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A radioimmunoassay for soluble HLA-A antigens in human serum
lmmunochemi.~try, 1977, Vol. 14, pp. 139 141. Pergamon Press. Printed in Great Britain
A RADIOIMMUNOASSAY FOR SOLUBLE HLA-A ANTIGENS IN H U M A N SERUM* M. J. G I P H A R T , E. D O Y E R a n d J. W. B R U N ' I N G Department of Immunohaematology, University Hospital, Leiden, The Netherlands (First received 15 July 1976; in revised form 23 September 1976) Abstract We developed a radioimmunoassay for the quantitation of soluble HLA molecules which are complexed with fl2-microglobulin (fl2MG) in human serum. With this type of assay one can overcome the drawbacks of complement dependent assays. The radioimmunoassay is based on the reaction of radiolabelled anti-HLA antibody with the soluble complex, which is then precipitated with rabbit anti-fl2MG and anti-rabbit IgG. INTRODUCTION
After the discovery of the presence of soluble histocompatibility antigens in h u m a n serum (van Rood et al., 1970; C h a r l t o n et al., 1970) it was found that the only way to detect these substances was to use their specific inhibitory activity on anti-HLA sera. This technique has some drawbacks, namely the low concentration of most of the serum H L A antigens, the dependence on complement, and the usual low titer of H L A alloantisera. To overcome these problems we developed a radioimmunoassay with radiolabelled H L A alloantibodies. We showed that the binding on lymphocytes of radiolabelled antiH L A antibodies can be inhibited specifically by solubilized HLA-antigens from spleen cells and by HLA antigens present in serum (Giphart et al., 1975). Based on these conclusions and on the observations by O h et al. (1975) and Reisfeld et al. (1975), who showed that HLA inhibitory activity in serum can be absorbed with an anti-fl2-microglobulin imm u n o a b s o r b e n t , we decided to develop a direct radioimmunoassay (RIA): If serum H L A molecules are naturally complexed with fl2MG, precipitation of radioactivity should be possible with anti-fl2MG after the H L A part of the complex is reacted with radiolabelled highly specific anti-HLA antibodies.
phocytes showed that the specific immunological activity was anti-HLA-A2 exclusively. There was no binding on HLA-A2 negative lymphocytes, whereas 15~o of the total radioactivity in the used preparations could be bound on HLA-A2 positive cells. Rabbit antiserum to human fl2MG (RAHu/fl2MG) was purchased as an immunoglobulin solution from Dakopatts, Denmark. Antiserum against Fc fragments of rabbit IgG was raised in pigs (SwAR/IgG[Fc]). After extensive absorption with human red blood cells and Sepharose-bound IgG, the antiserum gave in immunoelectrophoresis of normal rabbit serum a single IgG line and no visible precipitation of normal human serum. The radioimmunoassay (RIA) was carried out as follows: normal human serum was allowed to react with the ~25I-antiHLA-A2 preparation (2-10 nCi). Then RAHu/fl2MG was added and a cO-precipitate was formed with SwAR/ IgG(Fc). The precipitates were washed (3 times) with 1 ml of ice-cold phosphate buffered saline (PBS) and their radioactivity was measured in a Baird Atomic gamma counter. Details of the assay are in the legends to the tables. RESULTS Table 1 summarizes the results of the RIA when HLA-A2 positive serum was mixed with HLA-A2 Table 1. Radioimmunoassay of HLA-A2 serum antigen HLA-A2 positive serum (~l)
HLA-A2 negative serum (~l)
RIA radioactivity (04)
P valuea
4.8-5.0 5.8~.1 6.9-6.9 8.0-8.1 8.9-9.0 10.6-11.1 11.5-12.0
-<0.05 <0.001 <0.001 <0.001 <0.001 <0.001
MATERIALS AND METHODS
Human sera were taken from healthy HLA typed donors and stored at 4°C in the presence of 0.02~o NaN3; radiolabelled anti-HLA-A2 (~25I-anti-HLA-A2) was prepared as described elsewhere (Giphart et al., 1975); briefly: platelet eluates after antibody absorption were fractionated on a Sephadex G-200 column, the IgG fraction was radiolabelled with 125I using chloramine T and this preparation was absorbed and eluted again with HLA-A2 positive platelets. Alloantiserum (1 ml) yielded a radiolabelled antibody preparation of about 5/tCi. Binding assays on lym* Supported in part by the National Institute of Health (contract No. 1-AI-4-2508), the J. A. Cohen Institute of Radiopathology and Radiation Protection (IRS), the Dutch Foundation for Medical Research (FUNGO) which is subsidized by the Dutch Organization for the Advancement of Pure Research (ZWO) and the Dutch Organization for Health Research (TNO). 139
0 0.5 1 2 4 8 16
16 15.5 15 14 12 8 0
Varying amounts of HLA-A2 serum were diluted in nonHLA-A2 serum. Aliquots (16/A) of the mixtures were allowed to react with 2/A of a 12SI-anti-HLA-A2 preparation for 2 h r at 4°C. Then 15pl of RAHu/fl2MG was added and incubated for another hour at 4°C. Precipitation was carried out with 500/~1 SwAR/IgG(Fc) for 16 hr at 4°C. The precipitates were washed ( x 3) with 1 ml PBS and the radioactivity of the precipitate measured in a gamma counter, (duplicate determination). a The assay results of positive and negative serum were used in Student's t-test to obtain P values.
140
M.J. GIPHART, E. DOYER and J. W. BRUNING Table 2. Specificity controls of the RIA for HLA-A2 antigen complexed 32 microglobulin in human serum
Order of reagent addition
Percentage of radioactivity precipitated with serum of HLA type A2, BS, WI5 A1, 3, B7, 8
Type of experiment
a b
1, 4, 6 I, 5, 6
RIA
16.5 3.4
5.6 4.9
c
1, 2, 4, 6
Blocking with immune anti-HLA-A2 serum and control with non-immune serum
15.3
4.6
4.6 14.9
3.9 5.0
12.5
4.1
3.0
2.5
0.5
0.6
d 2, 1, 4, 6 e 1, 3, 4, 6 f
3. I. 4, 6
g 2, 8, 4, 6 h 2, 7, 4, 6
Inhibition by non-specific radiolabelled protein
Serum (10/A) was incubated at 4°C with a set of antisera and related or unrelated proteins. The varying order of addition of reagents is shown in the first column. The numbers correspond to the following antisera or proteins: 1, ~2SI-anti-HLA-A2 (23,000 counts/min) incubation for 2 hr. 2, anti-HLA-A2 serum (4#1) incubation for 2hr. 3, AB-serum (HLA type A29, W32, B7, 12) 4pl, incubation for 2hr. 4, RAHu/ /~2MG (15 td) incubation for l hr. 5, normal rabbit serum (15/A) incubation for 1 hr. 6, SwAR/IgG (0.5 ml) incubation for 16hr. 7, lZSl-labelled cytochrome c (315,000 counts/min) incubation for 2 hr. 8, ~25I-labelled human IgG (16,500 counts/min) incubation for 2 hr. The precipitates were washed 3 times with 1 ml of phosphate buffered saline (PBS) and the radioactivity of the precipitates measured in a gamma counter. The percentage of radioactivity in the precipitates was calculated (single determinations). negative serum. Serum volumes from 1 pl HLA-A2 positive serum led to statistically significant results. Up to 10pl of serum could be precipitated in the test and still have radiolabelled antibody excess. Table 2 summarizes control experiments on the specificity of the RIA with respect to the HLA type of the antigen source, the anti-32MG reagent and the specific blocking of the test with anti-HLA-A2 serum. The reactivity of RAHu/fl2MG is shown by replacement of RAHu//32MG by normal rabbit serum. The residual precipitation of radioactivity is no longer specific for HLA-A2 serum antigen (Table 2, a and b). The reactivity of the radioactive antibody preparation is shown by inhibition with immune antiHLA-A2 serum which inhibits precipitation of radioactivity only when added before the radiolabelled antibody (c and d). Non-immune serum does not inhibit specific precipitation (e and f). Precipitation of radioactivity with non-immune radiolabelled human IgG is low (g), however, not as low as with the unrelated protein cytochrome c (h). The residual precipitation of radioactivity with HLA-A2 negative serum (~f) may be ascribed to extra reactivities of normal rabbit serum, anti-/~2MG and SwAR/IgG (Fc). This non-specific precipitation with HLA-A2 negative serum could be reduced by prior addition of soluble human IgG (1 mg/ml) to the precipitating SwAR/IgG (Fc). Precipitation with serum of HLA type A2,-, B8, W15 and A1, 3, B7, 8 was then 12.85 and 2°//o, respectively. We conclude that the radioactivity will be precipitated only when HLA-A2 positive sera are reacted with ~ZSl-anti-HLA-A2, anti-/32MG and precipitating
antiserum. Specific blocking with anti-HLA-A2 serum can be observed. Non-immune rabbit serum precipitated amounts of radioactivity comparable to HLA-A2 negative serum. The RIA of 13 sera from HLA typed, healthy donors is summarized in Table 3. The amounts agree Table 3. Radioimmunoassay of HLA-A2 antigen complexed with /~zMG in serum from HLA typed donors Period of storage (months)
Precipitated radioactivity (~o)
7 8 12 I4 15
9.9-10.4 10.9 11.2 10.7 11.2 10.0-10.5 9.8-10.2
A1, 28, B8, 14 AW26, 28, B7, W17
3 14
9.5-9.8 9.0-9.1
A3, W30, B7, 13 All, W32, B5, 13 A1, 3, B8, W22 A3, W30, BWI6, 27 A1, W30, B13, 18 AI, W25, B18, 27
10 13 14 14 14 15
4.9-5.1 4.6-4.8 4.54.7 3.7 3.8 5.0-5.7 4.5~4.9
HLA type of serum donors
Group A2, A2, A2, A2, A2,
II
9, B12,W33, BW35, WI6 , B5, 27 3, BW35, 11, B27.
RIA for HLA-A2 antigen complexed with flzMG in serum from HLA typed donors: 10pl serum is incubated for 2 hr at 4~C with 2/~1 of 12SI-anti-HLA-A2 (4800 counts/ min) followed by incubation for 1 hr at 4°C with 15pl of RAHu/fl2MG and finally with 0.5 ml of SwAR/IgG (Fc) for 16 hr at 4°C. The precipitates were treated as described under Table 2 (duplicate determinations).
RIA for soluble HLA perfectly well with the HLA types of the serum donors, even with respect to the known cross-reactivity of HLA-A2 and A28. DISCUSSION The described RIA has been shown to be specific for HLA-A2. Table 1 shows that with the chosen quantities of reagents an antibody excess is still maintained, which enables all the antigens to be labelled with radioactive antibody and to be precipitated by anti-fl2MG and anti-rabbit IgG serum. Although some reactivity can be observed between precipitating swine anti-rabbit IgG and anti-/32MG with human IgG (Table 2), the difference between group I (HLA-A2 or A28 positive) and group II (HLA-A2 negative) is highly significant (P < 10 -s) using a Student's t-test (Table 3). We therefore conclude that HLA-A2 antigen in serum is complexed with /32MG, confirming the results of Oh et al. (1975). We did not 'observe false negative reactions contrary to Reisfeld et al. (1975) who have reported that in some sera HLA-antigens did not bind to fl2MG immunoadsorbents. A second conclusion from our results is that the complex is very stable: there is no apparent influence of storage time on the radioactivity in the precipitate. It seems therefore reasonable to assume that the HLA-flzMG complex is a stable entity in serum unless the sera are heat inactivated since this seems to destroy the antigenic activity. From our results it is not clear whether a 4-chain molecule is likely for the soluble complex antigens as has been suggested for membrane bound HLA antigens (Strominger et al., 1974; Cresswell et al., 1975). A multi-chain molecule can not be ruled out since Oh et al. (1975) have published
141
evidence that the tool. wt of the serum component carrying the HLA specificity is greater than 150,000. The question should be answered whether all HLA antigens in serum like those in the cell membrane are complexed with fl2MG. Experiments are designed to answer this question. The hypothetical possibility remains open that HLA antigens, upon shedding from the membrane, form complexes with serum fl2MG. We have described a powerful tool to study the soluble plasma HLA-antigens complexed with fl2MG, without the necessity of using complement dependent systems, or laborious purification devices. We report here only the results obtained with radiolabelled antiHLA-A2 antibodies, but radiolabelled antibody preparations of other HLA specificity have been prepared (Giphart et al.). The RIA is relatively easy, requires minute amounts of reagents, can be quantified and is very sensitive through the use of highly specific radiolabelled anti-HLA antibodies.
REFERENCES
Charlton R. K. & Zmijewski C. M. (1970) Science 170, 636. Cresswell P. & Dawson J. R. (1975) J. lmmun. 114, 523. Giphart M. J., Doyer E., Bruning J. W. & van Rood J. J. (1975) J. immunol. Meth. 9, 105. Oh S. K., Pellegrino M. A., Ferrone S., Sevier E. D. & Reisfeld R. A. (1975) Eur. J. lmmun. 5, 61l. Reisfeld R. A., Sevier E. D., Pellegrino M. A., Ferrone S. & Poulik M. D. (1975) Immunogenetics 2, 183. van Rood J. J., van Leeuwen A. & van Santen M. C. T. (1970) Nature 226, 366. Strominger J. L., Cresswell P., Grey H., Humphreys R. E., Mann D., McCune M., Parham P., Robb R., Sanderson A. R., Springer T. A., Terhorst C. & Turner M. J. (1974) Transplantn Rev. 21, 126.
A RADIOIMMUNOASSAY FOR SOLUBLE HLA-A ANTIGENS IN H U M A N SERUM* M. J. G I P H A R T , E. D O Y E R a n d J. W. B R U N ' I N G Department of Immunohaematology, University Hospital, Leiden, The Netherlands (First received 15 July 1976; in revised form 23 September 1976) Abstract We developed a radioimmunoassay for the quantitation of soluble HLA molecules which are complexed with fl2-microglobulin (fl2MG) in human serum. With this type of assay one can overcome the drawbacks of complement dependent assays. The radioimmunoassay is based on the reaction of radiolabelled anti-HLA antibody with the soluble complex, which is then precipitated with rabbit anti-fl2MG and anti-rabbit IgG. INTRODUCTION
After the discovery of the presence of soluble histocompatibility antigens in h u m a n serum (van Rood et al., 1970; C h a r l t o n et al., 1970) it was found that the only way to detect these substances was to use their specific inhibitory activity on anti-HLA sera. This technique has some drawbacks, namely the low concentration of most of the serum H L A antigens, the dependence on complement, and the usual low titer of H L A alloantisera. To overcome these problems we developed a radioimmunoassay with radiolabelled H L A alloantibodies. We showed that the binding on lymphocytes of radiolabelled antiH L A antibodies can be inhibited specifically by solubilized HLA-antigens from spleen cells and by HLA antigens present in serum (Giphart et al., 1975). Based on these conclusions and on the observations by O h et al. (1975) and Reisfeld et al. (1975), who showed that HLA inhibitory activity in serum can be absorbed with an anti-fl2-microglobulin imm u n o a b s o r b e n t , we decided to develop a direct radioimmunoassay (RIA): If serum H L A molecules are naturally complexed with fl2MG, precipitation of radioactivity should be possible with anti-fl2MG after the H L A part of the complex is reacted with radiolabelled highly specific anti-HLA antibodies.
phocytes showed that the specific immunological activity was anti-HLA-A2 exclusively. There was no binding on HLA-A2 negative lymphocytes, whereas 15~o of the total radioactivity in the used preparations could be bound on HLA-A2 positive cells. Rabbit antiserum to human fl2MG (RAHu/fl2MG) was purchased as an immunoglobulin solution from Dakopatts, Denmark. Antiserum against Fc fragments of rabbit IgG was raised in pigs (SwAR/IgG[Fc]). After extensive absorption with human red blood cells and Sepharose-bound IgG, the antiserum gave in immunoelectrophoresis of normal rabbit serum a single IgG line and no visible precipitation of normal human serum. The radioimmunoassay (RIA) was carried out as follows: normal human serum was allowed to react with the ~25I-antiHLA-A2 preparation (2-10 nCi). Then RAHu/fl2MG was added and a cO-precipitate was formed with SwAR/ IgG(Fc). The precipitates were washed (3 times) with 1 ml of ice-cold phosphate buffered saline (PBS) and their radioactivity was measured in a Baird Atomic gamma counter. Details of the assay are in the legends to the tables. RESULTS Table 1 summarizes the results of the RIA when HLA-A2 positive serum was mixed with HLA-A2 Table 1. Radioimmunoassay of HLA-A2 serum antigen HLA-A2 positive serum (~l)
HLA-A2 negative serum (~l)
RIA radioactivity (04)
P valuea
4.8-5.0 5.8~.1 6.9-6.9 8.0-8.1 8.9-9.0 10.6-11.1 11.5-12.0
-<0.05 <0.001 <0.001 <0.001 <0.001 <0.001
MATERIALS AND METHODS
Human sera were taken from healthy HLA typed donors and stored at 4°C in the presence of 0.02~o NaN3; radiolabelled anti-HLA-A2 (~25I-anti-HLA-A2) was prepared as described elsewhere (Giphart et al., 1975); briefly: platelet eluates after antibody absorption were fractionated on a Sephadex G-200 column, the IgG fraction was radiolabelled with 125I using chloramine T and this preparation was absorbed and eluted again with HLA-A2 positive platelets. Alloantiserum (1 ml) yielded a radiolabelled antibody preparation of about 5/tCi. Binding assays on lym* Supported in part by the National Institute of Health (contract No. 1-AI-4-2508), the J. A. Cohen Institute of Radiopathology and Radiation Protection (IRS), the Dutch Foundation for Medical Research (FUNGO) which is subsidized by the Dutch Organization for the Advancement of Pure Research (ZWO) and the Dutch Organization for Health Research (TNO). 139
0 0.5 1 2 4 8 16
16 15.5 15 14 12 8 0
Varying amounts of HLA-A2 serum were diluted in nonHLA-A2 serum. Aliquots (16/A) of the mixtures were allowed to react with 2/A of a 12SI-anti-HLA-A2 preparation for 2 h r at 4°C. Then 15pl of RAHu/fl2MG was added and incubated for another hour at 4°C. Precipitation was carried out with 500/~1 SwAR/IgG(Fc) for 16 hr at 4°C. The precipitates were washed ( x 3) with 1 ml PBS and the radioactivity of the precipitate measured in a gamma counter, (duplicate determination). a The assay results of positive and negative serum were used in Student's t-test to obtain P values.
140
M.J. GIPHART, E. DOYER and J. W. BRUNING Table 2. Specificity controls of the RIA for HLA-A2 antigen complexed 32 microglobulin in human serum
Order of reagent addition
Percentage of radioactivity precipitated with serum of HLA type A2, BS, WI5 A1, 3, B7, 8
Type of experiment
a b
1, 4, 6 I, 5, 6
RIA
16.5 3.4
5.6 4.9
c
1, 2, 4, 6
Blocking with immune anti-HLA-A2 serum and control with non-immune serum
15.3
4.6
4.6 14.9
3.9 5.0
12.5
4.1
3.0
2.5
0.5
0.6
d 2, 1, 4, 6 e 1, 3, 4, 6 f
3. I. 4, 6
g 2, 8, 4, 6 h 2, 7, 4, 6
Inhibition by non-specific radiolabelled protein
Serum (10/A) was incubated at 4°C with a set of antisera and related or unrelated proteins. The varying order of addition of reagents is shown in the first column. The numbers correspond to the following antisera or proteins: 1, ~2SI-anti-HLA-A2 (23,000 counts/min) incubation for 2 hr. 2, anti-HLA-A2 serum (4#1) incubation for 2hr. 3, AB-serum (HLA type A29, W32, B7, 12) 4pl, incubation for 2hr. 4, RAHu/ /~2MG (15 td) incubation for l hr. 5, normal rabbit serum (15/A) incubation for 1 hr. 6, SwAR/IgG (0.5 ml) incubation for 16hr. 7, lZSl-labelled cytochrome c (315,000 counts/min) incubation for 2 hr. 8, ~25I-labelled human IgG (16,500 counts/min) incubation for 2 hr. The precipitates were washed 3 times with 1 ml of phosphate buffered saline (PBS) and the radioactivity of the precipitates measured in a gamma counter. The percentage of radioactivity in the precipitates was calculated (single determinations). negative serum. Serum volumes from 1 pl HLA-A2 positive serum led to statistically significant results. Up to 10pl of serum could be precipitated in the test and still have radiolabelled antibody excess. Table 2 summarizes control experiments on the specificity of the RIA with respect to the HLA type of the antigen source, the anti-32MG reagent and the specific blocking of the test with anti-HLA-A2 serum. The reactivity of RAHu/fl2MG is shown by replacement of RAHu//32MG by normal rabbit serum. The residual precipitation of radioactivity is no longer specific for HLA-A2 serum antigen (Table 2, a and b). The reactivity of the radioactive antibody preparation is shown by inhibition with immune antiHLA-A2 serum which inhibits precipitation of radioactivity only when added before the radiolabelled antibody (c and d). Non-immune serum does not inhibit specific precipitation (e and f). Precipitation of radioactivity with non-immune radiolabelled human IgG is low (g), however, not as low as with the unrelated protein cytochrome c (h). The residual precipitation of radioactivity with HLA-A2 negative serum (~f) may be ascribed to extra reactivities of normal rabbit serum, anti-/~2MG and SwAR/IgG (Fc). This non-specific precipitation with HLA-A2 negative serum could be reduced by prior addition of soluble human IgG (1 mg/ml) to the precipitating SwAR/IgG (Fc). Precipitation with serum of HLA type A2,-, B8, W15 and A1, 3, B7, 8 was then 12.85 and 2°//o, respectively. We conclude that the radioactivity will be precipitated only when HLA-A2 positive sera are reacted with ~ZSl-anti-HLA-A2, anti-/32MG and precipitating
antiserum. Specific blocking with anti-HLA-A2 serum can be observed. Non-immune rabbit serum precipitated amounts of radioactivity comparable to HLA-A2 negative serum. The RIA of 13 sera from HLA typed, healthy donors is summarized in Table 3. The amounts agree Table 3. Radioimmunoassay of HLA-A2 antigen complexed with /~zMG in serum from HLA typed donors Period of storage (months)
Precipitated radioactivity (~o)
7 8 12 I4 15
9.9-10.4 10.9 11.2 10.7 11.2 10.0-10.5 9.8-10.2
A1, 28, B8, 14 AW26, 28, B7, W17
3 14
9.5-9.8 9.0-9.1
A3, W30, B7, 13 All, W32, B5, 13 A1, 3, B8, W22 A3, W30, BWI6, 27 A1, W30, B13, 18 AI, W25, B18, 27
10 13 14 14 14 15
4.9-5.1 4.6-4.8 4.54.7 3.7 3.8 5.0-5.7 4.5~4.9
HLA type of serum donors
Group A2, A2, A2, A2, A2,
II
9, B12,W33, BW35, WI6 , B5, 27 3, BW35, 11, B27.
RIA for HLA-A2 antigen complexed with flzMG in serum from HLA typed donors: 10pl serum is incubated for 2 hr at 4~C with 2/~1 of 12SI-anti-HLA-A2 (4800 counts/ min) followed by incubation for 1 hr at 4°C with 15pl of RAHu/fl2MG and finally with 0.5 ml of SwAR/IgG (Fc) for 16 hr at 4°C. The precipitates were treated as described under Table 2 (duplicate determinations).
RIA for soluble HLA perfectly well with the HLA types of the serum donors, even with respect to the known cross-reactivity of HLA-A2 and A28. DISCUSSION The described RIA has been shown to be specific for HLA-A2. Table 1 shows that with the chosen quantities of reagents an antibody excess is still maintained, which enables all the antigens to be labelled with radioactive antibody and to be precipitated by anti-fl2MG and anti-rabbit IgG serum. Although some reactivity can be observed between precipitating swine anti-rabbit IgG and anti-/32MG with human IgG (Table 2), the difference between group I (HLA-A2 or A28 positive) and group II (HLA-A2 negative) is highly significant (P < 10 -s) using a Student's t-test (Table 3). We therefore conclude that HLA-A2 antigen in serum is complexed with /32MG, confirming the results of Oh et al. (1975). We did not 'observe false negative reactions contrary to Reisfeld et al. (1975) who have reported that in some sera HLA-antigens did not bind to fl2MG immunoadsorbents. A second conclusion from our results is that the complex is very stable: there is no apparent influence of storage time on the radioactivity in the precipitate. It seems therefore reasonable to assume that the HLA-flzMG complex is a stable entity in serum unless the sera are heat inactivated since this seems to destroy the antigenic activity. From our results it is not clear whether a 4-chain molecule is likely for the soluble complex antigens as has been suggested for membrane bound HLA antigens (Strominger et al., 1974; Cresswell et al., 1975). A multi-chain molecule can not be ruled out since Oh et al. (1975) have published
141
evidence that the tool. wt of the serum component carrying the HLA specificity is greater than 150,000. The question should be answered whether all HLA antigens in serum like those in the cell membrane are complexed with fl2MG. Experiments are designed to answer this question. The hypothetical possibility remains open that HLA antigens, upon shedding from the membrane, form complexes with serum fl2MG. We have described a powerful tool to study the soluble plasma HLA-antigens complexed with fl2MG, without the necessity of using complement dependent systems, or laborious purification devices. We report here only the results obtained with radiolabelled antiHLA-A2 antibodies, but radiolabelled antibody preparations of other HLA specificity have been prepared (Giphart et al.). The RIA is relatively easy, requires minute amounts of reagents, can be quantified and is very sensitive through the use of highly specific radiolabelled anti-HLA antibodies.
REFERENCES
Charlton R. K. & Zmijewski C. M. (1970) Science 170, 636. Cresswell P. & Dawson J. R. (1975) J. lmmun. 114, 523. Giphart M. J., Doyer E., Bruning J. W. & van Rood J. J. (1975) J. immunol. Meth. 9, 105. Oh S. K., Pellegrino M. A., Ferrone S., Sevier E. D. & Reisfeld R. A. (1975) Eur. J. lmmun. 5, 61l. Reisfeld R. A., Sevier E. D., Pellegrino M. A., Ferrone S. & Poulik M. D. (1975) Immunogenetics 2, 183. van Rood J. J., van Leeuwen A. & van Santen M. C. T. (1970) Nature 226, 366. Strominger J. L., Cresswell P., Grey H., Humphreys R. E., Mann D., McCune M., Parham P., Robb R., Sanderson A. R., Springer T. A., Terhorst C. & Turner M. J. (1974) Transplantn Rev. 21, 126.