[42] Quantification of immune complexes by nephelometry

628

I M M U N E COMPLEXES

[42]

their constituents, and a comparison with tissue-fixed complexes. A monoclonal rheumatoid factor reagent, particularly because of its stability, seems to offer one method of approaching the problem. There is little doubt that this area needs to be examined closely if the pathogenetic mechanisms of IC disease are to be understood more clearly than they are at present. Acknowledgment This research was supported by Medical Research Council Grant No. G 978116.

[42] Quantification of Immune Complexes by Nephelometry By KLAUS HOFFKENand CARL G. SCHM1DT Introduction

The immunopathological and clinical significance of antigen-antibody complexes is widely recognized in a variety of benign diseases, e.g., rheumatoid arthritis, lupus erythematosus, nephritis, hepatitis. 1-4 Furthermore, immune complexes (ICs) have been detected in cancer patients and in tumor-bearing animals. 5-1° A range of reproducible tests are being used for the measurement of ICs in body fluids, but at present tests conform to no agreed-upon standards with respect to antigen or methodology, and their interpretation has F. J. Dixon, J. J. Vazquez, W. O. Weigle, and C. G. Cochrane, A M A Arch. Pathol. 65, 18 (1958). 2 Editorial, Lancet 1,580 (1977). a R. N. Maini and E. J. Holborow, Ann. Rheum. Dis. 36, Suppl. 1 (1977). a World Health Organization, "The Role of Immune Complexes in Disease." W.H.O. Tech. Rep. Ser. No. 606 (1977). 5 K. HSffken, I. D. Meredith, R. A. Robins, R. W. Baldwin, C. J. Davies, and R. W. Blamey, Br. Med. J. 2, 218 (1977). K. HStiken, I. D. Meredith, R. A. Robins, R. W. Baldwin, C. J. Davies, and R. W. Blamey, Lancet 1, 672 (1978). r K. HSffken, M. R. Price, P. J. McLaughlin, V. E. Moore, and R. W. Baldwin, Int. J. Cancer 21,496 (1978). s j. C. Jennette and J. D. Feldman, J. Immunol. 118, 2269 (1977). 9 R. D. Rossen, M. A. Reisberg, E. M. Hersh, andJ. U. Gutterman,JNCl, J. Natl. Cancer Inst. 58, 1205 (1977). to A. N. Theofilopoulos, B. S. Andrews, M. M. Urist, D. L. Morton, and F, J. Dixon, J. Immunol. 119, 657 (1977).

METHODS IN ENZYMOLOGY, VOL. 74

Copyright ~) 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181974-4

[42]

NEPHELOMETRIC QUANTIFICATION OF ICs

629

largely been a matter of individual experience in the laboratory performing them. The aims of tests for soluble ICs include (1) detection and quantification of ICs" (2) characterization of ICs: (3) identification of the antigen moiety: and (4) monitoring treatment of IC diseases. Most methods of detecting ICs are based on the ability of the complexes to fix complement components or to activate the complement sequence. Other tests make use of conformational changes induced by antigen-antibody interaction or of the molecular size of the ICs generated. With few exceptions, these methods are antigen nonspecific in that they quantify the antibody involved or complement components fixed by ICs. Most tests for measuring ICs require skillful handling of reagents or cells such as purified Clq, radiolabeled substances, or Raji cells. Consequently, only a few methods can be regarded as suitable for routine screening of sera or body fluids for ICs. An extensive WHO study 11 comparing various methods for detecting and quantifying ICs showed good correlation between some of the tests investigated, but it also confirmed the fact that distinct methods detect distinct ICs. With the exception of ultracentrifugation, ICs are quantified by virtue of their biological activity, such as interaction with complement, complement receptors on cells, conglutinin, and rheumatoid factors. Therefore, it was of interest to study another physicochemical feature of ICs, i.e., their capacity of light scattering. As early as 1938, Libby v2'13 reported on the measurement with a photon-reflectometer of turbidities caused by ICs and observed a linear relationship between galvanometer readings and concentrations of antiserum and antigen. Since then, nephelometric methods have greatly improved, especially after the introduction of a laser beam as the light source, and are now being used for the estimation of antigens, antibodies, and serum protein concentrations. We have demonstrated the applicability of nephelometry to quantification of ICs in native sera and believe that this method, though not competitive, represents a good alternative to conventional tests for measurement of ICs present in sera or body fluids. It is beyond the scope of this report to discuss the nephelometric measurement of ICs as a means of quantifying proteins in sera; the in~' P. H. Lambert, F. J. Dixon, R. H. Zubler, V. Agnello, C. Cambiaso, P. Casali, J. Clarke, J. S. Cowdery, F. C. McDuffie, F. C. Hay, I. C. M. MacLennan, P. Masson, H. J. MiillerEberhard, K. Penttinen, M. Smith, G. Tappeiner, A. N. Theofilopoulos, and P. Verroust, J. Clin. Lab. lmmunol. 1, 1 (1978). ~2 R. L. Libby, J. l m m u n o l . 34, 71 (1938a). ':~ R. L. Libby, J. lmmunol. 34, 269 (1938b).

630

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He-Ne Laser

IMMUNE COMPLEXES

T

Diaphragms

Cuvette Light'trap Lens system

[42]

Photodiode

FIG. 1. Optical system of the Behring Institute laser nephelometer. terested reader should see Sternberg, 14 Deaton et al., 15 Conrad et al., 16 and Ritchie. 1z Principle of the Method N e p h e l o m e t r y is based on the ability of ICs to induce in a liquid matrix a turbidity that causes scattering o f an incident light beam. The scattered light can be focused by an optical lens system and measured with a photomultiplier. Recently developed nephelometers use a helium-neon laser beam, which has the advantage o f being a monochromatic beam of constant intensity. Currently, three models are available, each offering slight but notable differences in operating principles and outfit. Behring Institute Laser N e p h e l o m e t e r (Behring Institute, Marburg, Federal Republic of Germany). Figure 1 shows the optical system o f the Behring nephelometer. Because o f the intensity of the laser beam used, a photomultiplier could be omitted. Forward scattered light is measured and displayed on a digital voltmeter. Special cuvettes with low background reading are recommended. All measurements are performed after the antigen-antibody reaction has reached equilibrium, e.g., when specific antibody is added to a known protein to quantify its concentration. This is not relevant for the detection of pre-formed ICs in, e.g., native sera, but it may b e c o m e important when the antigen moiety o f the ICs is to be measured by adding appropriate antibody. B e c k m a n N e p h e l o m e t e r (Beckman Instruments Inc., Fullerton, California). This instrument (Fig. 2) offers two ways o f measuring ICs. First, conventional light scattering may be quantified with ICs at equilibrium, as ~4j. C. Sternberg, Clin. Chem. 23, 1456 (1977). ~5C. D. Deaton, K. W. Maxwell, R. S. Smith, and R. L. Creveling, Clin. Chem. 22, 1465 (1976). ~sA. Conrad, J. Schiirmann, F. H. Kreutz, and A. Sieber, J. Clin. Chem. Clin. Biochem. 16, 299 (1978). ,7 R. F. Ritchie, ed., "Automated Immunoanalysis," Vols. 1 and 2. Dekker, New York, 1978.

[42]

N E P H E L O M E T R I C Q U A N T I F I C A T I O N OF I C s

631

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FtG. 2. Optical system of the Beckman nephelometer. with the other two instruments. Second, the kinetics of generation of ICs may be measured; at the time of addition of the antibody to the antigen-containing solution, a fluorescence signal (the fluorescent dye is in admixture with the antibody) marks time zero of the reaction. The turbidity of the reaction solution at this time is regarded as the reference point for the antigen-antibody reaction, allowing the nephelometer to always measure without blanks and--more importantly--to quantify ICs only during the increasing portion of the Heidelberger curve. Although not valid for the detection of ICs in native sera, it will be advantageous when proteins are to be quantified by their antigen-antibody reaction or when the antigen from preexisting ICs is to be detected and quantified. Any highly purified antiserum may be used. The samples are stirred throughout the reaction and measurement. As with the other two nephelometers, the procedure is quickly performed and fully automated. Instead of a laser beam, a tungsten/iodine light source is used, because this lamp has been found to provide better signal-to-noise ratios. TM Hyland Laser Nephelorneter PDQ (Hyland Division, Travenol Laboratories, Inc., Costa Mesa, California). This nephelometer also reads sam-

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pies containing ICs at equilibrium. As illustrated in Fig. 3, a differential circuit automatically subtracts blank values of buffer, antibody, and test sample, which have to be premeasured. Any antiserum may be used. Readings are expressed on a digital meter as percentage relative light scattering because the scale is adjusted (or rather extended) so that the highest standard sample reads 100%. This may increase sensitivity, but in the case of measuring preexisting ICs in native samples, it may lead to false positive results. Moreover, standards for ICs are still largely circumstantial because commonly used heat-aggregated immunoglobulin preparations have not proved satisfactorily reproducible and work on a

[42]

NEPHELOMETRIC QUANTIFICATION

OF ICs

633

standardized WHO antigen-antibody complex preparation is still in progress. Any expression of IC levels as equivalents of heat-aggregated IgG or IC standards has not been accepted unequivocally. Nephelometric Quantification of Immune Complexes Present in Native Sera In principle, any nephelometer should be applicable to the detection of ICs preexisting in sera, although the advantages and disadvantages described might favor the use of a specific nephelometer for this purpose. Work is underway in our laboratory to compare the three nephelometers. All results reported here have been obtained using the Behring Institute nephelometer. Material

NaC1, 0.15 M, pH 7.4 Human IgG (Cohn Fraction II) (Sigma Chemicals) Polyethylene glycol (MW 6000; dissolved in 0.15 M NaCI, pH 7.4) (Merck Chemicals, Darmstadt, Federal Republic of Germany) Protamine-HC1 "1000" (Hoffmann-La Roche, Grenzach, Federal Republic of Germany) Normal human serum (pooled sera from healthy blood donors) Cuvettes of low background reading (Behring Institute) Methods Nephelometry. Sera diluted 1/10 with 0.15 M NaCI and samples pretreated as described later were tested in the laser nephelometer. Light scattering was expressed as volts displayed on a digital meter ranging from 0 to 19.99 V. Two-tenths milliliter of sera or IC-containing samples were transferred into cuvettes of low background reading and measured for turbidity by light scattering of the laser beam. Measuring time was arbitrarily chosen as the time when the display on the digital voltmeter became stable. All tests were performed in duplicate. Because protamine has been found to increase the sensitivity of the method (A. Sieber, personal communication, 1979), samples were simultaneously tested after the addition of 25 units of protamine. Ten microliters of serum were mixed with 25/zl of protamine "1000" and 975/xl of 0.15 M NaC1, and 0.2 ml of the mixture was transferred into the measuring cuvettes. Calibration Curves. Although not essential for expression of results, calibration curves give insight into the sensitivity of a method used, so that they should be performed before routinely using nephelometry.

634

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201816-

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FIG. 4. Calibration curve. Relationship between nephelometric readings (volts _+ SE) and various amounts of heat-aggregated IgG (AIgG) in normal human serum with ( e O) or without (0 - - - - - 0) the addition of protamine. To 0.1 ml of serially diluted algG (highest concentration 3 mg/ml) 0.01 ml of normal human serum and, in one series, 0.025 ml of protamine were added. The mixture was made up to 1.0 ml with physiological saline and 0.2 ml were measured for light scattering. The upper limit of light scattering by normal human serum was 0.92 V. H e a t - a g g r e g a t e d I g G (63 ° for 20 m i n , f o l l o w e d b y a c e n t r i f u g a t i o n at 550 g for 15 m i n to r e m o v e i n s o l u b l e aggregates) is r e c o g n i z e d as b e i n g similar to I C s in t e r m s o f s o m e b i o l o g i c a l a n d c o n f o r m a t i o n a l f e a t u r e s . Figure 4 illustrates a typical calibration curve using various a m o u n t s of I g G a g g r e g a t e s in n o r m a l h u m a n s e r u m w i t h or w i t h o u t the a d d i t i o n o f p r o t a m i n e . W i t h a l o w e r limit o f d e t e c t i o n o f 7 5 / z g / m l I g G aggregates, the s e n s i t i v i t y o f n e p h e l o m e t r y c o m p a r e s well w i t h that o f o t h e r tests for m e a s u r i n g i m m u n e c o m p l e x e s , e.g., the C l q b i n d i n g a s s a y is a n d the '~ R. H. Zubler, G. Lange, P. H. Lambert, and P. A. Miescher, J. lmmunol. 116, 232 (1976).

[42]

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OF

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~lcjG (mcj/ml) FiG. 5. Calibration curve. Relationship between the percentage of 1125IICIq binding activity (percentage [~2~l]Clq BA ± SE) and various amounts of heat-aggregated IgG (,klgG) in normal human serum with (e - - - - - e) or without (e e) the addition of heparin. To 0.05 ml of serially diluted 5lgG, 0.05 ml of normal human serum (with or without 25 IU/ml heparin) and 0.05 ml of 0.2 M disodium ethylenediaminetetraacetic acid were added, and after incubation for 30 rain at 37°, 0.05 ml of [125I]Clq and 1.0 ml of 3c; polyethylene glycol in barbitone buffer were added. (For details see Zubler et al.l~) polyethylene glycol precipitation test ~9 (Figs. 5 and 6). After addition of protamine, light scattering is enhanced geometrically, i.e., samples with small amounts o f l g G aggregates display a lesser increase in turbidity than those containing large amounts of aggregates. In this context, it is interesting to note that sensitivity of the C l q binding assay can be augmented in a similar way by adding heparin to IC-containing samples (Fig. 5). Using samples containing ICs generated in vitro, similar calibration curves are obtained (Fig. 7). N o r m a l Range. The normal range should be assessed by using individual or pooled sera from fasting healthy blood donors. This appears to be of importance because lipids will cause increased light scattering of the samples. In addition, the normal serum pool used in this laboratory has been shown to contain no ICs when assayed in the C l q binding test. With our studies, nephelometric readings of 97 normal human blood ~9 I. Riha, V. H a s k o v a , J. Kaslik, M. Maierova, and J. Stransky, Mol. Immttm)l. 16, 489 (1979).

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tilgG (mg/ml) F[o. 6. Calibration curve. Relationship between optical density (OD -+ SE) and various amounts of heat-aggregated IgG (AIgG) in the polyethylene glycol precipitation test performed according to Riha et al.19 donor sera gave a mean value (-+ SD) o f 0.63 - 0.22 V without and 1.81 _ 1.76 V with protamine. Mean values (-4-- SE) for pooled normal serum tested on 12 consecutive days were 0.8 --- 0.06 V without and 1.24 --+ 0.16 V with the addition o f protamine, respectively. The variation coefficient calculated from eight independent tests measuring aggregated IgG was 3.9% without and 5.7% with the addition of protamine. Substances Interfering with Nephelometric Determination of Immune Complexes. Erroneous results may arise from two main sources when using nephelometry for measuring ICs in serum samples. First, as with most assays for quantification o f ICs, aggregated immunoglobulins give rise to false positive or increased nephelometric readings. Therefore, handling and storage of serum should be adapted so as to minimize generation o f aggregates. Second, lipids are known to increase light scattering. Despite collection of blood from fasting individuals, hyperlipemia is present in some

[42]

NEPHELOMETRIC QUANTIFICATION OF I C s

637

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FIG. 7. Nephelometric readings (volts -+ SE) for various amounts of ICs prepared in vitro (rat anti-BSA antiserum) compared with those after addition of bovine serum albumin (BSA) to normal rat serum (NRS). The shaded area represents the normal range + SE for NRS. Equal volumes of B S A in 0.15 M NaCI and rat anti-BSA antiserum (obtained from inbred D B A rats immunized by four intraperitoneal injections o f 100/xg B S A in admixture with 0.1 ml Freund's complete adjuvant) were incubated at 37° for 30 min before being diluted appropriately with physiological saline. Reproduced from H6ffken et al.2° by permission of the publisher.

samples, thus warranting attempts to distinguish between samples containing ICs and lipids. 2° As shown in Fig. 8, treatment with 1,1,2-trichlorofluoroethane (a substance causing accumulation of lipids at an interface) (Serva Chemicals, Heidelberg, Federal Republic of Germany) effectively cleared serum enriched with lipids. Likewise, it caused precipitation of ICs. Ultrafiltration of the samples using Millipore filters (0.45 tzm pore size) also cleared both lipids and IC-containing sera. By contrast, treatment of sera with 3% polyethylene glycol reproducibly precipitated ICs but caused an increase in the light scattering of sera containing lipids. One possible explanation for this phenomenon is that polyethylene glycol cross-links lipids or/3-1ipoproteins 21 without altering their solubility. Alternatively, polyethylene glycol might alter the surface tension of lipid 2. K. Hi~ffken, U. Bestek, U. Sperber, and C. G. Schmidt, J. Immunol. Methods 29, 237 (1979). 2~ p. H. Iverius and T. C. Laurent, Biochim. Biophys. Acta 133, 371 (1967).

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FIG. 8. Distinction between immune complexes (ICs) and lipids (lrNHS, lipid-rich normal human serum) in sera by pretreatment with polyethylene glycol (PEG), 1,1,2trichlorotrifluoroethane (TTE), and ultrafiltration (UF). Closed columns represent the nephelometric readings (volts ± SE) for untreated and hatched columns for treated serum samples, respectively. For comparison, normal rat serum (NRS) was treated identically. The shaded area represents the normal range + SE for NHS. Samples containing BSA-antiBSA ICs generated in vitro (see Fig. 7) or lipid-enriched NHS were treated with 3% final concentration of PEG (see Material) and centrifuged at 1500g for 20 min. Alternatively, one volume of IC-containing serum and lipid-enriched NHS, respectively, was added to 1.5 volumes of TTE. After shaking on a vortex mixer for 1 min, the samples were diluted to 1/10 of the original serum volume with 0.15 M NaCI and centrifuged at 2000 g for 15 min. Ultrafiltration was performed by passing samples through 0.45-~m Millipore filters before dilution to 1/10 with 0.15 M NaCI. Lipid-enriched NHS was prepared by centrifugation of NHS at 100,000g for 60 min and admixture of the lipid layer on top of this sample with a small amount of NHS. Reproduced from HiSffkenet al. 2° by permission of the publisher.

g l o b u l e s , so t h a t s m a l l e r b u t m o r e lipid p a r t i c l e s o c c u r , w h i c h m a y s c a t t e r the l a s e r b e a m . W h i l e the m e c h a n i s m o f p o l y e t h y l e n e glycol a c t i o n is as y e t u n r e s o l v e d , it w o u l d a p p e a r that t r e a t m e n t o f p o s i t i v e sera w i t h p o l y e t h y l e n e glycol allows the d i f f e r e n t i a t i o n b e t w e e n sera c o n t a i n i n g e i t h e r lipids o r ICs. It is p e r t i n e n t to n o t e , h o w e v e r , that this t r e a t m e n t m a y l e a v e u n d e t e c t e d I C s that are p r e s e n t in lipid-rich sera. N e v e r t h e l e s s , the risk o f e l i m i n a t i n g s a m p l e s as b e i n g n o n e v a l u a b l e a p p e a r s to be outw e i g h e d b y the a d v a n t a g e o f e x c l u d i n g false p o s i t i v e sera.

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[42]

NEPHELOMETRIC QUANTIFICATION OF ICs

641

Correlation of Nephelometry with Other Methods for Quantification of Serum-Borne Immune Complexes With artificial systems using beat-aggregated IgG or ICs generated in nephelometric quantification of ICs appears to correlate well with other tests, e.g., C l q binding assay, polyethylene glycol precipitation test (Figs. 4-6). This could be shown for a variety of tests when reference preparations of serum samples containing heat-aggregated human y-globulins were used, 11 whereas the same collaborative study indicated that with pathological sera different methods appear to detect different types of ICs. When 203 serum samples from patients with benign or malignant breast tumors were tested for the presence of immune complexes by nephelometry, the Clq binding assay, and polyethylene glycol precipitation (Fig. 9), we came to a similar conclusion: apparently each assay measures distinct types of immune complexes. This is also in agreement with the findings of Whitsed e t al., 2.~ who compared nephelometric detection of ICs using monoclonal rheumatoid factor with measurement by C lq binding. It should be noted, however, that the data of our ongoing study have not yet been evaluated as to clinical status, benign or malignant tumors, good or poor prognosis, and accompanying diseases. Therefore, at present, only the lack of correlation between the three assays can be demonstrated, but no conclusions can be drawn as to which test most reliably contributes to the determination of diagnosis and clinical course of, e.g., malignant disease. Interestingly, as did heparin with the Clq binding assay, protamine gave rise to an increase in light scattering of some serum samples, while leaving normal human serum unchanged (Fig. 10). This is in accordance with the finding of Baldwin e t al., .2..~who showed a clear distinction between the Clq binding activity of normal and pathological samples following the addition of heparin. With our study, it remains to be shown whether the addition of heparin in the Clq binding assay or protamine in the nephelometric test allows a distinction between normal and pathological sera. Although concordant when testing aggregated human immunoglobulin or normal human serum, the two assays did not correlate with each other either by the original or by the modified method when testing the pathological sera (r = -0.05; p = 0.5 for the original methods; r = -0.02; p = 0.7 after the addition of heparin and protamine, respectively) (Fig. 9a and d). Considering the admixture of serum samples with protamine for the nephelometric assay, a statistically significant correlation between original and modified test vitro,

~z H. Whitsed, W. H. McCarthy, and P. Hersey, .I. l m m u n o l . Methods 29, 311 (1979). 23 R. W. Baldwin, V. S. Byers, and R. A. Robins, Behrim,, Inst. Mitt. 64, 63 (1979).

642

IMMUNE COMPLEXES

[42]

(r = 0.34;p = 0.0001) indicates that the addition of protamine to samples containing ICs enhances the sensitivity of the method. Comments For most assays presently used to quantify ICs in body fluids, numerous reagents are required (e.g., purified radiolabeled Clq, purified conglutinin, viable Raji cells), whereas the nephelometric assay simply requires saline for diluting serum samples. Therefore, a number of variable factors that may lead to erroneous results can be eliminated. As with most other methods, nephelometry is adversely affected by the presence of immunoglobulin or other aggregates in serum samples, so that storage conditions should be standardized. Ideally, aliquoted serum should be kept frozen at - 7 0 ° and only thawed once before testing. One of the most frequent causes of false positive values in nephelometry is the increased turbidity of lipid-containing sera. One approach to differentiating between sera containing either ICs or lipids has been described using precipitation with polyethylene glycol. Alternatively, serum lipids have successfully been removed by extraction with heptane-butanol, 24 although this method appears to be time-consuming with respect to a routine screening test for detecting soluble ICs. Another approach 25 using precipitation of ICs with ammonium sulfate did not work in our hands. As to the expression of results in volts (Behring nephelometer), scatter units (Beckman nephelometer), and percentage relative light scattering (Hyland nephelometer), we consider this as being appropriate for the present. The attempt to refer nephelometric readings to a standard calibration curve of complexes of tetanol and anti-tetanol antiserum generated in v i t r o 2~ did not work reproducibly in our hands. Until standardized reference preparations are available, we do not see an advantage in expressing amounts of immune complexes measured as equivalents of reagents such as aggregated IgG or immune complexes generated in vitro, which can hardly be prepared in a reproducible way. The simplicity of the nephelometric assay allows large numbers of samples to be tested within a short time, which is one of the prerequisites of any screening test. All methods presently used for measuring immune complexes are antigen nonspecific, with one exception reported by Theofilopoulos e t al. lo.,,~ 24G. Schultz-Ellison,C. Charland, J. Driscoll, and W. Thayer,J. Immunol. Methods 31, 31 (1979). z5K. Helmke, M. Oppermann, C. Schatz, C. P. Sodomann, and J. Teuber,Behring Inst. Mitt. 64, 96 (1979). 24A. N. Theofilopoulos,R. A. Eisenberg, and F. J. Dixon,J. Clin. Invest. 61, 1570(1978).

[49-[

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They induced antibodies against tumor cell lines and observed a higher uptake of these antisera by Raji cell-bound ICs containing the relevant antigen, when compared with the uptake of the antiserum by ICs derived from patients with immunologically distinct tumors. Moreover, they were able to induce antibodies directed against the antigen moiety of ICs in IgG-tolerant rabbits. From these studies and from investigations on cell hybridization,'r it appears possible that appropriate antisera against the antigen moiety of ICs may become available. Preliminary results from this laboratory suggest that the nephelometric assay may have the potential of being an antigen-specific method for quantification of ICs. Admixture of additional antibody to a solution containing ICs in antibody excess led to an increase in the turbidity of the sample. The mechanism by which this was induced is not yet understood, and it appears to contradict the conception of lattice network formation by ICs in antibody excess. Nevertheless, it is pertinent to note in this context that an increase in turbidity after the addition of appropriate antisera to any IC-containing serum does not necessarily imply the presence of, e.g., tumor-specific ICs because it is not known a priori whether these ICs are in antibody excess. There still remains the possibility that free antigen in addition to epiphenomenal ICs may give rise to an increase in turbidity after admixture of the samples with antiserum. One way to increase the sensitivity of nephelometry has been described using protamine. Alternatively, polymeric buffers, such as polyethylene glycol, have been shown to enhance aggregation of immune complexes '~ and are now being used for nephelometric quantification of serum proteins. However, the problem already discussed, that polyethylene glycol also augments the turbidity of samples containing lipids, should be borne in mind. Considering again the main requirements of an assay for soluble ICs, the following conclusions may be drawn for the nephelometric test: 1. Immune complexes are readily detected and quantified in native sera with physiological saline being the sole reagent needed. 2. There is some suggestive evidence that characterization of ICs and identification of their antigen moieties may become possible. 3. Extensive clinical studies are required to demonstrate whether nephelometry may be a useful means for monitoring course and treatment of IC diseases. 4. Although not superior to other methods with respect to sensitivity

'-,7 G. Krhler and C. Milstein, Nature (London) 256, 495 (1975). z~ G. Virella, W. A. Hipp, J. F. John, Jr., B. Kahaleh, M. Ford, and H. H. Fudenberg, Int. Arch. Allergy Appl. hnm,nol. 58, 402 (1979).

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and specificity, the simplicity of nephelometry may favor its use as a screening test. Acknowledgments The excellent technical assistance of Mrs. U. Sperber and Mrs. U. Steih is gratefully acknowledged. Mr. B. Jacob is thanked for statistical analysis of data. This work was supported by grants from the Landesamut fiir Forschung, Ministerium fiJr Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Diisseldorf, Federal Republic of Germany.

[43] I s o l a t i o n o f S o l u b l e I m m u n e C o m p l e x e s f r o m H u m a n Serum: Combined Use of Polyethylene Glycol Precipitation, Gel Filtration, and Affinity Chromatography

on P r o t e i n

A- Sepharose

By GABRIEL VIRELLA,J. MICHAEL K I L P A T R I C K ,

FRANCOISE CHENAIS,

and H. HUGH FUDENBERG Introduction Several techniques have been introduced in recent years for the isolation of soluble immune complexes (ICs). The first technique reported used insolubilized Clq as a substrate for IC binding. 1 This technique has two drawbacks: the need for relatively large amounts of C lq (a protein that is difficult to purify) and rapid loss of adsorbing capacity with repeated use. Concanavalin A, a lectin that binds specifically to molecules containing a-o-mannopyranoside and a-o-glucopyranoside, has been used to isolate ICs containing antigens with those residues. 2 Raji cells have also been used for isolation of soluble ICs, by incubation of IC-containing samples with the cells and elution of the cell-bound material with acid buffer2 The most popular approach to isolation of ICs has been the use of immobilized staphylococcal protein A as a substrate. 4 Our initial protocol involved a combination of (1) precipitation of ICs with either polyethlene glycol (PEG) or ammonium sulfate; (2) gel filtration in AcA 34; and (3) S. E. Svehag and D. Burger, Acta Pathol, Microbiol. Scand., Sect. C 84, 45 (1976). 2 R. Heimer and G. Klein, Scand. J. lmmunol. 7, 315 (1978). 3 A. N. Theofilopoulos, R. A. Eisenberg, and F. J. Dixon, J. Clin. Invest. 61, 1570 (1978). 4 F. Chenais, G. Virella, C. C. Patrick, and H. H. Fudenberg, J. lmmunol. Methods 18, 133

(1977).

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