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Determination of α1-antitrypsin in fecal extracts by enzyme immunoassay
173
Ciinicu Chimica Acta, 189 (1990) 173-180 Elsevier
CCA 04755
Determination of a,-antitrypsin in fecal extracts by enzyme immunoassay Johan Brouwer and Frans Smekens Diagnostic Centre SSDZ,
De& (nie NetherIan&
(Received 30 November 1989; revision received 3 April 1990; accepted 9 April 1990) Key worak Fecal antitrypsin; Enzyme immunoassay; Reference interval
An enzyme immunoassay (EIA) has been developed for the determination of tY,-antitrypsin (AT). The assay can measure AT in fecal extracts (0.2 g wet feces/ml) from all healthy in~~du~s, Purified human AT was coupled to peroxidase by means of a heterobifunctional reagent and the conjugate was used as a labeled antigen in competitive immunoassays. Concentrations of AT in feces from healthy individuals were < 0.55 mg/g wet weight or < 2.2 mg/g dry weight. CV for the physiological day-to-day variance varied between 9 and 140%. After incubation for 48 h at 37*C, the average recovery rates for AT were 78% in ileostomy fluids, 88% in fecal extracts and 92% in feces. Results obtained with EIA correlated well with those obtained with a commercial radial immunodiffusion assay (r = 0.95).
Introduction Inflammatory bowel diseases are commonly associated with leakage of plasma into the gut lumen. Determination of fecal antitrypsin (AT) is a well-documented method for assessing protein-losing enteropathy in patients with Crohn’s disease, ulcerative colitis or inflammation due to enteric pathogens [l-4]. There is a significant correlation between ar,-AT excretion and disease activity [1,.5,6]. Because AT is resistant to proteolytic digestion within the intestinal tract its use as a marker for disease activity is independent of the site of the lesions [5,6]. Radial immunodiffusion (RID) is mostly used for the determination of AT in fecal extracts, because RID plates are commercially available [7]. As an alternative Correspondence to: Dr. J. Brouwer, Department of Clinical Chemistry, Diagnostic Centre SSDZ, P.O. Box 5010, 2600 GA Delft, The Netherlands. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers ‘B.V. (Biomedical Division)
174
to this relative insensitive and costly assay we have developed a sensitive enzyme immunoassay (EIA) which is completed within hours and can measure AT in fecal extracts (0.2 g wet wt/ml) of all normal healthy individuals. The described EIA has been used for the determination of the reference interval in healthy individuals and the physiological day-to-day variance. We also studied the in vitro stability of AT in ileostomy fluids, fecal extracts and feces at 37 ’ C. Materials and methods Materials Cohn fraction IV was a generous gift by Dr. H.G.J. Brummelhuis, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). DEAE-Sephacel, Sephacryl S-200, Sephadex G-25 and N-succinimidyl 3-(2pyridyldithio)propionate (SPDP) were obtained from Pharmacia (Woerden, The Netherlands), horseradish peroxidase (HRP, type VI) and taurocholic acid from Sigma (St. Louis, MO, USA). Materials used to perform enzyme immunoassays were the same as described previously [8]. The rabbit anti-q-antitrypsin was obtained from Dakopatts (Glostrup, Denmark). Radial immunodifusion (RID) was performed in LC-Partigenq-antitrypsin plates from Behringwerke (Marburg, FRG; Lot No. 055349A). The standards for EIA and RID were prepared from a pool of serum that contained 2.40 g AT/l as determined by nephelometry (BNA, Behringwerke). Preparation of AT-HRP conjugates AT was purified from Cohn fraction IV by the procedure described by Glaser et al. [9] which includes activation of AT, precipitation of contaminating proteins in the presence of dithiothreitol, fumed silica and ammonium sulfate, and chromatography on DEAE-Sephacel. As an additional step the preparation of AT was subjected to gel filtration on a 1.5 x 98 cm column of Sephacryl S-200 in PBS (50 mmol/l sodium phosphate, pH 7.4, 0.15 mol/l sodium chloride). The final AT preparation was pure as judged by SDS-polyacrylamide gel electrophoresis [lo]. AT was coupled to HRP by means of SPDP according to the procedure described by Carlsson et al. [ll]. The initial ratios were 24 pg SPDP/mg AT and 78 pg SPDP/mg HRP. After 30 min at room temperature the modified proteins were filtered through a column of Sephadex G-25. They contained 1.5 mol 2-pyridyl disulphide/mol AT and 1.4 mol 2-pyridyl disulphide/mol HRP, as measured spectrophotometrically at 280 and 343 nm according to the instructions supplied by the manufacturer of SPDP. Substituted HRP was reduced with dithiothreitol and a three-fold molar excess of modified HRP was mixed with substituted AT. AT-HRP conjugates and excess HRP were separated by gel filtration on a 1.5 X 98 cm column of Sephacryl S-200 in the PBS. Portions of eluted peaks were analysed by SDS slab gel electrophoresis [8]. Bovine serum albumin (BSA) was added to the
175
conjugate solution to a final concentration - 70°C.
of 10 g/l and portions were stored at
Samples
A total of 98 stool specimens were obtained from 36 healthy laboratory workers or their relatives: 18 women, aged 5-67 years (mean 34 years) and 18 men, aged 7-69 years (mean 31 years). The samples were stored at 4°C and assayed within 1 to 3 days. Portions of 1 to 3 g were lyophilized to determine the percentage of dry weight. From 28 donors we obtained portions on three non-consecutive days. These specimens were used for estimating the physiological day-to-day variance for fecal AT in healthy individuals. Ileostomy fluids were obtained from the Dept of Surgery, Reinier de Graaf Hospital (Delft, The Netherlands). PBS, containing 0.93 mmol/l taurocholic acid, was added to portions of feces (1 to 3 g wet wt) to give suspensions of 0.2 g wet wt/ml. After homogenization by vigorous shaking and vortexing for 30 min at room temperature, the suspensions were centrifuged for 5 min at 1500 X g and the supernatants were stored at - 70°C. After thawing samples were centrifuged for 5 min at 10000 x g and the final supematants were assayed in the EIA. Dilutions of standards and samples to be analysed in an EIA were made in PBS, containing BSA (10 g/l). Six dilutions of the fecal extracts were assayed in duplicate. Dilutions were mostly 1: 50 and further two-fold. The standard serum pool (see Materials) was diluted to 15 mg AT/l and stored in portions at -70°C. For the construction of standard curves in EIA this solution was diluted 1: 10 and further two-fold to concentrations of 1500, 750, 375, 188, 94 and 47 &l. A 1: 800 dilution of the standard serum (3.0 mg/l) was assayed on 30 different plates to estimate the between-run variance. Portions of the solution were stored at - 20°C and, after thawing, diluted further 1 : 10 before assay. Enzyme immunoassay
Anti-AT rabbit IgG was diluted to 10 mg/l in coating buffer (0.1 mol/l sodium carbonate, pH 9.6). Wells of PVC microtiter plates were filled with 150 ~1 of the solution, incubated for 16-20 h at room temperature and washed five times with the PBS, containing 0.05% Tween 20. For competition in an EIA 0.5 ml diluted sample was mixed with 60 ~1 diluted AT-HRP (the final dilution of the AT-HRP conjugate had been experimentally determined as the dilution in PBS, containing BSA (10 g/l), that gave an absorbance at 492 nm of about 1.8 in an EIA without competition by unlabeled AT) and 150 ~1 was added to microtiter plate wells in duplicate. Standard solutions were assayed in triplicate. Peripheral wells were filled with PBS, containing BSA (10 g/l). Plates were incubated for 2 h at 37” C and washed five times. Then 160 ~1 of chromogen solution was added: 3.7 mmol/l 0-phenylenediamine, 3.5 mmol/l hydrogen peroxide, 24 mmol/l citric acid and 51 mmol/l disodium hydrogen
176
phosphate. The reaction was allowed to proceed for 15 min in the dark at room temperature and stopped by adding 40 ,cll of 2.5 mol/l sulphuric acid. Absorbances at 492 nm were measured against wells of the first row of the plate which served as blanks.
Forty fecal extracts with AT ~n~tra~ons between 30 and 160 mg/l were assayed in RID plates. Each plate contained four standards which were prepared by diluting the standard serum in the PBS to 180, 120, 60 and 30 mg/l. A sample containing 80 mg/l was assayed in 15 different plates for estimating the between-run variance. Wells of plates were filled with 20 ~1 of the samples and incubation was for 3 days at room temperature or for 4 days at 4°C. Diameters of precipitin rings were measured to the nearest 0.1 mm and the squares of the diameters of the standards were plotted against the AT concentrations. In vitro incubation In order to study the stability of AT in vitro we incubated portions of feces, fecal extracts and ileostomy fluids for 2 days at 37OC. AT concentrations were determined before incubation and after 24 and 48 h of incubation. The fecal extracts (0.2 g wet wt/ml) were prepared as described above (see Samples). The following samples were studied: 10 portions of feces containing between 0.15 and 1.87 mg AT/g wet weight, 10 fecal extracts containing between 70 and 320 mg/l, and 3 portions of ileostomy fluids with 620, 720 and 68 mg AT/l. Other methods Electrophoresis was performed on SDS gel slabs [lo] containing a 6-20% gradient of polyacrylamide. Occasionally AT-HRP complexes were reduced by adding 50 $2-mercaptoethanol per ml sample buffer. As molecular weight markers we used proteins with M, values between 14 300 and 80 000 [8]. M, values > 80 000 were estimated by extrapolation. The standard serum used in this study was calibrated by repeated analyses in two different nephelometry systems, namely BNA (Behringwerke, Marburg, FRG) and ICS (Beckman Instruments, Mijdrecht, The Netherlands). Antibodies and standards were from the respective m~ufacturers. AT inundations in our standard serum were 2.40 g/l (BNA} and 1.81 g/l (ICS). Correlation between fecal AT and age was calculated by Spearman rank correlation and its associated test of statistical significance. Results
Analysis of the AT-HRP conjugate by means of SDS polyacrylamide gel electrophoresis showed that the conjugate contains complexes with h4, values of about 190000, 140000 and 100000. The composition of these molecules is probably
177
190
140 100 AT HRP
Fig. 1. SDS polyacrylamide gel electrophoresis of the AT-HRP conjugate in the absence (left) and in the presence (right) of a reducing agent. Numbers indicate molecular weight x 10A3.
1.6-
001
2o
I
I
1
10 [AT]
conceZLm
(pg/l)
2oo
Fig. 2. Standard curve for AT. Vertical bars represent *2 SD of each value. The part of the curve between arrows was actually used for calculating AT concentrations in fecal extracts.
178
(AT),-(HRP),, AT-(HRP), and AT-HRP. Although AT-HRP complexes and free HRP were completely separated by gel filtration on Sephacryl S-200, some free AT and HRP were seen after SDS gel electrophoresis of the conjugate (Fig. 1). This is probably due to a thiol-disulphide exchange process during preparation of the sample by heating in SDS [12]. When the mixture of the complexes between AT and HRP was used as a labeled antigen in competitive EIAs we obtained a standard curve as shown in Fig. 2. For the determination of the AT concentrations in fecal extracts we used the range between 50 and 1000 pg/l. CV for the between-run variance was 5.6% for the solution with 3.0 mg AT/l. The relation between results obtained with EIA and those with RID was AT (EIA) = 0.966 AT (RID) - 0.736 for fecal extracts containing between 30 and 160 mg AT/l. Correlation coefficient was 0.950. CV for the between-run variance in RID was 9.8% for the sample with 80 mg AT/l. AT concentrations in feces AT concentrations were measured in extracts prepared as described. Results were calculated as pg/wet weight of feces and as pg/g dry weight of feces. Ninety-eight portions of feces from 36 healthy individuals were analysed. Results in pg/g wet weight were as follows. Range: l-964; mean: 159; median: 68; SD: 193, mean + 2 SD: 545. Calculated as pg/g dry weight the results were: range: 5-3950; mean: 647; median: 287; SD: 788; mean + 2 SD: 2223. The relation between the concentrations in pg/g dry weight (y) and the concentrations in pg/g wet weight (x) was y = 3.99x + 13.65 with r = 0.977. The percentage of dry weight of the feces varied between 15 and 41% (mean 25%). The physiological day-to-day variation in fecal AT was measured for 28 individuals from whom we obtained portions on three different days. The coefficient of variation for fecal AT in different individuals varied between 9 and 140% (mean 66%). There was no statistical significant correlation between fecal AT and age of the donors. In vitro incubation studies A number of feces, fecal extracts and small bowel fluids were incubated at 37 o C. AT concentrations were determined before incubation and after incubation for 24 and 48 h. Recoveries of AT, expressed as a percentage of the initial concentrations, were as follows. In feces (n = 10) between 80 and 105% (mean 97) after 24 h and between 66 and 105% (mean 91) after 48 h. In fecal extracts (n = 10) between 80 and 105% (mean 92) after 24 h and between 72 and 105% (mean 88) after 48 h. In ileostomy fluids (n = 3) between 69 and 96% (mean 84) after 24 h and between 65 and 90% (mean 78) after 48 h. Discussion
The costs of reagents in the described EIA are very low and, therefore, the assay is particularly suited for processing large numbers of samples. Ail reagents are
179
co~erci~y available. AT can eventually be purified from Cohn fraction IV, a by-product during the production of albumin, IgG etc. [9]. Purified AT is not required in a sandwich EIA in which microtiter plates, after coating with anti-AT, are successively incubated with sample and peroxidase-labeled anti-AT. In preliminary experiments we have used such an assay and found it to be very sensitive, the detection limit being < 10 pg/l. Unfortunately the dilution curves obtained with different fecal extracts were not parallel to each other or to the standard curve. The described competitive EIA did not show that problem. Besides it requires oniy one incubation step. For the calculation of the normal upper limit (mean + 2 SD) for fecal AT we used the values determined in 98 different portions, although obtained from 36 donors, because the intra-indi~dual day-to-day variation is of the same order as the inter-indi~dual variation. In this way we determined a normal upper limit for fecal AT of 0.55 mg/g wet weight or 2.2 mg/g dry weight, which is in fair agreement with the values reported by other authors [1,3,5,7]. It should be realized that in almost all studies only RID plates of Behring have been used. Wilson et al. [7] have found that AT concentrations measured in RID plates of Calbiochem (Behring) were 30% higher than the values measured in RID plates of Helena. The AT concentration in our standard serum as determined in the Behring Nephelometer Analyzer was 1.33-times higher than the value measured in the Beckman system. So it seems that, compared with other manufacturers, Behring has assigned a relative high value for AT in their protein standard. The very good correlation between values for fecal AT in pg/g wet weight and those in pg/g dry weight is valid only for feces with a normal consistence. When diarrhoeal stool samples of patients are analysed fecal AT concentrations are preferably calculated as mg AT/g dry weight [7,13]. Resistance of AT to proteolytic digestion in the intestinal tract is one of the main reasons why fecal AT is used as a marker for protein-losing enteropathy. Our in vitro incubation studies with feces and fecal extracts confirmed previous described results 121.A more strong decrease of fecal AT was found during incubation of a few ileostomy fluids. In view of the physiological day-to-day variation in fecal AT this decrease was not such that it will affect the usefulness of fecal AT for following the activity of small bowel inflammation. References 1 Thomas DW, Sinatra FR, Merrit RI. Random fecal n,-antitrypsin concentration in children with gastrointestinal disease. Gastroenterology 1981;80:776-782. 2 Florent C, L’Hirondel C, Desmazures C, Aymes C, Bernier JJ. Intestinal clearance of a,-antitrypsin: a sensitive method for the detection of protein-losing enteropathy. Gastroenterology 1981;81:777-780. 3 Rybolt AH, Bennet RG, Laughon BE, Thomas DR, Greenough WB, Bartlett JG. Protein-losing enteropathy associated with Clostridium dif&cile infection. The Lancet 1989;i:1353-1355. 4 Bhan MK, Khoshoo V, Chowdhary D, et al. Increased fecal al-antitrypsin excretion in children with persistent diarrhoea associated with enteric pathogens. Acta Paediatr Stand 1989;78:265-267. 5 Meyers S, Wolke A, Field SP, Feuer El, Johnson JW, Janowitz HD. Fecal q-antitrypsin measurement: an indicator of Crohn’s disease activity. Castroenterology 1985;89:13-18.
180
6 Bohbout GE. Activity related abnormalities in inflammatory bowel disease. Ph.D. Thesis, State University L&den, The Netherlands, 1988. 7 Wilson CM, MC Gill&n K, Thomas DW. Determination of fecal a,-antitrypsin concentration by radial immunodiffusion: two systems compared. Clin Chem 1988;34:372-376. 8 Brouwer J, Van Leeuwen-Herberts GHHM, Otting van de Ruit MJ. Determination of lysozyme in serum, urine, cerebrospinal fluid and feces by enzyme immunoassay. Clin Chim Acta 1984;142:21-30. 9 Glaser CB, Chamorro M, Crowley R, Karic L, Childs A, Calderon M. The isolation of a,-protease inhibitor by a unique procedure designed for industrial application. Anal Biochem 1982;124:364-371. 10 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680-685. 11 Carlsson J, Drevin H, Axtn R. Protein thiolation and reversible protein-protein conjugation: a new heterobifunctional reagent. Biochem J N-succinimidyl 3-(2-pyridyldithio)propionate, 1978;173:723-737. 12 Victoria El, Mahan LC, Massouredis SP. Immunoglobuhn G disassembly during thermal denaturation in sodium dodecyl sulfate solutions. Biochemistry 1977;2566-2569. 13 Catassi C, Cardinah E, D’Angeio G, Coppa GV, Giorgi PL. Reliability of random fecal a,-antitrypsin determination on non-dried stools. J Pediatr 1986;109:500-502.
Ciinicu Chimica Acta, 189 (1990) 173-180 Elsevier
CCA 04755
Determination of a,-antitrypsin in fecal extracts by enzyme immunoassay Johan Brouwer and Frans Smekens Diagnostic Centre SSDZ,
De& (nie NetherIan&
(Received 30 November 1989; revision received 3 April 1990; accepted 9 April 1990) Key worak Fecal antitrypsin; Enzyme immunoassay; Reference interval
An enzyme immunoassay (EIA) has been developed for the determination of tY,-antitrypsin (AT). The assay can measure AT in fecal extracts (0.2 g wet feces/ml) from all healthy in~~du~s, Purified human AT was coupled to peroxidase by means of a heterobifunctional reagent and the conjugate was used as a labeled antigen in competitive immunoassays. Concentrations of AT in feces from healthy individuals were < 0.55 mg/g wet weight or < 2.2 mg/g dry weight. CV for the physiological day-to-day variance varied between 9 and 140%. After incubation for 48 h at 37*C, the average recovery rates for AT were 78% in ileostomy fluids, 88% in fecal extracts and 92% in feces. Results obtained with EIA correlated well with those obtained with a commercial radial immunodiffusion assay (r = 0.95).
Introduction Inflammatory bowel diseases are commonly associated with leakage of plasma into the gut lumen. Determination of fecal antitrypsin (AT) is a well-documented method for assessing protein-losing enteropathy in patients with Crohn’s disease, ulcerative colitis or inflammation due to enteric pathogens [l-4]. There is a significant correlation between ar,-AT excretion and disease activity [1,.5,6]. Because AT is resistant to proteolytic digestion within the intestinal tract its use as a marker for disease activity is independent of the site of the lesions [5,6]. Radial immunodiffusion (RID) is mostly used for the determination of AT in fecal extracts, because RID plates are commercially available [7]. As an alternative Correspondence to: Dr. J. Brouwer, Department of Clinical Chemistry, Diagnostic Centre SSDZ, P.O. Box 5010, 2600 GA Delft, The Netherlands. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers ‘B.V. (Biomedical Division)
174
to this relative insensitive and costly assay we have developed a sensitive enzyme immunoassay (EIA) which is completed within hours and can measure AT in fecal extracts (0.2 g wet wt/ml) of all normal healthy individuals. The described EIA has been used for the determination of the reference interval in healthy individuals and the physiological day-to-day variance. We also studied the in vitro stability of AT in ileostomy fluids, fecal extracts and feces at 37 ’ C. Materials and methods Materials Cohn fraction IV was a generous gift by Dr. H.G.J. Brummelhuis, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). DEAE-Sephacel, Sephacryl S-200, Sephadex G-25 and N-succinimidyl 3-(2pyridyldithio)propionate (SPDP) were obtained from Pharmacia (Woerden, The Netherlands), horseradish peroxidase (HRP, type VI) and taurocholic acid from Sigma (St. Louis, MO, USA). Materials used to perform enzyme immunoassays were the same as described previously [8]. The rabbit anti-q-antitrypsin was obtained from Dakopatts (Glostrup, Denmark). Radial immunodifusion (RID) was performed in LC-Partigenq-antitrypsin plates from Behringwerke (Marburg, FRG; Lot No. 055349A). The standards for EIA and RID were prepared from a pool of serum that contained 2.40 g AT/l as determined by nephelometry (BNA, Behringwerke). Preparation of AT-HRP conjugates AT was purified from Cohn fraction IV by the procedure described by Glaser et al. [9] which includes activation of AT, precipitation of contaminating proteins in the presence of dithiothreitol, fumed silica and ammonium sulfate, and chromatography on DEAE-Sephacel. As an additional step the preparation of AT was subjected to gel filtration on a 1.5 x 98 cm column of Sephacryl S-200 in PBS (50 mmol/l sodium phosphate, pH 7.4, 0.15 mol/l sodium chloride). The final AT preparation was pure as judged by SDS-polyacrylamide gel electrophoresis [lo]. AT was coupled to HRP by means of SPDP according to the procedure described by Carlsson et al. [ll]. The initial ratios were 24 pg SPDP/mg AT and 78 pg SPDP/mg HRP. After 30 min at room temperature the modified proteins were filtered through a column of Sephadex G-25. They contained 1.5 mol 2-pyridyl disulphide/mol AT and 1.4 mol 2-pyridyl disulphide/mol HRP, as measured spectrophotometrically at 280 and 343 nm according to the instructions supplied by the manufacturer of SPDP. Substituted HRP was reduced with dithiothreitol and a three-fold molar excess of modified HRP was mixed with substituted AT. AT-HRP conjugates and excess HRP were separated by gel filtration on a 1.5 X 98 cm column of Sephacryl S-200 in the PBS. Portions of eluted peaks were analysed by SDS slab gel electrophoresis [8]. Bovine serum albumin (BSA) was added to the
175
conjugate solution to a final concentration - 70°C.
of 10 g/l and portions were stored at
Samples
A total of 98 stool specimens were obtained from 36 healthy laboratory workers or their relatives: 18 women, aged 5-67 years (mean 34 years) and 18 men, aged 7-69 years (mean 31 years). The samples were stored at 4°C and assayed within 1 to 3 days. Portions of 1 to 3 g were lyophilized to determine the percentage of dry weight. From 28 donors we obtained portions on three non-consecutive days. These specimens were used for estimating the physiological day-to-day variance for fecal AT in healthy individuals. Ileostomy fluids were obtained from the Dept of Surgery, Reinier de Graaf Hospital (Delft, The Netherlands). PBS, containing 0.93 mmol/l taurocholic acid, was added to portions of feces (1 to 3 g wet wt) to give suspensions of 0.2 g wet wt/ml. After homogenization by vigorous shaking and vortexing for 30 min at room temperature, the suspensions were centrifuged for 5 min at 1500 X g and the supernatants were stored at - 70°C. After thawing samples were centrifuged for 5 min at 10000 x g and the final supematants were assayed in the EIA. Dilutions of standards and samples to be analysed in an EIA were made in PBS, containing BSA (10 g/l). Six dilutions of the fecal extracts were assayed in duplicate. Dilutions were mostly 1: 50 and further two-fold. The standard serum pool (see Materials) was diluted to 15 mg AT/l and stored in portions at -70°C. For the construction of standard curves in EIA this solution was diluted 1: 10 and further two-fold to concentrations of 1500, 750, 375, 188, 94 and 47 &l. A 1: 800 dilution of the standard serum (3.0 mg/l) was assayed on 30 different plates to estimate the between-run variance. Portions of the solution were stored at - 20°C and, after thawing, diluted further 1 : 10 before assay. Enzyme immunoassay
Anti-AT rabbit IgG was diluted to 10 mg/l in coating buffer (0.1 mol/l sodium carbonate, pH 9.6). Wells of PVC microtiter plates were filled with 150 ~1 of the solution, incubated for 16-20 h at room temperature and washed five times with the PBS, containing 0.05% Tween 20. For competition in an EIA 0.5 ml diluted sample was mixed with 60 ~1 diluted AT-HRP (the final dilution of the AT-HRP conjugate had been experimentally determined as the dilution in PBS, containing BSA (10 g/l), that gave an absorbance at 492 nm of about 1.8 in an EIA without competition by unlabeled AT) and 150 ~1 was added to microtiter plate wells in duplicate. Standard solutions were assayed in triplicate. Peripheral wells were filled with PBS, containing BSA (10 g/l). Plates were incubated for 2 h at 37” C and washed five times. Then 160 ~1 of chromogen solution was added: 3.7 mmol/l 0-phenylenediamine, 3.5 mmol/l hydrogen peroxide, 24 mmol/l citric acid and 51 mmol/l disodium hydrogen
176
phosphate. The reaction was allowed to proceed for 15 min in the dark at room temperature and stopped by adding 40 ,cll of 2.5 mol/l sulphuric acid. Absorbances at 492 nm were measured against wells of the first row of the plate which served as blanks.
Forty fecal extracts with AT ~n~tra~ons between 30 and 160 mg/l were assayed in RID plates. Each plate contained four standards which were prepared by diluting the standard serum in the PBS to 180, 120, 60 and 30 mg/l. A sample containing 80 mg/l was assayed in 15 different plates for estimating the between-run variance. Wells of plates were filled with 20 ~1 of the samples and incubation was for 3 days at room temperature or for 4 days at 4°C. Diameters of precipitin rings were measured to the nearest 0.1 mm and the squares of the diameters of the standards were plotted against the AT concentrations. In vitro incubation In order to study the stability of AT in vitro we incubated portions of feces, fecal extracts and ileostomy fluids for 2 days at 37OC. AT concentrations were determined before incubation and after 24 and 48 h of incubation. The fecal extracts (0.2 g wet wt/ml) were prepared as described above (see Samples). The following samples were studied: 10 portions of feces containing between 0.15 and 1.87 mg AT/g wet weight, 10 fecal extracts containing between 70 and 320 mg/l, and 3 portions of ileostomy fluids with 620, 720 and 68 mg AT/l. Other methods Electrophoresis was performed on SDS gel slabs [lo] containing a 6-20% gradient of polyacrylamide. Occasionally AT-HRP complexes were reduced by adding 50 $2-mercaptoethanol per ml sample buffer. As molecular weight markers we used proteins with M, values between 14 300 and 80 000 [8]. M, values > 80 000 were estimated by extrapolation. The standard serum used in this study was calibrated by repeated analyses in two different nephelometry systems, namely BNA (Behringwerke, Marburg, FRG) and ICS (Beckman Instruments, Mijdrecht, The Netherlands). Antibodies and standards were from the respective m~ufacturers. AT inundations in our standard serum were 2.40 g/l (BNA} and 1.81 g/l (ICS). Correlation between fecal AT and age was calculated by Spearman rank correlation and its associated test of statistical significance. Results
Analysis of the AT-HRP conjugate by means of SDS polyacrylamide gel electrophoresis showed that the conjugate contains complexes with h4, values of about 190000, 140000 and 100000. The composition of these molecules is probably
177
190
140 100 AT HRP
Fig. 1. SDS polyacrylamide gel electrophoresis of the AT-HRP conjugate in the absence (left) and in the presence (right) of a reducing agent. Numbers indicate molecular weight x 10A3.
1.6-
001
2o
I
I
1
10 [AT]
conceZLm
(pg/l)
2oo
Fig. 2. Standard curve for AT. Vertical bars represent *2 SD of each value. The part of the curve between arrows was actually used for calculating AT concentrations in fecal extracts.
178
(AT),-(HRP),, AT-(HRP), and AT-HRP. Although AT-HRP complexes and free HRP were completely separated by gel filtration on Sephacryl S-200, some free AT and HRP were seen after SDS gel electrophoresis of the conjugate (Fig. 1). This is probably due to a thiol-disulphide exchange process during preparation of the sample by heating in SDS [12]. When the mixture of the complexes between AT and HRP was used as a labeled antigen in competitive EIAs we obtained a standard curve as shown in Fig. 2. For the determination of the AT concentrations in fecal extracts we used the range between 50 and 1000 pg/l. CV for the between-run variance was 5.6% for the solution with 3.0 mg AT/l. The relation between results obtained with EIA and those with RID was AT (EIA) = 0.966 AT (RID) - 0.736 for fecal extracts containing between 30 and 160 mg AT/l. Correlation coefficient was 0.950. CV for the between-run variance in RID was 9.8% for the sample with 80 mg AT/l. AT concentrations in feces AT concentrations were measured in extracts prepared as described. Results were calculated as pg/wet weight of feces and as pg/g dry weight of feces. Ninety-eight portions of feces from 36 healthy individuals were analysed. Results in pg/g wet weight were as follows. Range: l-964; mean: 159; median: 68; SD: 193, mean + 2 SD: 545. Calculated as pg/g dry weight the results were: range: 5-3950; mean: 647; median: 287; SD: 788; mean + 2 SD: 2223. The relation between the concentrations in pg/g dry weight (y) and the concentrations in pg/g wet weight (x) was y = 3.99x + 13.65 with r = 0.977. The percentage of dry weight of the feces varied between 15 and 41% (mean 25%). The physiological day-to-day variation in fecal AT was measured for 28 individuals from whom we obtained portions on three different days. The coefficient of variation for fecal AT in different individuals varied between 9 and 140% (mean 66%). There was no statistical significant correlation between fecal AT and age of the donors. In vitro incubation studies A number of feces, fecal extracts and small bowel fluids were incubated at 37 o C. AT concentrations were determined before incubation and after incubation for 24 and 48 h. Recoveries of AT, expressed as a percentage of the initial concentrations, were as follows. In feces (n = 10) between 80 and 105% (mean 97) after 24 h and between 66 and 105% (mean 91) after 48 h. In fecal extracts (n = 10) between 80 and 105% (mean 92) after 24 h and between 72 and 105% (mean 88) after 48 h. In ileostomy fluids (n = 3) between 69 and 96% (mean 84) after 24 h and between 65 and 90% (mean 78) after 48 h. Discussion
The costs of reagents in the described EIA are very low and, therefore, the assay is particularly suited for processing large numbers of samples. Ail reagents are
179
co~erci~y available. AT can eventually be purified from Cohn fraction IV, a by-product during the production of albumin, IgG etc. [9]. Purified AT is not required in a sandwich EIA in which microtiter plates, after coating with anti-AT, are successively incubated with sample and peroxidase-labeled anti-AT. In preliminary experiments we have used such an assay and found it to be very sensitive, the detection limit being < 10 pg/l. Unfortunately the dilution curves obtained with different fecal extracts were not parallel to each other or to the standard curve. The described competitive EIA did not show that problem. Besides it requires oniy one incubation step. For the calculation of the normal upper limit (mean + 2 SD) for fecal AT we used the values determined in 98 different portions, although obtained from 36 donors, because the intra-indi~dual day-to-day variation is of the same order as the inter-indi~dual variation. In this way we determined a normal upper limit for fecal AT of 0.55 mg/g wet weight or 2.2 mg/g dry weight, which is in fair agreement with the values reported by other authors [1,3,5,7]. It should be realized that in almost all studies only RID plates of Behring have been used. Wilson et al. [7] have found that AT concentrations measured in RID plates of Calbiochem (Behring) were 30% higher than the values measured in RID plates of Helena. The AT concentration in our standard serum as determined in the Behring Nephelometer Analyzer was 1.33-times higher than the value measured in the Beckman system. So it seems that, compared with other manufacturers, Behring has assigned a relative high value for AT in their protein standard. The very good correlation between values for fecal AT in pg/g wet weight and those in pg/g dry weight is valid only for feces with a normal consistence. When diarrhoeal stool samples of patients are analysed fecal AT concentrations are preferably calculated as mg AT/g dry weight [7,13]. Resistance of AT to proteolytic digestion in the intestinal tract is one of the main reasons why fecal AT is used as a marker for protein-losing enteropathy. Our in vitro incubation studies with feces and fecal extracts confirmed previous described results 121.A more strong decrease of fecal AT was found during incubation of a few ileostomy fluids. In view of the physiological day-to-day variation in fecal AT this decrease was not such that it will affect the usefulness of fecal AT for following the activity of small bowel inflammation. References 1 Thomas DW, Sinatra FR, Merrit RI. Random fecal n,-antitrypsin concentration in children with gastrointestinal disease. Gastroenterology 1981;80:776-782. 2 Florent C, L’Hirondel C, Desmazures C, Aymes C, Bernier JJ. Intestinal clearance of a,-antitrypsin: a sensitive method for the detection of protein-losing enteropathy. Gastroenterology 1981;81:777-780. 3 Rybolt AH, Bennet RG, Laughon BE, Thomas DR, Greenough WB, Bartlett JG. Protein-losing enteropathy associated with Clostridium dif&cile infection. The Lancet 1989;i:1353-1355. 4 Bhan MK, Khoshoo V, Chowdhary D, et al. Increased fecal al-antitrypsin excretion in children with persistent diarrhoea associated with enteric pathogens. Acta Paediatr Stand 1989;78:265-267. 5 Meyers S, Wolke A, Field SP, Feuer El, Johnson JW, Janowitz HD. Fecal q-antitrypsin measurement: an indicator of Crohn’s disease activity. Castroenterology 1985;89:13-18.
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6 Bohbout GE. Activity related abnormalities in inflammatory bowel disease. Ph.D. Thesis, State University L&den, The Netherlands, 1988. 7 Wilson CM, MC Gill&n K, Thomas DW. Determination of fecal a,-antitrypsin concentration by radial immunodiffusion: two systems compared. Clin Chem 1988;34:372-376. 8 Brouwer J, Van Leeuwen-Herberts GHHM, Otting van de Ruit MJ. Determination of lysozyme in serum, urine, cerebrospinal fluid and feces by enzyme immunoassay. Clin Chim Acta 1984;142:21-30. 9 Glaser CB, Chamorro M, Crowley R, Karic L, Childs A, Calderon M. The isolation of a,-protease inhibitor by a unique procedure designed for industrial application. Anal Biochem 1982;124:364-371. 10 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680-685. 11 Carlsson J, Drevin H, Axtn R. Protein thiolation and reversible protein-protein conjugation: a new heterobifunctional reagent. Biochem J N-succinimidyl 3-(2-pyridyldithio)propionate, 1978;173:723-737. 12 Victoria El, Mahan LC, Massouredis SP. Immunoglobuhn G disassembly during thermal denaturation in sodium dodecyl sulfate solutions. Biochemistry 1977;2566-2569. 13 Catassi C, Cardinah E, D’Angeio G, Coppa GV, Giorgi PL. Reliability of random fecal a,-antitrypsin determination on non-dried stools. J Pediatr 1986;109:500-502.