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Quantitation of human complement fragment C4ai in physiological fluids by competitive inhibition radioimmune assay
Journal of Immunological Methods, 47 (1981) 61--73 Elsevier/North-Holland Biomedical Press
61
QUANTITATION OF HUMAN COMPLEMENT FRAGMENT C4ai IN PHYSIOLOGICAL FLUIDS BY COMPETITIVE INHIBITION RADIOIMMUNE ASSAY
J E F F R E Y P. GORSKI
Department of Cell Biology-Biochemistry, Mayo Clinic and Mayo Foundation, Rochester, MN 55901, U.S.A. (Received 6 May 1981, accepted 10 July 1981)
A method is described to quantitate complement fragment C4a i in human plasma, synovial fluid, and urine. Samples are first precipitated with 50% saturated (NH4)2SO4 to remove cross-reactive macromolecules C4 and pro-C4. Whereas greater than 97% of C4 is removed by this precipitation step, 88% of C4a i remains in solution. Second, the concentration of C4a i in supernatant fractions is determined by double antibody competitive inhibition radioimmunoassay. C4a was recently completely sequenced (Moon et al., 1981) and is readily available as a pure standard. Examination of the specificity of this method confirmed it was indeed specific for C4a antigenicity. Immunochemically depleted C4-deficient plasma and inulin-activated reconstituted C4-deficient plasma exhibited less than 0.1% of the immunoreactivity of untreated plasma. In addition, good agreement was observed in analyses of aggregated IgG activated serum between the experimentally determined concentration of C4a i and that expected from the initial concentration of C4. As a result, recovery and measurement of C4a i in physiological fluids with this method appear both quantitative and specific. Based on results from 17 adult volunteers, the average concentration of C4ai in normal plasma is 488 ng/ml. Interestingly, significant correlation could not be demonstrated between the levels of C4 and C4a i in normal plasma. The mean concentration of C4a i in human urine is 0.5 ng/ml.
INTRODUCTION
Activation of the classical p a t h w a y of complement is observed in many clinical pathologies, for example, serum sickness, myasthenia gravis, autoimmune hemolytic anemias, systemic lupus erythematosus, hereditary angioneurotic edema, and allograft rejection. Most studies have observed drops in hemolytic complement titers in these diseases (for reviews see: Shur and Austen, 1968; Ruddy et al., 1972). However, evaluation of these data in Abbreviations used in this paper: IgG, immunoglobulin G; PMN, polymorphonuclear leukocyte; C3a, C4a and C5a, activation peptides derived from the N-terminus of the ~-subunits of C3, C4 and C5; C3ai, C4a i and C5ai, products of carboxypeptidase B or N digestion of C3a, C4a and C5a; CIs0, amount of competing antigen required to attain half-maximal inhibition; CIs, calculated competitive inhibition slope; and B/B0, the quotient of the bound cpm to initial bound cpm ratio. 0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
62 a clinical setting has many uncertainties (Stites, 1980). First, an abnormally low hemolytic titer does not indicate a deficiency of this component, only a lack of its ability to complete a hemolytic sequence. Previously activated serum cannot be distinguished from deficient serum by this means. Second, a low titer may result from an increased rate of catabolic turnover. Third, low serum complement may result from increased tissue localization. And fourth, appearance of an inhibitor may give rise to an apparently low titer. Although hemolytic complement assays are of questionable reliability, the value of complement monitoring is nonetheless apparent. Assessment of complement status may assist in understanding the causes of cellular infiltration and humoral or PMN-mediated tissue destruction associated with immune complexes. Alternatives to hemolytic assays do exist, such as in vivo turnover measurement and complement fragment analysis. In vivo complement studies (Carpenter et al., 1969; Petz et al., 1977; Curd et al., 1979}, while worthwhile, are n o t practical on a routine basis. Immunochemical assays for complement fragments C3a, C5a, C3d, C4d and Bb have been developed and applied to patient populations (Perrin et al., 1975a, 1975b; Hugli and Chenoweth, 1980). The general principle of these latter assays is first to separate unactivated C3, C4, C5 and factor B from their respective fragments by selective precipitation. The samples are then analyzed by radial immunodiffusion (C3d, C4d, Bb), immunoelectrophoresis (C4d), or radioimmune assay (C3a, C5a) methods. Several criticisms may be raised against the C3d and C4d fragment assays. For example, C3d and C4d represent structural regions of C3b and C4b that mediate their binding to biological membranes (Schreiber and Mfiller-Eberhard, 1974; Law et al., 1979). As such, C3d and C4d may not be present in high concentrations in biological fluids. In the case of C4d, no pure standard is available and all previous measurements were compared to that of serum treated with aggregated IgG (Perrin et al., 1975b; Milgrom et al., 1980). Finally, the sensitivity of radial immunodiffusion and immunoelectrophoretic methods of detection are restricted to a lower limit of 1--5/~g of antigen protein. Current double antibody radioimmune assay methods display lower limits in the range of 1 ng antigen protein. In spite o f the fact the m e t h o d for quantitation of C5a relies upon radioimmune assay methodology, efforts to apply it to measurements of C5a in plasma have failed. Once formed, the majority of C5a binds tightly to blood neutrophils (Chenoweth and Hugli, 1978) and is n o t found free in plasma until this capacity apparently is exceeded (Chenoweth et al., 1981}. Development and application of a radioimmune assay for fragment C4a does n o t appear to suffer from the above-mentioned criticisms. Small size, lack of association with cell surfaces, availability of chemically characterized homogeneous standard (Gorski et al., 1981}, and direct kinetic relationship of its formation with C4 activation suggest an avoidance o f these problems.
63 MATERIALS
Normal human EDTA plasma was obtained fresh from the Mayo Clinic Blood Bank. Donkey anti-rabbit IgG serum and ~2SI-Boloton-Hunter reagent were purchased from Pel-Freez and Amersham, Inc., respectively. METHODS
Purification of human complement proteins and fragments C4a and C4b were isolated as previously described (Gorski et al., 1981). Alternatively, purified C4 (Bolotin et al., 1977) was converted to C4a and C4b by incubation with 5% (w/w) trypsin (Budzko and Miiller-Eberhard, 1970) for 2.5 min at 23°C in 0.1 M sodium bicarbonate buffer, pH 8.5, containing 0.15 M sodium chloride, 0.01 M EDTA, and 0.02% (w/v) sodium azide. A 5-fold weightexcess of soybean trypsin inhibitor was added to the digest to stop the reaction. Digestion of C4a with 1% (w/w) carboxypeptidase B for 60 min at 37°C quantitatively converted C4a to C4ai.
C4 hemolytic assay Plasma samples were assayed for C4
Method for quantitation of C4ai in physiological fluids One volume of physiological fluid was mixed with an equal volume of 0.2 M Tris-acetate buffer (pH 8.0), containing 35% (w/v) polyethylene glycol 6000, 0.04 mg/ml soybean trypsin inhibitor, 0.02 M EDTA, 0.02 M benzamidine hydrochloride, 0.2 M 2:-amino-n-caproic acid, and 0.04% (w/v) sodium azide. Alternatively, saturated ammonium sulfate was substituted for polyethylene glycol and used as a precipitant. After mixing for 2 h at 4°C, precipitated protein was removed by centrifugation. The supernatant fraction resulting from either ammonium sulfate or polyethylene glycol precipitation was subsequently analyzed by radioimmunoassay for C4a~ content. A 4-compartment radioimmunoassay system with a final volume of 1 ml was used: (1) assay buffer, 0.2 ml; (2) primary antiserum, 0.2 ml; (3) competing antigen preparation, 0.2 ml; and (4) secondary antiserum, 0.4 ml. The volume of radiolabeled antigen, 0.005-0.025 ml, was disregarded. Assay buffer consisted of 0.5 M Tris-acetate buffer (pH 8.0), containing 0.05 M EDTA, 0.2 M Y~-amino-n-caproic acid, 0~05 M benzamidine hydrochloride, 1.25 mg/ml PMSF, 0.04 mg/ml soybean trypsin inhibitor, 0.3% (w/v) Triton X-100, and 0.1% (w/v) sodium azide. Primary antiserum was diluted at least 50-fold with either saline or 50-fold diluted non-immune rabbit serum to keep the concentration of rabbit IgG at 0.2 mg/ml. Secondary antiserum,
64
d o n k e y anti-rabbit IgG serum, was pre-titered so as to bring a b o u t precipitation of at least 95% of rabbit IgG present in assays. Radioimmune assays c o m p o s e d of c o m p o n e n t s 1--3 and [12sI]C4ai were incubated initially for 16--20 h at 4°C on a rocking platform. Secondary antiserum, Component 4, was then added to assay tubes, which were re-incubated for a period of 6 h at 4°C. Immune aggregates were sedimented b y centrifugation at 8730 × gmax. Gamma counting was carried o u t first on the total assay volumes and subsequently on the immune pellets. Individual assay values (cpm) were corrected for experimental blank values, averaged, and calculated as a percentage of the experimental control value. Controls were prepared exactly as described above except that saline was substituted for competing antigen. Blanks, routinely 2% of input cpm, were prepared as described above except that 50-fold diluted non-immune rabbit serum was substituted for immune serum in c o m p a r t m e n t 2. Experimental points are the average of duplicate assays.
Miscellaneous The concentration of C4b, C4ai, or C4 protein in standard solutions was determined from the results of amino acid analysis. C4ai was radiolabeled with 12SI-Bolton-Hunter reagent (N-succinimidyl 3-(4-hydroxy, 5-[12sI]iodophenyl propionate) (Bolton and Hunter, 1973) according to instructions supplied b y Amersham Corp., except that 0.1 M sodium bicarbonate buffer (pH 8.5), containing 0.15 M sodium chloride, was substituted for sodium borate as the reaction buffer. Aggregated IgG was prepared by incubation of purified human IgG (10 mg/ml) in saline for 30 min at 63°C. RESULTS
Selective precipitation of C4 from plasma Direct immunochemical quantitation of activation fragment C4a in human plasma requires selective removal of cross-reacting proteins C4 and pro-C4 before analysis. C4a is a 77-residue polypeptide derived from the N-terminus of the ~-subunit of C4 (Gorski et al., 1981) and represents an internal sequence stretch of single-chain pro-C4 (Goldberger and Colten, 1980). Due to the large difference in molecular weights of C4a and of C4 or pro-C4, precipitation methods discriminating on the basis of size were examined. Soybean trypsin inhibitor, benzamidine hydrochloride, E-amino-n-caproic acid, and EDTA were included in the precipitation media to prevent activation of C4 during this step. The solubilities of plasma C4a and C4 in different concentrations of (NH4)2SO4 were determined and are plotted in Fig. 1. C4a remains in solution in all concentrations of (NH4)2SO4 tested. Specifically, an average of 88% of the total C4a remained in solution following precipitation of plasma with from 12.5 to 50% saturated (NH4)2SO4. As expected, plasma C4 is sus-
65
pectible to precipitation with salt. Whereas in 25% saturated (NH4)2SO4, 96% of the total plasma C4 remains in solution, less than 3% is soluble in 50% saturated (NH4)2SO4. It is assumed that the solubility o f pro-C4, which is the same size as C4, is similar to that observed for C4. The respective solubilities o f C4a and C4 in various concentrations of polyethylene glycol 6000 were also determined (not shown). Ninety-four percent of plasma C4a remained in solution following precipitation with from 7.5 to 20% (w/v) polyethylene glycol. In contrast, C4 was totally removed from solution with 15% (w/v) polyethylene glycol, in agreement with earlier studies (Perrin et al., 1975b; Bolotin et al., 1977). In addition to plasma, the differential solubilities of C4a and C4 in human synovial fluid were also studied (not shown). Results were identical to those described above for plasma C4a and C4 with (NH4)2SO4 and polyethylene glycol. In summary, C4 and pro-C4 may be separated from C4a in plasma or synovial fluid by specific precipitation with (NH4)2SO4 or polyethylene glycol 6000.
Characterization of the specificity of radioimmune assay for C4ai A double-antibody competitive inhibition radioimmunoassay for human C4a~ was developed with [ 12si] C4ai, rabbit anti-C4a serum, and donkey anti-
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Fig. 1. Solubilities of plasma C4a and C4 in different concentrations of (NH4)2SO4. Aliquots of normal human plasma with [ l : s I ] C 4 a (m) or [12SI]C4 (e) were added to equal volumes of 0.2 M Tris-acetate buffer (pH 8.0), containing 0.2 M Y-amino-n-caproic acid, 0.04 mg/ml soybean trypsin inhibitor, 0.02 M EDTA, 0.02 M benzamidine hydrochloride, 0.04% (w/v) sodium azide, and different percentages of saturated (NH4)2SO4. After mixing for 2 h at 4°C, the samples were centrifuged to remove precipitated protein and the supernatant fractions were counted. Fig. 2. Characterization o f the specificity of radioimmunoassay for C4a i. C4a (o), C4ai (m), C4 (A), normal human plasma (e), or C4b (D), was tested for its ability to competitively inhibit [12SI]C4ai precipitation by anti-C4a serum. 1.1 pmol o f [12sI]c4ai (7.6 ~Ci//~g) was present in each radioimmune assay. Results are calculated relative to control assays; controls bound 63.9% of input [ 12 s I ]C4ai in this experiment.
66 rabbit IgG serum (see Methods). It is known that the complement anaphylatoxins, once formed, are subject to the regulatory controlling action of carboxypeptidase N (EC 3.4.12.7) in blood (Bokisch et al., 1969). Therefore, [12sI]C4~ was used as radioligand in our studies because we believe it represents the form o f the C4a antigen found free in physiological fluids. Numerous checks on the specificity of the radioimmune assay for C4a were undertaken. The results o f some of these experiments are summarized in Fig. 2. Briefly, purified C4a was observed to c o m p e t e completely for [12sI]C4ai binding. A CIs0 value of 3.1 X 10 -13 M and a CIs of 0.611 characterize this competitive binding curve. As expected, C4ai also competes totally with its radiolabeled form. In quantitative terms, half-maximal binding was attained with 6.7 × 10 -13 M peptide and a CIs value of 0.623 was calculated from the results. Completing this survey of completely crossreacting antigens, purified C4 and plasma C4 gave rise to CIs0 values of 2.8 X 10 -12 M and 6.7 X 10 -12 M and, competitive inhibition slopes of 0.453 and 0.424, respectively. Purified C4b, the other product o f C4 activation, does n o t c o m p e t e specifically for antibody binding with [ 12sI]C4ai. On the contrary, a CIs value for C4b o f 0.372, which is similar to that for C4, and a CIs0 value for C4b o f 4 X 10 -l° M are interpreted as due to a 1% molar contamination of C4 in the C4b preparation. In support of this conclusion, wide variation was observed among different C4b preparations and their ability to competitively inhibit [12sI]C4a~ binding. Finally, C4
67 are presented graphically in Fig. 3. For normal serum and activated serum, B/B0 values are plotted versus mol of C4 as determined b y dilution from neat serum. The concentration of C4 in serum was assessed b y a single radial immunodiffusion method (Fahey and McKelvey, 1965; KShler and MfillerEberhard, 1967). For activated serum precipitated with (NH4)2SO4 or polyethylene glycol, B/B0 values are plotted against mol of C4a~ as determined from a standard curve. Two points should be emphasized with respect to the data depicted in Fig. 3. The C4ai c o n t e n t of activated serum is unchanged following (NH4)2SO4 precipitation, as evidenced b y identical CIso values for both competition curves. However, following polyethylene glycol 6000 precipitation of activated serum, only 25--30% of C4ai initially present was recovered in the supernatant fraction {not shown). This latter result is completely unexpected in view o f the observed solubility of plasma C4a in polyethylene glycol (Fig. 1). We conclude that polyethylene glycol is ineffective in breaking the non-covalent interaction of newly formed C4a for C4b. In this case C4a may be precipitated as a complex with C4b. In contrast, high salt concentrations, i.e., 50% saturated (NH4)2SO4, are known to dissociate the C4a-C4b complex and t h e r e b y allow for a true assessment of the C4~h concentration. (Fig. 3). As a result, (NH4)2SO4 was used routinely in all subsequent radioimmune assay determinations on physiological fluids. Finally, the reproducibility and accuracy of the radioimmune assay method as applied to physiological fluids were examined. To do this, 3 identical samples of serum activated with aggregated IgG were processed as unknowns. The a m o u n t of aggregated IgG added to serum was pre-titered so as to ensure complete activation of C4. The results of separate (NH4)2SO4 precipitation and radioimmune assay analyses at 5 separate dilutions of the supernatant fractions are noted below: (1) 1.87 -+ 0.35 X 10 -6 M C4a~ (2) 1.72 + 0.41 X 10 -6 M C4a~ (3) 1.86 +- 0.40 X 10 -6 M C4ai The average concentration o f C4ai present in the 3 activated serum samples is 1.82 X 10 -6 M. This value is in quite good agreement with the serum concentration of C4, 1.46 X 10 -6 M, determined b y single radial immunodiffusion. Complete activation of C4 should yield, as was measured, an equimolar a m o u n t of C4ai. Taken together these data demonstrate that the radioimmune assay method is able to accurately and reproducibly quantitate complement fragment C4a~ in serum and plasma. Since the (NH4)2SO4 precipitation step has also been shown to be effective when applied to synovial fluid, it is likely that the assay can be directly applied to C4ai measurements in parenteral fluids.
In vitro stability o f C4 in physiological fluids The in vitro lability of complement protein C4 in physiological fluids is well appreciated, forming fragments C4a and C4b. In spite of this general awareness, few studies have been published documenting this fact. As a
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Fig. 3. Radioimmunoassay analysis of C4a i in serum activated with aggregated IgG. Normal human serum (I), serum activated with aggregated IgG (&), or activated serum precipitated with 50% saturated (NH4)2SO4 (e) was analyzed for its ability to competitively inhibit [12sI]c4ai precipitation by anti-C4a serum. 2.03pmol of [12sI]c4ai (5.06 pCi/pg) was present in radioimmune assay. Results are calculated relative to control assays; controls bound 71.6% of input [ 12sI]c4ai in this experiment. Fig. 4. Stability of plasma C4 in vitro. Normal human EDTA-plasma was incubated at 0°C (e), 23°C (A), or 37°C (D). At timed intervals, aliquots were taken from the incubation mixtures and assayed immediately for residual C4 hemolytic activity.
result, we u n d e r t o o k an e x a m i n a t i o n o f t h e stability o f C4 as a c o m p o n e n t o f serum, synovial fluid, and plasma. Briefly, n o r m a l serum o r plasma samples were i n c u b a t e d f o r v a r y i n g periods o f t i m e at 0°C, 23°C, or 37°C. A t t i m e d intervals, aliquots were t a k e n f r o m t h e i n c u b a t i o n m i x t u r e s and assayed i m m e d i a t e l y f o r C4 h e m o l y t i c activity, an i n d i c a t o r o f the a m o u n t o f residual, u n c l e a v e d C4. Results o f t h e stability s t u d y on plasma C4 are p r e s e n t e d in Fig. 4. C4 h e m o l y t i c t i t e r is p l o t t e d versus the length o f i n c u b a t i o n in h o u r s at 0 ° C, 23°C, or 37°C. It can be seen t h a t C4 cleavage and inactivation are o b s e r v e d at all 3 t e m p e r a t u r e c o n d i t i o n s . H o w e v e r , it is also a p p a r e n t t h a t the stability o f C4 is m a r k e d l y i m p r o v e d at lower t e m p e r a t u r e s . Specifically, 83% o f plasma C4 r e m a i n e d u n c l e a v e d a f t e r 4 h at 0°C, whereas 67% and 26% r e m a i n e d a f t e r 4 h at 23°C and 37°C, respectively. A l t h o u g h g e n e r a t i o n o f C4a does o c c u r u p o n storage o f n o r m a l plasma, we suggest the m a i n t e n a n c e o f t h e sample at l o w e r e d t e m p e r a t u r e s and limiting t h e length o f storage t o less t h a n 4 h will k e e p this artifactual c o n t r i b u t i o n within m a n a g e a b l e limits. By way o f c o m p a r i s o n t o results with plasma C4, the stability o f C4 in serum and synovial fluid was greatly d e c r e a s e d even at 0°C ( n o t s h o w n ) . Only 66% o f C4 was f o u n d t o be u n c l e a v e d a f t e r 4 h at 0°C with n o r m a l serum. In the case o f synoviai fluid f r o m a p a t i e n t suffering f r o m r h e u m a t o i d arthritis ( R A ) , 24% o f t o t a l C4 r e m a i n e d following i n c u b a t i o n f o r 4 h at 0°C, even t h o u g h the synovial fluid c o n t a i n e d 0.01 M E D T A . Our initial results on the stability o f C4 in R A synovial fluid suggest t h a t great care m u s t be exercised
69
when applying c o m p l e m e n t fragment assays to abnormal patient fluids to prevent c o m p l e m e n t activation in vitro. On the basis of these stability studies, all radioimmune assay analyses on normal blood samples (see next section) have used EDTA plasma. In addition, all plasmas were aliquoted and frozen within 1 h of being drawn.
Quantitation o f C4a~ in normal human plasma and urine Seventeen normal adult plasmas were analyzed for their content C4ai and C4. Results of these studies are presented graphically in Fig. 5. The concentration of C4ai is plotted versus the concentration of C4 determined by radial immunodiffusion. It is apparent that the concentration of C4ai in our study group ranges from 256 ng/ml (2.92 × 10 -11 M) to 1620 ng/ml (1.85 × 10 -1° M), with 16 of the 17 values between 834 and 256 ng/ml. Of special interest is the fact that a volunteer with 1620 ng/ml of C4a~ possesses a family history of systemic lupus erythematosus. The average concentration of C4a~ and C4 in our study group is 488 ng/ml and 23.4 mg/dl, respectively. Thus, on average, free C4ai represents 0.2% of total C4a antigenicity (free C4~h plus C4) present in normal plasma. However, no evidence was obtained that indicated a strong relationship between the a m o u n t of free C4ai in plasma and the concentration of C4. Specifically, linear regression analysis of data presented in Fig. 5 gives a straight line with a correlation coefficient of 0.279. A Spearman's rank correlation coefficient of 0.373 was also calculated from the data. Neither of these statistical treatments supports a significant correlation between the levels of C4ai and C4 in normal plasma. Because of its small size, it is assumed that C4~, once formed, would readily pass through the glomerulus of the kidney. As a result, it was of interest to determine the level of C4a~ in normal urine and a t t e m p t to relate it to the concentration of C4ai in normal plasma. The half-life of C4 in plasma is 72 h ( R u d d y et al., 1975). Results of radioimmunoassay analysis of 8 normal urines is presented in Table 1. The uncorrected level of C4ai =
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Fig. 5. Comparison o f levels of C4a i and o f C4 in 17 normal plasmas. The concentrations o f C 4 ~ and o f C4 in normal plasma were measured by the r a d i o i m m u n o ~ y m e t h o d and by a single radial i m m u n o d i f f u s i o n method, respectively. A best-fit line for the data and correlation coefficient were determined by linear regression analysis.
70 TABLE 1 Quantitation of C4ai in normal human urine. An overnight urine specimen (100 ml)was obtained from 8 normal adult volunteers. After dialysis for 3 days against distilled water in 3500 MW cut-off dialysis tubing, the specimens were lyophilized to dryness. Urine concentrates were analyzed (see Methods) after reconstitution with 2.5 ml of saline. Protein concentration was determined according to Lowry et al. (1951). Donor
C4ai concentration (ng/ml)
Protein concentration (pg/ml)
Percent C4ai of total protein (x 10 -s)
1 2 3 4 5 6 7 8
0.40 0.35 0.15 0.58 0.24 0.42 1.38 0.53
492 710 128 502 358 222 748 605
8.1 4.9 12.0 11.0 6.7 19.0 18.0 8.7
ranged f r o m 0.15 to 1.38 ng/ml, However, after calculation as a percentage o f urine protein, the range of C4a~ c o n t e n t could be expressed as between 4.9 and 19.0 X 10-s%. T he average c o n c e n t r a t i o n of C4ai in our group was 0.5 ng/ml. This value for urine is 0.1% of that in normal plasma (Fig. 5). Based on the average adult excr e t i on volume, we estimate t hat a b o u t 0.1% of the total C4a~ in normal plasma is excr e t e d in the urine daily. This estimate agrees well with results for insulin, a protein of similar size. A p p r o x i m a t e l y 2% o f blood insulin is lost in the urine each 24 h period (Chamberlain and Stimmler, 1967). These data suggest t h a t C4a~, along with o t h e r low molecular constitutents of plasma (Strober and Waldmann, 1974), is filtered through the glomerulus, taken up by tubular cells, and degraded within tubular phagolysosomes. Thus, only a m i no r p r o p o r t i o n of filtered C4~ escapes degradation and is excreted. Conversely, it is e x p e c t e d t hat in chronic renal disease associated with damage t o the entire nephron the plasma c o n c e n t r a t i o n of C4a~ would be greatly elevated. DISCUSSION T he experimental results presented in this paper support the following points. First, C4ai m a y be q u a n t i t a t e d specifically in hum an plasma and synovial fluid by (NH4)2SO4 precipitation and radioimmunoassay. Second, based on measurements of 17 normal plasmas, the average c o n c e n t r a t i o n of C4a i is 4 8 8 ng/ml. However, the level of C4ai does n o t correlate significantly with the c o n c e n t r a t i o n of C4 in plasma. Third, at t ent i on must be paid t o the handling o f physiological fluid samples to prevent artifactual generation of C4a in vitro. And finally, C4ai m a y be d e t e c t e d in normal hum an urine.
71 However, the level of C4a~ is only 0.1% of that present in normal plasma. The range of C4 concentration in normal plasma is wide, 12--75 mg/dl. As a result, measurements of C4 levels in disease have been uninformative. It has been suggested that the wide range of C4 concentrations in normals is due to variable expression at the t w o co-dominant loci coding for C4 (O'Neill et al., 1978a, 1978b) on chromosome 6. All 4 possible types of expression are observed in the population, i.e., full haplotypes at one loci and null alleles at the other. C4a i in normal plasma is presumed to be the result of both catabolic turnover of C4 and low level activation. In our study group, however, the level of C4ai could n o t be correlated with the concentration of C4 in plasma. As a result, we believe that the rate of C4a formation is more dependent u p o n the concentration of the responsible protease than u p o n the concentration of C4. In contrast, it is expected that in the process of classical pathway activation the rate of C4a formation will be dependent both u p o n the initial concentration of C4 in plasma and the rate of C4 synthesis. Radioimmunoassay m e t h o d s for the detection of c o m p l e m e n t fragments C3a i and CSai have been published (Hugli and Chenoweth, 1980). The concentration of C3a i and C5ai in normal plasma is 104 ng/ml and 30 ng/ml, respectively. Furthermore, the level of C3ai in normal serum was 1000 ng/ ml. The higher level detected in serum may be due to enhanced conversion of C3 during clotting and to the greater instability of C3 in serum as compared to plasma. Radioimmunoassay measurement of C5ai is of limited usefulness when applied to p a t i e n t samples (Chenoweth et al., 1981). Once formed, CSa binds irreversibly to neutrophils and other blood-borne cells (Chenoweth and Hugli, 1978). Thus measurements of C5ai levels in plasma show no increase above normal values until apparently the cellular binding capacity is saturated. C3ai and C4eh show no such affinity for cellular constitutents of blood; radioimmunoassay analyses for C3ai and C4ai should produce accurate assessments of the concentration of these fragments. The application of the m e t h o d for quantitation of C4ai to patient samples should allow for simple and sensitive determination of the complement pathway undergoing activation. In the past, the degree of c o m p l e m e n t turnover was estimated by CHs0 measurements, individual c o m p o n e n t radial immunodiffusion analyses, and individual c o m p o n e n t hemolytic titrations. These laboratory tests are labor intensive and costly. In addition, interpretation of these data is difficult because the tests are based on static samplings of dynamic processes. The C4ai m e t h o d also represents a static measurement, however, C4a is a specific marker of prior or on-going C4 turnover. Thus, we suggest an alternative to hemolytic c o m p l e m e n t titrations: combined quantitation of C4ai, C3ai, C4 and C3. For example, low C4ai and C4 values are interpreted as decreased C4 synthesis. On the other hand, high C4ai and low C4 levels would be consistent with enhanced C4 turnover. Finally, a high C3a~ value coupled with a low C4ai value is indicative of alternative pathway activation. In summary, the development o f a radioimmunoassay m e t h o d for measurement of C4ai n o w allows for the simple,
72
specific and sensitive assessment of C4 activation in man. Combined analyses for C3ai, C4a~, C3 and C4 may result in a more accurate understanding of the dynamics of the c o m p l e m e n t system than current methods permit. ACKNOWLEDGEMENTS
I am indebted to Ms. Siri Fiebeger, Ms. Ruth Silversmith and Mrs. Sharon Jones for their valuable technical and secretarial assistance. In addition, I wish to thank Doctors Hunder, Michet and Luthra, Mayo Clinic, for their cooperation in obtaining patient samples. This work was supported by U.S. Public Health Service Grant AI 17511 and funds from the Mellon Foundation and the Mayo Foundation. REFERENCES Bokisch, V.A., H.J. Mfiller-Eberhard and C.G. Cochrane, 1969, J. Exp. Med. 129, 1109. Bolotin, C., S. Morris, B. Tack and J. Prahl, 1977, Biochemistry 16, 2008. Bolton, A.E. and W.M. Hunter, 1973, Biochem. J. 133,529. Boros, T., H.J. Rapp and M.M. Mayer, 1961, J. Immunol. 87,310. Budzko, D.B. and H.J. Mfiller-Eberhard, 1970, Immunochemistry 7,227. Carpenter, C.B., S. Ruddy, I.H. Shehadeh, H.J. Mfiller-Eberhard, J.P. Merrill and K.F. Austen, 1969, J. Clin. Invest. 48, 1495. Chamberlain, M.J. and L. Stimmler, 1967, J. Clin. Invest. 46,911. Chenoweth, D.E. and T.E. Hugli, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 3943. Chenoweth, E., S.W. Cooper, T.E. Hugli, R.W. Stewart, E.H. Blackstone and J.W. Kirklin, 1981, New Engl. J. Med. 304,497. Curd, J.G., H. Milgrom, D.D. Stevenson, D.A. Mathison and J.H. Vaughan, 1979, Ann. Intern. Med. 91,853. Fahey, J.L. and E.M. McKelvey, 1965, J. Immunol. 94, 84. Gaither, T.A., D.W. Alling and M.M. Frank, 1974, J. Immunol. 113,574. Goldberger, G. and H.R. Colten, 1980, Nature 286, 514. Gorski, J.P., T.E. Hugli and H.J. M~iller-Eberhard, 1981, J. Biol. Chem. 256, 2707. Hugli, T.E. and D.E. Chenoweth, 1980, in: Laboratory and Research Methods in Biology and Medicine, Vol. 4, eds. R.M. Nakamura, W.R. Dito and E.S. Tucker, III (Alan R. Liss, New York) p. 443. KShler, P.F. and H.J. Mfiller-Eberhard, 1967, J. Immunol. 99, 1211. Law, S.K., N.A. Lichtenberg and R.P. Levine, 1979, J. Immunol. 123, 1388. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Mayer, M.M., 1961, in: Experimental Immunochemistry, eds. E.S. Kabat and M.M. Mayer (Thomas, Springfield, IL) p. 150. Milgrom, H., J.C. Curd, R.A. Kaplan, H.J. Miiller-Eberhard and J. H. Vaughan, 1980, J. Immunoi. 124, 2780. Moon, K.E., J.P. Gorski and T.E. Hugli, 1981, J. Biol. Chem. 256, 8685. O'Neill, G.J., S.Y. Yang, J. Tegoli, R. Berger and B. Dupont, 1978a, Nature 273,668. O'Neill, G.J., S.Y. Yang and B. Dupont, 1978b, Proc. Natl. Acad. Sci. U.S.A. 75, 5165. Perrin, L.H., P.H. Lambert and P.A. Miescher, 1975a, J. Clin. Invest. 56, 165. Perrin, L.H., S. Shiraishi, R.M. Stroud and P.H. Lambert, 1975b, J. Immunol. 115, 32. Petz, L.D., R. Powers, J.R. Fries, N.R. Cooper and H.R. Holman, 1977, Arthr. Rheum. 20, 1304. Ruddy, S., I. Gigli and K.F. Austen, 1972, New Engl. J. Med. 287,642.
73 Ruddy, S., C.B. Carpenter, K.W. Chin, J.N. Knostman, N.A. Soter, O. Gotze, H.J. MiillerEberhard and K.F. Austen, 1975, M~edicine 54,165. Schreiber, R.D. and H.J. Miiller-Eberhard, 1974, J. Exp. Med. 140, 1324. Schur, P.H. and K.F. Austen, 1968, Ann. Rev. Med. 19, 1. Stites, D.P., 1980, in: Basic and Clinical Immunology, 3rd edn, eds. H.H. Fudenberg, D.P. Stites, J.L. Caldwell and J.V. Wells (Lange Medical Publications, Los Altos, CA) p. 343. Strober, W. and T.A. Waldmann, 1974, Nephron 13, 35.
61
QUANTITATION OF HUMAN COMPLEMENT FRAGMENT C4ai IN PHYSIOLOGICAL FLUIDS BY COMPETITIVE INHIBITION RADIOIMMUNE ASSAY
J E F F R E Y P. GORSKI
Department of Cell Biology-Biochemistry, Mayo Clinic and Mayo Foundation, Rochester, MN 55901, U.S.A. (Received 6 May 1981, accepted 10 July 1981)
A method is described to quantitate complement fragment C4a i in human plasma, synovial fluid, and urine. Samples are first precipitated with 50% saturated (NH4)2SO4 to remove cross-reactive macromolecules C4 and pro-C4. Whereas greater than 97% of C4 is removed by this precipitation step, 88% of C4a i remains in solution. Second, the concentration of C4a i in supernatant fractions is determined by double antibody competitive inhibition radioimmunoassay. C4a was recently completely sequenced (Moon et al., 1981) and is readily available as a pure standard. Examination of the specificity of this method confirmed it was indeed specific for C4a antigenicity. Immunochemically depleted C4-deficient plasma and inulin-activated reconstituted C4-deficient plasma exhibited less than 0.1% of the immunoreactivity of untreated plasma. In addition, good agreement was observed in analyses of aggregated IgG activated serum between the experimentally determined concentration of C4a i and that expected from the initial concentration of C4. As a result, recovery and measurement of C4a i in physiological fluids with this method appear both quantitative and specific. Based on results from 17 adult volunteers, the average concentration of C4ai in normal plasma is 488 ng/ml. Interestingly, significant correlation could not be demonstrated between the levels of C4 and C4a i in normal plasma. The mean concentration of C4a i in human urine is 0.5 ng/ml.
INTRODUCTION
Activation of the classical p a t h w a y of complement is observed in many clinical pathologies, for example, serum sickness, myasthenia gravis, autoimmune hemolytic anemias, systemic lupus erythematosus, hereditary angioneurotic edema, and allograft rejection. Most studies have observed drops in hemolytic complement titers in these diseases (for reviews see: Shur and Austen, 1968; Ruddy et al., 1972). However, evaluation of these data in Abbreviations used in this paper: IgG, immunoglobulin G; PMN, polymorphonuclear leukocyte; C3a, C4a and C5a, activation peptides derived from the N-terminus of the ~-subunits of C3, C4 and C5; C3ai, C4a i and C5ai, products of carboxypeptidase B or N digestion of C3a, C4a and C5a; CIs0, amount of competing antigen required to attain half-maximal inhibition; CIs, calculated competitive inhibition slope; and B/B0, the quotient of the bound cpm to initial bound cpm ratio. 0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
62 a clinical setting has many uncertainties (Stites, 1980). First, an abnormally low hemolytic titer does not indicate a deficiency of this component, only a lack of its ability to complete a hemolytic sequence. Previously activated serum cannot be distinguished from deficient serum by this means. Second, a low titer may result from an increased rate of catabolic turnover. Third, low serum complement may result from increased tissue localization. And fourth, appearance of an inhibitor may give rise to an apparently low titer. Although hemolytic complement assays are of questionable reliability, the value of complement monitoring is nonetheless apparent. Assessment of complement status may assist in understanding the causes of cellular infiltration and humoral or PMN-mediated tissue destruction associated with immune complexes. Alternatives to hemolytic assays do exist, such as in vivo turnover measurement and complement fragment analysis. In vivo complement studies (Carpenter et al., 1969; Petz et al., 1977; Curd et al., 1979}, while worthwhile, are n o t practical on a routine basis. Immunochemical assays for complement fragments C3a, C5a, C3d, C4d and Bb have been developed and applied to patient populations (Perrin et al., 1975a, 1975b; Hugli and Chenoweth, 1980). The general principle of these latter assays is first to separate unactivated C3, C4, C5 and factor B from their respective fragments by selective precipitation. The samples are then analyzed by radial immunodiffusion (C3d, C4d, Bb), immunoelectrophoresis (C4d), or radioimmune assay (C3a, C5a) methods. Several criticisms may be raised against the C3d and C4d fragment assays. For example, C3d and C4d represent structural regions of C3b and C4b that mediate their binding to biological membranes (Schreiber and Mfiller-Eberhard, 1974; Law et al., 1979). As such, C3d and C4d may not be present in high concentrations in biological fluids. In the case of C4d, no pure standard is available and all previous measurements were compared to that of serum treated with aggregated IgG (Perrin et al., 1975b; Milgrom et al., 1980). Finally, the sensitivity of radial immunodiffusion and immunoelectrophoretic methods of detection are restricted to a lower limit of 1--5/~g of antigen protein. Current double antibody radioimmune assay methods display lower limits in the range of 1 ng antigen protein. In spite o f the fact the m e t h o d for quantitation of C5a relies upon radioimmune assay methodology, efforts to apply it to measurements of C5a in plasma have failed. Once formed, the majority of C5a binds tightly to blood neutrophils (Chenoweth and Hugli, 1978) and is n o t found free in plasma until this capacity apparently is exceeded (Chenoweth et al., 1981}. Development and application of a radioimmune assay for fragment C4a does n o t appear to suffer from the above-mentioned criticisms. Small size, lack of association with cell surfaces, availability of chemically characterized homogeneous standard (Gorski et al., 1981}, and direct kinetic relationship of its formation with C4 activation suggest an avoidance o f these problems.
63 MATERIALS
Normal human EDTA plasma was obtained fresh from the Mayo Clinic Blood Bank. Donkey anti-rabbit IgG serum and ~2SI-Boloton-Hunter reagent were purchased from Pel-Freez and Amersham, Inc., respectively. METHODS
Purification of human complement proteins and fragments C4a and C4b were isolated as previously described (Gorski et al., 1981). Alternatively, purified C4 (Bolotin et al., 1977) was converted to C4a and C4b by incubation with 5% (w/w) trypsin (Budzko and Miiller-Eberhard, 1970) for 2.5 min at 23°C in 0.1 M sodium bicarbonate buffer, pH 8.5, containing 0.15 M sodium chloride, 0.01 M EDTA, and 0.02% (w/v) sodium azide. A 5-fold weightexcess of soybean trypsin inhibitor was added to the digest to stop the reaction. Digestion of C4a with 1% (w/w) carboxypeptidase B for 60 min at 37°C quantitatively converted C4a to C4ai.
C4 hemolytic assay Plasma samples were assayed for C4
Method for quantitation of C4ai in physiological fluids One volume of physiological fluid was mixed with an equal volume of 0.2 M Tris-acetate buffer (pH 8.0), containing 35% (w/v) polyethylene glycol 6000, 0.04 mg/ml soybean trypsin inhibitor, 0.02 M EDTA, 0.02 M benzamidine hydrochloride, 0.2 M 2:-amino-n-caproic acid, and 0.04% (w/v) sodium azide. Alternatively, saturated ammonium sulfate was substituted for polyethylene glycol and used as a precipitant. After mixing for 2 h at 4°C, precipitated protein was removed by centrifugation. The supernatant fraction resulting from either ammonium sulfate or polyethylene glycol precipitation was subsequently analyzed by radioimmunoassay for C4a~ content. A 4-compartment radioimmunoassay system with a final volume of 1 ml was used: (1) assay buffer, 0.2 ml; (2) primary antiserum, 0.2 ml; (3) competing antigen preparation, 0.2 ml; and (4) secondary antiserum, 0.4 ml. The volume of radiolabeled antigen, 0.005-0.025 ml, was disregarded. Assay buffer consisted of 0.5 M Tris-acetate buffer (pH 8.0), containing 0.05 M EDTA, 0.2 M Y~-amino-n-caproic acid, 0~05 M benzamidine hydrochloride, 1.25 mg/ml PMSF, 0.04 mg/ml soybean trypsin inhibitor, 0.3% (w/v) Triton X-100, and 0.1% (w/v) sodium azide. Primary antiserum was diluted at least 50-fold with either saline or 50-fold diluted non-immune rabbit serum to keep the concentration of rabbit IgG at 0.2 mg/ml. Secondary antiserum,
64
d o n k e y anti-rabbit IgG serum, was pre-titered so as to bring a b o u t precipitation of at least 95% of rabbit IgG present in assays. Radioimmune assays c o m p o s e d of c o m p o n e n t s 1--3 and [12sI]C4ai were incubated initially for 16--20 h at 4°C on a rocking platform. Secondary antiserum, Component 4, was then added to assay tubes, which were re-incubated for a period of 6 h at 4°C. Immune aggregates were sedimented b y centrifugation at 8730 × gmax. Gamma counting was carried o u t first on the total assay volumes and subsequently on the immune pellets. Individual assay values (cpm) were corrected for experimental blank values, averaged, and calculated as a percentage of the experimental control value. Controls were prepared exactly as described above except that saline was substituted for competing antigen. Blanks, routinely 2% of input cpm, were prepared as described above except that 50-fold diluted non-immune rabbit serum was substituted for immune serum in c o m p a r t m e n t 2. Experimental points are the average of duplicate assays.
Miscellaneous The concentration of C4b, C4ai, or C4 protein in standard solutions was determined from the results of amino acid analysis. C4ai was radiolabeled with 12SI-Bolton-Hunter reagent (N-succinimidyl 3-(4-hydroxy, 5-[12sI]iodophenyl propionate) (Bolton and Hunter, 1973) according to instructions supplied b y Amersham Corp., except that 0.1 M sodium bicarbonate buffer (pH 8.5), containing 0.15 M sodium chloride, was substituted for sodium borate as the reaction buffer. Aggregated IgG was prepared by incubation of purified human IgG (10 mg/ml) in saline for 30 min at 63°C. RESULTS
Selective precipitation of C4 from plasma Direct immunochemical quantitation of activation fragment C4a in human plasma requires selective removal of cross-reacting proteins C4 and pro-C4 before analysis. C4a is a 77-residue polypeptide derived from the N-terminus of the ~-subunit of C4 (Gorski et al., 1981) and represents an internal sequence stretch of single-chain pro-C4 (Goldberger and Colten, 1980). Due to the large difference in molecular weights of C4a and of C4 or pro-C4, precipitation methods discriminating on the basis of size were examined. Soybean trypsin inhibitor, benzamidine hydrochloride, E-amino-n-caproic acid, and EDTA were included in the precipitation media to prevent activation of C4 during this step. The solubilities of plasma C4a and C4 in different concentrations of (NH4)2SO4 were determined and are plotted in Fig. 1. C4a remains in solution in all concentrations of (NH4)2SO4 tested. Specifically, an average of 88% of the total C4a remained in solution following precipitation of plasma with from 12.5 to 50% saturated (NH4)2SO4. As expected, plasma C4 is sus-
65
pectible to precipitation with salt. Whereas in 25% saturated (NH4)2SO4, 96% of the total plasma C4 remains in solution, less than 3% is soluble in 50% saturated (NH4)2SO4. It is assumed that the solubility o f pro-C4, which is the same size as C4, is similar to that observed for C4. The respective solubilities o f C4a and C4 in various concentrations of polyethylene glycol 6000 were also determined (not shown). Ninety-four percent of plasma C4a remained in solution following precipitation with from 7.5 to 20% (w/v) polyethylene glycol. In contrast, C4 was totally removed from solution with 15% (w/v) polyethylene glycol, in agreement with earlier studies (Perrin et al., 1975b; Bolotin et al., 1977). In addition to plasma, the differential solubilities of C4a and C4 in human synovial fluid were also studied (not shown). Results were identical to those described above for plasma C4a and C4 with (NH4)2SO4 and polyethylene glycol. In summary, C4 and pro-C4 may be separated from C4a in plasma or synovial fluid by specific precipitation with (NH4)2SO4 or polyethylene glycol 6000.
Characterization of the specificity of radioimmune assay for C4ai A double-antibody competitive inhibition radioimmunoassay for human C4a~ was developed with [ 12si] C4ai, rabbit anti-C4a serum, and donkey anti-
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Fig. 1. Solubilities of plasma C4a and C4 in different concentrations of (NH4)2SO4. Aliquots of normal human plasma with [ l : s I ] C 4 a (m) or [12SI]C4 (e) were added to equal volumes of 0.2 M Tris-acetate buffer (pH 8.0), containing 0.2 M Y-amino-n-caproic acid, 0.04 mg/ml soybean trypsin inhibitor, 0.02 M EDTA, 0.02 M benzamidine hydrochloride, 0.04% (w/v) sodium azide, and different percentages of saturated (NH4)2SO4. After mixing for 2 h at 4°C, the samples were centrifuged to remove precipitated protein and the supernatant fractions were counted. Fig. 2. Characterization o f the specificity of radioimmunoassay for C4a i. C4a (o), C4ai (m), C4 (A), normal human plasma (e), or C4b (D), was tested for its ability to competitively inhibit [12SI]C4ai precipitation by anti-C4a serum. 1.1 pmol o f [12sI]c4ai (7.6 ~Ci//~g) was present in each radioimmune assay. Results are calculated relative to control assays; controls bound 63.9% of input [ 12 s I ]C4ai in this experiment.
66 rabbit IgG serum (see Methods). It is known that the complement anaphylatoxins, once formed, are subject to the regulatory controlling action of carboxypeptidase N (EC 3.4.12.7) in blood (Bokisch et al., 1969). Therefore, [12sI]C4~ was used as radioligand in our studies because we believe it represents the form o f the C4a antigen found free in physiological fluids. Numerous checks on the specificity of the radioimmune assay for C4a were undertaken. The results o f some of these experiments are summarized in Fig. 2. Briefly, purified C4a was observed to c o m p e t e completely for [12sI]C4ai binding. A CIs0 value of 3.1 X 10 -13 M and a CIs of 0.611 characterize this competitive binding curve. As expected, C4ai also competes totally with its radiolabeled form. In quantitative terms, half-maximal binding was attained with 6.7 × 10 -13 M peptide and a CIs value of 0.623 was calculated from the results. Completing this survey of completely crossreacting antigens, purified C4 and plasma C4 gave rise to CIs0 values of 2.8 X 10 -12 M and 6.7 X 10 -12 M and, competitive inhibition slopes of 0.453 and 0.424, respectively. Purified C4b, the other product o f C4 activation, does n o t c o m p e t e specifically for antibody binding with [ 12sI]C4ai. On the contrary, a CIs value for C4b o f 0.372, which is similar to that for C4, and a CIs0 value for C4b o f 4 X 10 -l° M are interpreted as due to a 1% molar contamination of C4 in the C4b preparation. In support of this conclusion, wide variation was observed among different C4b preparations and their ability to competitively inhibit [12sI]C4a~ binding. Finally, C4
67 are presented graphically in Fig. 3. For normal serum and activated serum, B/B0 values are plotted versus mol of C4 as determined b y dilution from neat serum. The concentration of C4 in serum was assessed b y a single radial immunodiffusion method (Fahey and McKelvey, 1965; KShler and MfillerEberhard, 1967). For activated serum precipitated with (NH4)2SO4 or polyethylene glycol, B/B0 values are plotted against mol of C4a~ as determined from a standard curve. Two points should be emphasized with respect to the data depicted in Fig. 3. The C4ai c o n t e n t of activated serum is unchanged following (NH4)2SO4 precipitation, as evidenced b y identical CIso values for both competition curves. However, following polyethylene glycol 6000 precipitation of activated serum, only 25--30% of C4ai initially present was recovered in the supernatant fraction {not shown). This latter result is completely unexpected in view o f the observed solubility of plasma C4a in polyethylene glycol (Fig. 1). We conclude that polyethylene glycol is ineffective in breaking the non-covalent interaction of newly formed C4a for C4b. In this case C4a may be precipitated as a complex with C4b. In contrast, high salt concentrations, i.e., 50% saturated (NH4)2SO4, are known to dissociate the C4a-C4b complex and t h e r e b y allow for a true assessment of the C4~h concentration. (Fig. 3). As a result, (NH4)2SO4 was used routinely in all subsequent radioimmune assay determinations on physiological fluids. Finally, the reproducibility and accuracy of the radioimmune assay method as applied to physiological fluids were examined. To do this, 3 identical samples of serum activated with aggregated IgG were processed as unknowns. The a m o u n t of aggregated IgG added to serum was pre-titered so as to ensure complete activation of C4. The results of separate (NH4)2SO4 precipitation and radioimmune assay analyses at 5 separate dilutions of the supernatant fractions are noted below: (1) 1.87 -+ 0.35 X 10 -6 M C4a~ (2) 1.72 + 0.41 X 10 -6 M C4a~ (3) 1.86 +- 0.40 X 10 -6 M C4ai The average concentration o f C4ai present in the 3 activated serum samples is 1.82 X 10 -6 M. This value is in quite good agreement with the serum concentration of C4, 1.46 X 10 -6 M, determined b y single radial immunodiffusion. Complete activation of C4 should yield, as was measured, an equimolar a m o u n t of C4ai. Taken together these data demonstrate that the radioimmune assay method is able to accurately and reproducibly quantitate complement fragment C4a~ in serum and plasma. Since the (NH4)2SO4 precipitation step has also been shown to be effective when applied to synovial fluid, it is likely that the assay can be directly applied to C4ai measurements in parenteral fluids.
In vitro stability o f C4 in physiological fluids The in vitro lability of complement protein C4 in physiological fluids is well appreciated, forming fragments C4a and C4b. In spite of this general awareness, few studies have been published documenting this fact. As a
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LENGTH OF INCUBATION, (H)
Fig. 3. Radioimmunoassay analysis of C4a i in serum activated with aggregated IgG. Normal human serum (I), serum activated with aggregated IgG (&), or activated serum precipitated with 50% saturated (NH4)2SO4 (e) was analyzed for its ability to competitively inhibit [12sI]c4ai precipitation by anti-C4a serum. 2.03pmol of [12sI]c4ai (5.06 pCi/pg) was present in radioimmune assay. Results are calculated relative to control assays; controls bound 71.6% of input [ 12sI]c4ai in this experiment. Fig. 4. Stability of plasma C4 in vitro. Normal human EDTA-plasma was incubated at 0°C (e), 23°C (A), or 37°C (D). At timed intervals, aliquots were taken from the incubation mixtures and assayed immediately for residual C4 hemolytic activity.
result, we u n d e r t o o k an e x a m i n a t i o n o f t h e stability o f C4 as a c o m p o n e n t o f serum, synovial fluid, and plasma. Briefly, n o r m a l serum o r plasma samples were i n c u b a t e d f o r v a r y i n g periods o f t i m e at 0°C, 23°C, or 37°C. A t t i m e d intervals, aliquots were t a k e n f r o m t h e i n c u b a t i o n m i x t u r e s and assayed i m m e d i a t e l y f o r C4 h e m o l y t i c activity, an i n d i c a t o r o f the a m o u n t o f residual, u n c l e a v e d C4. Results o f t h e stability s t u d y on plasma C4 are p r e s e n t e d in Fig. 4. C4 h e m o l y t i c t i t e r is p l o t t e d versus the length o f i n c u b a t i o n in h o u r s at 0 ° C, 23°C, or 37°C. It can be seen t h a t C4 cleavage and inactivation are o b s e r v e d at all 3 t e m p e r a t u r e c o n d i t i o n s . H o w e v e r , it is also a p p a r e n t t h a t the stability o f C4 is m a r k e d l y i m p r o v e d at lower t e m p e r a t u r e s . Specifically, 83% o f plasma C4 r e m a i n e d u n c l e a v e d a f t e r 4 h at 0°C, whereas 67% and 26% r e m a i n e d a f t e r 4 h at 23°C and 37°C, respectively. A l t h o u g h g e n e r a t i o n o f C4a does o c c u r u p o n storage o f n o r m a l plasma, we suggest the m a i n t e n a n c e o f t h e sample at l o w e r e d t e m p e r a t u r e s and limiting t h e length o f storage t o less t h a n 4 h will k e e p this artifactual c o n t r i b u t i o n within m a n a g e a b l e limits. By way o f c o m p a r i s o n t o results with plasma C4, the stability o f C4 in serum and synovial fluid was greatly d e c r e a s e d even at 0°C ( n o t s h o w n ) . Only 66% o f C4 was f o u n d t o be u n c l e a v e d a f t e r 4 h at 0°C with n o r m a l serum. In the case o f synoviai fluid f r o m a p a t i e n t suffering f r o m r h e u m a t o i d arthritis ( R A ) , 24% o f t o t a l C4 r e m a i n e d following i n c u b a t i o n f o r 4 h at 0°C, even t h o u g h the synovial fluid c o n t a i n e d 0.01 M E D T A . Our initial results on the stability o f C4 in R A synovial fluid suggest t h a t great care m u s t be exercised
69
when applying c o m p l e m e n t fragment assays to abnormal patient fluids to prevent c o m p l e m e n t activation in vitro. On the basis of these stability studies, all radioimmune assay analyses on normal blood samples (see next section) have used EDTA plasma. In addition, all plasmas were aliquoted and frozen within 1 h of being drawn.
Quantitation o f C4a~ in normal human plasma and urine Seventeen normal adult plasmas were analyzed for their content C4ai and C4. Results of these studies are presented graphically in Fig. 5. The concentration of C4ai is plotted versus the concentration of C4 determined by radial immunodiffusion. It is apparent that the concentration of C4ai in our study group ranges from 256 ng/ml (2.92 × 10 -11 M) to 1620 ng/ml (1.85 × 10 -1° M), with 16 of the 17 values between 834 and 256 ng/ml. Of special interest is the fact that a volunteer with 1620 ng/ml of C4a~ possesses a family history of systemic lupus erythematosus. The average concentration of C4a~ and C4 in our study group is 488 ng/ml and 23.4 mg/dl, respectively. Thus, on average, free C4ai represents 0.2% of total C4a antigenicity (free C4~h plus C4) present in normal plasma. However, no evidence was obtained that indicated a strong relationship between the a m o u n t of free C4ai in plasma and the concentration of C4. Specifically, linear regression analysis of data presented in Fig. 5 gives a straight line with a correlation coefficient of 0.279. A Spearman's rank correlation coefficient of 0.373 was also calculated from the data. Neither of these statistical treatments supports a significant correlation between the levels of C4ai and C4 in normal plasma. Because of its small size, it is assumed that C4~, once formed, would readily pass through the glomerulus of the kidney. As a result, it was of interest to determine the level of C4a~ in normal urine and a t t e m p t to relate it to the concentration of C4ai in normal plasma. The half-life of C4 in plasma is 72 h ( R u d d y et al., 1975). Results of radioimmunoassay analysis of 8 normal urines is presented in Table 1. The uncorrected level of C4ai =
=
i
=
i
1600,
z
"~
I.-
¢~
e:
~ 0 ~.)
~ o
1200.
r=0.279
800"
,-
~)
ee
0
0
I0
20
30
CONCENTRATION
40
50
OF
C4,
60
70
(rng/dl)
Fig. 5. Comparison o f levels of C4a i and o f C4 in 17 normal plasmas. The concentrations o f C 4 ~ and o f C4 in normal plasma were measured by the r a d i o i m m u n o ~ y m e t h o d and by a single radial i m m u n o d i f f u s i o n method, respectively. A best-fit line for the data and correlation coefficient were determined by linear regression analysis.
70 TABLE 1 Quantitation of C4ai in normal human urine. An overnight urine specimen (100 ml)was obtained from 8 normal adult volunteers. After dialysis for 3 days against distilled water in 3500 MW cut-off dialysis tubing, the specimens were lyophilized to dryness. Urine concentrates were analyzed (see Methods) after reconstitution with 2.5 ml of saline. Protein concentration was determined according to Lowry et al. (1951). Donor
C4ai concentration (ng/ml)
Protein concentration (pg/ml)
Percent C4ai of total protein (x 10 -s)
1 2 3 4 5 6 7 8
0.40 0.35 0.15 0.58 0.24 0.42 1.38 0.53
492 710 128 502 358 222 748 605
8.1 4.9 12.0 11.0 6.7 19.0 18.0 8.7
ranged f r o m 0.15 to 1.38 ng/ml, However, after calculation as a percentage o f urine protein, the range of C4a~ c o n t e n t could be expressed as between 4.9 and 19.0 X 10-s%. T he average c o n c e n t r a t i o n of C4ai in our group was 0.5 ng/ml. This value for urine is 0.1% of that in normal plasma (Fig. 5). Based on the average adult excr e t i on volume, we estimate t hat a b o u t 0.1% of the total C4a~ in normal plasma is excr e t e d in the urine daily. This estimate agrees well with results for insulin, a protein of similar size. A p p r o x i m a t e l y 2% o f blood insulin is lost in the urine each 24 h period (Chamberlain and Stimmler, 1967). These data suggest t h a t C4a~, along with o t h e r low molecular constitutents of plasma (Strober and Waldmann, 1974), is filtered through the glomerulus, taken up by tubular cells, and degraded within tubular phagolysosomes. Thus, only a m i no r p r o p o r t i o n of filtered C4~ escapes degradation and is excreted. Conversely, it is e x p e c t e d t hat in chronic renal disease associated with damage t o the entire nephron the plasma c o n c e n t r a t i o n of C4a~ would be greatly elevated. DISCUSSION T he experimental results presented in this paper support the following points. First, C4ai m a y be q u a n t i t a t e d specifically in hum an plasma and synovial fluid by (NH4)2SO4 precipitation and radioimmunoassay. Second, based on measurements of 17 normal plasmas, the average c o n c e n t r a t i o n of C4a i is 4 8 8 ng/ml. However, the level of C4ai does n o t correlate significantly with the c o n c e n t r a t i o n of C4 in plasma. Third, at t ent i on must be paid t o the handling o f physiological fluid samples to prevent artifactual generation of C4a in vitro. And finally, C4ai m a y be d e t e c t e d in normal hum an urine.
71 However, the level of C4a~ is only 0.1% of that present in normal plasma. The range of C4 concentration in normal plasma is wide, 12--75 mg/dl. As a result, measurements of C4 levels in disease have been uninformative. It has been suggested that the wide range of C4 concentrations in normals is due to variable expression at the t w o co-dominant loci coding for C4 (O'Neill et al., 1978a, 1978b) on chromosome 6. All 4 possible types of expression are observed in the population, i.e., full haplotypes at one loci and null alleles at the other. C4a i in normal plasma is presumed to be the result of both catabolic turnover of C4 and low level activation. In our study group, however, the level of C4ai could n o t be correlated with the concentration of C4 in plasma. As a result, we believe that the rate of C4a formation is more dependent u p o n the concentration of the responsible protease than u p o n the concentration of C4. In contrast, it is expected that in the process of classical pathway activation the rate of C4a formation will be dependent both u p o n the initial concentration of C4 in plasma and the rate of C4 synthesis. Radioimmunoassay m e t h o d s for the detection of c o m p l e m e n t fragments C3a i and CSai have been published (Hugli and Chenoweth, 1980). The concentration of C3a i and C5ai in normal plasma is 104 ng/ml and 30 ng/ml, respectively. Furthermore, the level of C3ai in normal serum was 1000 ng/ ml. The higher level detected in serum may be due to enhanced conversion of C3 during clotting and to the greater instability of C3 in serum as compared to plasma. Radioimmunoassay measurement of C5ai is of limited usefulness when applied to p a t i e n t samples (Chenoweth et al., 1981). Once formed, CSa binds irreversibly to neutrophils and other blood-borne cells (Chenoweth and Hugli, 1978). Thus measurements of C5ai levels in plasma show no increase above normal values until apparently the cellular binding capacity is saturated. C3ai and C4eh show no such affinity for cellular constitutents of blood; radioimmunoassay analyses for C3ai and C4ai should produce accurate assessments of the concentration of these fragments. The application of the m e t h o d for quantitation of C4ai to patient samples should allow for simple and sensitive determination of the complement pathway undergoing activation. In the past, the degree of c o m p l e m e n t turnover was estimated by CHs0 measurements, individual c o m p o n e n t radial immunodiffusion analyses, and individual c o m p o n e n t hemolytic titrations. These laboratory tests are labor intensive and costly. In addition, interpretation of these data is difficult because the tests are based on static samplings of dynamic processes. The C4ai m e t h o d also represents a static measurement, however, C4a is a specific marker of prior or on-going C4 turnover. Thus, we suggest an alternative to hemolytic c o m p l e m e n t titrations: combined quantitation of C4ai, C3ai, C4 and C3. For example, low C4ai and C4 values are interpreted as decreased C4 synthesis. On the other hand, high C4ai and low C4 levels would be consistent with enhanced C4 turnover. Finally, a high C3a~ value coupled with a low C4ai value is indicative of alternative pathway activation. In summary, the development o f a radioimmunoassay m e t h o d for measurement of C4ai n o w allows for the simple,
72
specific and sensitive assessment of C4 activation in man. Combined analyses for C3ai, C4a~, C3 and C4 may result in a more accurate understanding of the dynamics of the c o m p l e m e n t system than current methods permit. ACKNOWLEDGEMENTS
I am indebted to Ms. Siri Fiebeger, Ms. Ruth Silversmith and Mrs. Sharon Jones for their valuable technical and secretarial assistance. In addition, I wish to thank Doctors Hunder, Michet and Luthra, Mayo Clinic, for their cooperation in obtaining patient samples. This work was supported by U.S. Public Health Service Grant AI 17511 and funds from the Mellon Foundation and the Mayo Foundation. REFERENCES Bokisch, V.A., H.J. Mfiller-Eberhard and C.G. Cochrane, 1969, J. Exp. Med. 129, 1109. Bolotin, C., S. Morris, B. Tack and J. Prahl, 1977, Biochemistry 16, 2008. Bolton, A.E. and W.M. Hunter, 1973, Biochem. J. 133,529. Boros, T., H.J. Rapp and M.M. Mayer, 1961, J. Immunol. 87,310. Budzko, D.B. and H.J. Mfiller-Eberhard, 1970, Immunochemistry 7,227. Carpenter, C.B., S. Ruddy, I.H. Shehadeh, H.J. Mfiller-Eberhard, J.P. Merrill and K.F. Austen, 1969, J. Clin. Invest. 48, 1495. Chamberlain, M.J. and L. Stimmler, 1967, J. Clin. Invest. 46,911. Chenoweth, D.E. and T.E. Hugli, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 3943. Chenoweth, E., S.W. Cooper, T.E. Hugli, R.W. Stewart, E.H. Blackstone and J.W. Kirklin, 1981, New Engl. J. Med. 304,497. Curd, J.G., H. Milgrom, D.D. Stevenson, D.A. Mathison and J.H. Vaughan, 1979, Ann. Intern. Med. 91,853. Fahey, J.L. and E.M. McKelvey, 1965, J. Immunol. 94, 84. Gaither, T.A., D.W. Alling and M.M. Frank, 1974, J. Immunol. 113,574. Goldberger, G. and H.R. Colten, 1980, Nature 286, 514. Gorski, J.P., T.E. Hugli and H.J. M~iller-Eberhard, 1981, J. Biol. Chem. 256, 2707. Hugli, T.E. and D.E. Chenoweth, 1980, in: Laboratory and Research Methods in Biology and Medicine, Vol. 4, eds. R.M. Nakamura, W.R. Dito and E.S. Tucker, III (Alan R. Liss, New York) p. 443. KShler, P.F. and H.J. Mfiller-Eberhard, 1967, J. Immunol. 99, 1211. Law, S.K., N.A. Lichtenberg and R.P. Levine, 1979, J. Immunol. 123, 1388. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Mayer, M.M., 1961, in: Experimental Immunochemistry, eds. E.S. Kabat and M.M. Mayer (Thomas, Springfield, IL) p. 150. Milgrom, H., J.C. Curd, R.A. Kaplan, H.J. Miiller-Eberhard and J. H. Vaughan, 1980, J. Immunoi. 124, 2780. Moon, K.E., J.P. Gorski and T.E. Hugli, 1981, J. Biol. Chem. 256, 8685. O'Neill, G.J., S.Y. Yang, J. Tegoli, R. Berger and B. Dupont, 1978a, Nature 273,668. O'Neill, G.J., S.Y. Yang and B. Dupont, 1978b, Proc. Natl. Acad. Sci. U.S.A. 75, 5165. Perrin, L.H., P.H. Lambert and P.A. Miescher, 1975a, J. Clin. Invest. 56, 165. Perrin, L.H., S. Shiraishi, R.M. Stroud and P.H. Lambert, 1975b, J. Immunol. 115, 32. Petz, L.D., R. Powers, J.R. Fries, N.R. Cooper and H.R. Holman, 1977, Arthr. Rheum. 20, 1304. Ruddy, S., I. Gigli and K.F. Austen, 1972, New Engl. J. Med. 287,642.
73 Ruddy, S., C.B. Carpenter, K.W. Chin, J.N. Knostman, N.A. Soter, O. Gotze, H.J. MiillerEberhard and K.F. Austen, 1975, M~edicine 54,165. Schreiber, R.D. and H.J. Miiller-Eberhard, 1974, J. Exp. Med. 140, 1324. Schur, P.H. and K.F. Austen, 1968, Ann. Rev. Med. 19, 1. Stites, D.P., 1980, in: Basic and Clinical Immunology, 3rd edn, eds. H.H. Fudenberg, D.P. Stites, J.L. Caldwell and J.V. Wells (Lange Medical Publications, Los Altos, CA) p. 343. Strober, W. and T.A. Waldmann, 1974, Nephron 13, 35.