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Reutilization of 125I-labelled anti-IgE antibody and paper discs in prist and rast IgE determination
297
Clinica Chimica Acta, 90 (1978) 297-300 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CCA 9758
REUTILIZATION OF 12’1-LABELLED ANTI-IgE ANTIBODY DISCS IN PRIST AND RAST IgE DETERMINATION
P.L.B. BRUYNZEEL
*, W. VAN DEN BOGAARD
AND PAPER
and L. BERRENS
Laboratory of the Department of Pulmonary Disease and Division of Experimental University Hospital, Utrecht (The Netherlands) (Received
Allergy,
May 31st, 1978)
Summary In the paper radio~munosor~nt test (PRIST) ~ti-human IgE coupled paper discs are used for the estimation of total IgE in blood serum: in the radioallergosorbent test (RAST) allergen-coupled paper discs are used for the estimation of specific IgE in blood serum. The bound IgE or specific IgE is quantified by a ‘251-labelled anti-human IgE. The non-bound *2sI-labelled antihuman IgE can be collected and used in new assay. By a 4-h incubation of the used paper discs with 1 M glycine-HCl buffer (pH 2.7) the IgE-labelled anti-IgE complex can largely be removed. The paper discs treated in this manner can be used in a new assay.
Immunoglobulin E is known to occur in frequent association with allergic disease in human atopic subjects [If. Because of the very low concentration of IgE in the blood serum radioimmunolo~~~ methods, i.e. radioimmunosorbent test (RIST), paper radioimmunosorbent test (PRIST), and radioallergosorbent test (RAST) are commonly employed for its assessment [l--4 1. In PRIST and RAST a sandwich technique is used; IgE or specific IgE are bound to an antiIgE-paper disc or an allergen paper disc, respectively, and bound IgE is subsequently quantified by means of 12SI-labelled anti-IgE antibody. If both tests are performed strictly according to the manufacturer’s instructions (Pharmacia Diagnostics AB, Uppsala, Sweden), the percentage of bound radioactivity normally does not exceed about 40% of the added amount, The non-bound labelled anti-human IgE was very carefully sucked off with a disposable syringe
* Correspondence should be addressed to Dr. P.L.B. Bruynzeel. Department University Hospital, Catharijnesingel 101, Utrecht, The Netherlands.
of Pulmonary
Disease.
298
connected to a disposable flat-pointed needle, collected in a polystyrene vial and kept at -20°C until use. Then the discs were washed three times with saline according to the inst~ct~ons and counted in a y-counter. Immediately after estimation of the bound activity, the recycling procedure of the discs was started. The discs were suspended in about 100 ml of a 1 M glycine-HCl buffer (pH 2.7). This was followed by an incubation period of 4 h at room temperature, under vigorous stirring. During this incubation period the 1 M glycine-HCI buffer was replaced every hour. After termination of the incubation the discs were washed three times with saline and three times with RAST-buffer. The remaining activity bound to the discs was then estimated and the discs were stored in separate polystyrene vials in RAST-buffer at 4°C until use. In case the activity bound to the discs remained too high the recycling procedure was repeated for another 2 h. The residual activity attached to the disc was subtracted in a new assay. When PRIST and RAST discs were recycled in the way described, generally less than 1% of the total activity remained attached to the disc. Taking 1% of the total activity added as the limit of the activity allowed to remain bound to the disc, more than 95% of the recycled PRIST discs and more than 98% of the recycled RAST-discs could be reutilized. The recycled PRIST discs were tested in combination with the standard enclosed in the commercial PRIST kit and with freshly dissolved activity or collected activity. Furthermore, new PRIST discs were tested in combination with the standard enclosed in the kit and with freshly dissolved activity. The standard curves obtained in this way are presented in Fig. 1. The results obtained with several sera of bronchial asthma patients with these standard curves are presented in Table I. From the results presented in Fig. 1 and Table I it becomes obvious that collected non-bound
IgE In W/ml -k---1-,-,-, 1
10
100
Fig. 1. Standard curves obtained by PRIST using different reagents. (A) New discs and freshly prepared labelled antibody solution. lb) Recycled discs and freshly prepared labelled antibody solution. (C) Recycled discs and collected non-bound labelled antibody solution.
299 TABLE I RESULTS
OBTAINED
BY PRIST WITH SERA OF BRONCHIAL
ASTHMA
PATIENTS
The figures present the mean of duplicate values. SLXUm
Dilution factor
IgE in I.U./ml
Bi’742 B7745 B7150 B7761
20 20 20 10
120 440 130 18
540 410 102 16
560 360 98 16
A
B
C
Standardcurve (see Fig. 1)
labelled anti-human-IgE can be reutilized to some extent. In the high concentration range, i.e. above 25 I.U./ml standard curve and serum IgE values tend to deviate. This might be explained by partial denaturation caused by the collection procedure or by repeated freezing and thawing. More likely, however, seems that a relatively large proportion of high avidity antibodies will be removed during the original incubation step, leaving an increased proportion of relatively low avidity antibodies to be collected. This will lead to low IgE values, being more pronounced in the high IgE concentration range. Table I also illustrates that recycled anti-human-IgE discs can be used in a new assay. By treating the discs with a glycine-HCl buffer, damage of anti-IgE on the disc will probably occur. Reasoning that an excess of antibody is present, the reduction of effective binding places seems rather small. It could even be demonstrated that recycling could be performed twice, without greatly changing the binding capacity of the disc. In Table II the inter-assay variation for these four sera estimated by PRIST is presented. Table II shows that the standard deviation enlarges when the IgE concentration increases. From the results presented here it seems advisable to perform IgE estimations with recycled PRIST discs in the standard curve range between 2 and 25 I.U./ml (see also Fig. 1). A series of sera of allergic asthma patients was tested with different allergen paper discs, then the discs were recycled as described and the same sera tested again. The results of the RAST scores ((activity bound to allergen paper disc/ total activity added) corrected for the RAST score with pooled normal serum with that allergen paper disc) before and after recycling of the allergen paper discs are presented in Fig. 2. The Spearman rank correlation coefficient proved
TABLE II INTER-ASSAY
VARIATION
IgE values obtained with sera of bronchial asthma patients. n = 5. Serum
IgE level (I.U./ml;
B7742 B7745 Bl750 B7761
644 364 142 22.0
k 139 k 59 ? 13 ? 3.7
mean ? S.D.)
300
% bIndIng before
recycling
o”
0
ox+*
0
x
r=-90
p < .OOOl n: 57 after 210
LIO
recycling
bindlng
Fig. 2. Correlation Of RAST-scores obtained with 57 different allergen discs before and after recycling. Symbols of different allergens: 0, house dust (hz); 0, cat dander (el): +, lolium Perenne (gg); X, uVirradiated cat dander.
to be R = 0.90, which was significant at p < 10F4. The data in Fig. 2 clearly demonstrate that allergen-paper discs can be reutilized without loss of binding capacity. This once more shows the great stability of these allergenic preparations [ 51. Although only a selected number of lolium Perenne discs was tested, it is supposed that the recycling of paper discs coated with other allergens of pronounced protein nature can be similarly performed without appreciable damage. In conclusion, this study demonstrates the reutilization of labelled antihuman-IgE antibody and paper discs in PRIST and RAST IgE determination. Acknowledgements The authors tance.
wish to thank
Mr. L.A.M.J.
Houben
for skilled technical
assis-
References 1 Bennich, H. and Johansson, S.G.O. (1971) Adv. Immunol. 13,l 2 Wide, L. (1971) in Radioimmunoassay Methods. European Workshop (Kirkham, K.E. and Hunter, W.M.. eds.). Livingstone, Edinburgh 3 Johansson. S.G.O.. Berglund. A. and Kjellman, N.I.M. (1976) Clin. Allerg. 6.91 4 Johansson, S.G.O., Ben&h, H. and Berg, T. (1971) Int. Arch. Allerg. APP~. Immunol. 41,443 5 Berrens, L. (1971) The Chemist&y of Atopic Allergens. Monographs in Allergy. Vol. 7. Karger, Base1
Clinica Chimica Acta, 90 (1978) 297-300 0 Elsevier/North-Holland Biomedical Press
BRIEF TECHNICAL
NOTE
CCA 9758
REUTILIZATION OF 12’1-LABELLED ANTI-IgE ANTIBODY DISCS IN PRIST AND RAST IgE DETERMINATION
P.L.B. BRUYNZEEL
*, W. VAN DEN BOGAARD
AND PAPER
and L. BERRENS
Laboratory of the Department of Pulmonary Disease and Division of Experimental University Hospital, Utrecht (The Netherlands) (Received
Allergy,
May 31st, 1978)
Summary In the paper radio~munosor~nt test (PRIST) ~ti-human IgE coupled paper discs are used for the estimation of total IgE in blood serum: in the radioallergosorbent test (RAST) allergen-coupled paper discs are used for the estimation of specific IgE in blood serum. The bound IgE or specific IgE is quantified by a ‘251-labelled anti-human IgE. The non-bound *2sI-labelled antihuman IgE can be collected and used in new assay. By a 4-h incubation of the used paper discs with 1 M glycine-HCl buffer (pH 2.7) the IgE-labelled anti-IgE complex can largely be removed. The paper discs treated in this manner can be used in a new assay.
Immunoglobulin E is known to occur in frequent association with allergic disease in human atopic subjects [If. Because of the very low concentration of IgE in the blood serum radioimmunolo~~~ methods, i.e. radioimmunosorbent test (RIST), paper radioimmunosorbent test (PRIST), and radioallergosorbent test (RAST) are commonly employed for its assessment [l--4 1. In PRIST and RAST a sandwich technique is used; IgE or specific IgE are bound to an antiIgE-paper disc or an allergen paper disc, respectively, and bound IgE is subsequently quantified by means of 12SI-labelled anti-IgE antibody. If both tests are performed strictly according to the manufacturer’s instructions (Pharmacia Diagnostics AB, Uppsala, Sweden), the percentage of bound radioactivity normally does not exceed about 40% of the added amount, The non-bound labelled anti-human IgE was very carefully sucked off with a disposable syringe
* Correspondence should be addressed to Dr. P.L.B. Bruynzeel. Department University Hospital, Catharijnesingel 101, Utrecht, The Netherlands.
of Pulmonary
Disease.
298
connected to a disposable flat-pointed needle, collected in a polystyrene vial and kept at -20°C until use. Then the discs were washed three times with saline according to the inst~ct~ons and counted in a y-counter. Immediately after estimation of the bound activity, the recycling procedure of the discs was started. The discs were suspended in about 100 ml of a 1 M glycine-HCl buffer (pH 2.7). This was followed by an incubation period of 4 h at room temperature, under vigorous stirring. During this incubation period the 1 M glycine-HCI buffer was replaced every hour. After termination of the incubation the discs were washed three times with saline and three times with RAST-buffer. The remaining activity bound to the discs was then estimated and the discs were stored in separate polystyrene vials in RAST-buffer at 4°C until use. In case the activity bound to the discs remained too high the recycling procedure was repeated for another 2 h. The residual activity attached to the disc was subtracted in a new assay. When PRIST and RAST discs were recycled in the way described, generally less than 1% of the total activity remained attached to the disc. Taking 1% of the total activity added as the limit of the activity allowed to remain bound to the disc, more than 95% of the recycled PRIST discs and more than 98% of the recycled RAST-discs could be reutilized. The recycled PRIST discs were tested in combination with the standard enclosed in the commercial PRIST kit and with freshly dissolved activity or collected activity. Furthermore, new PRIST discs were tested in combination with the standard enclosed in the kit and with freshly dissolved activity. The standard curves obtained in this way are presented in Fig. 1. The results obtained with several sera of bronchial asthma patients with these standard curves are presented in Table I. From the results presented in Fig. 1 and Table I it becomes obvious that collected non-bound
IgE In W/ml -k---1-,-,-, 1
10
100
Fig. 1. Standard curves obtained by PRIST using different reagents. (A) New discs and freshly prepared labelled antibody solution. lb) Recycled discs and freshly prepared labelled antibody solution. (C) Recycled discs and collected non-bound labelled antibody solution.
299 TABLE I RESULTS
OBTAINED
BY PRIST WITH SERA OF BRONCHIAL
ASTHMA
PATIENTS
The figures present the mean of duplicate values. SLXUm
Dilution factor
IgE in I.U./ml
Bi’742 B7745 B7150 B7761
20 20 20 10
120 440 130 18
540 410 102 16
560 360 98 16
A
B
C
Standardcurve (see Fig. 1)
labelled anti-human-IgE can be reutilized to some extent. In the high concentration range, i.e. above 25 I.U./ml standard curve and serum IgE values tend to deviate. This might be explained by partial denaturation caused by the collection procedure or by repeated freezing and thawing. More likely, however, seems that a relatively large proportion of high avidity antibodies will be removed during the original incubation step, leaving an increased proportion of relatively low avidity antibodies to be collected. This will lead to low IgE values, being more pronounced in the high IgE concentration range. Table I also illustrates that recycled anti-human-IgE discs can be used in a new assay. By treating the discs with a glycine-HCl buffer, damage of anti-IgE on the disc will probably occur. Reasoning that an excess of antibody is present, the reduction of effective binding places seems rather small. It could even be demonstrated that recycling could be performed twice, without greatly changing the binding capacity of the disc. In Table II the inter-assay variation for these four sera estimated by PRIST is presented. Table II shows that the standard deviation enlarges when the IgE concentration increases. From the results presented here it seems advisable to perform IgE estimations with recycled PRIST discs in the standard curve range between 2 and 25 I.U./ml (see also Fig. 1). A series of sera of allergic asthma patients was tested with different allergen paper discs, then the discs were recycled as described and the same sera tested again. The results of the RAST scores ((activity bound to allergen paper disc/ total activity added) corrected for the RAST score with pooled normal serum with that allergen paper disc) before and after recycling of the allergen paper discs are presented in Fig. 2. The Spearman rank correlation coefficient proved
TABLE II INTER-ASSAY
VARIATION
IgE values obtained with sera of bronchial asthma patients. n = 5. Serum
IgE level (I.U./ml;
B7742 B7745 Bl750 B7761
644 364 142 22.0
k 139 k 59 ? 13 ? 3.7
mean ? S.D.)
300
% bIndIng before
recycling
o”
0
ox+*
0
x
r=-90
p < .OOOl n: 57 after 210
LIO
recycling
bindlng
Fig. 2. Correlation Of RAST-scores obtained with 57 different allergen discs before and after recycling. Symbols of different allergens: 0, house dust (hz); 0, cat dander (el): +, lolium Perenne (gg); X, uVirradiated cat dander.
to be R = 0.90, which was significant at p < 10F4. The data in Fig. 2 clearly demonstrate that allergen-paper discs can be reutilized without loss of binding capacity. This once more shows the great stability of these allergenic preparations [ 51. Although only a selected number of lolium Perenne discs was tested, it is supposed that the recycling of paper discs coated with other allergens of pronounced protein nature can be similarly performed without appreciable damage. In conclusion, this study demonstrates the reutilization of labelled antihuman-IgE antibody and paper discs in PRIST and RAST IgE determination. Acknowledgements The authors tance.
wish to thank
Mr. L.A.M.J.
Houben
for skilled technical
assis-
References 1 Bennich, H. and Johansson, S.G.O. (1971) Adv. Immunol. 13,l 2 Wide, L. (1971) in Radioimmunoassay Methods. European Workshop (Kirkham, K.E. and Hunter, W.M.. eds.). Livingstone, Edinburgh 3 Johansson. S.G.O.. Berglund. A. and Kjellman, N.I.M. (1976) Clin. Allerg. 6.91 4 Johansson, S.G.O., Ben&h, H. and Berg, T. (1971) Int. Arch. Allerg. APP~. Immunol. 41,443 5 Berrens, L. (1971) The Chemist&y of Atopic Allergens. Monographs in Allergy. Vol. 7. Karger, Base1