Quantitation of Canine Plasma von Willebrand Factor Antigen Using a Commercial Enzyme-linked Immunosorbent Assay I.B. Johnstone and S. Crane
ABSTRACT The purpose of this study was to evaluate a commercial enzyme-linked immunosorbent assay (ELISA) for human von Willebrand factor antigen (vWF:Ag) with respect to its potential value in quantitating the protein in canine plasma. The assay was a sandwich technique using F(ab')2 fragments specific for von Willebrand factor (vWF) and a peroxidase conjugated rabbit anti-vWF second antibody, with a microplate as the support surface. Canine plasmas were assayed by ELISA, and by Laurell electroimmunoassay (EIA), our reference methodology. The ELISA had a within-day variation of 1.21-4.44% and a between-day variation of 0.854.88% depending on the level of vWF:Ag. The sensitivity of the assay was less than 0.1% vWF:Ag. The range of vWF:Ag concentrations in plasmas from 24 clinically normal dogs compared favorably with the range for the same plasmas when assayed by EIA (ELISA = 60-152% of normal; EIA = 50-142% of normal). In 121 canine plasmas with vWF:Ag concentrations (as assessed by EIA) ranging from undetectable levels (< 6% of normal) to 142% of normal, there was good correlation with measurements made by ELISA (correlation coefficient = 0.835). It was concluded that this commercial ELISA technique could be used to provide reliable, same-day measurements of canine plasma vWF:Ag. Since it requires no special equipment other than a microplate reader and washer it is particularly suitable for
laboratories lacking the electrophoretic expertise or equipment required for EIA. RESUME Le but de cette etude etait d'evaluer test ELISA (enzyme-linked immunosorbent assay) commercial pour la detection de l'antigene du facteur von Willebrand humain (Ag: vWF) en rapport avec sa valeur potentielle de quantifier cette proteine dans du plasma canin. L'essai fut une technique dite "sandwich", utilisant le fragment F(ab')2, specifique pour le facteur von Willebrand (vWF) et un anticorps de lapin anti-vWF conjugue, avec une microplaque comme support de surface. Les echantillons de plasma furent testes avec la technique ELISA et la technique EIA (electroimmunoassay) de Laurell, utilisee comme technique de reference. L'ELISA a montre une variation intrajournaliere de 1,21-4,44% et une variation interjournaliere de 0,854,88%, dependamment du niveau d'antigenes Ag:vWF. La sensibilite du test fut de moins de 0,1% d'Ag:vWF. Les niveaux de concentration d'Ag:vWF dans le plasma de 24 chiens cliniquement normaux se sont favorablement compares avec les niveaux obtenus pour les memes echantillons lorsque testes avec la technique EIA (ELISA = 60-152% du plasma de reference normal; EIA = 50-142% du plasma de reference normal). Dans 121 echantillons de plasma, les concentrations d'Ag:vWF (comme lorsque testes par EIA) se situant entre un
les niveaux non-detectables (< 6% du plasma de reference normal) jusqu'a 142% du plasma de reference normal, il y eut une bonne correlation avec les mesures obtenues par ELISA (coefficient de correlation = 0.835). II fut conclu que la technique commerciale ELISA pourrait etre utilisee pour obtenir des mesures flables, dans un delai de 24 heures, d'Ag:vWF, a partir de plasma de chiens. Comme cette technique ne necessite aucun equipement specifique mis a part un lecteur et un laveur de microplaque, elle est particulierement designee pour les laboratoires ne possedant pas d'expertise electrophoretique ou l'equipement necessaire pour realiser la technique EIA. (Traduit par Dr A nne Provencher)
INTRODUCTION Von Willebrand's disease (vWD) and classical hemophilia (Factor VIII deficiency) are the two most common inherited bleeding disorders of dogs (1,2). Quantitation of plasma von Willebrand factor antigen (vWF:Ag) is critical to the diagnosis of vWD, and is useful in confirming factor VIII deficiency and in predicting the asymptomatic female carriers of hemophilia (3). Presently the most common method of quantitating canine vWF:Ag is by Laurell electroimmunoassay (EIA). This technique however requires expertise in electrophoresis, tends to be labor intensive, and may be limited by gel size to a relatively small sample load. The sensitivity of the EIA is
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario NI G 2W 1. This research was supported in part by a grant from the Ontario Veterinary College Pet Trust Fund. Submitted June 10, 1990.
Can J Vet Res 1991; 55: 11-14
I1I
usually only about 3-6% of normal plasma vWF:Ag concentrations. In laboratories which test human samples, attempts have been made to increase the sensitivity of the EIA procedure by concomitant enzyme enhancement or radiolabelling (6-8). Fluoroimmunoassays and radioimmunoassays have also been described but these too require special equipment and expertise (7,9,10). Enzyme-linked immunosorbent assays (ELISA), using equipment common to most diagnostic laboratories, offer a more practical means of quantitating vWF:Ag, and are being used with increasing frequency to quantitate human vWF:Ag (11,12). Commercially available ELISA kits for measuring human vWF:Ag are now available, but kits using canine specific reagents are not. Since there appears to be good cross-reactivity between canine vWF:Ag and antibodies produced against human vWF, we undertook to evaluate a commercially available ELISA test kit for its potential application in quantitating canine plasma vWF:Ag (5,13).
MATERIALS AND METHODS PLASMA SAMPLES
Canine blood samples were collected in 3.8% trisodium citrate at a ratio of 9:1 (blood:anticoagulant). The citrated blood was immediately centrifuged at 40 C for 15 min at 2500 g and the supernatant platelet-poor plasma (PPP) was removed. This PPP was divided into aliquots and held at -70° C until assayed by ELISA and EIA. The reference plasma was a preparation of PPP pooled from 12 clinically normal dogs and frozen in aliquots at -70° C. This plasma was arbitrarily designated as containing 100% vWF:Ag. Individual plasmas were measured against this standard and vWF:Ag was expressed as a percent of normal. ENZYME-LINKED IMMUNOSORBENT ASSAY
Quantitation of canine vWF:Ag by ELISA was carried out using a commercial reagent kit for measuring human vWF:Ag (Asserachrome vWF; 12
to each well. After exactly 3.5 min of incubation at room temperature, the
Diagnostica Stago, Asnieres-SurSeine, France). This kit consisted of five components: a buffered coating solution containing rabbit anti-vWF F(ab')2 fragments (freeze-dried), concentrated diluent and buffer solutions, anti-vWF rabbit immunoglobulins coupled to peroxidase (freeze-dried), and orthophenylene diamine (OPD) substrate (freezedried). The procedure was carried out by the manufacturers' directions with only minor modifications. Briefly, a microplate (Nunc Immunoplate IF, Nunc, Copenhagen, Denmark) was coated by placing 200 IAL of reconstituted coating solution in each well and incubating overnight at room temperature in the covered microplate. Each well was then washed five times with diluted washing solution using a manual plate washer (Model EL 40108, Bio-Tek Corp., Burlington, Vermont). The assay consisted first of the addition of 200 ,uL of diluted plasma to each of two coated wells (all plasma samples were assayed in duplicate). After a 2 h incubation at room temperature the plate was washed five times with washing solution, then 200 ,L of reconstituted enzymeantibody conjugate were added to each well. After a further 2 h incubation at room temperature the plate was washed as before and 200 ,uL of OPD substrate solution were added
conjugated antibody enzymesubstrate reaction was stopped by the addition of 50 ,L of 3M H2SO4. Ten minutes later the optical density was measured at 490 nm using a microplate reader (Model EL 308, Bio-Tek Corp., Burlington, Vermont). Initial dose-response curves were generated using the canine reference plasma at plasma dilutions ranging from 1:25 to 1:6400 (Fig. 1). Dilutions of 1:50, 1:100, 1:200, 1:500 and 1:1000 were ultimately used to prepare standard curves in the vWF:Ag assay, and individual plasmas were assayed in duplicate at dilutions of 1:50 or 1:100. ELECTROIMMUNOSSAY
Plasma vWF:Ag was assayed using a Laurell electroimmunoassay with rabbit antihuman antiserum as previously described (13).
RESULTS DOSE RESPONSE CURVE
A typical dose-response curve using a log-log plot is shown in Fig. 1. The response was linear over a wide range of plasma dilutions, however the range 1:50-1:1000 was selected for establishing the calibration curve in subsequent vWF:Ag assays.
10.00
1.00 E 0
0.10±
0.01
i
10
.
.
.
..0 10
.
.
.
.
.
1
i
i
1 000
i
i
i i
1 E4
RECIPROCAL OF PLASMA DILUTION Fig. 1. Typical dose-response curve for canine vWF:Ag quantitated by enzyme-linked immunosorbent assay (normal reference plasma; n = 6).
PRECISION STUDIES
Samples of plasma with three different levels of vWF:Ag (as previously determined by EIA) were used in the precision studies. These plasmas were selected to cover low, intermediate, and normal levels of vWF:Ag, particularly relevant in testing for vWF:Ag deficiency. (1,2,14). Within-day variability was assessed from 12 replicate determinations on the same plasma. As shown in Table I, intra-assay variation ranged from 1.21% to 4.44% (coefficient of variation). Between-day variability was assessed from four determinations on aliquots of the same plasma assayed at weekly intervals. Interassay variability ranged from 0.85% to 4.88% (Table I).
TABLE II. Plasma von Willebrand factor antigen in 24 clinically normal dogs as quantitated by enzyme-linked immunosorbent assay (ELISA) and Laurell electroimmunoassay (EIA)
Assay
Meana ELISA 95 EIA 90 aExpressed as % of normal reference plasma
ELISA. For purposes of data analysis, samples having undetectable vWF:Ag by EIA were designated as having a level of 6% of normal (limit of sensitivity). As shown in Fig. 2 there was good correlation (r = 0.835) between the two methods of vWF:Ag quantitation. Nonagreement was most evident at the extreme high and low ends of the vWF:Ag range studied. At NORMAL RANGE low vWF:Ag concentrations, the When plasma samples from 24 ELISA values tended to be higher clinically normal adult dogs were than the EIA values with 11 of the 12 assayed for vWF:Ag by ELISA , the samples which had undetectable levels vWF:Ag levels ranged from 60% to of vWF:Ag (< 6% of normal) by EIA 152% of normal, with a mean of 95% showing measurable levels in excess of of normal (Table II). In comparison, 6% of normal by ELISA. Of the seven EIA determinations on the same samples with vWF:Ag concentrations plasmas yielded a range of 50% to above 130% of normal (by EIA), all 142% of normal, with a mean of 90% had somewhat lower levels by ELISA of normal. The correlation coefficient but still in excess of 100% of the level for measurements by these two in the reference plasma. methods was r = 0.767.
Correlation coefficient (r)
Rangea 60-152 50-142
0.767
EIA and ELISA techniques. There good correlation between the two methods. The ELISA technique produced reliable same-day results, was easy to perform and, with a microplate as the coating support, was suitable for large sample numbers. Up to 42 plasma samples could be assayed in duplicate with appropriate controls at one time. The only major equipment required was a microplate reader (spectrophotometer). These were significant advantages over the EIA technique which required special electrophoresis expertise and equipment, and needed two days to was
complete. The precision of the ELISA was good with coefficients of variation ranging from 1.21% to 4.44% within batch and 0.85% to 4.88% between batch. These variations compare
favorably with results for fluoroim-
COMPARISON OF ELISA AND EIA MEASUREMENTS ON PLASMAS WITH NORMAL AND SUBNORMAL vWF:Ag CONCENTRATIONS
One hundred and twenty-one plasma samples from clinically normal dogs and dogs previously determined to have subnormal plasma vWF:Ag concentrations (< 50% of normal by EIA) were assayed by both EIA and
munoassays
and other types of
DISCUSSION
ELISA, and are better than those generally reported for EIA techniques In this study, vWF:Ag in canine (8,10,12,15,16). The sensitivity of this plasma was quantitated using both ELISA was less than 0.1% of normal 200
150 lL
TABLE I. Precision studies for enzyme-linked immunosorbent assay quantitation of von Willebrand factor antigen
Sample #1 #2 a) Within-day variation (n = 12) Mean 108a 52 SD 4.1 2.3 %CVb 3.80 4.44 b) Between-day variation (n = 4) Mean 54 131a SD 1.0 1.3 %CV 0.85 2.31 aPlasma vWF:Ag (% of normal) bCoefficient of variation
I-
100 z
#3 CLi 0m
50
25 0.3 1.21 0 24 1.2 4.88
50
100
PERCENT vWF:Ag
150 -
200
ELISA
Fig. 2. Plasma vWF:Ag concentrations in 121 canine plasmas as quantitated by enzyme-linked immunosorbent assay (ELISA) and Laurell electroimmunoassay (EIA) techniques.
13
plasma vWF:Ag, a considerable improvement over the sensitivity of most EIA techniques. The ranges of vWF:Ag in normal canine plasmas compared favorably by both methods. The ELISA range (60-152% of normal) was however slightly higher than that obtained for the same samples using EIA (50-142% of normal). In most cases there was good agreement for ELISA and EIA measurements on the same individual plasmas. As noted earlier nonagreement was most evident at the upper and lower ends of the vWF:Ag range studied. Ingerslev (12) reported similar results with ELISA/EIA determinations on human plasmas. He reported slightly higher ELISA values than EIA values in plasmas with vWF:Ag concentrations below 50% of normal, and similar or slightly lower ELISA than EIA readings at over the 50% of normal. Quantitative differences in proteins assessed by different types of immunoassays are known to occur (9,11). An important reason likely relates to differences in the antibodies used. Another reason for disagreement in vWF:Ag assays may be the molecular size of the vWF in the sample being studied. Since the same plasmas were assayed by both techniques in this study it is unlikely that sample differences were a factor in this case. Different antibodies were used in the ELISA and EIA procedures described. It is possible therefore that differences in the quantitative results could have occurred as a result of the different antisera recognizing different
antigenic determinants, possibly related to molecular size (9). In addition, in EIA techniques the magnitude of the rocket formed is influenced by both molecular size and
14
electrical charge of the migrating 3. JOHNSTONE IB, NORRIS AM. A moderately severe expression of classical protein whereas ELISA expresses the in a family of German shepherd hemophilia number of available epitopes and is dogs. Can Vet J 1984; 25: 191-194. thus influenced by protein mass. 4. ZIMMERMAN TS, ROBERTS JR, RUGGERI MZ. Factor VIII-related antigen: The diagnosis of vWD is usually characterization by electrophoretic techbased on evidence of subnormal levels niques. In: Bloom AL, ed. The Haemophiof vWF:Ag supplemented by approplias. London: Churchill Livingston, 1982: riate historical/genetic/ clinical infor88-89. mation. In this study there was good 5. BENSON RE, JONES DW, DODDS WJ. Efficiency and precision of electroimmunoagreement when plasmas were characassay for canine factor VIII-related antigen. terized as having either normal or Am J Vet Res 1983; 44: 399-403. subnormal concentrations of 6. HAN P, TEO S-H, WONG H-B. Electroimmunoassay of vWF:Ag: Increased vWF:Ag. Of the 61 plasmas having sensitivity with enzyme enhancement. subnormal vWF:Ag (< 50% of norRes 1987; 47: 113-116. mal by EIA), 47 (77%) of these 7. Thromb HOYER LW. Immunological studies of samples were also classified as being antihemophiliac factor (AHF, factor VIII). VI. Radioimmunoassay of AHF antigen. J subnormal by ELISA. The remaining Lab Clin Med 1972; 80: 822-833. 14 samples would have been charac- 8. WANG HX, GEORGE I, THORPE GHG, terized as "low normal" by ELISA. Of STOTT RA, KRICKA LJ, WHITEHEAD the 60 plasmas testing normal by EIA TP. Enhanced luminescence enzyme immunoassay for factor VIII related (> 50% of normal), 58 (97%) also J Clin Pathol 1985; 38: 317-319. tested normal by ELISA. The ability 9. antigen. YODER JM, SCHICK LA, MOORE RP. of the ELISA technique to discrimiA convenient, rapid fluoroimmunoassay nate between normal and subnormal for factor VIII related antigen. Thromb Res 1981; 24: 51-59. levels of vWF:Ag compared favorably 10. RUDZKI Z, TUNBRIDGE LJ, LLOYD with the EIA. JV. A new simple assay for factor VIII In conclusion, the ELISA technique related antigen. Thromb Res 1979; 16: 577described here appears to be of value 586. in quantitating vWF:Ag in canine 11. BARTLETT A, DORMANDY KM, HAWKEY CM, STABLEFORTH P, plasma. The procedure does not VOLLER A. Factor-VIII-related antigen: require specialized electrophoresis Measurement of enzyme immunoassay. Br expertise or equipment and can be Med J 1976; 1: 994-996. done with equipment available in most 12. INGERSLEV J. A sensitive ELISA for von Willebrand factor (vWF:Ag). Scand J Clin veterinary diagnostic laboratories. Lab Invest 1987; 47: 143-149. Measurements of vWF:Ag by this 13. JOHNSTONE IB, CRANE S. Determintechnique compare favorably to those ation of canine factor VIII-related antigen using commercial antihuman factor VIII obtained by EIA. REFERENCES 1. JOHNSTONE IB. Inherited defects of hemostasis. Compend Contin Educ Pract Vet 1982; 4: 483-488. 2. DODDS WJ. Testing for canine von Willebrands disease and hemophilia. Purebred Dogs/ American Kennel Gazette 1983; 100: 65-66.
serum. Vet Clin Pathol 1980; 9: 31-35. 14. HACKNEY JR, CEMBROWSKI GS. Need for improved instrument and kit evaluations. Am J Clin Pathol 1986; 86: 39 1-393. 15. NILSSON IM. Report of the working party on factor VIII-related antigen. Thromb Haemost 1978; 39: 511-520. 16. SCHLINK GT, JOHNSON GS. A sensitive autoradiographic procedure for factor VIII-related antigen in canine plasma. Vet Clin Pathol 1983; 12: 21-27.
ABSTRACT The purpose of this study was to evaluate a commercial enzyme-linked immunosorbent assay (ELISA) for human von Willebrand factor antigen (vWF:Ag) with respect to its potential value in quantitating the protein in canine plasma. The assay was a sandwich technique using F(ab')2 fragments specific for von Willebrand factor (vWF) and a peroxidase conjugated rabbit anti-vWF second antibody, with a microplate as the support surface. Canine plasmas were assayed by ELISA, and by Laurell electroimmunoassay (EIA), our reference methodology. The ELISA had a within-day variation of 1.21-4.44% and a between-day variation of 0.854.88% depending on the level of vWF:Ag. The sensitivity of the assay was less than 0.1% vWF:Ag. The range of vWF:Ag concentrations in plasmas from 24 clinically normal dogs compared favorably with the range for the same plasmas when assayed by EIA (ELISA = 60-152% of normal; EIA = 50-142% of normal). In 121 canine plasmas with vWF:Ag concentrations (as assessed by EIA) ranging from undetectable levels (< 6% of normal) to 142% of normal, there was good correlation with measurements made by ELISA (correlation coefficient = 0.835). It was concluded that this commercial ELISA technique could be used to provide reliable, same-day measurements of canine plasma vWF:Ag. Since it requires no special equipment other than a microplate reader and washer it is particularly suitable for
laboratories lacking the electrophoretic expertise or equipment required for EIA. RESUME Le but de cette etude etait d'evaluer test ELISA (enzyme-linked immunosorbent assay) commercial pour la detection de l'antigene du facteur von Willebrand humain (Ag: vWF) en rapport avec sa valeur potentielle de quantifier cette proteine dans du plasma canin. L'essai fut une technique dite "sandwich", utilisant le fragment F(ab')2, specifique pour le facteur von Willebrand (vWF) et un anticorps de lapin anti-vWF conjugue, avec une microplaque comme support de surface. Les echantillons de plasma furent testes avec la technique ELISA et la technique EIA (electroimmunoassay) de Laurell, utilisee comme technique de reference. L'ELISA a montre une variation intrajournaliere de 1,21-4,44% et une variation interjournaliere de 0,854,88%, dependamment du niveau d'antigenes Ag:vWF. La sensibilite du test fut de moins de 0,1% d'Ag:vWF. Les niveaux de concentration d'Ag:vWF dans le plasma de 24 chiens cliniquement normaux se sont favorablement compares avec les niveaux obtenus pour les memes echantillons lorsque testes avec la technique EIA (ELISA = 60-152% du plasma de reference normal; EIA = 50-142% du plasma de reference normal). Dans 121 echantillons de plasma, les concentrations d'Ag:vWF (comme lorsque testes par EIA) se situant entre un
les niveaux non-detectables (< 6% du plasma de reference normal) jusqu'a 142% du plasma de reference normal, il y eut une bonne correlation avec les mesures obtenues par ELISA (coefficient de correlation = 0.835). II fut conclu que la technique commerciale ELISA pourrait etre utilisee pour obtenir des mesures flables, dans un delai de 24 heures, d'Ag:vWF, a partir de plasma de chiens. Comme cette technique ne necessite aucun equipement specifique mis a part un lecteur et un laveur de microplaque, elle est particulierement designee pour les laboratoires ne possedant pas d'expertise electrophoretique ou l'equipement necessaire pour realiser la technique EIA. (Traduit par Dr A nne Provencher)
INTRODUCTION Von Willebrand's disease (vWD) and classical hemophilia (Factor VIII deficiency) are the two most common inherited bleeding disorders of dogs (1,2). Quantitation of plasma von Willebrand factor antigen (vWF:Ag) is critical to the diagnosis of vWD, and is useful in confirming factor VIII deficiency and in predicting the asymptomatic female carriers of hemophilia (3). Presently the most common method of quantitating canine vWF:Ag is by Laurell electroimmunoassay (EIA). This technique however requires expertise in electrophoresis, tends to be labor intensive, and may be limited by gel size to a relatively small sample load. The sensitivity of the EIA is
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario NI G 2W 1. This research was supported in part by a grant from the Ontario Veterinary College Pet Trust Fund. Submitted June 10, 1990.
Can J Vet Res 1991; 55: 11-14
I1I
usually only about 3-6% of normal plasma vWF:Ag concentrations. In laboratories which test human samples, attempts have been made to increase the sensitivity of the EIA procedure by concomitant enzyme enhancement or radiolabelling (6-8). Fluoroimmunoassays and radioimmunoassays have also been described but these too require special equipment and expertise (7,9,10). Enzyme-linked immunosorbent assays (ELISA), using equipment common to most diagnostic laboratories, offer a more practical means of quantitating vWF:Ag, and are being used with increasing frequency to quantitate human vWF:Ag (11,12). Commercially available ELISA kits for measuring human vWF:Ag are now available, but kits using canine specific reagents are not. Since there appears to be good cross-reactivity between canine vWF:Ag and antibodies produced against human vWF, we undertook to evaluate a commercially available ELISA test kit for its potential application in quantitating canine plasma vWF:Ag (5,13).
MATERIALS AND METHODS PLASMA SAMPLES
Canine blood samples were collected in 3.8% trisodium citrate at a ratio of 9:1 (blood:anticoagulant). The citrated blood was immediately centrifuged at 40 C for 15 min at 2500 g and the supernatant platelet-poor plasma (PPP) was removed. This PPP was divided into aliquots and held at -70° C until assayed by ELISA and EIA. The reference plasma was a preparation of PPP pooled from 12 clinically normal dogs and frozen in aliquots at -70° C. This plasma was arbitrarily designated as containing 100% vWF:Ag. Individual plasmas were measured against this standard and vWF:Ag was expressed as a percent of normal. ENZYME-LINKED IMMUNOSORBENT ASSAY
Quantitation of canine vWF:Ag by ELISA was carried out using a commercial reagent kit for measuring human vWF:Ag (Asserachrome vWF; 12
to each well. After exactly 3.5 min of incubation at room temperature, the
Diagnostica Stago, Asnieres-SurSeine, France). This kit consisted of five components: a buffered coating solution containing rabbit anti-vWF F(ab')2 fragments (freeze-dried), concentrated diluent and buffer solutions, anti-vWF rabbit immunoglobulins coupled to peroxidase (freeze-dried), and orthophenylene diamine (OPD) substrate (freezedried). The procedure was carried out by the manufacturers' directions with only minor modifications. Briefly, a microplate (Nunc Immunoplate IF, Nunc, Copenhagen, Denmark) was coated by placing 200 IAL of reconstituted coating solution in each well and incubating overnight at room temperature in the covered microplate. Each well was then washed five times with diluted washing solution using a manual plate washer (Model EL 40108, Bio-Tek Corp., Burlington, Vermont). The assay consisted first of the addition of 200 ,uL of diluted plasma to each of two coated wells (all plasma samples were assayed in duplicate). After a 2 h incubation at room temperature the plate was washed five times with washing solution, then 200 ,L of reconstituted enzymeantibody conjugate were added to each well. After a further 2 h incubation at room temperature the plate was washed as before and 200 ,uL of OPD substrate solution were added
conjugated antibody enzymesubstrate reaction was stopped by the addition of 50 ,L of 3M H2SO4. Ten minutes later the optical density was measured at 490 nm using a microplate reader (Model EL 308, Bio-Tek Corp., Burlington, Vermont). Initial dose-response curves were generated using the canine reference plasma at plasma dilutions ranging from 1:25 to 1:6400 (Fig. 1). Dilutions of 1:50, 1:100, 1:200, 1:500 and 1:1000 were ultimately used to prepare standard curves in the vWF:Ag assay, and individual plasmas were assayed in duplicate at dilutions of 1:50 or 1:100. ELECTROIMMUNOSSAY
Plasma vWF:Ag was assayed using a Laurell electroimmunoassay with rabbit antihuman antiserum as previously described (13).
RESULTS DOSE RESPONSE CURVE
A typical dose-response curve using a log-log plot is shown in Fig. 1. The response was linear over a wide range of plasma dilutions, however the range 1:50-1:1000 was selected for establishing the calibration curve in subsequent vWF:Ag assays.
10.00
1.00 E 0
0.10±
0.01
i
10
.
.
.
..0 10
.
.
.
.
.
1
i
i
1 000
i
i
i i
1 E4
RECIPROCAL OF PLASMA DILUTION Fig. 1. Typical dose-response curve for canine vWF:Ag quantitated by enzyme-linked immunosorbent assay (normal reference plasma; n = 6).
PRECISION STUDIES
Samples of plasma with three different levels of vWF:Ag (as previously determined by EIA) were used in the precision studies. These plasmas were selected to cover low, intermediate, and normal levels of vWF:Ag, particularly relevant in testing for vWF:Ag deficiency. (1,2,14). Within-day variability was assessed from 12 replicate determinations on the same plasma. As shown in Table I, intra-assay variation ranged from 1.21% to 4.44% (coefficient of variation). Between-day variability was assessed from four determinations on aliquots of the same plasma assayed at weekly intervals. Interassay variability ranged from 0.85% to 4.88% (Table I).
TABLE II. Plasma von Willebrand factor antigen in 24 clinically normal dogs as quantitated by enzyme-linked immunosorbent assay (ELISA) and Laurell electroimmunoassay (EIA)
Assay
Meana ELISA 95 EIA 90 aExpressed as % of normal reference plasma
ELISA. For purposes of data analysis, samples having undetectable vWF:Ag by EIA were designated as having a level of 6% of normal (limit of sensitivity). As shown in Fig. 2 there was good correlation (r = 0.835) between the two methods of vWF:Ag quantitation. Nonagreement was most evident at the extreme high and low ends of the vWF:Ag range studied. At NORMAL RANGE low vWF:Ag concentrations, the When plasma samples from 24 ELISA values tended to be higher clinically normal adult dogs were than the EIA values with 11 of the 12 assayed for vWF:Ag by ELISA , the samples which had undetectable levels vWF:Ag levels ranged from 60% to of vWF:Ag (< 6% of normal) by EIA 152% of normal, with a mean of 95% showing measurable levels in excess of of normal (Table II). In comparison, 6% of normal by ELISA. Of the seven EIA determinations on the same samples with vWF:Ag concentrations plasmas yielded a range of 50% to above 130% of normal (by EIA), all 142% of normal, with a mean of 90% had somewhat lower levels by ELISA of normal. The correlation coefficient but still in excess of 100% of the level for measurements by these two in the reference plasma. methods was r = 0.767.
Correlation coefficient (r)
Rangea 60-152 50-142
0.767
EIA and ELISA techniques. There good correlation between the two methods. The ELISA technique produced reliable same-day results, was easy to perform and, with a microplate as the coating support, was suitable for large sample numbers. Up to 42 plasma samples could be assayed in duplicate with appropriate controls at one time. The only major equipment required was a microplate reader (spectrophotometer). These were significant advantages over the EIA technique which required special electrophoresis expertise and equipment, and needed two days to was
complete. The precision of the ELISA was good with coefficients of variation ranging from 1.21% to 4.44% within batch and 0.85% to 4.88% between batch. These variations compare
favorably with results for fluoroim-
COMPARISON OF ELISA AND EIA MEASUREMENTS ON PLASMAS WITH NORMAL AND SUBNORMAL vWF:Ag CONCENTRATIONS
One hundred and twenty-one plasma samples from clinically normal dogs and dogs previously determined to have subnormal plasma vWF:Ag concentrations (< 50% of normal by EIA) were assayed by both EIA and
munoassays
and other types of
DISCUSSION
ELISA, and are better than those generally reported for EIA techniques In this study, vWF:Ag in canine (8,10,12,15,16). The sensitivity of this plasma was quantitated using both ELISA was less than 0.1% of normal 200
150 lL
TABLE I. Precision studies for enzyme-linked immunosorbent assay quantitation of von Willebrand factor antigen
Sample #1 #2 a) Within-day variation (n = 12) Mean 108a 52 SD 4.1 2.3 %CVb 3.80 4.44 b) Between-day variation (n = 4) Mean 54 131a SD 1.0 1.3 %CV 0.85 2.31 aPlasma vWF:Ag (% of normal) bCoefficient of variation
I-
100 z
#3 CLi 0m
50
25 0.3 1.21 0 24 1.2 4.88
50
100
PERCENT vWF:Ag
150 -
200
ELISA
Fig. 2. Plasma vWF:Ag concentrations in 121 canine plasmas as quantitated by enzyme-linked immunosorbent assay (ELISA) and Laurell electroimmunoassay (EIA) techniques.
13
plasma vWF:Ag, a considerable improvement over the sensitivity of most EIA techniques. The ranges of vWF:Ag in normal canine plasmas compared favorably by both methods. The ELISA range (60-152% of normal) was however slightly higher than that obtained for the same samples using EIA (50-142% of normal). In most cases there was good agreement for ELISA and EIA measurements on the same individual plasmas. As noted earlier nonagreement was most evident at the upper and lower ends of the vWF:Ag range studied. Ingerslev (12) reported similar results with ELISA/EIA determinations on human plasmas. He reported slightly higher ELISA values than EIA values in plasmas with vWF:Ag concentrations below 50% of normal, and similar or slightly lower ELISA than EIA readings at over the 50% of normal. Quantitative differences in proteins assessed by different types of immunoassays are known to occur (9,11). An important reason likely relates to differences in the antibodies used. Another reason for disagreement in vWF:Ag assays may be the molecular size of the vWF in the sample being studied. Since the same plasmas were assayed by both techniques in this study it is unlikely that sample differences were a factor in this case. Different antibodies were used in the ELISA and EIA procedures described. It is possible therefore that differences in the quantitative results could have occurred as a result of the different antisera recognizing different
antigenic determinants, possibly related to molecular size (9). In addition, in EIA techniques the magnitude of the rocket formed is influenced by both molecular size and
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electrical charge of the migrating 3. JOHNSTONE IB, NORRIS AM. A moderately severe expression of classical protein whereas ELISA expresses the in a family of German shepherd hemophilia number of available epitopes and is dogs. Can Vet J 1984; 25: 191-194. thus influenced by protein mass. 4. ZIMMERMAN TS, ROBERTS JR, RUGGERI MZ. Factor VIII-related antigen: The diagnosis of vWD is usually characterization by electrophoretic techbased on evidence of subnormal levels niques. In: Bloom AL, ed. The Haemophiof vWF:Ag supplemented by approplias. London: Churchill Livingston, 1982: riate historical/genetic/ clinical infor88-89. mation. In this study there was good 5. BENSON RE, JONES DW, DODDS WJ. Efficiency and precision of electroimmunoagreement when plasmas were characassay for canine factor VIII-related antigen. terized as having either normal or Am J Vet Res 1983; 44: 399-403. subnormal concentrations of 6. HAN P, TEO S-H, WONG H-B. Electroimmunoassay of vWF:Ag: Increased vWF:Ag. Of the 61 plasmas having sensitivity with enzyme enhancement. subnormal vWF:Ag (< 50% of norRes 1987; 47: 113-116. mal by EIA), 47 (77%) of these 7. Thromb HOYER LW. Immunological studies of samples were also classified as being antihemophiliac factor (AHF, factor VIII). VI. Radioimmunoassay of AHF antigen. J subnormal by ELISA. The remaining Lab Clin Med 1972; 80: 822-833. 14 samples would have been charac- 8. WANG HX, GEORGE I, THORPE GHG, terized as "low normal" by ELISA. Of STOTT RA, KRICKA LJ, WHITEHEAD the 60 plasmas testing normal by EIA TP. Enhanced luminescence enzyme immunoassay for factor VIII related (> 50% of normal), 58 (97%) also J Clin Pathol 1985; 38: 317-319. tested normal by ELISA. The ability 9. antigen. YODER JM, SCHICK LA, MOORE RP. of the ELISA technique to discrimiA convenient, rapid fluoroimmunoassay nate between normal and subnormal for factor VIII related antigen. Thromb Res 1981; 24: 51-59. levels of vWF:Ag compared favorably 10. RUDZKI Z, TUNBRIDGE LJ, LLOYD with the EIA. JV. A new simple assay for factor VIII In conclusion, the ELISA technique related antigen. Thromb Res 1979; 16: 577described here appears to be of value 586. in quantitating vWF:Ag in canine 11. BARTLETT A, DORMANDY KM, HAWKEY CM, STABLEFORTH P, plasma. The procedure does not VOLLER A. Factor-VIII-related antigen: require specialized electrophoresis Measurement of enzyme immunoassay. Br expertise or equipment and can be Med J 1976; 1: 994-996. done with equipment available in most 12. INGERSLEV J. A sensitive ELISA for von Willebrand factor (vWF:Ag). Scand J Clin veterinary diagnostic laboratories. Lab Invest 1987; 47: 143-149. Measurements of vWF:Ag by this 13. JOHNSTONE IB, CRANE S. Determintechnique compare favorably to those ation of canine factor VIII-related antigen using commercial antihuman factor VIII obtained by EIA. REFERENCES 1. JOHNSTONE IB. Inherited defects of hemostasis. Compend Contin Educ Pract Vet 1982; 4: 483-488. 2. DODDS WJ. Testing for canine von Willebrands disease and hemophilia. Purebred Dogs/ American Kennel Gazette 1983; 100: 65-66.
serum. Vet Clin Pathol 1980; 9: 31-35. 14. HACKNEY JR, CEMBROWSKI GS. Need for improved instrument and kit evaluations. Am J Clin Pathol 1986; 86: 39 1-393. 15. NILSSON IM. Report of the working party on factor VIII-related antigen. Thromb Haemost 1978; 39: 511-520. 16. SCHLINK GT, JOHNSON GS. A sensitive autoradiographic procedure for factor VIII-related antigen in canine plasma. Vet Clin Pathol 1983; 12: 21-27.
