Journal of Immunological Methods, 126 (1990) 13-20 Elsevier
13
JIM 05413
Measurement of the 'fast' or complexed form of a 2 macroglobulin in biological fluids using a sandwich enzyme immunoassay R.E. Banks 1, S.W. Evans 1, F. V a n L e u v e n 2, D. A l e x a n d e r 3, M.J. M c M a h o n 3 a n d J.T. W h i c h e r 1 1 Department of Chemical Pathology, University of Leeds, Leeds, U.K., : Department of Human Genetics, University of Leuven, Gasthuisberg ON.G, B 3000 Leuven, Belgium, and 3 Department of Surgery, The General Infirmary at Leeds, Leeds, U.K. (Received 6 July 1989, revised received 8 September 1989, accepted 11 September 1989)
A sandwich enzyme immunoassay has been developed for measuring the 'fast' or complexed form of a 2 macroglobulin using a complex-specific monoclonal antibody. The working range of this assay is 1.5-15 /xg/1 and is suitable for use with various biological fluids. Using this assay the normal plasma levels were found to range from 4.2 rag/1 to 14.4 m g / l (0.17%-0.70% of total a2 macroglobulin) with a mean value of 7.6 mg/1 + 2.6 (0.37% ___0.12% of total a 2 macroglobulin). Elevated levels were seen in plasma samples taken on the day of admission from patients with acute pancreatitis and in some synovial fluid samples from patients with various arthritides. Key words: ELISA; ctz Macroglobulin-proteinase complex; Clinical assay; Pancreatitis; Arthritis; Monoclonal antibody
Introduction The glycoprotein a 2 macroglobulin (a2-M) is one of the major proteinase inhibitors in plasma (for reviews see Travis and Salvesen, 1983; Roberts, 1986). It has a molecular weight of approximately 720 000 and is assembled from four identical subunits which are covalently associated in pairs by disulfide bonds and the resulting dimers linked non-covalently. A broad spectrum of proteinases of different specificities, catalytic mechanisms and tissue sources react with a2-M. Uniquely, the proteolytic activity of the bound
Correspondence to: R.E. Banks, Department of Chemical Pathology, University of Leeds, Leeds, LS2 9JT, U.K. Abbreviations." a2-M, a 2 macroglobulin: ELISA, enzymelinked immunosorbent assay; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.1% Tween 20, OPD = o-phenylenediamine dihydrochloride; HRP, horseradishperoxidase,
proteinases is retained although only towards low molecular weight substrates. This may be explained by the 'trap' hypothesis (Barrett and Starkey, 1973) which proposes that the proteinase attacks a 'bait' region in the a2-M molecule causing a proteolytic cleavage and a rapid conformational change. This results in the physical entrapment of the proteinase molecule within the macroglobulin molecule thus preventing reactivity of the bound proteinase with large molecular weight substrates and, in most cases, with antibodies. The apparently irreversible change from an active to a complexed or inactive form of a2-M is accompanied by a change in electrophoretic mobility from a 'slow' form to a 'fast' form (Barrett et al., 1979). A similar transition to the complexed or 'fast' form is also produced by the covalent binding to a2-M of primary amines such as methylamine (Barrett et al., 1979; Van Leuven et al., 1981a,b), and during prolonged storage (Barrett et al., 1979). For the purpose of this paper the term
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14
'complexed' will be used to describe the 'fast' form of a2-M, irrespective of whether produced by proteinase binding, methylamine binding or storage, The use of monoclonal antibodies has revealed the existence of antigenic determinants which are present only on the complexed from of a2-M (Marynen et al., 1981). The epitopes recognised by these antibodies have in many cases been mapped to the receptor recognition site of a2-M (Van Leuven et al., 1986a,b, 1988) which is expressed only on the complexed form and allows its recognition by receptors present on various cell types including fibroblasts (Van Leuven et al., 1979) and macrophages (Debanne et al., 1975). Complexed aE-M is removed rapidly from the circulation by receptor-mediated endocytosis with a reported half-life in mice of < 5 rain compared with several hours for the native form (Imber and Pizzo, 1981). 19/2 macroglobulin has been reported to exhibit various immuno-modulatory actions including suppression of cell-mediated cytotoxicity by the native form, possibly via an inhibition of T cell surface-associated proteinases involved in target recognition (Ades et al., 1982; Petersen et al., 1989), and the inhibition of activated T cell proliferation by both the native and complexed forms (Mannhalter et al., 1986; Petersen et al., 1989). This suppressive effect of complexed a2-M in proliferative assays may possibly be explained by the degradative activity associated with the bound proteinase following the finding by Borth and Teodorescu (1986) that ct2-M bound proteinase is able to degrade IL-2 (T cell growth factor). Additionally it has been suggested that aE-M-protease complexes may play a role in polyclonal B cell activation (Teodorescu et al., 1981; Chang et al., 1983) and recently a2-M has been identified as a carrier protein for IL-6 (Matsuda et al., 1989). Various methods have been used to determine the amount of complexed CtE-Mpresent in biological samples including gradient gel electrophoresis of samples before and after reduction with dithiothreitol which dissociates native aE-M (Levine et al., 1987), radioimmunoassay with a monoclonal antibody specific to the complexed form of t~E-M (Marynen et al., 1984), the measurement of trypsin-binding capacity of solid-phase im-
munoadsorbed ot2-M using radiolabelled trypsin (Borth et al., 1986), and measurement of the hydrolytic activity of immunoadsorbed plasma ot2-M against Chromozym-Try (Boehringer Corporation, Lewes, U.K.), a broad-spectrum trypsin substrate (Gaspar et al,. 1984; Teodorescu et al., 1984). We report here the development of a sensitive ELISA method incorporating a complex-specific monoclonal antibody which permits the measurement of the complexed form of a2-M , and investigate the levels present in controls and various clinical conditions.
Materials and methods
Materials Nunc Immunoplate I microwell plates were obtained from Gibco BRL (Paisley, U.K.). Purified a2-M, Tween 20 and OPD were purchased from Sigma (Poole, U.K.). Rabbit anti-human CtE-M and HRP-labelled swine anti-rabbit Ig were purchased from Dako (High Wycombe, U.K.). Nephelometric grade antiserum to a2-M and standard serum were obtained from Behring Diagnostics (Hounslow, U.K.). The monoclonal antibody F44GA2 to o~2-M (complex specific) was prepared and characterised by Van Leuven et al. as previously described (1988). All other chemicals were obtained from BDH (Poole, U.K.).
Biologicalsamples The effect of various anticoagulants on levels of complexed ct2-M was investigated by taking blood samples from five healthy subjects and dividing them into tubes containing either potassium EDTA, sodium citrate, lithium heparin or no anticoagulant. The samples were then centrifuged at 1500 × g for 10 rain and the plasma or serum removed and stored in aliquots at - 4 0 °C until assayed. A comparison of levels of complexed a2-M in various clinical conditions was performed by collecting plasma samples, obtained by centrifugation of citrated whole blood at 1500 × g for 10 min, from 22 healthy controls (seven males aged 24-69 years, mean 37.1 + 15.2, and 15 females aged 29-77 years, mean 59.1 _+ 14.7), and from 11 patients with pancreatitis of differing aetiology,
15
Measurementof total a2-M
severity and outcome, on the day of hospital admission (seven males and four females aged 44-96 years, mean 68.2 + 14.3). In addition citrated synovial fluid was obtained from two patients with osteoarthritis, two patients with seropositive rheumatoid arthritis, one patient with psoriatic arthritis, one patient with seronegative polyarthritis and one patient with probable Reiter's disease (three females and four males aged 33-72 years, mean 56.5 4- 12.2), and centrifuged at 1500 × g for 10 min. Plasma and synovial fluid samples were aliquoted and stored at - 4 0 ° C prior to analysis, Stability during storage was assessed by analysing aliquots from several samples on two or three later occasions,
Total levels of a2-M in samples were measured using a Behring nephelometric analyzer together with Behring nephelometric grade anti-a2-M and Behring N Protein Standard serum calibrated against a WHO International Reference Preparation.
Enzymeimmunoassayfor complexed ae-M The principle of the assay is illustrated in Fig. 1 with the appropriate antibody dilutions being determined by checkerboard titration. Nunc Immunoplate I microwell plates were incubated with F44GA2 antibody in PBS, pH 7.2 (100 #l/well at a concentration of 1.3 /zg/ml) at 4 ° C overnight. Plates were rinsed 3 × in PBS-T and triplicate 100 #1 aliquots of complexed a2-M standards and biological samples diluted a minimum of 1000 × in PBS-T were added to the wells. The plates were sealed and incubated at 15 °C for 2 h. Following the incubation the plates were rinsed 3 × with PBS-T and rabbit anti-human a2-M diluted in PBS-T was added to each well. After 1 h at room temperature, the plates were washed a further 3 × and HRP-conjugated swine anti-rabbit immunoglobulin (affinity purified) was added to the plates
Complexed ('fast')a2-Mstandards A stock solution of the purified aE-M was prepared and standardised by nephelometry as described below. This preparation was judged to contain a2-M which was 100% in the complexed or 'fast' form since no difference was found in the ELISA before and after overnight treatment with methylamine as previously described (Barrett et al., 1979). Standards ranging from 1.5 # g / l to 15 #g/1 were prepared using PBS-T as a diluent,
Rabb.
Complexed C~2-M
Anti0(2- M
HRP-linked Swine antirabbit Ig
F44GA2 antiC~2-M (complexspecific)
~HRP COLOUR '
Subatrate
÷
~~lr
Fig. 1. Pfincip|e of the sandwich ELISA for comp]cxed aE-macrog]obu]in.
16 which were then incubated at room temperature for 1 h. Unbound conjugate was then removed by washing 3 × with PBS-T, 100 /zl of substrate solution (0.1 M citric acid-phosphate buffer, p H 5.0, containing 8 mg O P D and 5 /~1 of 30% H 2 0 2 / 1 2 ml of buffer) were added to each well and the plates left in the dark for 15 min. The reaction was terminated by the addition of 1 M H2SO 4. Absorbance was read at 492 nm on a Titertek Multiskan M C C plate reader. The concentration of complexed a2-M in the samples w a s calculated from the standard curve for each plate,
TABLEI COMPARISONOF LEVELS OF COMPLEXED a2-M IN PLASMA (VARIOUS ANTICOAGULANTS) AND SERUM OF A REPRESENTATIVE SAMPLE Statisticalanalysis was by Student's t test against the citrated sample. Sample Citrated plasma EDTA plasma Heparinised plasma
Serum
Complexed a2-M (mg/1) (mean + SD, n = 3) 5.2+ 0.1 5.3+ 0.3 11.8 + 0.2 236.5 ___25.0
Significance level NS P < 0.001 P < 0.001
Results A typical standard curve obtained using purlfled a2-M in the complexed form is shown in Fig. 2 together with a representative curve obtained with serial dilutions of a plasma sample. Similar parallel dilution curves were obtained with several plasma and synovial fluid samples.
e-O_PlasmaStandard P,a~a 1
1 1 8000 4000
20,00
u,otioos 1
15100
1
10100
~.4. ....'"'" 12
1.o ~.z 0.8 •~ 0.6 < 04
.
0.2 °o
~
~
~ ~ 1'0 1'2 1'4 1'8 com0~e*e~ O%-M(~g/~) Fig. 2. Representative standard curves obtained using purified a2-M in the complexed form and serial dilutions of a normal plasma sample,
The working range of the assay was 1.5-15 /~g/1. Within-run CVs ranged from 2.9% to 5.9% (n = 9) for the four samples analysed (two plasma and two synovial fluid samples), with an overall mean of 3.8% + 1.4%. Between-run CVs were 6.4% + 0.1% (n = 6) at the top end of the range to 8.7% + 0.1% (n = 8) at the bottom half of the range. Rheumatoid factor was judged not to interfere with the assay as samples containing high levels of rheumatoid factor assayed with nonspecific rabbit Ig in place of the specific rabbit Ig produced readings no different from the blanks. Recovery studies were performed using five assorted plasma samples, one of which contained a high level of rheumatoid factor, and a rheumatoid synovial fluid. The initial levels of complexed a2-M ranged from 4.2 mg/1 to 108 m g / l and purified complexed az-M was spiked into the samples to give a theoretical increase of either 22.5 mg/1 or 224.5 m g / l . Recovery figures ranged from 99.1% to 115.4% (mean = 108% _ 5.7%, n = 6) for the low spike and 96.9% to 116.4% (mean = 106.3% + 6.5%, n --- 6) for the high spike. The effects of various anticoagulants on complexed a2-M levels are illustrated in Table I for a representative control plasma sample. Although there was no significant difference between the E D T A and citrated plasma, heparinised plasma levels were significantly greater in most samples (in this case P < 0.001, Student's t test) compared with the citrated sample. Levels in serum were greatly elevated (in this case P < 0.001). Levels of complexed a2-M appeared to increase gradually in samples stored at - 4 0 ° C . Out of
17
140. 120 loo -Z v
80
:~ Ioj 60'
•
xQ 40
Z
T
,~, 20.
• -lk • Pancreatitis Arthritides •
Controls (n=22)
(n=11)
,
(synovial ,u~d)
with a mean value of 7.6 mg/1 + 2.6 (0.37% + 0.12% of total a2-M ). N o significant correlation was seen between age and levels of complexed a2-M. The patients with pancreatitis had plasma levels of complexed a2-M on the day of hospital admission which ranged from 5.3 m g / l to 133.7 mg/1 (0.47%-16.2% of total a2-M), with the majority of samples lying outside the range of the control group. Although the patient with the highest level of complexed a2-M on admission was classified as severe pancreatitis (alcohol-induced), for the remainder of the patients there was no clear relationship between the amount of ot2-M present in the complexed form and the clinical severity or the outcome of the illness. Of the synovial fluid samples, levels ranged from 1.6 mg/1 to 64.3 mg/1 (0.26%-5.2% of total ot2-M) with all but two of the samples lying within the normal
(n=7)
Fig. 3. Complexed a2-M levels in plasma samples from a control group, pancreatitis patients on the day of admission to hospital, and synovial fluid samples from patients with various arthritides. , represents the median.
plasma concentrations. The two samples outside
18'
three plasma samples stored for 2.5 weeks and six plasma samples stored for 3.5 weeks to 1 month, seven appeared to be stable over the period of storage. The remaining two samples had complexed a2-M levels which increased from 4.8 mg/1 to 5.9 mg/1 (0.22%-0.27% of total a2-M ) by 3.5 weeks, and from 4.8 mg/1 to 8.3 mg/1 (0.17%-0.29% of total OtE-M) by 2.5 weeks respectively. Two of the plasma samples which had been stable for upto at least 3 - 4 weeks were reanalysed after 3-3.5 months and exhibited increases in complexed a2-M levels of between 180% and > 500% of initial levels. Of four synovial fluid samples, all appeared stable over a period of at least 4-5 weeks of storage at - 4 0 o C. The levels of complexed a2-M in citrated plasma from control subjects and patients with pancreatitis on the day of admission are shown in Fig. 3 together with the levels in synovial fluid samples from patients with various arthritides. All samples were analysed within 1 month of being taken, many being analysed within 2 weeks. Fig. 4 shows the same results expressed as a percentage of the total a2-M. The control samples ranged from 4.2 mg/1 to 14.4 mg/1 (0.17%-0.70% of total a2-M )
:~ 16'
,,~ 14'
12
~ 10 ~ a ;' oEo 6
j_ 2. 0
~ Controls cn=22)
"
-tP"
Pancreatitis (n=l I)
Arthritides (synovial fluid)
I
¢n.7) Fig. 4. Complexed a2-M levels expressed as % of total a2-M in plasma samples from controls, pancreatitis patients on the day of admission to hospital, and synovial fluid Samples from patients with various a r t h r i f i d e s . - - , represents the
median.
:'
18 the normal plasma range were from the patients with seronegative polyarthyritis and probable Reiter's disease,
Discussion In the present study, a sensitive and specific immunoassay has been developed which permits the quantitation of complexed a2-M. This includes a2-M which has undergone a conformational change after complexing with a proteinase or following prolonged storage. It is evident that the type of sample and the length of storage are critical in obtaining valid results. Serum has greatly elevated levels of complexed a2-M compared with plasma due to the fact that a2-M reacts with coagulation enzymes(reviewedby Roberts, 1 9 8 6 ) . The lability of samples during prolonged storage has already been noted by Barrett et al. (1979), and Gressner and Peltzer (1984) who reported serum samples to be stable over a 3 week period, In the study reported here the majority of plasma and synovial fluid samples examined appeared to be stable for a month at least although there was one marked exception to this. Ideally samples should be analysed when fresh, but since this is often not feasible we would reco~nmend storage of not more than a month and the use of a control group of samples which have been stored for a similar length of time in order to make valid comparisons, It is also important that the levels of complexed a2-M are expressed as a percentage of the total a2-M levels since we and others have found that total levels vary widely in different clinical conditions, being reduced in many cases of pancreatitis (McMahon et al., 1984), and are usually lower in synovial fluid than plasma. Interestingly, although total a2-M levels are generally accepted as showing variation with age and sex (Roberts, 1986), complexed ct2-M levels do not appear to correlate with age or sex, either when expressed as absolute amounts or as a percentage of total levels. This needs to be confirmed using a larger group of subjects, The levels of complexed a2-M in the control group in this study are slightly higher than those reported by Marynen et al. (1984) of between
0.09%-0.3% of total ot2-M, using a radioimmunoassay. This may be due to the fact that they used only fresh samples or alternatively may be due to the smaller size of their control group (n = 11). However both these sets of measurements are considerably lower than the 0%-21.4% reported by Virca et al. (1984) when relating trypsin-reactive levels with immune-reactive levels of a2-M. The elevated levels of complexed ct2-M in the patients with pancreatitis presumably reflect the complexing of a2-M with pancreatic enzymes released in this illness. Although levels of complexed a2-M did not appear to predict the course or severity of the attack in this study, the group was very small and only three patients had a severe attack. Although the number of synovial fluids analysed in this study is small with only two samples being from seropositive rheumatoid patients and two from osteoarthritic patients, all four of these had levels of inactive a2-M within the normal plasma range. This contrasts with the findings of Virca et al. (1984) where active a2-M levels were found to range from 3% to 48% (i.e., complexed levels of 52%-97%) of the total levels in synovial fluid samples from eight patients with rheumatoid arthritis. They also reported high levels of complexed a2-M in synovial fluid of patients with osteoarthritis although not to the same extent as in the rheumatoid patients. Greater levels of a2-M proteinase complex in synovial fluid from patients with rheumatoid arthritis compared with osteoarthritis have also been reported by Borth et al. (1986) and although levels did not appear to be as elevated as those reported by Virca et al. (1984), the total levels of a2-M were not provided so that comparisons are difficult. Abnormally high plasma concentrations of complexes of a2-M with a trypsin-like proteinase have been found in patients with sero-positive rheumatoid arthritis (Gaspar et al., 1984; Teodorescu et al., 1984). However these were detected by measuring the hydrolytic activity of immunoadsorbed plasma a2-M against Chromozym-Try, a broad-spectrum trypsin substrate, and Davies et al. (1987) have subsequently found that the elevated levels of activity were due to contamination of the anti-a2-M antibody with a trypsin-like proteinase.
19 W h e t h e r d i f f e r e n c e s b e t w e e n studies r e f l e c t diff e r e n c e s in d i s e a s e state o f t h e p a t i e n t s , o r diff e r e n c e s in t e c h n i q u e , l e n g t h o f s a m p l e s t o r a g e , o r the relatively small number of patients examined in m o s t cases is n o t clear. T h i s q u e s t i o n will b e a d d r e s s e d f u r t h e r a n d t h e p o s s i b l e use o f cornp l e x e d a 2 - M as a p r o g n o s t i c m a r k e r will b e e v a l u a t e d in v a r i o u s clinical c o n d i t i o n s .
Acknowledgements W e are g r a t e f u l to t h e A r t h r i t i s a n d R h e u m a t i s m C o u n c i l for t h e i r f i n a n c i a l s u p p o r t a n d w o u l d like to t h a n k D r . M. R o b e r t s a n d the D e p a r t m e n t of Rheumatism and Rehabilitation of the General I n f i r m a r y at L e e d s for p r o v i d i n g the s y n o v i a l f l u i d s a m p l e s , a n d the v o l u n t a r y p e r s o n n e l w i t h i n the h o s p i t a l f o r a c t i n g as c o n t r o l s ,
References Ades, E.W., Hinson, A., Chapuis-Cellier, C. and Arnaud, P. (1982) Modulation of the immune response by plasma protease inhibitors: I. Alpha2-macroglobulin and alpha 1antitrypsin inhibit natural killing and antibody-dependent cell-mediated cytotoxicity. Scand. J. Immunol. 15, 109. Barrett, A.J. and Starkey, P.M. (1973) The interation of a 2macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. J. 133, 709. Borth, W., Dunky, A. and Kleesiek, K. (1986) a2-macroglobulin-proteinase complexes as correlated with al-proteinase inhibitor-elastase complexes in synovial fluids of rheumatoid arthritis patients. Arthritis Rheum. 29, 319. Borth, W. and Teodorescu, M. (1986) Inactivation of human interleukin-2 (IL-2) by a2-macroglobulin-trypsin complexes. Immunology 57, 367. Chang, J.-L., Ganea, D., Dray, S. and Teodorescu, M. (1983) Blast transformation of B cells induced by an a-macroglobulin-associated lymphokine produced in crowded lymphoid cell cultures. J. Immunol. 130, 267. Davies, M.E., Coughlan, R. and Barrett, A.J. (1987) Plasma from rheumatoid arthritis patients does not contain abnormally high levels of a2-macroglobulin-proteinase complexes. Arthritis Rheum. 30, 872. Debanne, M.T., Bell, R. and Dolovich, J. (1975) Uptake of proteinase-a-macroglobulin complexes by macrophages. Biochim. Biophys. Acta 411,295. Gaspar, A., Skosey, J.L., Sequeira, W. and Teodorescu, M. (1984) Detection of a2-macroglobulin-associated proteases in the plasma of patients with rheumatoid arthritis. Clin. Chem. 30, 1517.
Gressner, A.M. and Peltzer, B. (1984) Amidolytic and immuno-nephelometric determination of aa-proteinase inhibitor and a2-macroglobulin in serum with calculation of specific inhibitor activities in health and disease. J. Clin. Chem. Clin. Biochem. 22, 633. Imber, M.J. and Pizzo, S.V. (1981) Clearance and binding of two electrophoretic 'fast' forms of human a2-macroglobulin. J. Biol. Chem. 256, 8134. Levine, J.J., Udall, J.N., Evernden, B.A. and Epstein, M.F. (1987) Elevated levels of a2-macroglobulin-protease complexes in infants. Biol. Neonate 51, 149. McMahon, M.J., Bowen, M., Mayer, A.D. and Cooper, E.H. (1984) Relation of a2-macroglobulin and other antiproteases to the clinical features of acute pancreatitis. Am. J. Surgery 147, 164. Mannhalter, J.W., Borth, W. and Eibl, M.M. (1986) Modulation of antigen-induced T cell proliferation by 0t2Mtrypsin complexes. J. Immunol. 136, 2792. Marynen, P., Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981) A monoclonal antibody to a neo-antigen on a2-macroglobulin complexes inhibits receptor-mediated endocytosis. J. Immunol. 127, 1782. Marynen, P., Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1984) a2-macroglobulin-proteinase complexes in cystic fibrosis serum. Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts. Biochim. Biophys. Acta 797, 187. Matsuda, T., Hirano, T., Nagasawa, S. and Kishimoto, T. (1989) Identification of a2-macroglobulin as a carrier protein for IL-6. J. Immunol. 142, 148. Petersen, C.M., Ejlersen, E,~ Moestrup, S.K., Jensen, P.H., Sand, O. and Sottrup-Jensen, L. (1989) Immunosuppressive properties of electrophoretically 'slow' and 'fast' form a 2macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation. J. Immunol. 142, 629. Roberts, R.C. (1986) Alpha-2-macroglobulin. J. Med. 16, 129. Teodorescu, M., Chang, J.-L. and Skosey, J.L. (1981) Polyclonal B cell activator associated with alpha-2-macroglobulin in the serum of patients with rheumatoid arthritis. Int. Arch. Allergy Appl. Immunol. 66, 1. Teodorescu, M., Gaspar, A., Spear, G., Skosey, J.L. and Ganea, D. (1984) Degradation of a chromogenic substrate by a 2macroglobulin from plasma of patients with rheumatoid arthritis. Arthritis Rheum. 27, 1122. Travis, J. and Salvesen, G.S. (1983) Human plasma proteinase inhibitors. Annu. Rev. Biochem. 52, 655. Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1979) Demonstration of an a2-macroglobulin receptor in human fibroblasts, absent in tumor-derived cell line. J. Biol. Chem. 254, 5155. Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981a) Functional modifications of a2-macroglobulin by primary amines. I. Characterization of a2M after derivatization by methylamine and by factor XIII. J. Biol. Chem. 256, 9016. Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981b) Functional modifications of a2-macroglobulin by
20 primary amines. II. Inhibition of covalent binding of trypsin to aEM by methylamine and other primary amines. J. Biol. Chem. 256, 9023. Van Leuven, F., Marynen, P., Cassiman, J.-J. and Van Den Berghe, H. (1986a) The epitopes of two complex-specific monoclonal antibodies, related to the receptor recognition site, map to the COOH-terminal end of human aE-macroglobulin. J. Biol. Chem. 261, 6933. Van Leuven, F., Marynen, P., Cassiman, J.-J. and Van Den Berg,he, H. (1986b) Reversed dot-blotting in hybridoma screening and epitope mapping. A model study with human
~x2-macroglobulin to select complex-specific monoclonal antibodies. J. Immunol. Methods 90, 125. Van Leuven, F., Marynen, P., Cassiman, J.-J. and Van Den Berghe, H. (1988) Mapping of structure-function relationships in proteins with a panel of monoclonal antibodies. J. Immunol. Methods 111, 39. Virca, G.D., Mallya, R.K., Pepys, M.B. and Schnebli, H.P. (1984) Quantitation of human leukocyte elastase, cathepsin G, a-2-macroglobulin and a-l-proteinase inhibitor in osteoarthrosis and rheumatoid arthritis synovial fluids. Adv. Exp. Med. Biol. 164, 345.
13
JIM 05413
Measurement of the 'fast' or complexed form of a 2 macroglobulin in biological fluids using a sandwich enzyme immunoassay R.E. Banks 1, S.W. Evans 1, F. V a n L e u v e n 2, D. A l e x a n d e r 3, M.J. M c M a h o n 3 a n d J.T. W h i c h e r 1 1 Department of Chemical Pathology, University of Leeds, Leeds, U.K., : Department of Human Genetics, University of Leuven, Gasthuisberg ON.G, B 3000 Leuven, Belgium, and 3 Department of Surgery, The General Infirmary at Leeds, Leeds, U.K. (Received 6 July 1989, revised received 8 September 1989, accepted 11 September 1989)
A sandwich enzyme immunoassay has been developed for measuring the 'fast' or complexed form of a 2 macroglobulin using a complex-specific monoclonal antibody. The working range of this assay is 1.5-15 /xg/1 and is suitable for use with various biological fluids. Using this assay the normal plasma levels were found to range from 4.2 rag/1 to 14.4 m g / l (0.17%-0.70% of total a2 macroglobulin) with a mean value of 7.6 mg/1 + 2.6 (0.37% ___0.12% of total a 2 macroglobulin). Elevated levels were seen in plasma samples taken on the day of admission from patients with acute pancreatitis and in some synovial fluid samples from patients with various arthritides. Key words: ELISA; ctz Macroglobulin-proteinase complex; Clinical assay; Pancreatitis; Arthritis; Monoclonal antibody
Introduction The glycoprotein a 2 macroglobulin (a2-M) is one of the major proteinase inhibitors in plasma (for reviews see Travis and Salvesen, 1983; Roberts, 1986). It has a molecular weight of approximately 720 000 and is assembled from four identical subunits which are covalently associated in pairs by disulfide bonds and the resulting dimers linked non-covalently. A broad spectrum of proteinases of different specificities, catalytic mechanisms and tissue sources react with a2-M. Uniquely, the proteolytic activity of the bound
Correspondence to: R.E. Banks, Department of Chemical Pathology, University of Leeds, Leeds, LS2 9JT, U.K. Abbreviations." a2-M, a 2 macroglobulin: ELISA, enzymelinked immunosorbent assay; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.1% Tween 20, OPD = o-phenylenediamine dihydrochloride; HRP, horseradishperoxidase,
proteinases is retained although only towards low molecular weight substrates. This may be explained by the 'trap' hypothesis (Barrett and Starkey, 1973) which proposes that the proteinase attacks a 'bait' region in the a2-M molecule causing a proteolytic cleavage and a rapid conformational change. This results in the physical entrapment of the proteinase molecule within the macroglobulin molecule thus preventing reactivity of the bound proteinase with large molecular weight substrates and, in most cases, with antibodies. The apparently irreversible change from an active to a complexed or inactive form of a2-M is accompanied by a change in electrophoretic mobility from a 'slow' form to a 'fast' form (Barrett et al., 1979). A similar transition to the complexed or 'fast' form is also produced by the covalent binding to a2-M of primary amines such as methylamine (Barrett et al., 1979; Van Leuven et al., 1981a,b), and during prolonged storage (Barrett et al., 1979). For the purpose of this paper the term
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14
'complexed' will be used to describe the 'fast' form of a2-M, irrespective of whether produced by proteinase binding, methylamine binding or storage, The use of monoclonal antibodies has revealed the existence of antigenic determinants which are present only on the complexed from of a2-M (Marynen et al., 1981). The epitopes recognised by these antibodies have in many cases been mapped to the receptor recognition site of a2-M (Van Leuven et al., 1986a,b, 1988) which is expressed only on the complexed form and allows its recognition by receptors present on various cell types including fibroblasts (Van Leuven et al., 1979) and macrophages (Debanne et al., 1975). Complexed aE-M is removed rapidly from the circulation by receptor-mediated endocytosis with a reported half-life in mice of < 5 rain compared with several hours for the native form (Imber and Pizzo, 1981). 19/2 macroglobulin has been reported to exhibit various immuno-modulatory actions including suppression of cell-mediated cytotoxicity by the native form, possibly via an inhibition of T cell surface-associated proteinases involved in target recognition (Ades et al., 1982; Petersen et al., 1989), and the inhibition of activated T cell proliferation by both the native and complexed forms (Mannhalter et al., 1986; Petersen et al., 1989). This suppressive effect of complexed a2-M in proliferative assays may possibly be explained by the degradative activity associated with the bound proteinase following the finding by Borth and Teodorescu (1986) that ct2-M bound proteinase is able to degrade IL-2 (T cell growth factor). Additionally it has been suggested that aE-M-protease complexes may play a role in polyclonal B cell activation (Teodorescu et al., 1981; Chang et al., 1983) and recently a2-M has been identified as a carrier protein for IL-6 (Matsuda et al., 1989). Various methods have been used to determine the amount of complexed CtE-Mpresent in biological samples including gradient gel electrophoresis of samples before and after reduction with dithiothreitol which dissociates native aE-M (Levine et al., 1987), radioimmunoassay with a monoclonal antibody specific to the complexed form of t~E-M (Marynen et al., 1984), the measurement of trypsin-binding capacity of solid-phase im-
munoadsorbed ot2-M using radiolabelled trypsin (Borth et al., 1986), and measurement of the hydrolytic activity of immunoadsorbed plasma ot2-M against Chromozym-Try (Boehringer Corporation, Lewes, U.K.), a broad-spectrum trypsin substrate (Gaspar et al,. 1984; Teodorescu et al., 1984). We report here the development of a sensitive ELISA method incorporating a complex-specific monoclonal antibody which permits the measurement of the complexed form of a2-M , and investigate the levels present in controls and various clinical conditions.
Materials and methods
Materials Nunc Immunoplate I microwell plates were obtained from Gibco BRL (Paisley, U.K.). Purified a2-M, Tween 20 and OPD were purchased from Sigma (Poole, U.K.). Rabbit anti-human CtE-M and HRP-labelled swine anti-rabbit Ig were purchased from Dako (High Wycombe, U.K.). Nephelometric grade antiserum to a2-M and standard serum were obtained from Behring Diagnostics (Hounslow, U.K.). The monoclonal antibody F44GA2 to o~2-M (complex specific) was prepared and characterised by Van Leuven et al. as previously described (1988). All other chemicals were obtained from BDH (Poole, U.K.).
Biologicalsamples The effect of various anticoagulants on levels of complexed ct2-M was investigated by taking blood samples from five healthy subjects and dividing them into tubes containing either potassium EDTA, sodium citrate, lithium heparin or no anticoagulant. The samples were then centrifuged at 1500 × g for 10 rain and the plasma or serum removed and stored in aliquots at - 4 0 °C until assayed. A comparison of levels of complexed a2-M in various clinical conditions was performed by collecting plasma samples, obtained by centrifugation of citrated whole blood at 1500 × g for 10 min, from 22 healthy controls (seven males aged 24-69 years, mean 37.1 + 15.2, and 15 females aged 29-77 years, mean 59.1 _+ 14.7), and from 11 patients with pancreatitis of differing aetiology,
15
Measurementof total a2-M
severity and outcome, on the day of hospital admission (seven males and four females aged 44-96 years, mean 68.2 + 14.3). In addition citrated synovial fluid was obtained from two patients with osteoarthritis, two patients with seropositive rheumatoid arthritis, one patient with psoriatic arthritis, one patient with seronegative polyarthritis and one patient with probable Reiter's disease (three females and four males aged 33-72 years, mean 56.5 4- 12.2), and centrifuged at 1500 × g for 10 min. Plasma and synovial fluid samples were aliquoted and stored at - 4 0 ° C prior to analysis, Stability during storage was assessed by analysing aliquots from several samples on two or three later occasions,
Total levels of a2-M in samples were measured using a Behring nephelometric analyzer together with Behring nephelometric grade anti-a2-M and Behring N Protein Standard serum calibrated against a WHO International Reference Preparation.
Enzymeimmunoassayfor complexed ae-M The principle of the assay is illustrated in Fig. 1 with the appropriate antibody dilutions being determined by checkerboard titration. Nunc Immunoplate I microwell plates were incubated with F44GA2 antibody in PBS, pH 7.2 (100 #l/well at a concentration of 1.3 /zg/ml) at 4 ° C overnight. Plates were rinsed 3 × in PBS-T and triplicate 100 #1 aliquots of complexed a2-M standards and biological samples diluted a minimum of 1000 × in PBS-T were added to the wells. The plates were sealed and incubated at 15 °C for 2 h. Following the incubation the plates were rinsed 3 × with PBS-T and rabbit anti-human a2-M diluted in PBS-T was added to each well. After 1 h at room temperature, the plates were washed a further 3 × and HRP-conjugated swine anti-rabbit immunoglobulin (affinity purified) was added to the plates
Complexed ('fast')a2-Mstandards A stock solution of the purified aE-M was prepared and standardised by nephelometry as described below. This preparation was judged to contain a2-M which was 100% in the complexed or 'fast' form since no difference was found in the ELISA before and after overnight treatment with methylamine as previously described (Barrett et al., 1979). Standards ranging from 1.5 # g / l to 15 #g/1 were prepared using PBS-T as a diluent,
Rabb.
Complexed C~2-M
Anti0(2- M
HRP-linked Swine antirabbit Ig
F44GA2 antiC~2-M (complexspecific)
~HRP COLOUR '
Subatrate
÷
~~lr
Fig. 1. Pfincip|e of the sandwich ELISA for comp]cxed aE-macrog]obu]in.
16 which were then incubated at room temperature for 1 h. Unbound conjugate was then removed by washing 3 × with PBS-T, 100 /zl of substrate solution (0.1 M citric acid-phosphate buffer, p H 5.0, containing 8 mg O P D and 5 /~1 of 30% H 2 0 2 / 1 2 ml of buffer) were added to each well and the plates left in the dark for 15 min. The reaction was terminated by the addition of 1 M H2SO 4. Absorbance was read at 492 nm on a Titertek Multiskan M C C plate reader. The concentration of complexed a2-M in the samples w a s calculated from the standard curve for each plate,
TABLEI COMPARISONOF LEVELS OF COMPLEXED a2-M IN PLASMA (VARIOUS ANTICOAGULANTS) AND SERUM OF A REPRESENTATIVE SAMPLE Statisticalanalysis was by Student's t test against the citrated sample. Sample Citrated plasma EDTA plasma Heparinised plasma
Serum
Complexed a2-M (mg/1) (mean + SD, n = 3) 5.2+ 0.1 5.3+ 0.3 11.8 + 0.2 236.5 ___25.0
Significance level NS P < 0.001 P < 0.001
Results A typical standard curve obtained using purlfled a2-M in the complexed form is shown in Fig. 2 together with a representative curve obtained with serial dilutions of a plasma sample. Similar parallel dilution curves were obtained with several plasma and synovial fluid samples.
e-O_PlasmaStandard P,a~a 1
1 1 8000 4000
20,00
u,otioos 1
15100
1
10100
~.4. ....'"'" 12
1.o ~.z 0.8 •~ 0.6 < 04
.
0.2 °o
~
~
~ ~ 1'0 1'2 1'4 1'8 com0~e*e~ O%-M(~g/~) Fig. 2. Representative standard curves obtained using purified a2-M in the complexed form and serial dilutions of a normal plasma sample,
The working range of the assay was 1.5-15 /~g/1. Within-run CVs ranged from 2.9% to 5.9% (n = 9) for the four samples analysed (two plasma and two synovial fluid samples), with an overall mean of 3.8% + 1.4%. Between-run CVs were 6.4% + 0.1% (n = 6) at the top end of the range to 8.7% + 0.1% (n = 8) at the bottom half of the range. Rheumatoid factor was judged not to interfere with the assay as samples containing high levels of rheumatoid factor assayed with nonspecific rabbit Ig in place of the specific rabbit Ig produced readings no different from the blanks. Recovery studies were performed using five assorted plasma samples, one of which contained a high level of rheumatoid factor, and a rheumatoid synovial fluid. The initial levels of complexed a2-M ranged from 4.2 mg/1 to 108 m g / l and purified complexed az-M was spiked into the samples to give a theoretical increase of either 22.5 mg/1 or 224.5 m g / l . Recovery figures ranged from 99.1% to 115.4% (mean = 108% _ 5.7%, n = 6) for the low spike and 96.9% to 116.4% (mean = 106.3% + 6.5%, n --- 6) for the high spike. The effects of various anticoagulants on complexed a2-M levels are illustrated in Table I for a representative control plasma sample. Although there was no significant difference between the E D T A and citrated plasma, heparinised plasma levels were significantly greater in most samples (in this case P < 0.001, Student's t test) compared with the citrated sample. Levels in serum were greatly elevated (in this case P < 0.001). Levels of complexed a2-M appeared to increase gradually in samples stored at - 4 0 ° C . Out of
17
140. 120 loo -Z v
80
:~ Ioj 60'
•
xQ 40
Z
T
,~, 20.
• -lk • Pancreatitis Arthritides •
Controls (n=22)
(n=11)
,
(synovial ,u~d)
with a mean value of 7.6 mg/1 + 2.6 (0.37% + 0.12% of total a2-M ). N o significant correlation was seen between age and levels of complexed a2-M. The patients with pancreatitis had plasma levels of complexed a2-M on the day of hospital admission which ranged from 5.3 m g / l to 133.7 mg/1 (0.47%-16.2% of total a2-M), with the majority of samples lying outside the range of the control group. Although the patient with the highest level of complexed a2-M on admission was classified as severe pancreatitis (alcohol-induced), for the remainder of the patients there was no clear relationship between the amount of ot2-M present in the complexed form and the clinical severity or the outcome of the illness. Of the synovial fluid samples, levels ranged from 1.6 mg/1 to 64.3 mg/1 (0.26%-5.2% of total ot2-M) with all but two of the samples lying within the normal
(n=7)
Fig. 3. Complexed a2-M levels in plasma samples from a control group, pancreatitis patients on the day of admission to hospital, and synovial fluid samples from patients with various arthritides. , represents the median.
plasma concentrations. The two samples outside
18'
three plasma samples stored for 2.5 weeks and six plasma samples stored for 3.5 weeks to 1 month, seven appeared to be stable over the period of storage. The remaining two samples had complexed a2-M levels which increased from 4.8 mg/1 to 5.9 mg/1 (0.22%-0.27% of total a2-M ) by 3.5 weeks, and from 4.8 mg/1 to 8.3 mg/1 (0.17%-0.29% of total OtE-M) by 2.5 weeks respectively. Two of the plasma samples which had been stable for upto at least 3 - 4 weeks were reanalysed after 3-3.5 months and exhibited increases in complexed a2-M levels of between 180% and > 500% of initial levels. Of four synovial fluid samples, all appeared stable over a period of at least 4-5 weeks of storage at - 4 0 o C. The levels of complexed a2-M in citrated plasma from control subjects and patients with pancreatitis on the day of admission are shown in Fig. 3 together with the levels in synovial fluid samples from patients with various arthritides. All samples were analysed within 1 month of being taken, many being analysed within 2 weeks. Fig. 4 shows the same results expressed as a percentage of the total a2-M. The control samples ranged from 4.2 mg/1 to 14.4 mg/1 (0.17%-0.70% of total a2-M )
:~ 16'
,,~ 14'
12
~ 10 ~ a ;' oEo 6
j_ 2. 0
~ Controls cn=22)
"
-tP"
Pancreatitis (n=l I)
Arthritides (synovial fluid)
I
¢n.7) Fig. 4. Complexed a2-M levels expressed as % of total a2-M in plasma samples from controls, pancreatitis patients on the day of admission to hospital, and synovial fluid Samples from patients with various a r t h r i f i d e s . - - , represents the
median.
:'
18 the normal plasma range were from the patients with seronegative polyarthyritis and probable Reiter's disease,
Discussion In the present study, a sensitive and specific immunoassay has been developed which permits the quantitation of complexed a2-M. This includes a2-M which has undergone a conformational change after complexing with a proteinase or following prolonged storage. It is evident that the type of sample and the length of storage are critical in obtaining valid results. Serum has greatly elevated levels of complexed a2-M compared with plasma due to the fact that a2-M reacts with coagulation enzymes(reviewedby Roberts, 1 9 8 6 ) . The lability of samples during prolonged storage has already been noted by Barrett et al. (1979), and Gressner and Peltzer (1984) who reported serum samples to be stable over a 3 week period, In the study reported here the majority of plasma and synovial fluid samples examined appeared to be stable for a month at least although there was one marked exception to this. Ideally samples should be analysed when fresh, but since this is often not feasible we would reco~nmend storage of not more than a month and the use of a control group of samples which have been stored for a similar length of time in order to make valid comparisons, It is also important that the levels of complexed a2-M are expressed as a percentage of the total a2-M levels since we and others have found that total levels vary widely in different clinical conditions, being reduced in many cases of pancreatitis (McMahon et al., 1984), and are usually lower in synovial fluid than plasma. Interestingly, although total a2-M levels are generally accepted as showing variation with age and sex (Roberts, 1986), complexed ct2-M levels do not appear to correlate with age or sex, either when expressed as absolute amounts or as a percentage of total levels. This needs to be confirmed using a larger group of subjects, The levels of complexed a2-M in the control group in this study are slightly higher than those reported by Marynen et al. (1984) of between
0.09%-0.3% of total ot2-M, using a radioimmunoassay. This may be due to the fact that they used only fresh samples or alternatively may be due to the smaller size of their control group (n = 11). However both these sets of measurements are considerably lower than the 0%-21.4% reported by Virca et al. (1984) when relating trypsin-reactive levels with immune-reactive levels of a2-M. The elevated levels of complexed ct2-M in the patients with pancreatitis presumably reflect the complexing of a2-M with pancreatic enzymes released in this illness. Although levels of complexed a2-M did not appear to predict the course or severity of the attack in this study, the group was very small and only three patients had a severe attack. Although the number of synovial fluids analysed in this study is small with only two samples being from seropositive rheumatoid patients and two from osteoarthritic patients, all four of these had levels of inactive a2-M within the normal plasma range. This contrasts with the findings of Virca et al. (1984) where active a2-M levels were found to range from 3% to 48% (i.e., complexed levels of 52%-97%) of the total levels in synovial fluid samples from eight patients with rheumatoid arthritis. They also reported high levels of complexed a2-M in synovial fluid of patients with osteoarthritis although not to the same extent as in the rheumatoid patients. Greater levels of a2-M proteinase complex in synovial fluid from patients with rheumatoid arthritis compared with osteoarthritis have also been reported by Borth et al. (1986) and although levels did not appear to be as elevated as those reported by Virca et al. (1984), the total levels of a2-M were not provided so that comparisons are difficult. Abnormally high plasma concentrations of complexes of a2-M with a trypsin-like proteinase have been found in patients with sero-positive rheumatoid arthritis (Gaspar et al., 1984; Teodorescu et al., 1984). However these were detected by measuring the hydrolytic activity of immunoadsorbed plasma a2-M against Chromozym-Try, a broad-spectrum trypsin substrate, and Davies et al. (1987) have subsequently found that the elevated levels of activity were due to contamination of the anti-a2-M antibody with a trypsin-like proteinase.
19 W h e t h e r d i f f e r e n c e s b e t w e e n studies r e f l e c t diff e r e n c e s in d i s e a s e state o f t h e p a t i e n t s , o r diff e r e n c e s in t e c h n i q u e , l e n g t h o f s a m p l e s t o r a g e , o r the relatively small number of patients examined in m o s t cases is n o t clear. T h i s q u e s t i o n will b e a d d r e s s e d f u r t h e r a n d t h e p o s s i b l e use o f cornp l e x e d a 2 - M as a p r o g n o s t i c m a r k e r will b e e v a l u a t e d in v a r i o u s clinical c o n d i t i o n s .
Acknowledgements W e are g r a t e f u l to t h e A r t h r i t i s a n d R h e u m a t i s m C o u n c i l for t h e i r f i n a n c i a l s u p p o r t a n d w o u l d like to t h a n k D r . M. R o b e r t s a n d the D e p a r t m e n t of Rheumatism and Rehabilitation of the General I n f i r m a r y at L e e d s for p r o v i d i n g the s y n o v i a l f l u i d s a m p l e s , a n d the v o l u n t a r y p e r s o n n e l w i t h i n the h o s p i t a l f o r a c t i n g as c o n t r o l s ,
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Gressner, A.M. and Peltzer, B. (1984) Amidolytic and immuno-nephelometric determination of aa-proteinase inhibitor and a2-macroglobulin in serum with calculation of specific inhibitor activities in health and disease. J. Clin. Chem. Clin. Biochem. 22, 633. Imber, M.J. and Pizzo, S.V. (1981) Clearance and binding of two electrophoretic 'fast' forms of human a2-macroglobulin. J. Biol. Chem. 256, 8134. Levine, J.J., Udall, J.N., Evernden, B.A. and Epstein, M.F. (1987) Elevated levels of a2-macroglobulin-protease complexes in infants. Biol. Neonate 51, 149. McMahon, M.J., Bowen, M., Mayer, A.D. and Cooper, E.H. (1984) Relation of a2-macroglobulin and other antiproteases to the clinical features of acute pancreatitis. Am. J. Surgery 147, 164. Mannhalter, J.W., Borth, W. and Eibl, M.M. (1986) Modulation of antigen-induced T cell proliferation by 0t2Mtrypsin complexes. J. Immunol. 136, 2792. Marynen, P., Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981) A monoclonal antibody to a neo-antigen on a2-macroglobulin complexes inhibits receptor-mediated endocytosis. J. Immunol. 127, 1782. Marynen, P., Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1984) a2-macroglobulin-proteinase complexes in cystic fibrosis serum. Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts. Biochim. Biophys. Acta 797, 187. Matsuda, T., Hirano, T., Nagasawa, S. and Kishimoto, T. (1989) Identification of a2-macroglobulin as a carrier protein for IL-6. J. Immunol. 142, 148. Petersen, C.M., Ejlersen, E,~ Moestrup, S.K., Jensen, P.H., Sand, O. and Sottrup-Jensen, L. (1989) Immunosuppressive properties of electrophoretically 'slow' and 'fast' form a 2macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation. J. Immunol. 142, 629. Roberts, R.C. (1986) Alpha-2-macroglobulin. J. Med. 16, 129. Teodorescu, M., Chang, J.-L. and Skosey, J.L. (1981) Polyclonal B cell activator associated with alpha-2-macroglobulin in the serum of patients with rheumatoid arthritis. Int. Arch. Allergy Appl. Immunol. 66, 1. Teodorescu, M., Gaspar, A., Spear, G., Skosey, J.L. and Ganea, D. (1984) Degradation of a chromogenic substrate by a 2macroglobulin from plasma of patients with rheumatoid arthritis. Arthritis Rheum. 27, 1122. Travis, J. and Salvesen, G.S. (1983) Human plasma proteinase inhibitors. Annu. Rev. Biochem. 52, 655. Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1979) Demonstration of an a2-macroglobulin receptor in human fibroblasts, absent in tumor-derived cell line. J. Biol. Chem. 254, 5155. Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981a) Functional modifications of a2-macroglobulin by primary amines. I. Characterization of a2M after derivatization by methylamine and by factor XIII. J. Biol. Chem. 256, 9016. Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981b) Functional modifications of a2-macroglobulin by
20 primary amines. II. Inhibition of covalent binding of trypsin to aEM by methylamine and other primary amines. J. Biol. Chem. 256, 9023. Van Leuven, F., Marynen, P., Cassiman, J.-J. and Van Den Berghe, H. (1986a) The epitopes of two complex-specific monoclonal antibodies, related to the receptor recognition site, map to the COOH-terminal end of human aE-macroglobulin. J. Biol. Chem. 261, 6933. Van Leuven, F., Marynen, P., Cassiman, J.-J. and Van Den Berg,he, H. (1986b) Reversed dot-blotting in hybridoma screening and epitope mapping. A model study with human
~x2-macroglobulin to select complex-specific monoclonal antibodies. J. Immunol. Methods 90, 125. Van Leuven, F., Marynen, P., Cassiman, J.-J. and Van Den Berghe, H. (1988) Mapping of structure-function relationships in proteins with a panel of monoclonal antibodies. J. Immunol. Methods 111, 39. Virca, G.D., Mallya, R.K., Pepys, M.B. and Schnebli, H.P. (1984) Quantitation of human leukocyte elastase, cathepsin G, a-2-macroglobulin and a-l-proteinase inhibitor in osteoarthrosis and rheumatoid arthritis synovial fluids. Adv. Exp. Med. Biol. 164, 345.
