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AN IMMUNOASSAY METHOD FOR URINARY ALBUMIN AT LOW CONCENTRATIONS
913
AN IMMUNOASSAY METHOD FOR URINARY ALBUMIN AT LOW CONCENTRATIONS H. KEEN Lond., M.R.C.P. C. CHLOUVERAKIS
M.B.
M.D. Athens OF THE DEPARTMENT OF EXPERIMENTAL
MEDICINE,
GUY’S HOSPITAL, LONDON, S.E.1
ALTERATIONS in the permeability of the renal glomerular membrane may be reflected by changes in the amount of albumin in the urine. The ordinary clinical tests for albuminuria, however, become positive only after a twenty to hundred-fold increase over the normal. Most methods of measuring albumin at low concentrations are laborious because they depend upon the separation of protein by electrophoresis after the concentration of large volumes of urine. Immunoelectrophoresis or geldiffusion techniques, although requiring smaller volumes
and less preparation, give only semiquantitative estimates. The method described here overcomes most of the difficulties outlined above: it is specific, it requires very small volumes of urine only, it is accurate and sensitive over a selected concentration range, and it is capable of dealing with batches of a hundred or so samples of urine at each " run ".
Principle of Method The method, based on isotope-saturation analysis, employs the same principle as the methods of Hales and Randle (1963) and Morgan and Lazarow (1963) for measuring insulin in human plasma. A known amount (W’) of isotopically labelled human albumin (1251-albumin) is added to a measured volume of the urine under investigation, containing the unknown quantity of albumin (W) to be measured. To this mixture is added antihuman-albumin serum (raised in the guineapig) in such a quantity that the 1251-albumin is present in excess. After equilibration between the albumin in the urine and the added antibody, a part of the albumin is bound and some remains in the free form. An anti-guineapig-globulin serum (raised in the rabbit) serves to precipitate the bound albumin which is then separated from the free albumin by filtration. The radioactivity found in the precipitate is related to the amount of endogenous albumin (W). Theoretically, if a is the radioactivity of the precipitate in the absence of endogenous albumin (i.e., when W=O), and x is the radioactivity of the precipitate in the of endoeenous albumin (i.e.. when W >
Dresence
-being
measurable and W’
a
unknown
being known,
0). then
Fig. 1-Standard
precipitated to
proportion
curve relating the the concentration of albumin.
of radioactivity ,
Guineapig antibody against human albumin was obtained injected with Lister albumin and Freund’s adjuvant. Anti-serum to guineapig globulin was produced by injecting a globulin preparation from normal guineapig serum (made by ammonium-sulphate precipitation) into rabbits. The dilutions
from animals
of both anti-sera and of the labelled albumin were made in Michaelis’ barbitone buffer (pH=8-4) containing 200 mg. per 100 ml. gelatin. Reactions were carried out in small disposable polystyrene precipitin tubes (Baird & Tatlock, 11/2 X 1/4 in.), and reactants were adequately mixed with a homemade microvortex mixer. The urine (0-1 ml.), with admixed 125I-albumin (0-1 ml.), was incubated for 18 hours at room temperature with 0-1 ml. of appropriately diluted guineapig antihuman-albumin serum. The precipitating antibody (0-1 ml. of appropriately diluted rabbit anti-guineapig-globulin serum) was added, and the reactants were mixed and incubated for a further hour at the same temperature. The precipitated complex was then separated from the free albumin in solution by filtering the contents of the tubes through cellulose-acetate-membrane filters (Oxoid). The dilutions of the two antibodies, selected as a result of preliminary experiments, were such that the albumin antiserum complexed with 30-50% only of the added 125I-albumin, and the precipitating serum brought down all of the complex. During each experiment a standard albumin curve was constructed, substituting for the urine 0-1 ml. of suitable
the value for the
quantity, W, is calculable:
This formulation would apply if the system conform
were found to In practice it departs and the value for the unknown is derived standard curve.
strictly to the dilution principle.
significantly from it, by interpolation in a
Materials and Methods Human albumin prepared at the Lister Institute was used for radioiodination, for immunisation of guineapigs, and for the standard albumin solutions. The iodination of the albumin with 1251 was carried out by McFarlane’s method (1958), free iodine being removed by passage through an ion-exchange resin (’ De-acidite FF’, Permutit Ltd.). The concentration of this albumin and its specific activity were adjusted to the desired level by mixing with buffered solutions of unlabelled albumin. The final specific activity of the 125I-a1bumin solution varied between 100 and 200 fLC per mg., and the concentration between 0-3 and 1 mg. per 100 ml. approximately.
Fig. 2-Curve relating the reciprocal of the proportion of radioactivity precipitated to the albumin concentration. The solid line indicates the experimental curve, and the broken line indicates the theoretical line based on the dilution principle.
914 albumin solutions in buffer, in concentrations from 0 to 60 mg. per 100 ml. From this curve the amount of albumin in the urine under investigation was inferred. Duplicate measurements were performed on all specimens of urine and standards, and the precipitates were counted in a
well-type scintillation
counter.
Results 1 shows
curve with albumin concentrations which vary from 0 to 60 mg. per 100 ml. While the curve is extremely sensitive at low concentrations of albumin, large variations at higher concentrations result in only small changes in the radioactivity precipitated. For this reason urine specimens that gave a positive reaction with ’Albustix’ (i.e., concentration > 30 mg. per 100 ml.) were diluted with distilled water before assay until the reaction with albustix became
Fig.
a
typical standard
negative. In fig. 2 the reciprocal of the proportion of the precipitated radioactivity shown in fig. 1 is plotted against the albumin content of the standards. The broken line represents the theoretical relationship calculated on the assumption that the dilution principle alone applies. The experimental line approximates to it over the very lowest range of albumin concentration only. At higher concentrations the manner in which the experimental line diverges from the theoretical reflects the fact that the radioactivity actually precipitated exceeds that predicted on the basis of simple dilution. This probably indicates that, as the
Preliminary
Communications
DEVELOPMENT OF VACCINIA VIRUS RESISTANT TO 5-IODO-21-DEOXYURIDINE PREVIOUS experiments have shown that poliovirus is inhibited by guanidine and by 2-(a-hydroxybenzyl) benzimidazole (H.B.B.), but the virus rapidly acquires resistance to both drugs.1-5 Melnick et al.have suggested that drug resistance might explain the failure of guanidine in the treatment of poliovirus infection in monkeys. An investigation of the inhibitory effect of 5-iodo-21deoxyuridine (I.D.U.R.) on vaccinia virus was undertaken to discover whether drug resistance occurred in other 1. 2. 3.
4. 5. 6.
albumin concentration increases, so does the combining power of the antibody. Unless this change in the valency " of the antibody with albumin concentration can be defined, it is not possible to apply the dilution principle, and the unknown albumin concentration is found empirically by interpolation into the standard curve. The reproducibility which is obtained by the method is indicated by the following values of albumin concentration- 1 - 08, 1-04, 0-99, and 1-07 mg. per 100 ml. -obtained for the same sample of urine assayed along with successive batches of urine on four different days and, on each occasion, with a new standard curve. "
Summary A
simple immunological method for estimating the
albumin concentration in human urine is described. It is sensitive and reproducible down to very low concentrations, and it requires less than 1 ml. of urine. Fifty to a hundred urine samples can be estimated in each experimental run ". "
We should like to acknowledge the assistance of Mr. N. Veall, B.SC., F.INST.P., who prepared the 1251-albumin; and to thank Mr. C. Arden, Miss R. Emerson, and Mr. M. Shaw for technical help. The work was supported by a grant from the United States Public Health Service and a research grant from the British Diabetic Association. REFERENCES
Hales, O. N., Randle, P. J. (1963) Biochem. J. 88, 137. McFarlane, A. S. (1958) Nature, Lond. 182, 53. Morgan, C. R., Lazarow, A. (1963) Diabetes, 12, 115.
viruses besides enteroviruses, and with other chemotherapeutic agents. Vaccinia virus was grown in the continuous line of human-amnion cells (Mascoli) in the presence of I.D.U.R. between 1 g. to 2 mg. per ml. The results are shown in the tables. It will be seen that vaccinia virus which had been passed in the presence of I.D.U.R. became resistant to I.D.u.R. Thus whilst normal vaccinia virus is inhibited by 2-5 or 5 g. of I.D.U.R. per ml., the strain which had been passed in I.D.U.R. was able to grow in 20 {ig. of I.D.U.R. per ml. Similar results were obtained 5-I.D.U.R. ON THE YIELDS OF 5-I.D.U.R.-SENSITIVE 5-I.D.U.R.-RESISTANT VACCINIA VIRUS
TABLE II-EFFECT OF AND
Loddo, B. Boll. Soc. ital. Biol. sper. 1962, 38, 1043. Loddo, B. ibid. p. 1047. Loddo, B., Ferrari, W., Spanedda, A., Brotzu, G. Experientia, 1962, 18, 518. Loddo, B. Boll. Soc. ital. Biol. sper. 1963, 39, 102. Loddo, B. ibid. p. 104. Melnick, J. L., Crowther, D., Barrera Oro, J. Science, 1961, 134, 557
TABLE I-EFFECT OF
5-I.D.U.R.
ON CYTOPATHIC EFFECT OF I.D.U.R.-
SENSITIVE AND I.D.U.R.-RESISTANT VACCINIA
VIRUS* *
3 hours after inoculation the cell monolayers were washed 4 times with Hanks’ solution, and then 4 ml. of maintenance medium containing
+
30 hours after the infection the cells were cryolised. Cytopathic units were titrated in stationary tube cultures of human amnion cells; readings
tris-hydroxymethyl-aminomethane 0-03% were
made after 5
See table
was
added.
days.
I.
TABLE III-INHIBITION BY THYMIDINE OF THE ANTIVIRAL ACTION OF
5-I.D.U.R., VIRUS
Test performed with human amniotic-cell cultures, in petri dishes. The compound was added 2 hours after the viral inoculum in 3 ml. of maintenance medium containing tris-hydroxymethyl-aminomethane
0.03 °o. At 30 hours after cell-culture infection. The diameter of the petri dishes used
was
40
mm.
This virus was transferred in human amnion cells (Mascoli) in the presence of the following 5-i.D.u.R. concentrations (us. per ml.): once 1, once 2, once 4, twice 10, twice 20, twice 50, twice 100, twice 200, twice 500, three times 1000 and four times 2000.
IN I.D.U.R.-SENSITIVE AND I.D.U.R.-RESISTANT VACCINIA
AN IMMUNOASSAY METHOD FOR URINARY ALBUMIN AT LOW CONCENTRATIONS H. KEEN Lond., M.R.C.P. C. CHLOUVERAKIS
M.B.
M.D. Athens OF THE DEPARTMENT OF EXPERIMENTAL
MEDICINE,
GUY’S HOSPITAL, LONDON, S.E.1
ALTERATIONS in the permeability of the renal glomerular membrane may be reflected by changes in the amount of albumin in the urine. The ordinary clinical tests for albuminuria, however, become positive only after a twenty to hundred-fold increase over the normal. Most methods of measuring albumin at low concentrations are laborious because they depend upon the separation of protein by electrophoresis after the concentration of large volumes of urine. Immunoelectrophoresis or geldiffusion techniques, although requiring smaller volumes
and less preparation, give only semiquantitative estimates. The method described here overcomes most of the difficulties outlined above: it is specific, it requires very small volumes of urine only, it is accurate and sensitive over a selected concentration range, and it is capable of dealing with batches of a hundred or so samples of urine at each " run ".
Principle of Method The method, based on isotope-saturation analysis, employs the same principle as the methods of Hales and Randle (1963) and Morgan and Lazarow (1963) for measuring insulin in human plasma. A known amount (W’) of isotopically labelled human albumin (1251-albumin) is added to a measured volume of the urine under investigation, containing the unknown quantity of albumin (W) to be measured. To this mixture is added antihuman-albumin serum (raised in the guineapig) in such a quantity that the 1251-albumin is present in excess. After equilibration between the albumin in the urine and the added antibody, a part of the albumin is bound and some remains in the free form. An anti-guineapig-globulin serum (raised in the rabbit) serves to precipitate the bound albumin which is then separated from the free albumin by filtration. The radioactivity found in the precipitate is related to the amount of endogenous albumin (W). Theoretically, if a is the radioactivity of the precipitate in the absence of endogenous albumin (i.e., when W=O), and x is the radioactivity of the precipitate in the of endoeenous albumin (i.e.. when W >
Dresence
-being
measurable and W’
a
unknown
being known,
0). then
Fig. 1-Standard
precipitated to
proportion
curve relating the the concentration of albumin.
of radioactivity ,
Guineapig antibody against human albumin was obtained injected with Lister albumin and Freund’s adjuvant. Anti-serum to guineapig globulin was produced by injecting a globulin preparation from normal guineapig serum (made by ammonium-sulphate precipitation) into rabbits. The dilutions
from animals
of both anti-sera and of the labelled albumin were made in Michaelis’ barbitone buffer (pH=8-4) containing 200 mg. per 100 ml. gelatin. Reactions were carried out in small disposable polystyrene precipitin tubes (Baird & Tatlock, 11/2 X 1/4 in.), and reactants were adequately mixed with a homemade microvortex mixer. The urine (0-1 ml.), with admixed 125I-albumin (0-1 ml.), was incubated for 18 hours at room temperature with 0-1 ml. of appropriately diluted guineapig antihuman-albumin serum. The precipitating antibody (0-1 ml. of appropriately diluted rabbit anti-guineapig-globulin serum) was added, and the reactants were mixed and incubated for a further hour at the same temperature. The precipitated complex was then separated from the free albumin in solution by filtering the contents of the tubes through cellulose-acetate-membrane filters (Oxoid). The dilutions of the two antibodies, selected as a result of preliminary experiments, were such that the albumin antiserum complexed with 30-50% only of the added 125I-albumin, and the precipitating serum brought down all of the complex. During each experiment a standard albumin curve was constructed, substituting for the urine 0-1 ml. of suitable
the value for the
quantity, W, is calculable:
This formulation would apply if the system conform
were found to In practice it departs and the value for the unknown is derived standard curve.
strictly to the dilution principle.
significantly from it, by interpolation in a
Materials and Methods Human albumin prepared at the Lister Institute was used for radioiodination, for immunisation of guineapigs, and for the standard albumin solutions. The iodination of the albumin with 1251 was carried out by McFarlane’s method (1958), free iodine being removed by passage through an ion-exchange resin (’ De-acidite FF’, Permutit Ltd.). The concentration of this albumin and its specific activity were adjusted to the desired level by mixing with buffered solutions of unlabelled albumin. The final specific activity of the 125I-a1bumin solution varied between 100 and 200 fLC per mg., and the concentration between 0-3 and 1 mg. per 100 ml. approximately.
Fig. 2-Curve relating the reciprocal of the proportion of radioactivity precipitated to the albumin concentration. The solid line indicates the experimental curve, and the broken line indicates the theoretical line based on the dilution principle.
914 albumin solutions in buffer, in concentrations from 0 to 60 mg. per 100 ml. From this curve the amount of albumin in the urine under investigation was inferred. Duplicate measurements were performed on all specimens of urine and standards, and the precipitates were counted in a
well-type scintillation
counter.
Results 1 shows
curve with albumin concentrations which vary from 0 to 60 mg. per 100 ml. While the curve is extremely sensitive at low concentrations of albumin, large variations at higher concentrations result in only small changes in the radioactivity precipitated. For this reason urine specimens that gave a positive reaction with ’Albustix’ (i.e., concentration > 30 mg. per 100 ml.) were diluted with distilled water before assay until the reaction with albustix became
Fig.
a
typical standard
negative. In fig. 2 the reciprocal of the proportion of the precipitated radioactivity shown in fig. 1 is plotted against the albumin content of the standards. The broken line represents the theoretical relationship calculated on the assumption that the dilution principle alone applies. The experimental line approximates to it over the very lowest range of albumin concentration only. At higher concentrations the manner in which the experimental line diverges from the theoretical reflects the fact that the radioactivity actually precipitated exceeds that predicted on the basis of simple dilution. This probably indicates that, as the
Preliminary
Communications
DEVELOPMENT OF VACCINIA VIRUS RESISTANT TO 5-IODO-21-DEOXYURIDINE PREVIOUS experiments have shown that poliovirus is inhibited by guanidine and by 2-(a-hydroxybenzyl) benzimidazole (H.B.B.), but the virus rapidly acquires resistance to both drugs.1-5 Melnick et al.have suggested that drug resistance might explain the failure of guanidine in the treatment of poliovirus infection in monkeys. An investigation of the inhibitory effect of 5-iodo-21deoxyuridine (I.D.U.R.) on vaccinia virus was undertaken to discover whether drug resistance occurred in other 1. 2. 3.
4. 5. 6.
albumin concentration increases, so does the combining power of the antibody. Unless this change in the valency " of the antibody with albumin concentration can be defined, it is not possible to apply the dilution principle, and the unknown albumin concentration is found empirically by interpolation into the standard curve. The reproducibility which is obtained by the method is indicated by the following values of albumin concentration- 1 - 08, 1-04, 0-99, and 1-07 mg. per 100 ml. -obtained for the same sample of urine assayed along with successive batches of urine on four different days and, on each occasion, with a new standard curve. "
Summary A
simple immunological method for estimating the
albumin concentration in human urine is described. It is sensitive and reproducible down to very low concentrations, and it requires less than 1 ml. of urine. Fifty to a hundred urine samples can be estimated in each experimental run ". "
We should like to acknowledge the assistance of Mr. N. Veall, B.SC., F.INST.P., who prepared the 1251-albumin; and to thank Mr. C. Arden, Miss R. Emerson, and Mr. M. Shaw for technical help. The work was supported by a grant from the United States Public Health Service and a research grant from the British Diabetic Association. REFERENCES
Hales, O. N., Randle, P. J. (1963) Biochem. J. 88, 137. McFarlane, A. S. (1958) Nature, Lond. 182, 53. Morgan, C. R., Lazarow, A. (1963) Diabetes, 12, 115.
viruses besides enteroviruses, and with other chemotherapeutic agents. Vaccinia virus was grown in the continuous line of human-amnion cells (Mascoli) in the presence of I.D.U.R. between 1 g. to 2 mg. per ml. The results are shown in the tables. It will be seen that vaccinia virus which had been passed in the presence of I.D.U.R. became resistant to I.D.u.R. Thus whilst normal vaccinia virus is inhibited by 2-5 or 5 g. of I.D.U.R. per ml., the strain which had been passed in I.D.U.R. was able to grow in 20 {ig. of I.D.U.R. per ml. Similar results were obtained 5-I.D.U.R. ON THE YIELDS OF 5-I.D.U.R.-SENSITIVE 5-I.D.U.R.-RESISTANT VACCINIA VIRUS
TABLE II-EFFECT OF AND
Loddo, B. Boll. Soc. ital. Biol. sper. 1962, 38, 1043. Loddo, B. ibid. p. 1047. Loddo, B., Ferrari, W., Spanedda, A., Brotzu, G. Experientia, 1962, 18, 518. Loddo, B. Boll. Soc. ital. Biol. sper. 1963, 39, 102. Loddo, B. ibid. p. 104. Melnick, J. L., Crowther, D., Barrera Oro, J. Science, 1961, 134, 557
TABLE I-EFFECT OF
5-I.D.U.R.
ON CYTOPATHIC EFFECT OF I.D.U.R.-
SENSITIVE AND I.D.U.R.-RESISTANT VACCINIA
VIRUS* *
3 hours after inoculation the cell monolayers were washed 4 times with Hanks’ solution, and then 4 ml. of maintenance medium containing
+
30 hours after the infection the cells were cryolised. Cytopathic units were titrated in stationary tube cultures of human amnion cells; readings
tris-hydroxymethyl-aminomethane 0-03% were
made after 5
See table
was
added.
days.
I.
TABLE III-INHIBITION BY THYMIDINE OF THE ANTIVIRAL ACTION OF
5-I.D.U.R., VIRUS
Test performed with human amniotic-cell cultures, in petri dishes. The compound was added 2 hours after the viral inoculum in 3 ml. of maintenance medium containing tris-hydroxymethyl-aminomethane
0.03 °o. At 30 hours after cell-culture infection. The diameter of the petri dishes used
was
40
mm.
This virus was transferred in human amnion cells (Mascoli) in the presence of the following 5-i.D.u.R. concentrations (us. per ml.): once 1, once 2, once 4, twice 10, twice 20, twice 50, twice 100, twice 200, twice 500, three times 1000 and four times 2000.
IN I.D.U.R.-SENSITIVE AND I.D.U.R.-RESISTANT VACCINIA