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Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay
ANALYTICAL
BIOCHEMISTRY
133,283-287
(1983)
Quantitation of Serum Retinol-Binding Enzyme-Linked lmmunosorbent EJAL BosIN*,‘,* *Departments
of Oncology
and tpathology,
AND NOBUO
University
of Miami
Protein by an Assay
MONJI?~ School
of Medicine,
Miami,
Florida
33101
Received December 10, 1982 An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assaydetects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and pnitrophenyl phosphate (sub strate). Calorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coe&zients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assaycan be performed in less than 7 h and has a sensitivity in the nanogram range (3-48 rig/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50-398 &ml) and urine RBP levels (mean 14, range l-80 &ml) than normal donors (mean serum 43, range 30-60 prglml; mean urine RBP 0.06, range 0.04-0.13 &ml). Liver disease patients had lower than normal serum RBP values (mean 22, range IO-43 &ml).
Human RBP“ is the specific plasma carrier for retinol (vitamin A alcohol). It is a small protein of ~2 1,000 Da and transports retinol from the liver to other tissues. As such, its major portion in plasma circulates in a complex bound to retinol and to an additional -55,000-Da protein, prealbumin (1,2). RBP is synthesized in the liver, the catabolism occurring mostly in the kidney (3). Plasma RBP concentration is reduced in vi’ To whom correspondence and reprint requests should be addressed. ’ Present address: 617 Alcazar Avenue, Coral Gables Pla. 33134. 3 Present address: Medical Research Division, Lederle Laboratories, American Cyanamid Co., Pearl River, N.Y. 10965. 4 Abbreviations used: RBP, retinol-binding protein; ELISA, enzyme-linked immunosorbcnt assay; Ig, immunoglobulin; BSA, bovine serum albumin; PNP, pnitrophenyl phosphate; DEA, diethanolamine; CBC, carbonate-bicarbonate buffer, PBS, phosphate-buffered saline; PBS-T, PBS plus 0.05% Tween-20; RT, room temperature
tamin A and protein deficiency and by hepatocellular dysfunction, whereas in renal failure it is higher than normal (3-5). Like many small proteins, RBP is partly filtered at the glomerular level and subsequently reabsorbed by the renal tubules (3). The small amounts of RBP present in normal urine are reported to increase from nano- to microand milligram levels in a variety of renal diseases, genetic and acquired (6,7). RBP recovered from urine was shown to have functional and immunologic similarities to serum RBP (S-lo). Radial immunodiffusion and radioimmunoassay are the most widely used methods for repetitive RBP analysis. The radial immunodiffusion method has an operative extent in the microgram range, precluding its use when body fluids other than blood are to be analyzed without special concentrating procedures; the latter method can be employed only at facilities where y-radiation use is routinely allowed. 283
0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved
284
BOSIN
AND
The ELISA assay for human RBP described here is simple, sensitive, low cost and precise; it can be carried out in any laboratory lacking radiation-counting facilities. MATERIALS
AND
METHODS
Monospecific Ig fraction of rabbit antisera to human RBP (145 pg/ml anti-RBP Ig) and normal rabbit serum were purchased from Accurate Chemical and Scientific Company, Hicksville, New York. Goat antibody to rabbit Ig ( 10 &ml Ig) and standard stabilized human serum containing 48-51 &ml RBP were purchased from Calbiochem-Behring Company, La Jolla, California. Bovine serum albumin (BSA), pnitrophenyl phosphate (PNP), diethanolamine (DEA), glutaraldehyde (25%), and alkaline phosphatase type VII were purchased from Sigma Chemical Company, St. Louis, Missouri. All other chemicals were obtained from Fisher Scientific Company, Fairlawn, New Jersey. Sera and spot urine specimens collected at the same time from patients diagnosed with alcohol-related liver cirrhosis and chronic renal failure and from healthy donors were provided by the Department of Pathology, Veterans Administration Medical Center, Miami, Florida, in accordance with the human research guidelines of the University. The specimens were preserved with NaN3 and stored at -20°C until ready to use. Solutions requiredfor the assay. Carbonatebicarbonate buffer (CBC), pH 9.6 (0.05 M containing 0.03 M NaN3), was used as the antibody-coating buffer. Phosphate-buffered saline (PBS), pH 7.4 (0.02 M containing 0.15 M NaCl and 0.03 M NaNJ), was used both as the washing and as the antigen buffer. PBST (PBS containing 0.05% Tween 20) was used as the enzyme buffer. The conjugate solution was freshly prepared prior to the assay by diluting the enzyme conjugate 500-fold in PBST containing 0.5% BSA. The enzyme-substrate solution was prepared by dissolving 1 mg PNP in 1 ml of a 10% DEA solution (containing 0.02% NaNr and 0.01% MgC12 6H20), pH adjusted to 9.8 with 6 N HCl.
MONJI
Preparation of conjugate. Conjugation of alkaline phosphatase to the anti-RBP Ig was carried out according to a method previously described by Voller et al. (11). Alkaline phosphatase (1 .O mg) was added to 0.6 ml of PBS containing 0.4 mg anti-RBP antibody, and the solution was dialyzed against PBS for 24 h at 4°C. To the dialyzed antibody-enzyme mixture, 2.5% glutaraldehyde was added to obtain a final concentration of 0.2%; after a thorough mixing, the solution was incubated for 2 h at room temperature (RT). The reaction mixture was dialyzed overnight against PBS at 4°C followed by further 24 h of dialysis against a 0.05 M Tris-HCl buffer, pH 8.0, at 4°C. At the end of this step, the dialysate was centrifuged for 20 min at 1OOOgand 4°C; the supematant was saved and diluted to 1.0 ml with 0.05 M Tris-HCl buffer, pH 8.0, containing 1.O% BSA and 0.02% NaNr . The prepared conjugate was stored protected from light at 4°C; under these conditions the conjugate was stable for at least 6 months. The conjugation was confirmed as follows: 0.1 ml of normal rabbit serum diluted 1:20 with 0.05 M PBS was mixed with 0.1 ml of the enzyme conjugate, itself diluted 1: 1000 with the same buffer. To above mixture, 0.2 ml of goat anti-rabbit antiserum was added and the solution was incubated for 6 h at RT. Following the incubation, the solution was centrifuged for 20 min at 1OOOg and 4°C. More than 90% of the enzyme activity was present in the precipitate. ELISA method. We first attached anti-human RBP antibody to 12 X 75-mm polystyrene tubes (W. Sarstedt, Princeton, N.J.) by passive adsorption as follows: 1.O ml of antiRBP Ig diluted 2000-fold in CBC buffer was added to a tube and incubated at least overnight at 4°C. Under these conditions, the tubes could be stored for about 2 weeks; longer storage resulted in a decrease in precision between the tubes. Just prior to the assay, the antibody solution was removed by aspiration and the tubes were washed three times with PBS. Aliquots of 0.9 ml of standard and unknown sera diluted lOOO- to 32,000-fold or urine
IMMUNOSORBENT
ASSAY FOR SERUM RETINOGBINDING
samples diluted 25-fold with PBS were added to anti-RBP-coated tubes and incubated for 2 h at RT. At the end of this step, the RBP unbound to antibody was removed by three washings with PBS, and the enzyme-conjugate solution (0.9 ml/tube) was added and incubated for 2 h at RT. After incubation, excess enzyme-conjugate was removed by washing with PBS-T (X5). Bound enzyme was assayed by adding 0.9 ml/tube of the enzyme-substrate solution followed by a 2-h incubation at 37°C. The enzymatic reaction was stopped by adding 0.1 ml/tube of a 3 N NaOH solution and the absorbance of the mixture was measured at 405 nm. The obtained data were analyzed using the Prophet system ( 12), and the standard curve was plotted as the log of RBP concen-
”
PROTEIN
tration versus net absorbance (net absorbance = sample absorbance - background absorbance). RESULTS AND DISCUSSION
The proportional displacement of the conjugated antibody by various known concentrations of RBP (3-48 @ml) is shown in Fig. 1. Four samples containing various RBP concentrations (2.5-51 @ml) were analyzed in separate experiments of at least 10 samples/ experiment; the results are shown in Table la. Seven standard curves performed on different occasions were also analyzed and the results are presented in Table 1b. The slight discrepancy between the intra- and interassay data may be due to different antigen and enzyme-
1,
5
285
10 CONCENTRATION
20
50
(nglml)
FIG. 1. Standard calibration curve of RBP. ELISA assay using standard serum (3-48 n&ml). Equation of regression: y = 0.32x + 0.08; r (correlation coefficient) = 0.98; number of points = 15.
286
BOSIN AND MONJI TABLE QUALITY
1
CONTROL DATA OF INTRAINTERASSAY VARIABILW
RBP (n&ml)
Absorbance”
No.
AND
Coefficient of variation (%I
a. Intraasay
2.5 5 25 51 b. Interassaysb 3 6 12 24 48
0.27 0.29 0.44 0.53
+ 0.01 + 0.01 +- 0.03 k 0.03
0.24 0.29 0.34 0.40 0.45
k + + + +
0.03 0.03 0.03 0.05 0.04
10 10 14 10 7 7 7 7 7
12 9 9 12 9
’ Average absorbance k SD. * Standard curves performed on different days.
conjugate preparations employed in those experiments. The coefficients of variation for intra- and interassay variability ranged between 4 and 7 and 9 and 12%, respectively. The presence of significant dilutional factors and the assay specificity were tested in experiments consisting of parallel dilutions A4
(1: 1000 to 1:32,000) of standard and clinical serum specimens. Results of such an experiment are shown in Fig. 2. Statistical analysis did not reveal a significant difference between the curves. Experiments with urine specimens showed similar behavior. The clinical samples analyzed for RBP content included serum and urine aliquots from 20 healthy donors and 17 chronic renal failure patients; 20 serum specimens from patients with alcohol related liver cirrhosis were also analyzed for RBP content. The results of these analyses are presented in Table 2. They are similar to RBP values reported in literature and obtained with different methods (4,7). We believe the assay reported here represents an improvement over the existing methodologies for RBP analysis. With respect to the radioimmunoassay technique (13), the present assay eliminates the need for radioactive counting and disposal, for a second antibody, and for high-speed centrifugation; the time constraint imposed by the inherent radioactive decay is also eliminated. The radial immunodiffision technique requires a minimum of 3 days incubation time and most of all requires sample concentration for expected low ranges with possible loss of antigen.
T
+
standard
SS
clinical
sample
+ 88
.18
>> 0
10,000
20,000
32,000
DILUTION FIG. 2. Parallel dilutions study. + = Absorbance of the standard serum; ss = ?)sorbauce of clinical specimen.
IMMUNOSORBENT
ASSAY
FOR
SERUM RETINOLBINDING
PROTEIN
IN CLINICAL
287
REFERENCES
TABLE 2 RETINOL-BINDING
PROTEIN
SPECIMENS
Serum (&ml)
Urine btiml)
Normals N= 20
43 + 12” (30-65)”
0.06 + 0.03 (0.04-O. 12)
Renal patients N= 17
158 + 95 (52-398)
14 f 21 (l-79)
Liver patients N = 20
22 + 9 (10-43)
a Mean + SD. * Range.
In conclusion, we believe the assay reported here is simple, reproducible, sensitive, and can be performed in less than 7 h with good precision. ACKNOWLEDGMENTS The technical support and assistance provided by the Department of Pathology, Veterans Administration Medical Center, Miami, Florida, gratefully acknowledged. The use of laboratory facilities of the Comprehensive Cancer Center for the State of Florida is greatly appreciated. Partial support for this research was provided by NC1 Grant 5T32CA092 12 1.
1. Lanai M., Raz, A., and Goodman De, W. S. (1968) J. C/in. Invest. 47, 2025-2044. 2. Goodman De, W. S. (1980)Ann. N. Y. Acad. Sci. 348, 378-390. 3. Peterson, P. A., Nilsson, F. S., O&erg, L., Rask, L., and Vahlquist, A. (1974) Vitam. Horm. 32, 18 l214. 4. Smith, F. R., and Goodman De, W. S. (1971) J. Clin. Invest. SO, 2426-2436. 5. Ingenbleek, Y., Van Der Schrieck, H-G., De Nayer, P., and De Visscher, M. (1975) Metabolism 24, 633-641. 6. Kanai, M., Iwanaha, T., Hagino, N., and Muto, Y. (1978) Wld. Rev. Nutr. Diet. 31, 3 l-36. 7. Scarpioni, L., DaBAngelo, P., Poisetti, P. G., and Buzio, G. (1976) Clin. Chim. Acta 68, 107-l 13. 8. Peterson, P. A., and Bergard, I. (197 1) J. Biol. Chem. 246, 25-33. 9. Fex, G., and Hansson, B. (1979) Eur. J. Biochem. 94, 307-3 13. 10. Fex, G., Albertsson, P-A., and Hansson, B. (1979) Eur. J. Biochem. 99, 353-360. 11. Voller, A., Bidwell, D., and Bartlett, A. (1976) in Manual of Clinical Immunology (Rose, N. R., and Friedman, H., eds.), Chap. 69, American Society of Microbiology, Washington, D.C. 12. Raub, W. F. (1974) Fed. Proc. 33, 2390-2392. 13. Smith, F. R., Raz, A., andGoodman De, W. S. (1970) J. Clin. Invest. 49, 1754-176 1
BIOCHEMISTRY
133,283-287
(1983)
Quantitation of Serum Retinol-Binding Enzyme-Linked lmmunosorbent EJAL BosIN*,‘,* *Departments
of Oncology
and tpathology,
AND NOBUO
University
of Miami
Protein by an Assay
MONJI?~ School
of Medicine,
Miami,
Florida
33101
Received December 10, 1982 An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assaydetects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and pnitrophenyl phosphate (sub strate). Calorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coe&zients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assaycan be performed in less than 7 h and has a sensitivity in the nanogram range (3-48 rig/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50-398 &ml) and urine RBP levels (mean 14, range l-80 &ml) than normal donors (mean serum 43, range 30-60 prglml; mean urine RBP 0.06, range 0.04-0.13 &ml). Liver disease patients had lower than normal serum RBP values (mean 22, range IO-43 &ml).
Human RBP“ is the specific plasma carrier for retinol (vitamin A alcohol). It is a small protein of ~2 1,000 Da and transports retinol from the liver to other tissues. As such, its major portion in plasma circulates in a complex bound to retinol and to an additional -55,000-Da protein, prealbumin (1,2). RBP is synthesized in the liver, the catabolism occurring mostly in the kidney (3). Plasma RBP concentration is reduced in vi’ To whom correspondence and reprint requests should be addressed. ’ Present address: 617 Alcazar Avenue, Coral Gables Pla. 33134. 3 Present address: Medical Research Division, Lederle Laboratories, American Cyanamid Co., Pearl River, N.Y. 10965. 4 Abbreviations used: RBP, retinol-binding protein; ELISA, enzyme-linked immunosorbcnt assay; Ig, immunoglobulin; BSA, bovine serum albumin; PNP, pnitrophenyl phosphate; DEA, diethanolamine; CBC, carbonate-bicarbonate buffer, PBS, phosphate-buffered saline; PBS-T, PBS plus 0.05% Tween-20; RT, room temperature
tamin A and protein deficiency and by hepatocellular dysfunction, whereas in renal failure it is higher than normal (3-5). Like many small proteins, RBP is partly filtered at the glomerular level and subsequently reabsorbed by the renal tubules (3). The small amounts of RBP present in normal urine are reported to increase from nano- to microand milligram levels in a variety of renal diseases, genetic and acquired (6,7). RBP recovered from urine was shown to have functional and immunologic similarities to serum RBP (S-lo). Radial immunodiffusion and radioimmunoassay are the most widely used methods for repetitive RBP analysis. The radial immunodiffusion method has an operative extent in the microgram range, precluding its use when body fluids other than blood are to be analyzed without special concentrating procedures; the latter method can be employed only at facilities where y-radiation use is routinely allowed. 283
0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved
284
BOSIN
AND
The ELISA assay for human RBP described here is simple, sensitive, low cost and precise; it can be carried out in any laboratory lacking radiation-counting facilities. MATERIALS
AND
METHODS
Monospecific Ig fraction of rabbit antisera to human RBP (145 pg/ml anti-RBP Ig) and normal rabbit serum were purchased from Accurate Chemical and Scientific Company, Hicksville, New York. Goat antibody to rabbit Ig ( 10 &ml Ig) and standard stabilized human serum containing 48-51 &ml RBP were purchased from Calbiochem-Behring Company, La Jolla, California. Bovine serum albumin (BSA), pnitrophenyl phosphate (PNP), diethanolamine (DEA), glutaraldehyde (25%), and alkaline phosphatase type VII were purchased from Sigma Chemical Company, St. Louis, Missouri. All other chemicals were obtained from Fisher Scientific Company, Fairlawn, New Jersey. Sera and spot urine specimens collected at the same time from patients diagnosed with alcohol-related liver cirrhosis and chronic renal failure and from healthy donors were provided by the Department of Pathology, Veterans Administration Medical Center, Miami, Florida, in accordance with the human research guidelines of the University. The specimens were preserved with NaN3 and stored at -20°C until ready to use. Solutions requiredfor the assay. Carbonatebicarbonate buffer (CBC), pH 9.6 (0.05 M containing 0.03 M NaN3), was used as the antibody-coating buffer. Phosphate-buffered saline (PBS), pH 7.4 (0.02 M containing 0.15 M NaCl and 0.03 M NaNJ), was used both as the washing and as the antigen buffer. PBST (PBS containing 0.05% Tween 20) was used as the enzyme buffer. The conjugate solution was freshly prepared prior to the assay by diluting the enzyme conjugate 500-fold in PBST containing 0.5% BSA. The enzyme-substrate solution was prepared by dissolving 1 mg PNP in 1 ml of a 10% DEA solution (containing 0.02% NaNr and 0.01% MgC12 6H20), pH adjusted to 9.8 with 6 N HCl.
MONJI
Preparation of conjugate. Conjugation of alkaline phosphatase to the anti-RBP Ig was carried out according to a method previously described by Voller et al. (11). Alkaline phosphatase (1 .O mg) was added to 0.6 ml of PBS containing 0.4 mg anti-RBP antibody, and the solution was dialyzed against PBS for 24 h at 4°C. To the dialyzed antibody-enzyme mixture, 2.5% glutaraldehyde was added to obtain a final concentration of 0.2%; after a thorough mixing, the solution was incubated for 2 h at room temperature (RT). The reaction mixture was dialyzed overnight against PBS at 4°C followed by further 24 h of dialysis against a 0.05 M Tris-HCl buffer, pH 8.0, at 4°C. At the end of this step, the dialysate was centrifuged for 20 min at 1OOOgand 4°C; the supematant was saved and diluted to 1.0 ml with 0.05 M Tris-HCl buffer, pH 8.0, containing 1.O% BSA and 0.02% NaNr . The prepared conjugate was stored protected from light at 4°C; under these conditions the conjugate was stable for at least 6 months. The conjugation was confirmed as follows: 0.1 ml of normal rabbit serum diluted 1:20 with 0.05 M PBS was mixed with 0.1 ml of the enzyme conjugate, itself diluted 1: 1000 with the same buffer. To above mixture, 0.2 ml of goat anti-rabbit antiserum was added and the solution was incubated for 6 h at RT. Following the incubation, the solution was centrifuged for 20 min at 1OOOg and 4°C. More than 90% of the enzyme activity was present in the precipitate. ELISA method. We first attached anti-human RBP antibody to 12 X 75-mm polystyrene tubes (W. Sarstedt, Princeton, N.J.) by passive adsorption as follows: 1.O ml of antiRBP Ig diluted 2000-fold in CBC buffer was added to a tube and incubated at least overnight at 4°C. Under these conditions, the tubes could be stored for about 2 weeks; longer storage resulted in a decrease in precision between the tubes. Just prior to the assay, the antibody solution was removed by aspiration and the tubes were washed three times with PBS. Aliquots of 0.9 ml of standard and unknown sera diluted lOOO- to 32,000-fold or urine
IMMUNOSORBENT
ASSAY FOR SERUM RETINOGBINDING
samples diluted 25-fold with PBS were added to anti-RBP-coated tubes and incubated for 2 h at RT. At the end of this step, the RBP unbound to antibody was removed by three washings with PBS, and the enzyme-conjugate solution (0.9 ml/tube) was added and incubated for 2 h at RT. After incubation, excess enzyme-conjugate was removed by washing with PBS-T (X5). Bound enzyme was assayed by adding 0.9 ml/tube of the enzyme-substrate solution followed by a 2-h incubation at 37°C. The enzymatic reaction was stopped by adding 0.1 ml/tube of a 3 N NaOH solution and the absorbance of the mixture was measured at 405 nm. The obtained data were analyzed using the Prophet system ( 12), and the standard curve was plotted as the log of RBP concen-
”
PROTEIN
tration versus net absorbance (net absorbance = sample absorbance - background absorbance). RESULTS AND DISCUSSION
The proportional displacement of the conjugated antibody by various known concentrations of RBP (3-48 @ml) is shown in Fig. 1. Four samples containing various RBP concentrations (2.5-51 @ml) were analyzed in separate experiments of at least 10 samples/ experiment; the results are shown in Table la. Seven standard curves performed on different occasions were also analyzed and the results are presented in Table 1b. The slight discrepancy between the intra- and interassay data may be due to different antigen and enzyme-
1,
5
285
10 CONCENTRATION
20
50
(nglml)
FIG. 1. Standard calibration curve of RBP. ELISA assay using standard serum (3-48 n&ml). Equation of regression: y = 0.32x + 0.08; r (correlation coefficient) = 0.98; number of points = 15.
286
BOSIN AND MONJI TABLE QUALITY
1
CONTROL DATA OF INTRAINTERASSAY VARIABILW
RBP (n&ml)
Absorbance”
No.
AND
Coefficient of variation (%I
a. Intraasay
2.5 5 25 51 b. Interassaysb 3 6 12 24 48
0.27 0.29 0.44 0.53
+ 0.01 + 0.01 +- 0.03 k 0.03
0.24 0.29 0.34 0.40 0.45
k + + + +
0.03 0.03 0.03 0.05 0.04
10 10 14 10 7 7 7 7 7
12 9 9 12 9
’ Average absorbance k SD. * Standard curves performed on different days.
conjugate preparations employed in those experiments. The coefficients of variation for intra- and interassay variability ranged between 4 and 7 and 9 and 12%, respectively. The presence of significant dilutional factors and the assay specificity were tested in experiments consisting of parallel dilutions A4
(1: 1000 to 1:32,000) of standard and clinical serum specimens. Results of such an experiment are shown in Fig. 2. Statistical analysis did not reveal a significant difference between the curves. Experiments with urine specimens showed similar behavior. The clinical samples analyzed for RBP content included serum and urine aliquots from 20 healthy donors and 17 chronic renal failure patients; 20 serum specimens from patients with alcohol related liver cirrhosis were also analyzed for RBP content. The results of these analyses are presented in Table 2. They are similar to RBP values reported in literature and obtained with different methods (4,7). We believe the assay reported here represents an improvement over the existing methodologies for RBP analysis. With respect to the radioimmunoassay technique (13), the present assay eliminates the need for radioactive counting and disposal, for a second antibody, and for high-speed centrifugation; the time constraint imposed by the inherent radioactive decay is also eliminated. The radial immunodiffision technique requires a minimum of 3 days incubation time and most of all requires sample concentration for expected low ranges with possible loss of antigen.
T
+
standard
SS
clinical
sample
+ 88
.18
>> 0
10,000
20,000
32,000
DILUTION FIG. 2. Parallel dilutions study. + = Absorbance of the standard serum; ss = ?)sorbauce of clinical specimen.
IMMUNOSORBENT
ASSAY
FOR
SERUM RETINOLBINDING
PROTEIN
IN CLINICAL
287
REFERENCES
TABLE 2 RETINOL-BINDING
PROTEIN
SPECIMENS
Serum (&ml)
Urine btiml)
Normals N= 20
43 + 12” (30-65)”
0.06 + 0.03 (0.04-O. 12)
Renal patients N= 17
158 + 95 (52-398)
14 f 21 (l-79)
Liver patients N = 20
22 + 9 (10-43)
a Mean + SD. * Range.
In conclusion, we believe the assay reported here is simple, reproducible, sensitive, and can be performed in less than 7 h with good precision. ACKNOWLEDGMENTS The technical support and assistance provided by the Department of Pathology, Veterans Administration Medical Center, Miami, Florida, gratefully acknowledged. The use of laboratory facilities of the Comprehensive Cancer Center for the State of Florida is greatly appreciated. Partial support for this research was provided by NC1 Grant 5T32CA092 12 1.
1. Lanai M., Raz, A., and Goodman De, W. S. (1968) J. C/in. Invest. 47, 2025-2044. 2. Goodman De, W. S. (1980)Ann. N. Y. Acad. Sci. 348, 378-390. 3. Peterson, P. A., Nilsson, F. S., O&erg, L., Rask, L., and Vahlquist, A. (1974) Vitam. Horm. 32, 18 l214. 4. Smith, F. R., and Goodman De, W. S. (1971) J. Clin. Invest. SO, 2426-2436. 5. Ingenbleek, Y., Van Der Schrieck, H-G., De Nayer, P., and De Visscher, M. (1975) Metabolism 24, 633-641. 6. Kanai, M., Iwanaha, T., Hagino, N., and Muto, Y. (1978) Wld. Rev. Nutr. Diet. 31, 3 l-36. 7. Scarpioni, L., DaBAngelo, P., Poisetti, P. G., and Buzio, G. (1976) Clin. Chim. Acta 68, 107-l 13. 8. Peterson, P. A., and Bergard, I. (197 1) J. Biol. Chem. 246, 25-33. 9. Fex, G., and Hansson, B. (1979) Eur. J. Biochem. 94, 307-3 13. 10. Fex, G., Albertsson, P-A., and Hansson, B. (1979) Eur. J. Biochem. 99, 353-360. 11. Voller, A., Bidwell, D., and Bartlett, A. (1976) in Manual of Clinical Immunology (Rose, N. R., and Friedman, H., eds.), Chap. 69, American Society of Microbiology, Washington, D.C. 12. Raub, W. F. (1974) Fed. Proc. 33, 2390-2392. 13. Smith, F. R., Raz, A., andGoodman De, W. S. (1970) J. Clin. Invest. 49, 1754-176 1