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IMMUNOLOGICAL MEASUREMENT OF PROTEINS
770 of the individual curve. For practical analysis T is the time taken to regain 63-22% of -the temperature drop. It is apparent that the T of Dr. Allen (=50%) represents an empirical first approximation of 7. But only by means of the T value can the course of these skin-rewarming curves be exactly described. Department of Dermatology and Section of Electronics,
HANS MEFFERT NIELS SÖNNICHSEN BEATE MEFFERT.
Humboldt University, 104 Berlin.
IMMUNOLOGICAL MEASUREMENT OF PROTEINS
SiR,-The technique of single radial immunodiffusion is widely applied to the measurement of proteins in bodyfluids. The reproducibility and accuracy are excellent.1 Related methods of quantitative immunoelectrophoresis and electroimmunodiffusion are also increasingly used to measure proteins in body-fluids. 2, Unsatisfactory results have been reported when such techniques have been used with the immunoglobulins. These proteins have different molecular weights but are antigenically related. There are major differences in structure, chemical and antigenic, which are readily distinguished by conventional immunological techniques, and specific antisera can be raised to the different immunoglobulin classes. Finer distinctions can be made within these classes using specific antisera. Monoclonal immunoglobulins, which may seem antigenically normal by immunoelectrophoresis, may behave abnormally in quantitative techniques. This has been attributed to antigenic deficiency ". In a collaborative report on immunoglobulin estimation, including purified immunoglobulins, an international reference preparation (of freeze-dried serum), and normal "
human serum, it was recommended that serum concentrations of IgG, IgA, and IgM should be estimated by comparison with the reference preparation and expressed in terms of international units per millilitre. Comparisons using purified immunoglobulin standards lead to divergent results " consistent with the present lack of uniformity of the numerous immunoglobulin standards prepared in In other words inconsistent different laboratories". results can be obtained in comparative immunoassays of
highly purified immunoglobulin preparations. It seemsto us that immunoassays using the reference standard may still give results which are meaningless and not reproducible if the characteristics of the specific antisera
not known. similar but antigenically less complex system, Kr0ll and Thambiah 6 concluded that " one condition is that the protein is [for immunological measurement] antigenically homogeneous or that the antiserum applied only contains antibodies against common antigens [antigenic determinants] ". For the antigenically heterogeneous immunoglobulin classes the latter condition is unlikely (and, moreover, is not intended) to be satisfied in the normal way. Current methods of raising antisera by the injection of myeloma proteins or serum fractionsinto foreign hosts of uncertain genetic constitution, selected from a small number of species, can give only ill-defined antisera, whose deficiencies are not eliminated by pooling. Consistent results in immunoassays comparing a normal serum with a reference preparation can be expected, on
For
are
a
...
2. 3. 4.
Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. Minchin Clarke, H. G., Freeman, T Clin. Sci. 1968, 35, 403. Merrill, D., Hartley, T. F. J. Lab. clin. Med. 1967, 69, 151. Chazot, G., Poncet, J., Creyssel, R. Revue fr. Étud. clin. biol. 1971,
5. 6. 7.
16, 943. J. Immun. 1971, 107, 1798. Krøll, J., Thambiah. Protides biol. Fluids, 1969, 17, 533. Skvaril, F., Morell, A. J. Immun. 1970, 104, 1310.
1.
purely statistical grounds, from any group of immunoglobulin-class-specific antisera, regardless of their differences in determinant specificity. Different antisera, which are nevertheless class specific by current tests, cannot give meaningful assays of purified immunoglobulins, abnormal sera, or isolated antibodies, such as rheumatoid factors. Commercial quantitative immunoglobulin plates can give multiple precipitin rings with abnormal sera, which do not contain myeloma proteins.8 Techniques of raising antisera to proteins which are antigenically heterogeneous and of defining precisely their determinant specificity may be inadequate for the immunological determination of such proteins. Robert Jones and Agnes Hunt
Orthopædic Hospital, Oswestry, Shropshire SY10
D. J. LEA D. J. WARD.
7AG.
VALUE OF PLASMA-DIGOXIN ESTIMATION SIR,—Dr. Stewart and Dr. Simpson (Sept. 2, p. 493) raise the important question of the timing of blood-samples for measurement of plasma-digoxin. In routine cases we try to obtain a sample at 8 hours after the last dose. This interval was chosen to record the digoxin level during a steady-state period after the absorption/distribution period and before the main part of the elimination phase. A blood-level at the peak of absorption will not accurately reflect tissue-levels, just as, in amore extreme situation, the very high blood-levels immediately after the intravenous injection of digoxin do not correspond to the level at the receptor sites or hence to clinical effect. For a patient with normal renal function the half-life of digoxin in the plasma is approximately 36 hours and a blood taken at 24 hours after the last dose will give a digoxin level significantly lower than that recorded at 8 hours. Since the rate -of digoxin excretion varies from patient to patient the extent of the fall from 8 to 24 hours will vary. It is also likely that in some patients minor toxicity present at 8 hours will have disappeared by 24 hours. Moreover, 8 hours is the approximate time interval from the last dose in patients attending an afternoon outpatient clinic. For these reasons we have sought samples at the 8-hour interval and found it a useful guide to therapeutic and toxic effects of digoxin. As a steady-state level at this time is the result of the final balance between absorption and excretion, further investigation of digoxin kinetics is needed only when this level is unsatisfactory. The figure accompanying the letter by Stewart and Simpson shows that plasma-digoxin fell only slowly in the period 8-16 hours after the oral dose, and that by 24 hours the level had returned to near the original value. We suggest that this supports the validity at a single 8-10 hour level. The trough which they recorded after the absorption peak is interesting and difficult to explain. The type of tablet used was not specified, but, from the blood-levels obtained, it seems very likely that the tablets were of low biological availability. In 12 of our patients who had plasma-digoxin levels followed for 6-8 hours after an 0-5 mg. dose using tablets of low biological availability a small second rise at 0-2 ng. per ml. or more was seen in 8 of the total of 16 absorption curves. With our assay the standard deviation of repeated estimations of the same plasmadigoxin standard is 0-05-0-1 ng. per ml. in the range studied. These second peaks were of short duration and occurred at intervals ranging from 2½ to 7 hours after the dose. They were not always seen when the absorption curve was repeated with tablets of a different brand of similar bioavailability. These secondary peaks of short duration may represent delayed dissolution of part of the 8.
Mulder, J., Sloots, L. C. E., Verhaar, M. 1972, 1, 211.
A.
T. J. Immun. Meth.
HANS MEFFERT NIELS SÖNNICHSEN BEATE MEFFERT.
Humboldt University, 104 Berlin.
IMMUNOLOGICAL MEASUREMENT OF PROTEINS
SiR,-The technique of single radial immunodiffusion is widely applied to the measurement of proteins in bodyfluids. The reproducibility and accuracy are excellent.1 Related methods of quantitative immunoelectrophoresis and electroimmunodiffusion are also increasingly used to measure proteins in body-fluids. 2, Unsatisfactory results have been reported when such techniques have been used with the immunoglobulins. These proteins have different molecular weights but are antigenically related. There are major differences in structure, chemical and antigenic, which are readily distinguished by conventional immunological techniques, and specific antisera can be raised to the different immunoglobulin classes. Finer distinctions can be made within these classes using specific antisera. Monoclonal immunoglobulins, which may seem antigenically normal by immunoelectrophoresis, may behave abnormally in quantitative techniques. This has been attributed to antigenic deficiency ". In a collaborative report on immunoglobulin estimation, including purified immunoglobulins, an international reference preparation (of freeze-dried serum), and normal "
human serum, it was recommended that serum concentrations of IgG, IgA, and IgM should be estimated by comparison with the reference preparation and expressed in terms of international units per millilitre. Comparisons using purified immunoglobulin standards lead to divergent results " consistent with the present lack of uniformity of the numerous immunoglobulin standards prepared in In other words inconsistent different laboratories". results can be obtained in comparative immunoassays of
highly purified immunoglobulin preparations. It seemsto us that immunoassays using the reference standard may still give results which are meaningless and not reproducible if the characteristics of the specific antisera
not known. similar but antigenically less complex system, Kr0ll and Thambiah 6 concluded that " one condition is that the protein is [for immunological measurement] antigenically homogeneous or that the antiserum applied only contains antibodies against common antigens [antigenic determinants] ". For the antigenically heterogeneous immunoglobulin classes the latter condition is unlikely (and, moreover, is not intended) to be satisfied in the normal way. Current methods of raising antisera by the injection of myeloma proteins or serum fractionsinto foreign hosts of uncertain genetic constitution, selected from a small number of species, can give only ill-defined antisera, whose deficiencies are not eliminated by pooling. Consistent results in immunoassays comparing a normal serum with a reference preparation can be expected, on
For
are
a
...
2. 3. 4.
Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. Minchin Clarke, H. G., Freeman, T Clin. Sci. 1968, 35, 403. Merrill, D., Hartley, T. F. J. Lab. clin. Med. 1967, 69, 151. Chazot, G., Poncet, J., Creyssel, R. Revue fr. Étud. clin. biol. 1971,
5. 6. 7.
16, 943. J. Immun. 1971, 107, 1798. Krøll, J., Thambiah. Protides biol. Fluids, 1969, 17, 533. Skvaril, F., Morell, A. J. Immun. 1970, 104, 1310.
1.
purely statistical grounds, from any group of immunoglobulin-class-specific antisera, regardless of their differences in determinant specificity. Different antisera, which are nevertheless class specific by current tests, cannot give meaningful assays of purified immunoglobulins, abnormal sera, or isolated antibodies, such as rheumatoid factors. Commercial quantitative immunoglobulin plates can give multiple precipitin rings with abnormal sera, which do not contain myeloma proteins.8 Techniques of raising antisera to proteins which are antigenically heterogeneous and of defining precisely their determinant specificity may be inadequate for the immunological determination of such proteins. Robert Jones and Agnes Hunt
Orthopædic Hospital, Oswestry, Shropshire SY10
D. J. LEA D. J. WARD.
7AG.
VALUE OF PLASMA-DIGOXIN ESTIMATION SIR,—Dr. Stewart and Dr. Simpson (Sept. 2, p. 493) raise the important question of the timing of blood-samples for measurement of plasma-digoxin. In routine cases we try to obtain a sample at 8 hours after the last dose. This interval was chosen to record the digoxin level during a steady-state period after the absorption/distribution period and before the main part of the elimination phase. A blood-level at the peak of absorption will not accurately reflect tissue-levels, just as, in amore extreme situation, the very high blood-levels immediately after the intravenous injection of digoxin do not correspond to the level at the receptor sites or hence to clinical effect. For a patient with normal renal function the half-life of digoxin in the plasma is approximately 36 hours and a blood taken at 24 hours after the last dose will give a digoxin level significantly lower than that recorded at 8 hours. Since the rate -of digoxin excretion varies from patient to patient the extent of the fall from 8 to 24 hours will vary. It is also likely that in some patients minor toxicity present at 8 hours will have disappeared by 24 hours. Moreover, 8 hours is the approximate time interval from the last dose in patients attending an afternoon outpatient clinic. For these reasons we have sought samples at the 8-hour interval and found it a useful guide to therapeutic and toxic effects of digoxin. As a steady-state level at this time is the result of the final balance between absorption and excretion, further investigation of digoxin kinetics is needed only when this level is unsatisfactory. The figure accompanying the letter by Stewart and Simpson shows that plasma-digoxin fell only slowly in the period 8-16 hours after the oral dose, and that by 24 hours the level had returned to near the original value. We suggest that this supports the validity at a single 8-10 hour level. The trough which they recorded after the absorption peak is interesting and difficult to explain. The type of tablet used was not specified, but, from the blood-levels obtained, it seems very likely that the tablets were of low biological availability. In 12 of our patients who had plasma-digoxin levels followed for 6-8 hours after an 0-5 mg. dose using tablets of low biological availability a small second rise at 0-2 ng. per ml. or more was seen in 8 of the total of 16 absorption curves. With our assay the standard deviation of repeated estimations of the same plasmadigoxin standard is 0-05-0-1 ng. per ml. in the range studied. These second peaks were of short duration and occurred at intervals ranging from 2½ to 7 hours after the dose. They were not always seen when the absorption curve was repeated with tablets of a different brand of similar bioavailability. These secondary peaks of short duration may represent delayed dissolution of part of the 8.
Mulder, J., Sloots, L. C. E., Verhaar, M. 1972, 1, 211.
A.
T. J. Immun. Meth.