Clini. exp. Inmunol. (1979) 36, 250-255.
A comparison of two 125I Clq binding tests to detect soluble immune complexes in serum of patients with malignant disease P. S. SHEPHERD The Division of Tumour Immunolog)', The Chester Beatty Research Institute, The Royal Marsden Hospital, Sutton, Surrey
(Accepted for publication 31 October 1978) SUMMARY
The "2I5 C lq deviation test and the modified "25I Clq PEG precipitation test were compared in their ability to detect soluble immune complexes in serum using a model system of HSA-rabbitanti-HSA, and were then applied to sera collected from patients with malignant and nonmalignant conditions. Despite close agreement in the model system, the two tests gave divergent results for the presence of Clq binding substances in individual serum samples collected from patients. The inherent complexities of interpreting Cl q binding in serum, in terms of the presence of soluble immune complexes, makes it questionable Whether either test can be relied upon to provide a means of identifying these complexes in the sera from patients with malignant disease.
INTRODUCTION In the search for soluble immune complexes in serum a number of biological and immuno-chemical techniques (Theofilopoulos, Wilson & Dixon, 1976; Onyexxotu, Holborows & Johnson, 1974; Hay, Nineham & Roitt, 1976; Casali et al., 1977) are being used. In the absence of a 'standard amount of soluble immune complex' the results given by individual techniques vary according to which types of immune complexes they are capable of measuring. Complement components are known to attach themselves to soluble immune complexes, and radioimmunoassays utilizing purified radiolabelled Clq subcomponent have been developed to measure circulating levels of these complexes in serum. The Clq deviation test of Sobel, Bokisch & Muller-Eberhard (1975) is based on the inhibition by immune complexes of the uptake of radiolabelled Clq by sensitized sheep erythrocytes. It requires the heat inactivation of test samples to destroy natural Clq and the separation of free from bound Cl q is done on a 40% sucrose gradient. In the modified Clq binding test described by Zubler et al. (1976), there is no heat inactivation step and the separation of bound from unbound radiolabelled C I q is achieved by precipitation with 2.50 0 polyethylene glycol. The present study compares these tx o methods of measuring radiolabelled Clq bound to immune complexes on serum samples collected from patients xith malignant and nonmalignant disease.
MATERIALS AND METHODS Sera. These were collected from healthy indixiduals and from patients not on treatment and stored at either -20 C or in liquid nitrogen. Each serum sample was frozen and thawed only once, and spun at 6000 g for 10 min at 23 C before use in any test. Estimations on serum samples were done without prior knowledge of the clinical status of the patient. Subjects. Immune complexes were sought in single serum samples from 184 patients and twenty healthy individuals. Patients with malignant disease were classified as havting localized disease or clinical eidence of metastases. Clinical staging of Hodgkin's lymphomas used the Ann Arbor classification (Carbone et al., 1971). Bluffers. Veronal buffer (VB) containing 8-3 g NaCl and 1 019 g/1 sodium barbital was adjusted to pH 7-4 with 1 0 N HC1.
Correspondence: Dr P. S. Shepherd, Department of Chemical Pathology, Guy's Hospital Medical School, London Bridge SEl 9RT. 0099-9104/79/0050-0250S02.00 C 1979 Blacklxell Scientific Publications
250
125I Clq binding to imnmnuine complexes in serum
251
Sucrose veronal buffer (SVB) contained 9.25% sucrose, 0-025 M sodium barbital, 1-5 x 10-4M Ca+ +, 5 x 10-4 M Mg+ +; the pH was adjusted to 7 2 with 1-0 N HCl and the specific conductance to 3 mmhos/cm2 with 9% sodium chloride. Clq subcomponent. The Clq subcomponent of human complement was isolated from serum using the method of Assimeh, Bing & Painter (1974), and radioiodinated by a modification of the chloramine T method (McConahey & Dixon, 1966). Specific radioactivity ranged between 0-4 and 0-8 pCi/pg Clq. The 1251I Clq PEG precipitation test. The modified 1251 Clq binding test was performed as described by Zubler et al. (1976). Briefly, 50 pl serum samples were added to 100 pl of 0-2 M EDTA (adjusted to pH 7 5 with NaOH) in 1-5 ml polypropylene test tubes (W. Sarstedt, UK Ltd.) and incubated for 30 min at 37 C. The mixtures were then transferred to an ice bath and 50 ul' 251 Clq in VBS (pH 7 4) (1 0 pg 125I Clq) added, followed by 1 0 ml of 300 (w/v) polyethylene glycol (PEG) (BDH, MW 6000) dissolved in 0 1 M boric acid containing 0-025 M disodium tetraborate, 0 075 M NaCl at PH 8 3. After 2 hr, the tubes were centrifuged at 1500 g for 45 min in a Mistral 6L set at 4 C. The supernatants were discarded and the radioactivity of the tubes counted in a Packard autogamma spectrometer. Results are expressed as a percentage of the same amount of 1251 Clq in 100 pl normal human serum (NHS) precipitated by 1-0 ml of 10% trichloroacetic acid. The 25I Clq deviation test. The method described by Sobel et al. (1975) was used. For sensitization of erythrocytes (E), a dilution of rat antiserum to sheep red blood cells which gave an uptake of 50-65°, of the available radiolabelled Clq was used. The test consisted of the following steps: (a) 50 pl serum+ 100 pl isotonic saline+ 100 pl SVB at pH 7 2 and specific conductivity of 9 mmhos/cm2 were mixed at room temperature in LP/3 polystyrene tubes (Luckham Ltd., UK); (b) the mixtures Xwere heated at 56 C for 30 min; (c) 10 pg 1251 Clq in 10 Al VBS containing 0.1% bovine serum albumen was added to each tube, and the mixture left at room temperature for 30 min before the addition of 109 sensitized sheep erythrocytes (EA) in 200 pl of SVB; (d) after 60 min, 200 pll of the reaction mixture was layered on to 200 pl of 40% sucrose soluinto in 0 05 M sodium phosphate buffer pH 7 2 in a 400 pl polyethylene microfuge tube (Sarstedt (UK) Ltd.), and centrifuged at 1500 g for 20 min; (e) the tubes were clamped with a haemostat 5 0 mm above the tip and the pellet cut off with scissors into a disposable counting tube. The radioactivity was counted in the supernatant and in the pellet and the percentage 1251 Clq uptake on EA calculated by the formula given below: cpm in pellet X 100. cpm in pellet cpm in supernatant The inhibition of l2251 Clq uptake by NHS was measured at the same time as that of the test samples in exery experiment. The percentage inhibition of the test samples was calculated using the formula: percentage uptake in control-percentage uptake in test 100 percentage uptake in control Normal aiid positive controls. A single serum sample was used as the normal control throughout all tests. It was chosen after determinations on twenty normal subjects and frozen in 250 pl samples which were thawed only once. The positive controls consisted of soluble complexes of HSA (A. B. Kabi, Stockholm, Sweden) and rabbit anti-HSA (kindly provided by Dr D. Darcy), formed in antigen excess. Preparation of soluble rabbit alti-HSA/HS.4 complexes. HSA (0.05-2 0 mg in 1 0 ml PBS) was added to 1 0 ml portions of the antiserum in glass tubes. After 2 hr at 37"C and 16 hr at 4VC the tubes were centrifuged at 1500 g and the supernatants removed. The precipitates were washed three times in cold PBS, redissolved in 0 1 N NaOH and their absorbance read at 280 nm. The supernatants were tested by the two 1251 Clq binding tests.
RESULTS All serum samples under investigation were assayed for i251 Clq binding by the deviation test and the PEG precipitation test on the same day, using the same positive and negative controls and 125J Clq preparation. The two tests gave similar results in the HSA-rabbit-anti-HSA model system shown in Fig. 1. Maximum binding of 1251 Clq was obtained in both tests using the supernatant from the tube which had contained 04 mg HSA, twice the antigen level giving optimum precipitation of specific antibody. At the higher concentrations of HSA (three to ten times equivalence), 1251 Clq binding was substantially reduced. No binding was detected in the supernatants containing antibody excess. Doubling dilutions made in normal rabbit serum (NRS) of the supernatant giving maximum binding were tested by both methods and the results are shown in Table 1. Significant binding was seen to a dilution of 1: 4, corresponding to approximately 6O0 fig of soluble complex. The results in Fig. 1 and Table 1 show that both the l251 Clq tests detect the same types of immune complexes, with the same degree of sensitivity. The normal range of i251 Clq binding in serum from twenty healthy individuals is given in Table 2. When comparing the results given by the two tests on serum samples from patients, the 'normal range' was defined as being within 2 s.d. of the normal control included in each set of assays.
P. S. Shepherd
252
i
.6
0114
70
0'E ~~~~~~~~~~~~~~E 60~~~~~~~~~~~~~~~~~~~~~0
40~~~~~~~~~~~~~~~~~~~~~~~~~0 - ~~~~~~~~~0*8 CL 0
30
~ ~ ~
*
l25I~ Cl bidn ~~'-
~
~~~~~~~~*
rprcnaepeiiain f.0
l
a
A on
n5
ape
C
0
0.2
0.4
0.6
Human
0.8
serum
.-0
12
albumen/ tube
1.4
1.6
1.8
(mg)
FIG. 1.
"'IJ
Immunoprecipitation curves of rabbit anti-HSA/HSA (± *)or percentage precipitation of Clq binding Clq
±) and percentage inhibition of A)done on 50 pI samples
of supernatants.
TABLE
1.
A comparison of both 125I Clq tests to detect dilutions of soluble immune complexes in two-fold antigen excess in normal rabbit serum
Detection of soluble complexes by the binding of 1251 Clq Dilution of soluble complexes*
Percentage inhibition in deviation test
Percentage precipitation in PEG precipitation test
1/1 1/2 1/4 NRS
57-1 29-0 13-0 0
43-1 18-1 10-8 10 0
* The complexes were rabbit-anti-HSA in two-fold excess of HSA; dilutions were made in normal rabbit serum (NRS). TABLE 2. Mean values for 1251 Clq binding from assays on twenty healthy individuals*
Clq deviation test Percentage uptake on EA+ 2 s.d. Percentage precipitation by PEG+ 2 s.d. *
Clq PEG precipitation test
62-5+ 6-2 -
5.4+ 3-8
Mean age 32-7 years, range 19-59 years.
The results of assays on serum samples from patients with either non-malignant or malignant disease are shown in Table 3. They show that there were a number of patients in whose sera there were greater than normal levels of 1251 Clq binding. There was, however, no concordance in the numbers of such 'positive' samples detected by the two Clq tests in any of the disease states examined. For example, in rheumatoid arthritis only one out of twelve patients was positive in both tests; of the fifty-five patients with carcinoma of the breast not one was positive in both tests, although a total of twenty-one cases with abnormal binding were detected. The discrepancies between the two tests do not seem to be entirely random: the PEG precipitation
125I Clq binding to immune complexes in serum
253
TABLE 3. A comparison of the 125I Clq deviation test and the 125I Clq PEG precipitation test in some disease states
Numbers within normal range
Non-malignant Coeliac disease Rheumatoid arthritis Malignant Carcinoma of breast: (a) No clinical evidence of distant metastases (b) Clinical evidence of distant metastases Melanoma All stages Hodgkin's lymphoma All stages Acute myeloid leukaemia
Numbers greater than normal range
Individual sample numbers greater PEG than normal in Deviation test both tests precipitation
Number of patients tested
Deviation test
PEG precipitation
6 12
5 11
5 1
1 1
1 11
0 1
33
31
30
2
3
0
22
22
6
0
16
0
38
34
21
4
17
4
40 33
19 12
35 11
21 21
5 22
5 14
test gave a much larger number of positive results in rheumatoid arthritis, carcinoma of the breast and melanoma (Table 3), while the reverse was true in Hodgkin's lymphomas. In the Hodgkin's lymphomas classified according to clinical stage (Table 4), the number of sera found to be positive in each test differed but they were all associated with the more advanced stages of the disease. However, in malignant melanoma and carcinoma of the breast with clinical evidence of metastases, the 125I Clq PEG precipitation test detected a greater number of positive results than that shown by the deviation test. TABLE 4. Relation of clinical staging in Hodgkin's lymphoma and the presence of soluble complexes measured by 1251 Clq
binding Stage I A+B
II A+B III A+3B IV A+B
Number in 125I Clq deviation test > 2 s.d.
Number in 1251 Clq PEG precipitation > 2 s.d.
Number in both tests > 2 s.d.
0/6 7/10 10/16 4/8
0/6 2/10 2/16 1/8
2 2 1
DISCUSSION The two 125I Clq binding tests under study here gave similar results when soluble immune complexes of HSA and rabbit anti-HSA formed in approximately two-fold antigen excess were assayed. The 125I Clq used throughout was isolated and iodinated by a different method to that used in the original PEG precipitation method. However, both methods, using the same 125I Clq, gave similar results in the model system, which would suggest that the 125I Clq had not altered the behaviour of either test. Their sensitivity was similar and both tests showed less binding at higher antigen concentrations; this was due to the increasing proportion of complexes consisting of one antibody molecule linked to two HSA molecules, which cannot bind Cl.
254
P. S. Shepherd
When the tests were applied to pathological sera under conditions designed to reduce experimental errors to a minimum, the level of agreement between them was much less than that obtained in the model system. If both tests were indeed measuring immune complexes in the patients' sera, then the different results given by each test must be due to the effect of the different experimental conditions of the two tests on these complexes. Alternatively, the discrepancies may be due to Clq binding by substances other than immune complexes. Acute phase proteins, e.g. C-reactive protein (CRP), polyanions, e.g. DNA, and polycations are known to bind the Clq subcomponent (Claus et al., 1977), as are certain lipopolysaccharides and various bacterial endotoxins. By the addition of several bacterial and phage products to serum, inhibition of Clq binding in the deviation test has been shown (Sobel et al., 1975) and single stranded DNA at 5-0 pg/ml can be detected. It is unlikely that serum collected from patients by normal methods would contain large quantities of DNA. In systemic lupus erythematosis where DNA-anti-DNA complexes are known to occur, Isui, Lambert & Miescher (1977) have shown, using two radioimmunoassays designed specifically to detect these complexes, that only 6% of all sera tested were positive, but the Clq PEG precipitation test detected 52% of them as positive. C-reactive protein is known to be elevated in a large number of malignant and non-malignant conditions. Levels of CRP were estimated by single radial immuno-diffusion (Mancini, Carbonara & Heremans, 1965) in all the serum samples from patients with Hodgkin's lymphoma, carcinoma of the breast and malignant melanoma, and no correlation between Clq binding and CRP was found. The presence of cryoglobulins in serum would be detected by the 125I Clq precipitation test but not the deviation test. Normal human serum incubated with aggregates of human IgG at 370C and then heated at 560C for 30 min showed a reduction in 125I Clq binding to these aggregates in the PEG precipitation test (Zubler et al., 1976). It is difficult to assess the amount of damage caused by heating serum containing soluble immune complexes instead of aggregates of immunoglobulins. Heating the complexes formed in the rabbit anti-HSA model system did not greatly affect Clq binding as shown by these two tests. Apart from the effects of heat on immune complexes, the changes induced by chelating agents and pH may be important. The 125I Clq PEG precipitation test is performed in a final concentration of 17 mM EDTA and at pH 8-3. Soluble immune complexes formed in the inhibition zone of the precipitation reaction between egg albumen and rabbit-anti-egg albumen were unaffected by pH 6&25-8&45, but inhibition of precipitate formation was practically complete at pH 4-2 and 9.5 (Kleinschmidt & Boyer, 1951). The precipitation of a population of homologous sheep antibodies against ax-poly (Glu60 Ala30 Tyr"0) (GAT) has been shown to specifically require calcium ions, (Liberti, Maurer & Clark, 1971), but whether immune complexes produced in man are similarly affected under these conditions is unknown. The problems raised by this study are important because they show that two 125I Clq binding tests can fail to agree on what they are detecting in the same serum sample. For clinical purposes, relating the results given by these two tests to clinical diagnosis or management of soluble immune complexes in malignant diseases is clearly unjustified. The author wishes to express his sincere appreciation to Professor P. Alexander and Dr E. Orlans for their help and advice in the preparation of this paper, and to Drs T. and R. Powles, Professor M. Peckham and Dr D. W. Hedley, and Sister R. Grabow who helped in the collection of serum samples from patients. This work was carried out during the tenure of a Royal Marsden Hospital Fellowship.
REFERENCES (1977) Solid-phase enzyme immunoassay or radioimmunoASSIMEH, S.N., BING, D.H. & PAINTER, R.H. (1974) A assay for the detection of immune complexes based on simple method for the isolation of the subcomponents of their recognition by conglutinin: conglutinin-binding the first component of complement by affinity chromatotest. A comparative study with '25I-labelled Clq binding graphy. J. Immunol. 113, 225. and Raji cell RIA tests. Clin. exp. Immunol. 29, 242. CARBONE, P.P., KAPLAN, H.S., MUSHOFF, K., SMITHER S, S.W. & TUBIANA, M. (1971) Report of the committee on CLAUS, D.R., SIEGEL, J., PETRAS, K., SKOR, D., OsmAND, A.P. & GEwuaz, H. (1977) Complement activation by Hodgkin's disease staging procedures. Cancer Res. 31, interaction of polyanions and polycations. J. Immunol. 1860. 118, 83. CASALI, P., Bossus, A., CARPENTIER, N.A. & LAMBERT, P.H.
25I Clq binding to immune complexes in serum HAY, F.C., NINEHAM, L.J. & RoITT, I.M. (1976) Routine assay for the detection of immune complexes of known immunoglobulin class using solid phase Clq. Clin. exp. Immunol. 24, 396. Izui, S., LAMBERT, P.H. & MIESCHER, P.A. (1977) Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosis. Clin. exp. Immunol. 30, 384. KLEINSCHMIDT, W.J. & BOYER, P.D. (1951) Interaction of protein antigens and antibodies. J. Immunol. 69, 247. LIBERTI, P.A., MAURER, P.H. & CLARK, L.G. (1971) Antigenicity of polypeptides (poly-a-amino acids). Physicochemical studies of a calcium-dependent antigenantibody reaction. Biochemistry, 10, 1632. MANCINI, G., CARBONARA, A.O. & HEREMANS, J.F. (1965) Immuno-chemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235. MCCONAHEY, P.J. & DIXON, F.J. (1966) A method of trace
255
iodination of proteins for immunologic studies. Int. Arch. Allergy Appl. Immunol. 29, 185. ONYEWOTU, I.I., HOLBOROW, E.J. & JOHNSON, G.D. (1974) Detection and radioassay of soluble circulating immune complexes using guinea-pig peritoneal exudate cells. Nature (Lond.), 248, 156. SOBEL, A.T., BOKISCH, V.A. & MULLER-EBERHARD, H.J. (1975) Clq deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum. J. exp. Med. 142, 139. THEOFILOPOULOUS, AN., WILSON, C.B. & DIXON, P.J. (1976) The Raji cell radioimmune assay for detecting immune complexes in human sera. 5. c/in. Invest. 57, 169. ZUBLER, R.H., LANGE, G., LAMBERT, P.H. & MIESCHER, P.A. (1976) Detection of immune complexes in unheated sera by a modified '25I-labelled Clq binding test. 5. Immunol. 116, 232.
A comparison of two 125I Clq binding tests to detect soluble immune complexes in serum of patients with malignant disease P. S. SHEPHERD The Division of Tumour Immunolog)', The Chester Beatty Research Institute, The Royal Marsden Hospital, Sutton, Surrey
(Accepted for publication 31 October 1978) SUMMARY
The "2I5 C lq deviation test and the modified "25I Clq PEG precipitation test were compared in their ability to detect soluble immune complexes in serum using a model system of HSA-rabbitanti-HSA, and were then applied to sera collected from patients with malignant and nonmalignant conditions. Despite close agreement in the model system, the two tests gave divergent results for the presence of Clq binding substances in individual serum samples collected from patients. The inherent complexities of interpreting Cl q binding in serum, in terms of the presence of soluble immune complexes, makes it questionable Whether either test can be relied upon to provide a means of identifying these complexes in the sera from patients with malignant disease.
INTRODUCTION In the search for soluble immune complexes in serum a number of biological and immuno-chemical techniques (Theofilopoulos, Wilson & Dixon, 1976; Onyexxotu, Holborows & Johnson, 1974; Hay, Nineham & Roitt, 1976; Casali et al., 1977) are being used. In the absence of a 'standard amount of soluble immune complex' the results given by individual techniques vary according to which types of immune complexes they are capable of measuring. Complement components are known to attach themselves to soluble immune complexes, and radioimmunoassays utilizing purified radiolabelled Clq subcomponent have been developed to measure circulating levels of these complexes in serum. The Clq deviation test of Sobel, Bokisch & Muller-Eberhard (1975) is based on the inhibition by immune complexes of the uptake of radiolabelled Clq by sensitized sheep erythrocytes. It requires the heat inactivation of test samples to destroy natural Clq and the separation of free from bound Cl q is done on a 40% sucrose gradient. In the modified Clq binding test described by Zubler et al. (1976), there is no heat inactivation step and the separation of bound from unbound radiolabelled C I q is achieved by precipitation with 2.50 0 polyethylene glycol. The present study compares these tx o methods of measuring radiolabelled Clq bound to immune complexes on serum samples collected from patients xith malignant and nonmalignant disease.
MATERIALS AND METHODS Sera. These were collected from healthy indixiduals and from patients not on treatment and stored at either -20 C or in liquid nitrogen. Each serum sample was frozen and thawed only once, and spun at 6000 g for 10 min at 23 C before use in any test. Estimations on serum samples were done without prior knowledge of the clinical status of the patient. Subjects. Immune complexes were sought in single serum samples from 184 patients and twenty healthy individuals. Patients with malignant disease were classified as havting localized disease or clinical eidence of metastases. Clinical staging of Hodgkin's lymphomas used the Ann Arbor classification (Carbone et al., 1971). Bluffers. Veronal buffer (VB) containing 8-3 g NaCl and 1 019 g/1 sodium barbital was adjusted to pH 7-4 with 1 0 N HC1.
Correspondence: Dr P. S. Shepherd, Department of Chemical Pathology, Guy's Hospital Medical School, London Bridge SEl 9RT. 0099-9104/79/0050-0250S02.00 C 1979 Blacklxell Scientific Publications
250
125I Clq binding to imnmnuine complexes in serum
251
Sucrose veronal buffer (SVB) contained 9.25% sucrose, 0-025 M sodium barbital, 1-5 x 10-4M Ca+ +, 5 x 10-4 M Mg+ +; the pH was adjusted to 7 2 with 1-0 N HCl and the specific conductance to 3 mmhos/cm2 with 9% sodium chloride. Clq subcomponent. The Clq subcomponent of human complement was isolated from serum using the method of Assimeh, Bing & Painter (1974), and radioiodinated by a modification of the chloramine T method (McConahey & Dixon, 1966). Specific radioactivity ranged between 0-4 and 0-8 pCi/pg Clq. The 1251I Clq PEG precipitation test. The modified 1251 Clq binding test was performed as described by Zubler et al. (1976). Briefly, 50 pl serum samples were added to 100 pl of 0-2 M EDTA (adjusted to pH 7 5 with NaOH) in 1-5 ml polypropylene test tubes (W. Sarstedt, UK Ltd.) and incubated for 30 min at 37 C. The mixtures were then transferred to an ice bath and 50 ul' 251 Clq in VBS (pH 7 4) (1 0 pg 125I Clq) added, followed by 1 0 ml of 300 (w/v) polyethylene glycol (PEG) (BDH, MW 6000) dissolved in 0 1 M boric acid containing 0-025 M disodium tetraborate, 0 075 M NaCl at PH 8 3. After 2 hr, the tubes were centrifuged at 1500 g for 45 min in a Mistral 6L set at 4 C. The supernatants were discarded and the radioactivity of the tubes counted in a Packard autogamma spectrometer. Results are expressed as a percentage of the same amount of 1251 Clq in 100 pl normal human serum (NHS) precipitated by 1-0 ml of 10% trichloroacetic acid. The 25I Clq deviation test. The method described by Sobel et al. (1975) was used. For sensitization of erythrocytes (E), a dilution of rat antiserum to sheep red blood cells which gave an uptake of 50-65°, of the available radiolabelled Clq was used. The test consisted of the following steps: (a) 50 pl serum+ 100 pl isotonic saline+ 100 pl SVB at pH 7 2 and specific conductivity of 9 mmhos/cm2 were mixed at room temperature in LP/3 polystyrene tubes (Luckham Ltd., UK); (b) the mixtures Xwere heated at 56 C for 30 min; (c) 10 pg 1251 Clq in 10 Al VBS containing 0.1% bovine serum albumen was added to each tube, and the mixture left at room temperature for 30 min before the addition of 109 sensitized sheep erythrocytes (EA) in 200 pl of SVB; (d) after 60 min, 200 pll of the reaction mixture was layered on to 200 pl of 40% sucrose soluinto in 0 05 M sodium phosphate buffer pH 7 2 in a 400 pl polyethylene microfuge tube (Sarstedt (UK) Ltd.), and centrifuged at 1500 g for 20 min; (e) the tubes were clamped with a haemostat 5 0 mm above the tip and the pellet cut off with scissors into a disposable counting tube. The radioactivity was counted in the supernatant and in the pellet and the percentage 1251 Clq uptake on EA calculated by the formula given below: cpm in pellet X 100. cpm in pellet cpm in supernatant The inhibition of l2251 Clq uptake by NHS was measured at the same time as that of the test samples in exery experiment. The percentage inhibition of the test samples was calculated using the formula: percentage uptake in control-percentage uptake in test 100 percentage uptake in control Normal aiid positive controls. A single serum sample was used as the normal control throughout all tests. It was chosen after determinations on twenty normal subjects and frozen in 250 pl samples which were thawed only once. The positive controls consisted of soluble complexes of HSA (A. B. Kabi, Stockholm, Sweden) and rabbit anti-HSA (kindly provided by Dr D. Darcy), formed in antigen excess. Preparation of soluble rabbit alti-HSA/HS.4 complexes. HSA (0.05-2 0 mg in 1 0 ml PBS) was added to 1 0 ml portions of the antiserum in glass tubes. After 2 hr at 37"C and 16 hr at 4VC the tubes were centrifuged at 1500 g and the supernatants removed. The precipitates were washed three times in cold PBS, redissolved in 0 1 N NaOH and their absorbance read at 280 nm. The supernatants were tested by the two 1251 Clq binding tests.
RESULTS All serum samples under investigation were assayed for i251 Clq binding by the deviation test and the PEG precipitation test on the same day, using the same positive and negative controls and 125J Clq preparation. The two tests gave similar results in the HSA-rabbit-anti-HSA model system shown in Fig. 1. Maximum binding of 1251 Clq was obtained in both tests using the supernatant from the tube which had contained 04 mg HSA, twice the antigen level giving optimum precipitation of specific antibody. At the higher concentrations of HSA (three to ten times equivalence), 1251 Clq binding was substantially reduced. No binding was detected in the supernatants containing antibody excess. Doubling dilutions made in normal rabbit serum (NRS) of the supernatant giving maximum binding were tested by both methods and the results are shown in Table 1. Significant binding was seen to a dilution of 1: 4, corresponding to approximately 6O0 fig of soluble complex. The results in Fig. 1 and Table 1 show that both the l251 Clq tests detect the same types of immune complexes, with the same degree of sensitivity. The normal range of i251 Clq binding in serum from twenty healthy individuals is given in Table 2. When comparing the results given by the two tests on serum samples from patients, the 'normal range' was defined as being within 2 s.d. of the normal control included in each set of assays.
P. S. Shepherd
252
i
.6
0114
70
0'E ~~~~~~~~~~~~~~E 60~~~~~~~~~~~~~~~~~~~~~0
40~~~~~~~~~~~~~~~~~~~~~~~~~0 - ~~~~~~~~~0*8 CL 0
30
~ ~ ~
*
l25I~ Cl bidn ~~'-
~
~~~~~~~~*
rprcnaepeiiain f.0
l
a
A on
n5
ape
C
0
0.2
0.4
0.6
Human
0.8
serum
.-0
12
albumen/ tube
1.4
1.6
1.8
(mg)
FIG. 1.
"'IJ
Immunoprecipitation curves of rabbit anti-HSA/HSA (± *)or percentage precipitation of Clq binding Clq
±) and percentage inhibition of A)done on 50 pI samples
of supernatants.
TABLE
1.
A comparison of both 125I Clq tests to detect dilutions of soluble immune complexes in two-fold antigen excess in normal rabbit serum
Detection of soluble complexes by the binding of 1251 Clq Dilution of soluble complexes*
Percentage inhibition in deviation test
Percentage precipitation in PEG precipitation test
1/1 1/2 1/4 NRS
57-1 29-0 13-0 0
43-1 18-1 10-8 10 0
* The complexes were rabbit-anti-HSA in two-fold excess of HSA; dilutions were made in normal rabbit serum (NRS). TABLE 2. Mean values for 1251 Clq binding from assays on twenty healthy individuals*
Clq deviation test Percentage uptake on EA+ 2 s.d. Percentage precipitation by PEG+ 2 s.d. *
Clq PEG precipitation test
62-5+ 6-2 -
5.4+ 3-8
Mean age 32-7 years, range 19-59 years.
The results of assays on serum samples from patients with either non-malignant or malignant disease are shown in Table 3. They show that there were a number of patients in whose sera there were greater than normal levels of 1251 Clq binding. There was, however, no concordance in the numbers of such 'positive' samples detected by the two Clq tests in any of the disease states examined. For example, in rheumatoid arthritis only one out of twelve patients was positive in both tests; of the fifty-five patients with carcinoma of the breast not one was positive in both tests, although a total of twenty-one cases with abnormal binding were detected. The discrepancies between the two tests do not seem to be entirely random: the PEG precipitation
125I Clq binding to immune complexes in serum
253
TABLE 3. A comparison of the 125I Clq deviation test and the 125I Clq PEG precipitation test in some disease states
Numbers within normal range
Non-malignant Coeliac disease Rheumatoid arthritis Malignant Carcinoma of breast: (a) No clinical evidence of distant metastases (b) Clinical evidence of distant metastases Melanoma All stages Hodgkin's lymphoma All stages Acute myeloid leukaemia
Numbers greater than normal range
Individual sample numbers greater PEG than normal in Deviation test both tests precipitation
Number of patients tested
Deviation test
PEG precipitation
6 12
5 11
5 1
1 1
1 11
0 1
33
31
30
2
3
0
22
22
6
0
16
0
38
34
21
4
17
4
40 33
19 12
35 11
21 21
5 22
5 14
test gave a much larger number of positive results in rheumatoid arthritis, carcinoma of the breast and melanoma (Table 3), while the reverse was true in Hodgkin's lymphomas. In the Hodgkin's lymphomas classified according to clinical stage (Table 4), the number of sera found to be positive in each test differed but they were all associated with the more advanced stages of the disease. However, in malignant melanoma and carcinoma of the breast with clinical evidence of metastases, the 125I Clq PEG precipitation test detected a greater number of positive results than that shown by the deviation test. TABLE 4. Relation of clinical staging in Hodgkin's lymphoma and the presence of soluble complexes measured by 1251 Clq
binding Stage I A+B
II A+B III A+3B IV A+B
Number in 125I Clq deviation test > 2 s.d.
Number in 1251 Clq PEG precipitation > 2 s.d.
Number in both tests > 2 s.d.
0/6 7/10 10/16 4/8
0/6 2/10 2/16 1/8
2 2 1
DISCUSSION The two 125I Clq binding tests under study here gave similar results when soluble immune complexes of HSA and rabbit anti-HSA formed in approximately two-fold antigen excess were assayed. The 125I Clq used throughout was isolated and iodinated by a different method to that used in the original PEG precipitation method. However, both methods, using the same 125I Clq, gave similar results in the model system, which would suggest that the 125I Clq had not altered the behaviour of either test. Their sensitivity was similar and both tests showed less binding at higher antigen concentrations; this was due to the increasing proportion of complexes consisting of one antibody molecule linked to two HSA molecules, which cannot bind Cl.
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P. S. Shepherd
When the tests were applied to pathological sera under conditions designed to reduce experimental errors to a minimum, the level of agreement between them was much less than that obtained in the model system. If both tests were indeed measuring immune complexes in the patients' sera, then the different results given by each test must be due to the effect of the different experimental conditions of the two tests on these complexes. Alternatively, the discrepancies may be due to Clq binding by substances other than immune complexes. Acute phase proteins, e.g. C-reactive protein (CRP), polyanions, e.g. DNA, and polycations are known to bind the Clq subcomponent (Claus et al., 1977), as are certain lipopolysaccharides and various bacterial endotoxins. By the addition of several bacterial and phage products to serum, inhibition of Clq binding in the deviation test has been shown (Sobel et al., 1975) and single stranded DNA at 5-0 pg/ml can be detected. It is unlikely that serum collected from patients by normal methods would contain large quantities of DNA. In systemic lupus erythematosis where DNA-anti-DNA complexes are known to occur, Isui, Lambert & Miescher (1977) have shown, using two radioimmunoassays designed specifically to detect these complexes, that only 6% of all sera tested were positive, but the Clq PEG precipitation test detected 52% of them as positive. C-reactive protein is known to be elevated in a large number of malignant and non-malignant conditions. Levels of CRP were estimated by single radial immuno-diffusion (Mancini, Carbonara & Heremans, 1965) in all the serum samples from patients with Hodgkin's lymphoma, carcinoma of the breast and malignant melanoma, and no correlation between Clq binding and CRP was found. The presence of cryoglobulins in serum would be detected by the 125I Clq precipitation test but not the deviation test. Normal human serum incubated with aggregates of human IgG at 370C and then heated at 560C for 30 min showed a reduction in 125I Clq binding to these aggregates in the PEG precipitation test (Zubler et al., 1976). It is difficult to assess the amount of damage caused by heating serum containing soluble immune complexes instead of aggregates of immunoglobulins. Heating the complexes formed in the rabbit anti-HSA model system did not greatly affect Clq binding as shown by these two tests. Apart from the effects of heat on immune complexes, the changes induced by chelating agents and pH may be important. The 125I Clq PEG precipitation test is performed in a final concentration of 17 mM EDTA and at pH 8-3. Soluble immune complexes formed in the inhibition zone of the precipitation reaction between egg albumen and rabbit-anti-egg albumen were unaffected by pH 6&25-8&45, but inhibition of precipitate formation was practically complete at pH 4-2 and 9.5 (Kleinschmidt & Boyer, 1951). The precipitation of a population of homologous sheep antibodies against ax-poly (Glu60 Ala30 Tyr"0) (GAT) has been shown to specifically require calcium ions, (Liberti, Maurer & Clark, 1971), but whether immune complexes produced in man are similarly affected under these conditions is unknown. The problems raised by this study are important because they show that two 125I Clq binding tests can fail to agree on what they are detecting in the same serum sample. For clinical purposes, relating the results given by these two tests to clinical diagnosis or management of soluble immune complexes in malignant diseases is clearly unjustified. The author wishes to express his sincere appreciation to Professor P. Alexander and Dr E. Orlans for their help and advice in the preparation of this paper, and to Drs T. and R. Powles, Professor M. Peckham and Dr D. W. Hedley, and Sister R. Grabow who helped in the collection of serum samples from patients. This work was carried out during the tenure of a Royal Marsden Hospital Fellowship.
REFERENCES (1977) Solid-phase enzyme immunoassay or radioimmunoASSIMEH, S.N., BING, D.H. & PAINTER, R.H. (1974) A assay for the detection of immune complexes based on simple method for the isolation of the subcomponents of their recognition by conglutinin: conglutinin-binding the first component of complement by affinity chromatotest. A comparative study with '25I-labelled Clq binding graphy. J. Immunol. 113, 225. and Raji cell RIA tests. Clin. exp. Immunol. 29, 242. CARBONE, P.P., KAPLAN, H.S., MUSHOFF, K., SMITHER S, S.W. & TUBIANA, M. (1971) Report of the committee on CLAUS, D.R., SIEGEL, J., PETRAS, K., SKOR, D., OsmAND, A.P. & GEwuaz, H. (1977) Complement activation by Hodgkin's disease staging procedures. Cancer Res. 31, interaction of polyanions and polycations. J. Immunol. 1860. 118, 83. CASALI, P., Bossus, A., CARPENTIER, N.A. & LAMBERT, P.H.
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iodination of proteins for immunologic studies. Int. Arch. Allergy Appl. Immunol. 29, 185. ONYEWOTU, I.I., HOLBOROW, E.J. & JOHNSON, G.D. (1974) Detection and radioassay of soluble circulating immune complexes using guinea-pig peritoneal exudate cells. Nature (Lond.), 248, 156. SOBEL, A.T., BOKISCH, V.A. & MULLER-EBERHARD, H.J. (1975) Clq deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum. J. exp. Med. 142, 139. THEOFILOPOULOUS, AN., WILSON, C.B. & DIXON, P.J. (1976) The Raji cell radioimmune assay for detecting immune complexes in human sera. 5. c/in. Invest. 57, 169. ZUBLER, R.H., LANGE, G., LAMBERT, P.H. & MIESCHER, P.A. (1976) Detection of immune complexes in unheated sera by a modified '25I-labelled Clq binding test. 5. Immunol. 116, 232.
