin concentrations were measured medium to give 10 different concentrations, by laser nephelometry, using a DISC 120
-
Downloaded from acb.sagepub.com by guest on June 4, 2016
Kappa and lambda light chains Nephelometer (Hyland, Nivelles, Belgium) and rabbit antisera against human y, a and p chains and x and X light chains (Dako, Glostrup, Denmark).
Procedure The microtitre plates were coated with 200pL coating solution, containing either anti-y, anti-a or anti-p antibody as the first antibody, and incubated for at least 18 h at 4°C. The plates were thereafter washed four times with incubation buffer. Subsequently, l00pL of the serum samples in three different dilutions, standard serum in 10 different dilutions and control serum in three different dilutions were added, in duplicate, to the wells. To avoid edge effects, incubation buffer alone was added to the edge wells. The plate was incubated again for at least 18 h at 4°C and washed four times. The second antiserum (anti-x-Po, anti-A-Po, anti-y-Po, anti-a-Po or anti-p-Po) was then added, 100pL to each well in the different dilutions according to the antisera used and incubated for 90min at 37°C. The plates were then washed four times with incubation buffer. Subsequently, 100 pL of fresh substrate solution was added and incubation was carried out at room temperature for 5 to 7 min,dependent on the colour development. Finally, the reaction was stopped with 15OpL of 2mol/L sulphuric acid and the absorbance was measured at 492 nm. Statistical procedures To compare our results to earlier methods the x plus X values of immunoglobulin G , A and M were compared to the total IgG, IgA or IgM. respectively, as measured by ELISA and by immunoephelometry. For the comparison of the two different measurement techniques we followed the procedure of Bland and Altman'l in which the 'limits of agreement' are the mean difference ? 2SD of the differences. To visualize the degree of agreement, plots are made of the differences versus the average value of the two methods after log (natural logarithm, In) transformation. RESULTS
The standard curves were made over the range 0-1 U/L for total IgG, IgG x and IgG X, 0-9 U/L for total IgA, IgA x and IgA X and 0- 15 U/L for total IgM, IgM x and IgM X, the actual range for each measurement depending on the most accurate part of the curve. The detection limit for total IgG, IgG x and IgG X, was 0.06 U/L, for total
463
TABLE1. Between-run precision of the ELISA Mean
(KUIL) 78 39 118 64 57 122 72 44
118
SD (KU/L)
cv (Yo)
n
7-0 3.7 10.9 3.2 3.1 5.6 2.6 2.1 4.5
8.9 9.6 9.3 4.9 5.5 4.6 3.6 4.7 3.8
32 33 33 29 30 30 28 29 29
IgA, IgA x and IgA X 0.2 U/L and for total IgM, IgM x and IgM X O.SU/L. The within-run precision of the method was calculated using the differences of the duplicates for all measurements. The coefficient of variation ranged from 2.0 to 4.5% (n = 50). The between-run precision is shown in Table 1. When measuring serum samples from patients with IgG deficiency, no IgG x or X light chains were detected. The same negative result was found when measuring IgA x and IgA X from IgA deficient patients. Measurement of the pooled serum revealed X A ratios of 2 - 0 for IgG, 1.1 for IgA and 1.7 for IgM. The measurements of 20 healthy individuals revealed comparable results with an acceptable standard deviation. The results of the measurements in serum of healthy adults and in pooled serum of 500 healthy Dutch blood donors are shown in Table 2. One hundred and fourteen serum samples were used when comparing our method to earlier techniques. A high degree of agreement was found between the x plus X for IgG, IgA or IgM and the total IgG, IgA and IgM, respectively, as measured by the ELISA. The mean differences of the two methods after log (natural logarithm, In) transformation within the limits of agreement expressed as k 2SD were 0.00& 0 - 14 for IgG, 0.01&0-17 for IgA and 0-OlkO.10 for IgM TABLE 2. Zmmunoglobulin x A ratio in ZgG, IgA and ZgM in pooled serum of SW healthy Dutch blood donors and mean x A ratio in 20 healthy adults. 10 men and 10 women (statistics are calculated from 10 healthy individuals)
Pooled serum IgG IgA
IgM
Total
2.0 1.1 1.7 2.0
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20 individuals (SD) Men (n = 10)
Women (n = 10)
2.05 1.01 1.65 2.01
2.07 1.12 1.76 2.02
(0.36) (0.20) (0.26) (0.32)
(0.34) (0.24) (0.39) (0.37)
Haraldsson et al.
464
I
-0 3 4 4
and - 1.4 and for IgM 0.99, 1.01 and 6.3, respectively. The agreement between the x plus X for IgG, IgA and IgM as measured by ELISA and immunonephelometry was lower revealing a mean difference after log (In) transformation of - 0 - 0 3 + 0 - 2 3 for IgG, 0*14?0-28 for IgA and 0-08+_0-28 for Igh4 (Fig. 2). This implies that the quotient of IgG ELISA divided by IgG x plus IgG X lies between 0.77 and 1-22. For IgA the
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FIGURE 1 . Agreement between measurements of total immunoglobulin as measured by ELISA and x plus X of that particular immunoglobulin as measured by our method, expressed after log (natural logarithm) transformation as mean difference -t 2SD. (a) Total IgC versus IgC x plus IgG A. (b) Total IgA versus IgA x plus 1gA X. (c) Total IgM versus IgM x plus IgM X.
(Fig. 1). This implies that the quotient of IgG ELISA divided by IgG x plus IgG X lies between 0.87 and 1.15. For IgA the quotient lies between 0-85 and 1.20 and for IgM between 0.91 and 1.12. The difference between these two methods was not statistically significant (P>0-05). When comparing the x plus X for IgG, I g A or IgM to the total IgG, IgA and IgM, respectively, all measured by ELISA, the correlation coefficient, slope and intercept for IgG was 0-98,0-98and 2.3, for IgA 0-99, 1-07
..
0 4 ............................................................................
-0-4
Averoge In of the t w o methods
FIGURE 2. Agreement between measurements of total immunoglobulin as measured by imrnunonephelometry and x plus X of that particular immunoglobulin as measured by our method, expressed after log (natural logarithm) transformation as mean difference 250. (a) Total IgC versus IgC x plus IgG X. (b) Total IgA versus IgA x plus IgA A. (c) Total ISM versus IgM x plus IgM X.
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Kappa and lambda light chains quotient lies between 0.87 and 1.52 and for IgM between 0.82 and 1 .43. The correlation coefficient, slope and intercept for the comparison of the measured x plus X values of the separate immunoglobulins and the immunonephelometry was 0-95 and 3.0 for IgG, 0.96,1-06 and 6.1 for IgA and 0.97, 1-12and - 1.9 for IgM, respectively. The serum samples showing the lowest agreement between the different methods could not be correlated with any age or patients' groups. O m % ,
DISCUSSION
465
Recently, light chain ratios of IgG, IgA and IgM measured by an ELISA method using rabbit antisera have been de~cribed.'~The results obtained in that study reveal definitely higher x/X ratios of IgG, IgA and IgM than ours. We question the way of calculating absorbance ratios by simply adding the absorbances of the different immunoglobulin x and X values. We clearly prefer using different standards for each determination. However, both studies show the X Aratio for I g A to be the lowest and the ratio for IgG the highest. In certain diseases and in autoimmune disorders x or X preference has been implicated. Some infections have been found to carry with them a high total x/X ratioI2 and a high total x/X ratio has also been found in systemic lupus erythematosus, juvenile rheumatoid arthritis and some other autoimmune disorders. 1~3-7*8,12 In addition, an imbalanced total x/X ratio has been described in hypogammaglobulinaemia,2 agammaglobulinaemia and in ataxia telangiectasia.12An abnormal x concentration has also been detected in IgA deficiency.l Imbalanced x or X deposits have also been demonstrated in various tissues in certain diseases such as IgA nephropathy6 and Sjdgren d i ~ e a s e . ~ The bulk of the total immunoglobulin concentration in serum of healthy individuals derives from IgG. Therefore, IgG x and X accounts for the majority of the light chains in serum. The ratio of x and X in other immunoglobulins is therefore partly overshadowed by the IgG light chains. Measuring the x/X ratios separately in IgG, IgA and IgM may provide a means for understanding better the function of the immunoglobulin light chains in health and disease.
Concentrations of x plus X of the separate immunoglobulin classes IgG, I g A or IgM, measured by our method, compared well with the total immunoglobulins measured by ELISA. The difference between the two methods was not statistically significant and a good agreement was consistent. Agreement was poorer when comparison was made with immunonephelometry. This was to be expected. The sensitivity of the nephelometric method causes greater deviation for the measurement of IgA and IgM than for IgG. Measuring IgM by nephelometry requires a particularly high precision and sensitivity. Furthermore, since many of the serum samples obtained from children contained low values of IgA, high precision was needed for IgA as well. This possibly explains a certain bias in these measurements. Additionally, as goat antisera were used in the ELISA and rabbit antisera in the immunonephelometry a slight difference in results due to these antisera may be expected. Our values show a distinct difference between the x/X ratios of the different immunoglobulins. Interestingly, IgA shows a lower x/X ratio (1.1) Acknowledgements than IgG (2.0) and IgM (1.7). The reason for this The work was supported in part by a research difference is not known. The total x/X ratio has earlier been reported to be 1.7 to 2.0.1-4*9*'2*13grant from the Icelandic Council of Science. As IgG comprises the majority of total immunoglobulins it was expected that the IgG x/X ratio REFERENCES would resemble that of the total immunoglobulins. 1 Renckens ALJM, Jansen MJH, Van Munster PJJ, Studies of the x/X ratio in different immunoWeemaes CMR, Bakkeren JAJM. Nephelometry of the globulin classes have been carried out5 in patients kappa/lambda light chain ratio in serum of normal with monoclonal production of immunoglobulins. and diseased children. CIin Chem 1986; 32: 2147 These results were clearly different from ours 2 Skvaril F, Barandun S, Morel1 A, Probst M. showing a IgM x/x ratio of 3.2 and a IgA x/x ratio Imbalances of k/l immunoglobulin light chain ratios of 1.4-1.6. This variation underlines the differin normal individuals and in immunodeficient patients. In: Peters H, ed. Twenty-second Colloquium ence between light chain ratios of monoclonal and Bruges: Protides of Biological Fluids. New York: polyclonal immunoglobulins. The results gained Pergamon Press, 1975; 415 from monoclonal immunoglobulins can hardly be 3 Normansell DE. Use of kappa and lambda chain compared to those obtained in healthy individuals. quantitation for the detection of immunoglobulin Hitherto, healthy individuals have scarcely been abnormalities in serum. Diagn CIin Imrnunol 1987; 5: loo investigated.
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4 Ford RP, Mitchell PEG, Fraser CG. Desirable performance characteristics and clinical utility of immunoglobulin and light-chain assays derived from data on biological variation. CIin Chem 1988; 34: 1733 5 Eisen HN. Antibody structure: The immunoglobulins. In: Davis BD, Dulbecco RO, Eisen HN, Ginsberg HS, Wood WB Jr, eds. Microbiology 2nd ed. Maryland: Harper International Edition, 1988; 446 6 Lai KN, Chan KW, Mac-Moune Lai F, et al. The immunochemical characterization of the light chains in the mesangial IgA deposits in IgA nephropathy. Am J Clin Pathol 1986; 85: 548 7 Knight J, Laing P, Knight A, Adams D, Ling N. Thyroid-stimulation autoantibodies usually contain only L-light chains: evidence for the ‘forbidden clone’ theory. J CIin Endocrinol Metab 1986; 62: 342 8 Knight JG, Laing P , Adams DD, Bray JJ, Ling NR. Autoantibodies to acetylcholine receptor in myasthenia gravis: light chains. Neurology 1986; 36: 1531 9 Jasani B. Immunohistologically definable light chain
restriction in autoimmune disease. J Pathol 1988; 154: 1 10 Reimer CB, Smith SJ, Hannon WH, et al. Progress towards international reference standards for human serum proteins. J Biol Stand 1978; 6 : 133 11 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurements. Lancet 1986; i: 307 12 McKelvey EM, Fahey JL. Immunoglobulin changes in disease: quantitation on the basis of heavy polypeptide chains, IgG (gG), IgA (gA)and IgM (gM), and of light polypeptide chains, type K(I) and type L(I1). J CIin Invest 1965; 44: 1778 13 Barandun S, Skvaril F, Morel1 A. Imbalances of the k/l ratio of human immunoglobulins. CIin Immunol Biol 1976; 3: 57 14 Chui SH. Lam CWK, Lai KN. Light-chain ratios of immunoglobulins G, A and M determined by Enzyme immunoassay. CIin Chem 1990; 36: 501 Accepted for publication 2 April 1991
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