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Brief study of a myeloma protein
283
CLINICA CHIMICA ACTA
BRIEF
NOTES
Brief study of a myeloma protein The study of the abnormal proteins in two sera which had shown a sharp M peak in paper and cellulose acetate pherogram scans, is reported here. The sera were from patients with confirmed multiple myeloma and the study was made using quantitative immunoelectrophoresisl with some technical refinements described later2. Collateral identity patterns obtained as described earlier3 and their coaxial variant and displaced overlapping were used for establishing the immunological nature of the abnormal proteins.
Fig. I, Patterns of undiluted sera (2 ,ul) developed with 8 pi/ml of antiserum. A = myeloma pattern; B = normal serum pattern; AB = collateral identity pattern of A and B. Fractions numbered are: I = prealbumin, 2 = albumin, 3 = c(~L, 4 = a1 antitrypsin 5 = CQ (unidentified), 6 = haptoglobin, 7 = CC~M, 8 = transferrin and 9 = &.
The two sera gave strikingly similar patterns by the quantitative technique. Pattern A of Fig. I shows the bizarre configuration of the myeloma pattern as compared to normal serum (pattern B, Fig. I). The normal vs. myeloma collateral identity pattern AB (Fig. I) established the identification of IgG which has a very low concentration in the pathological serum. The identity of the abnormal protein (marked Clin. Chim.
Acta,
21
(1968) 283-287
BRIEF NOTES
284
M in Fig. I) is not clear because of weak and hazy precipitation. As it seemed that poor precipitation was due to excess antigen, collateral identity patterns were obtained with undiluted normal OS. diluted myeloma sera (Fig. 2). Patterns B and D of Fig. 2 show clear precipitation lines of the abnormal proteins. IgG of the myeloma sera has become so diluted that no clear precipitation is seen in the isolated patterns of the dilutions used (patterns A and B, Fig. 2). Enlarged photographs of pattern B, as shown in Fig. 3 helped in defining the tracing of the abnormal proteins M precipitation lines (here, M does not denote macroglobulin). On the cathodic side the M lines seem to be intercepted by the normal IgG (arrow X, Fig. 3) and on the anodic side by the normal IgA (arrow Y). As the normal lines are not interrupted at the intercept, this identity pattern could be significative of genuine or apparent partial identity, in the sense that the abnormal protein is either (I) immunologically identical with a fiart of the popula-
Fig. 2. A, C patterns of myeloma serum, diluted identity patterns vs. undiluted normal serum. CZin. Chim. Acta, ZI (1968) 283-287
I
: 3 and I : I with buffer. B, D corresponding
BRIEF NOTES
Fig. 3. Enlarged photograph A of Fig. I.
285
of pattern B of Fig. 2. Numbers refer to fractions
Fig. 4. A, pattern of normal serum. B, pattern of I
as in pattern
: I mixture of myeloma and normal sera.
tions of IgG and IgA, or (2) it has a number of antigenic groups in common with normal IgG and IgA. This type of intercepts correspond to the two alternatives of the single spur identity patterns of Ouchterlony. Clin.
Chim.
Acta,
ZI (1968)
283-287
286
BRIEF NOTES
In order to investigate further the above two alternatives, it was reasoned that a pattern of a mixture of myeloma sera and normal sera would be helpful, giving a coaxial identity which could be analysed by comparing with the normal serum pattern. Pattern B of Fig. 4 shows the coaxial identity. The M lines fuse with the outermost precipitation rim of normal IgG (at point marked by arrow X). This outer rim precipitation is well visible in the normal pattern (arrows R, pattern A, Fig. 4). The identity M lines, after fusion, are independent from the main IgG line at the cathodic apex and become much clearer and heavier than in the pathological pattern of equal dilution (arrow P, Fig. 5). All these characteristics mean that there is genuine partial identity between the outer-rim IgG components and the myeloma protein, i.e., they have common antigenic groups. It seems that the partially identical populations of IgG contribute to reinforcement of the cathodic apex precipitation. Such M-like components of normal IgG are often found increased in some non-myeloma sera which give a “double” IgG line which is specially clear at the apex. Even the normal pattern A of Fig. 4 shows a faint haziness at the apex (marked M?). The anodic intercept (arrow Y, pattern B, Fig. 4) is also clearly seen as a fusion of the M lines with normal IgA, which prolong into the cc*range instead of stopping abruptly within the p1 range, as in the myeloma pattern C (Fig. 2).
Fig.
5.
As some authors4+ reported association of albumin with some myeloma proteins, collateral overlapping was obtained by depositing normal serum in a well at the required place of the gel plate, on the cathodic side of the myeloma serum well, so that after migration, normal albumin would overlap with the abnormal protein. Pattern of Fig. 5, obtained in this way shows that there is undisturbed cross-over (arrow X) and no loss of symmetry of the M lines, showing absence of association. In brief, the abnormal M-peak protein of the two myeloma cases analysed is therefore immunologically partially identical to IgG and IgA. This partial identity is of the genuine type, i.e., normal IgA and some populations of normal IgG have antigenic groups in common with the abnormal protein, besides different groups for which antibodies are present in the anti-horse serum used, which is a total horse CL&. Chim. Acta,
21
(1968) 283-287
287
BRIEF NOTES
antiserum for normal human serum proteins (Netherlands Red Cross A.H.S. in equo, Batch THO-12-P3). The abnormal protein in its turn would also probably have differing antigenic groups for which antibodies would be present in a corresponding anti-myeloma serum, but these are irrelevant in the present analysis. Laboratory
of Health Services, Goa (India)
EMIDIO AFONSO
I E. AFONSO, Clin. Chim. Acta, 13 (1966) 107. 2 E. AFONSO, Technical Bulletin No. I, Sadananda Press, Pangim, Goa, India, 1967. 3 E. AFONSO, C&n. Chim. Acta, 17 (1967) 131. 4 J. F. HEREMANS, Les Globulins Se’riques du SystLme Gamma, Arscia, Brussels; Masson, Paris, 1960. 5 R. E. BALLIEUX, J. W. IMHOFAND AND G. H. NIEHANS, Nature, 189 (1961) 768.
Received March 4, 1968 C&z. Chim. Acta, 21 (1968) 283-287
Serum creatine phosphokinase and haemolysis In a recent paper Shaw et al. l comment that serum creatine phosphokinase (CPK) has no special merits as compared with other enzymes, such as aldolase, in the investigation of muscle disease. In support of this contention they quote the work of Hess et al.8 who found that the presence of red cell haemolysate had a marked inhibitory effect on the apparent activity of serum CPK when assayed by a procedure based on that of Tanzer and Gilvarga. At concentrations in the range of 40-180 mg% the apparent inhibition was at times as much as 40%. At around 220 mg% the highest haemoglobin concentration for which they give data, activity was reduced to about 20% of its initial value. Clearly these observations have an important bearing on the problem of detecting small serum abnormalities, such as those found in carriers of Duchenne muscular dystrophy, in the presence of red cell haemolysate. I have studied the effect of added red cell haemolysate on serum CPK activity, using the procedure of Hughes4 to assay CPK except that, in the calorimetric estimation of creatine, the I-naphthol reagent was added last so that full colour was developed in 30 min as compared with the 60 min required in the original technique. A stock haemoglobin solution (kindly estimated by Dr. D. Donaldson) prepared from washed, lysed and centrifuged human red cells was suitably diluted with heatdeactivated (56” for 15 min) pooled human serum containing initially about 8 mg% haemoglobin and to each haemoglobin dilution the same amount of creatine phosphokinase activity was added. In experiment I the enzyme source was serum from a case of Duchenne dystrophy diluted with pooled serum, and in experiment 2 it was a centrifuged homogenate of human skeletal muscle obtained post-mortem, and also diluted with pooled serum. An amount of diluted enzyme was added such that the final activity was within the linear range of the method. The results are presented in C&Z.Chim. Acta, 21 (1968) 287-288
CLINICA CHIMICA ACTA
BRIEF
NOTES
Brief study of a myeloma protein The study of the abnormal proteins in two sera which had shown a sharp M peak in paper and cellulose acetate pherogram scans, is reported here. The sera were from patients with confirmed multiple myeloma and the study was made using quantitative immunoelectrophoresisl with some technical refinements described later2. Collateral identity patterns obtained as described earlier3 and their coaxial variant and displaced overlapping were used for establishing the immunological nature of the abnormal proteins.
Fig. I, Patterns of undiluted sera (2 ,ul) developed with 8 pi/ml of antiserum. A = myeloma pattern; B = normal serum pattern; AB = collateral identity pattern of A and B. Fractions numbered are: I = prealbumin, 2 = albumin, 3 = c(~L, 4 = a1 antitrypsin 5 = CQ (unidentified), 6 = haptoglobin, 7 = CC~M, 8 = transferrin and 9 = &.
The two sera gave strikingly similar patterns by the quantitative technique. Pattern A of Fig. I shows the bizarre configuration of the myeloma pattern as compared to normal serum (pattern B, Fig. I). The normal vs. myeloma collateral identity pattern AB (Fig. I) established the identification of IgG which has a very low concentration in the pathological serum. The identity of the abnormal protein (marked Clin. Chim.
Acta,
21
(1968) 283-287
BRIEF NOTES
284
M in Fig. I) is not clear because of weak and hazy precipitation. As it seemed that poor precipitation was due to excess antigen, collateral identity patterns were obtained with undiluted normal OS. diluted myeloma sera (Fig. 2). Patterns B and D of Fig. 2 show clear precipitation lines of the abnormal proteins. IgG of the myeloma sera has become so diluted that no clear precipitation is seen in the isolated patterns of the dilutions used (patterns A and B, Fig. 2). Enlarged photographs of pattern B, as shown in Fig. 3 helped in defining the tracing of the abnormal proteins M precipitation lines (here, M does not denote macroglobulin). On the cathodic side the M lines seem to be intercepted by the normal IgG (arrow X, Fig. 3) and on the anodic side by the normal IgA (arrow Y). As the normal lines are not interrupted at the intercept, this identity pattern could be significative of genuine or apparent partial identity, in the sense that the abnormal protein is either (I) immunologically identical with a fiart of the popula-
Fig. 2. A, C patterns of myeloma serum, diluted identity patterns vs. undiluted normal serum. CZin. Chim. Acta, ZI (1968) 283-287
I
: 3 and I : I with buffer. B, D corresponding
BRIEF NOTES
Fig. 3. Enlarged photograph A of Fig. I.
285
of pattern B of Fig. 2. Numbers refer to fractions
Fig. 4. A, pattern of normal serum. B, pattern of I
as in pattern
: I mixture of myeloma and normal sera.
tions of IgG and IgA, or (2) it has a number of antigenic groups in common with normal IgG and IgA. This type of intercepts correspond to the two alternatives of the single spur identity patterns of Ouchterlony. Clin.
Chim.
Acta,
ZI (1968)
283-287
286
BRIEF NOTES
In order to investigate further the above two alternatives, it was reasoned that a pattern of a mixture of myeloma sera and normal sera would be helpful, giving a coaxial identity which could be analysed by comparing with the normal serum pattern. Pattern B of Fig. 4 shows the coaxial identity. The M lines fuse with the outermost precipitation rim of normal IgG (at point marked by arrow X). This outer rim precipitation is well visible in the normal pattern (arrows R, pattern A, Fig. 4). The identity M lines, after fusion, are independent from the main IgG line at the cathodic apex and become much clearer and heavier than in the pathological pattern of equal dilution (arrow P, Fig. 5). All these characteristics mean that there is genuine partial identity between the outer-rim IgG components and the myeloma protein, i.e., they have common antigenic groups. It seems that the partially identical populations of IgG contribute to reinforcement of the cathodic apex precipitation. Such M-like components of normal IgG are often found increased in some non-myeloma sera which give a “double” IgG line which is specially clear at the apex. Even the normal pattern A of Fig. 4 shows a faint haziness at the apex (marked M?). The anodic intercept (arrow Y, pattern B, Fig. 4) is also clearly seen as a fusion of the M lines with normal IgA, which prolong into the cc*range instead of stopping abruptly within the p1 range, as in the myeloma pattern C (Fig. 2).
Fig.
5.
As some authors4+ reported association of albumin with some myeloma proteins, collateral overlapping was obtained by depositing normal serum in a well at the required place of the gel plate, on the cathodic side of the myeloma serum well, so that after migration, normal albumin would overlap with the abnormal protein. Pattern of Fig. 5, obtained in this way shows that there is undisturbed cross-over (arrow X) and no loss of symmetry of the M lines, showing absence of association. In brief, the abnormal M-peak protein of the two myeloma cases analysed is therefore immunologically partially identical to IgG and IgA. This partial identity is of the genuine type, i.e., normal IgA and some populations of normal IgG have antigenic groups in common with the abnormal protein, besides different groups for which antibodies are present in the anti-horse serum used, which is a total horse CL&. Chim. Acta,
21
(1968) 283-287
287
BRIEF NOTES
antiserum for normal human serum proteins (Netherlands Red Cross A.H.S. in equo, Batch THO-12-P3). The abnormal protein in its turn would also probably have differing antigenic groups for which antibodies would be present in a corresponding anti-myeloma serum, but these are irrelevant in the present analysis. Laboratory
of Health Services, Goa (India)
EMIDIO AFONSO
I E. AFONSO, Clin. Chim. Acta, 13 (1966) 107. 2 E. AFONSO, Technical Bulletin No. I, Sadananda Press, Pangim, Goa, India, 1967. 3 E. AFONSO, C&n. Chim. Acta, 17 (1967) 131. 4 J. F. HEREMANS, Les Globulins Se’riques du SystLme Gamma, Arscia, Brussels; Masson, Paris, 1960. 5 R. E. BALLIEUX, J. W. IMHOFAND AND G. H. NIEHANS, Nature, 189 (1961) 768.
Received March 4, 1968 C&z. Chim. Acta, 21 (1968) 283-287
Serum creatine phosphokinase and haemolysis In a recent paper Shaw et al. l comment that serum creatine phosphokinase (CPK) has no special merits as compared with other enzymes, such as aldolase, in the investigation of muscle disease. In support of this contention they quote the work of Hess et al.8 who found that the presence of red cell haemolysate had a marked inhibitory effect on the apparent activity of serum CPK when assayed by a procedure based on that of Tanzer and Gilvarga. At concentrations in the range of 40-180 mg% the apparent inhibition was at times as much as 40%. At around 220 mg% the highest haemoglobin concentration for which they give data, activity was reduced to about 20% of its initial value. Clearly these observations have an important bearing on the problem of detecting small serum abnormalities, such as those found in carriers of Duchenne muscular dystrophy, in the presence of red cell haemolysate. I have studied the effect of added red cell haemolysate on serum CPK activity, using the procedure of Hughes4 to assay CPK except that, in the calorimetric estimation of creatine, the I-naphthol reagent was added last so that full colour was developed in 30 min as compared with the 60 min required in the original technique. A stock haemoglobin solution (kindly estimated by Dr. D. Donaldson) prepared from washed, lysed and centrifuged human red cells was suitably diluted with heatdeactivated (56” for 15 min) pooled human serum containing initially about 8 mg% haemoglobin and to each haemoglobin dilution the same amount of creatine phosphokinase activity was added. In experiment I the enzyme source was serum from a case of Duchenne dystrophy diluted with pooled serum, and in experiment 2 it was a centrifuged homogenate of human skeletal muscle obtained post-mortem, and also diluted with pooled serum. An amount of diluted enzyme was added such that the final activity was within the linear range of the method. The results are presented in C&Z.Chim. Acta, 21 (1968) 287-288