Journal o f Immunological Methods, 18 (1977) 33--44
© Elsevier/North-Holland Biomedical Press
P A R T I C L E C O U N T I N G IMMUNOASSAY (PACIA). I. A G E N E R A L M E T H O D F O R T H E D E T E R M I N A T I O N O F A N T I B O D I E S , ANTIGENS, AND HAPTENS
C.L. CAMBIASO, A.E. LEEK, F. DE STEENWINKEL, J. BILLEN and P.L. MASSON Unit o f Experimental Medicine, International Institute o f Cellular and Molecular Pathology, and Universitd Catholique de Louvain, 74, avenue Hippocrate UCL-ICP 7430 B-1200 Brussels, Belgium
(Received 23 February 1977, accepted 14 May 1977)
By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8p) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g. human rheumatoid factor or rabbit anti-bovine serum albumin antibodies. Macromolecules with multiple antigenic determinants agglutinate particles carrying specific antibodies. This system has been applied for determining HPL and ~t-fetoprotein with a threshold of sensitivity of about 10 pg/1. However the agglutination was decreased by serum factors which led to a 10-fold loss of sensitivity. The interference of rheumatoid factor which agglutinated the particles coated with rabbit or goat immunoglobulins could be avoided by reduction of the serum to be analyzed with 5 mM dithiothreitol for 5 min. Haptens, i.e. DNP-lysine and T4, were determined by their inhibitory activities toward their specific antibodies, the agglutinator being a hapten-macromolecule conjugate or the antibodies themselves.
INTRODUCTION F o r m a n y years, a n t i g e n - - a n t i b o d y r e a c t i o n s have been studied b y agglut i n a t i o n t e c h n i q u e s . T h e particles w h i c h are m o s t f r e q u e n t l y used f o r this p u r p o s e are red cells or p o l y s t y r e n e beads, generally called ' l a t e x ' particles. Slide tests based o n latex agglutination have been d e v e l o p e d f o r d e t e r m i n i n g a n t i b o d i e s , e.g. r h e u m a t o i d f a c t o r (Singer and Plotz, 1 9 5 6 ) or a n t i - D N A antibodies (Christian et al., 1 9 5 8 ) , and antigens such as f i b r i n o g e n d e g r a d a t i o n p r o d u c t s (Allington, 1 9 7 1 ) , h u m a n c h o r i o n i c g o n a d o t r o p h i n ( R o b b i n s et al., 1 9 6 2 ; H o r w i t z et al., 1 9 7 1 ) , viral (Carvajal et al., 1 9 7 6 ) or bacterial (Fleck a n d E v e n c h i k , 1 9 6 2 ) antigens, and various plasma p r o t e i n s (Singer et al., 1 9 5 7 ; S c h m i d t et al., 1975). T h e d r a w b a c k o f these tests is the d i f f i c u l t y o f d e t e r m i n i n g the end p o i n t precisely b y eye. I n s t r u m e n t a l reading based o n t u r b i d i m e t r y (De~elid et al., 1971} or light scattering (Hoign6 et al., 1 9 5 9 ; B l u m e and G r e e n b e r g , 1 9 7 5 ;
34 Cohen and Benedek, 1975) has been proposed but is used only on a small scale. In the present work, we will show that a device designed for counting blood cells is particularly suitable for the study of the agglutination process of latex, and can become a practical and sensitive tool for determining not only antibodies and antigens but also haptens. The principle is based on the reduction of the total number of particles when they are agglutinated. MATERIALS AND METHODS
An tisera Goat antiserum against h u m a n placental lactogen (HPL) was supplied by ILS Ltd, L o n d o n , U.K. (Letchworth et al., 1971), and goat anti-al-fetoprotein serum by Mrs. M.J. Kitau of St. Bartholomew's Hospital, London, U.K. (Leek et al., 1975). Anti-DNP antibodies were purified as previously described (Cambiaso et al., 1974) by passing the goat antiserum through a column of DNP-lysine coupled to Sepharose 4B, the antibodies being eluted by 5 M guanidinium hydrochloride. Antisera against L-thyroxine (T4) were raised in rabbits by immunization with T4 (Fluka AG, Buchs, Switzerland) coupled to bovine serum albumin (BSA) by means of 1-ethyl-3(3-dimethylamino-propyl)-carbodiimide (Sigma, St. Louis, MO, U.S.A.) as described by Gharib et al. (1971). Serum from each bleed was tested for its ability to bind 12SI-labelled T4, and the best were pooled. This pool was used as the source of anti-BSA, and of anti-T4 after passage through an immunoadsorbent column of BSA-Sepharose (Cambiaso et al., 1975). The immunoglobulins (Ig) of antisera were prepared as described by Schultze and Heremans (1966) by precipitation with Rivanol (6,9-diaminoacridine lactate, Federa, Brussels, Belgium). The Ig concentration was estimated by measuring optical absorbance at /E 1% 280 nm ~ 2 8 0 n m = 13.4). Sera containing rheumatoid factor were submitted to reduction for 5 min at room temperature by 5 mM dithiothreitol (DTT, Calbiochem, San Diego, CA, U.S.A.).
Hapten-carrier conjugates and antigens A conjugate of T4 and Dextran T70 (Pharmacia Fine Chemicals, Uppsala, Sweden) was prepared by the cyanogen bromide technique of Porath et al. (1973). Five ml of 0.2 M sodium carbonate buffer, pH 11, containing 100 mg Dextran T70 was stirred with 22 mg CNBr, and 10 ml of carbonate buffer containing 30 mg disodium T4 was added. The reaction was allowed to proceed for 90 min at room temperature, then the solution was dialysed overnight at 4°C against 2 1 of 0.9% NaC1, to remove unreacted T4. Human serum albumin (HSA) and bovine ~/-globulins (BGG) were dinitro-
35 p h e n y l a t e d by means of 2,4-dinitrobenzene sulfonate (Eisen, 1964). The HSA conjugate contained 48 DNP residues, and that of BGG, 23 residues. HPL was purchased f r om ILS Ltd ( L o n d o n , U.K.).
Particles P o ly s ty r en e latex particles, 0.794 p diameter, were m a n u f a c t u r e d by Dow Chemical Co., Indianapolis, IN, U.S.A., and purchased as a 10% suspension either from Dow or from Serva, Heidelberg, G.F.R. All handling and use of latex particles was carried out in glycine/NaC1 buffer, pH 9.0. A stock solution, 1.0 M in glycine, 1.7 M in NaC1 and 76 mM in NaN3 was made up, and was diluted 10 times for use as an assay buffer (GBS). Uncoated latex particles can be unstable in solutions of such a high ionic strength, and so for the steps of the coating procedure, the stock solution was diluted 50 times (dGBS). This coating procedure was basically the same for all the proteins used, and in all cases the first step was to wash the latex particles in 20 volumes of dGBS to remove any residual surfactant from the medium. The washed latex was then incubated for 15 to 45 rain at r o o m t e m p e r a t u r e with occasional mixing in a dilute solution of the protein with which it was to be coated in dGBS, washed with this buffer, and finally resuspended in GBS usually containing 10 g/1 human or bovine serum albumin to stabilize the suspension. Even at low ionic strength, the addition of certain proteins, e.g. hum an and rabbit IgG, caused an agglutination of latex, through prot ei n--prot ei n interaction (Heimer and Abruzzo, 1972). To overcome this difficulty, bovine or h u man serum albumin was mixed with the protein to be adsorbed, to dilute it. To find the optimal p r o p o r t i o n of active to ballast proteins on the latex, aliquots o f latex were incubated with solutions of different amounts o f the two species.
Assay technique F o r the agglutination of latex particles and its inhibition, we have used a manual technique and also an a u t o m a t e d one. In the manual technique, small aliquots of the reagents are pipetted by hand into test tubes, and incubated at r o o m t e m p e r a t u r e on a vibrating mixer, at 10--50 Hz, with a vibration amplitude of 2--5 mm. After incubation, 5 ml of GBS is added to each tube, and an aliquot of this dilution is furt her diluted with the same b u f f er before passing the sample through the flow-cell of a Technicon AutoC o u n ter ( T a r r y t o w n , New York, U.S.A.) to c o u n t the n u m b e r of particles. The final dilution is such that a m a x i m u m of 4000 particles/sec pass through the flow-cell. R h e u m a t o i d factor, anti-BSA and HPL were all assayed by the a u t o m a t e d technique. F o r this, we used a manifold (fig. 1) incorporating an incubation coil agitated at 50 Hz with an amplitude of 1 mm, giving an incubation time o f 15 min before dilution and counting. The manifold was fed by a Tech-
I Upper Levee ..... I
VIBRATING MIXING COIL MIXING COIL
WASTE WASTE ~
BOFFE 20 042
BUFFER i AIR
PROPORTIONING PUMP . ~ ( ~ 20
Fig. 1. Diagram of the continuous flow system for the automated particle counting immunoassay.
n i c o n Sampler IV m o d i f i e d with an extra arm and pick-up needle to aspirate the latex preparation at the same time as the sample. The sampler was run at 2 0 / h with a sample/wash ratio o f 4 / 5 for r h e u m a t o i d factor, and at 30/h w i t h a sample/wash ratio of 1/1 for anti-BSA and HPL. RESULTS
The determination of antibodies The agglutinating activity o f serial dilutions o f ten r h e u m a t o i d sera on a 0.05% suspension of h u m a n IgG-coated latex was measured with the automated system. A typical e x a m p l e of such titration is s h o w n in fig. 2. In this case, a significant decrease in the n u m b e r of particles was recorded with a dilution of the r h e u m a t o i d serum of 1 / 5 1 2 0 , whereas the agglutination titre with the latex agglutination slide test was 1 / 3 2 0 . A p r o z o n e effect was observed with the 1 / 1 0 to 1 / 4 0 dilutions w h i c h caused less agglutination than the 1 / 8 0 and 1 / 1 6 0 dilutions. Hence, the practical range o f determina-
1 J 1
Fig. 2. A u t o c o u n t e r recording of the agglutination of h u m a n IgG-coated p o l y s t y r e n e particles by a r h e u m a t o i d serum at various dilutions. The two highest peaks on the right (buffer) correspond to the suspension of particles w i t h o u t agglutinating agent.
tion e x t e n d e d f r o m a dilution of 1/80 to 1/5120. R h e u m a t o i d sera were also f o u n d to agglutinate latex coated with goat IgG, but to a lesser extent. In genera/, to reach the same agglutination with goat IgG-coated latex, rheumatoid sera were ten times less diluted. We have tested the agglutinating activity o f six sero-negative r he um at oi d sera. F o u r displayed in the PACIA system clear agglutinating properties on hum an IgG-coated latex, b u t n o t on goat IgG-coated latex. One hundr ed sera from blood donors were analyzed at a dilution o f 1/5 in GBS, and seven were f o u n d to agglutinate latex. Decomp l e m e n t a t i o n o f these sera by heating did n o t decrease the agglutination.
38 After reduction with 5 mM DTT for 5 min, the rheumatoid sera completely lost their agglutinating abilities. A second experiment was carried out using latex coated with BSA, and dilutions of a rabbit antiserum in normal rabbit serum as agglutinator (fig. 3). Reduction with DTT failed to reduce the height of the peaks, indicating that only IgG antibodies were involved. A significant agglutination was detected at a 1/10,000 dilution, which has to be compared to 1/200 dilution representing the lower threshold for the slide agglutination test. The anti-BSA antibodies were purified by adsorption to BSA covalently linked to Sepharose, followed by elution with 3 M NaSCN. This purified preparation was chromatographed on Ultrogel AcA 2-2, and each fraction assayed for its ability to agglutinate BSA-coated latex. No reaction was seen with those fractions corresponding to the elution volume of IgM, which also contained no detectable protein; agglutinating activity was restricted to the region corresponding to IgG.
The determination of antigens The PACIA system was first applied to BGG-DNP23, using the automated procedure. The particles, which were coated with purified goat anti-DNP antibodies, were agglutinated by BGG-DNP23 dissolved in increasing concentrations (10 to 100 pg/1) in GBS containing 10 g/1 BSA (fig. 4). When the
Fig. 3. A g g l u t i n a t i o n in t h e P A C I A s y s t e m o f B S A - e o a t e d p a r t i c l e s w i t h v a r i o u s d i l u t i o n s of a rabbit anti-BSA antiserum.
T 0 N T
< w o_
aGG - DNP
Fig. 4. A g g l u t i n a t i o n curves in t h e P A C I A s y s t e m applied to d i n i t r o p h e n y l a t e d B G G used as a g g l u t i n a t o r , t h e particles b e i n g c o a t e d w i t h goat a n t i - D N P a n t i b o d i e s . T h e curves were o b t a i n e d w i t h B G G - D N P dissolved in GBS, fresh h u m a n s e r u m , or h u m a n s e r u m h e a t e d at 56°C for 30 rain. Fig. 5. A g g l u t i n a t i o n in t h e P A C I A s y s t e m of particles c o a t e d w i t h increasing a m o u n t s of goat a n t i - D N P a n t i b o d i e s by d i n i t r o p h e n y l a t e d BGG. One curve was o b t a i n e d w i t h BGGD N P dissolved in G B S (o), a n d t h e o t h e r in fresh h u m a n s e r u m (e). T h e abscissae corres p o n d t o t h e c o n c e n t r a t i o n s of a n t i - D N P a n t i b o d i e s used for c o a t i n g t h e particles.
experiment was repeated with BGG-DNP in fresh normal human serum, a marked reduction in the agglutination was observed (fig. 4). Since complem e n t factors could be responsible for this inhibition by reacting with the immunoglobulins coating the particles and making them unavailable to the antigen, the samples were heated at 56°C for 30 min. After decomplementation, the agglutination was only slightly improved (fig. 4). In order to improve the agglutinability of latex in serum, we have also studied the effect of the density of antibodies bound to the particles on the agglutination process in buffer or in serum (fig. 5). For this purpose the particles were incubated with solutions of anti-DNP antibodies containing from 50 to 1000 mg/1. When tested in buffer, the particles loaded with the highest amounts of antibodies were less agglutinated. In serum, a stronger agglutination was at first obtained when the density of antibodies on the latex was increased. However, the agglutinability of the latex prepared with more than 125 mg/1 antibodies was not improved and the preparation corresponding to 1000 mg/l gave less agglutination than the latex coated with fewer antibodies. The PACIA system was used manually for the determination of al-fetoprotein. The suspension of latex (25 pl) coated with goat anti-a r f e t o p r o t e i n immunoglobulins was incubated for 2 h with an equal volume of hepatoblastoma serum diluted in GBS containing 10 g BSA/1, then diluted and counted. The serum contained 3 g/1 a~-fetoprotein, as determined by latex
agglutination against dilutions of an amniotic fluid standardized by radioimmunoassay. Agglutination was observed at 30 pg/1 and increased up to 200 pg/1. Above this concentration, a clear-cut prozone phenomenon was observed with antigen excess (fig. 6). For HPL, we used the automated system. A series of dilutions of HPL in 1% human serum was tested for its ability to agglutinate latex coated with goat anti-HPL antibodies, and produced the standard curve shown in fig. 7. A significant agglutination was observed with 10 pg/1, while above 100 pg/1 the curve tended to flatten out.
The determination of haptens In an experimental system, the concentration of the hapten was measured by its inhibitory activity on the agglutination of hapten-coated latex by the specific antiserum. We have used DNP-lysine as a model, the latex being coated with HSA-DNP. Since the whole antiserum gave little specific agglutination, the IgM fraction was first separated by gel filtration on an Ultrogel AcA 22 column. An inhibition experiment with DNP-lysine and the specific IgM antibodies was performed using the manual procedure. Various dilutions (25 pl) of a DNP-lysine solution in GBS containing 10 g HSA/1 were incubated for 10 min with 25 pl of the IgM fraction of anti-DNP serum. This IgM fraction contained 450 mg protein per litre. The suspension of latex coated
I o LU I ,z"
a I - FETOPROTEIN
Fig. 6. A g g l u t i n a t i o n in the P A C I A s y s t e m o f particles c o a t e d with g o a t ~ l - f e t o p r o t e i n a n t i b o d i e s by a h e p a t o b l a s t o m a s e r u m diluted in GBS c o n t a i n i n g 10 g BSA/1. The s e r u m sample c o n t a i n e d 3 g/1 ~ l - f e t o p r o t e i n -
60 }:E ~9 tO 2:
x~ tO 0_ 20
Fig. 7. A g g l u t i n a t i o n in the P A C I A s y s t e m o f the particles c o a t e d w i t h goat anti-HPL a n t i b o d i e s by a series o f dilutions o f HPL in 1% h u m a n s e r u m in GBS. 100
6o FT C9
lr ~c 4 0
,( i,i It
20I o D N P - LYS
Fig. 8. D e t e r m i n a t i o n o f DNP-lysine in GBS, by t h e P A C I A s y s t e m . Increasing a m o u n t s o f DNP- lysine were a d d e d to goat IgM a n t i b o d i e s d i r e c t e d against DNP, and t h e residual agglutinating activity o f these a n t i b o d i e s was m e a s u r e d on particles c o a t e d w i t h dinitrop h e n y l a t e d HSA.
with HSA-DNP (25 pl) was then added for a further 15-min incubation before dilution and counting. A standard curve (fig. 8) ranging from 1 #g/1 to 64 pg/1 was obtained. In a second system, the latex was coated with antibody, and agglutinated by the hapten-conjugate in solution; the free hapten was determined by its ability to inhibit this agglutination. As a test hapten we have used T4, with a conjugate of T4 coupled to dextran (MW 70,000) as agglutinator, containing an average of 6.5 molecules of T4 per molecule of dextran. Samples of serum (12.5 pl) with various concentrations of T4 were mixed with an equal volume of a solution of 1.3 mM 8-anilino-l-naphthalene sulfonic acid in 75 mM sodium veronal buffer, pH 8.6, in order to dissociate T4 from serum proteins (Chopra et al., 1972), with 25/ll of latex coated with rabbit anti-T4 immunoglobulins, and with 2 5 / l l of dextran-T4 conjugate. After 30 min incubation, the latex suspension was diluted and counted. In the absence of free T4,300 pg/1 of dextran-T4 caused substantial agglutination. With increasing concentrations of T4, a standard curve for inhibition over the range of 10 to 300 pg/1 T4 in serum was obtained (fig. 9).
~J & 40~
Fig. 9. D e t e r m i n a t i o n o f T4 in h u m a n s e r u m b y t h e P A C I A s y s t e m . T h e s e r u m samples c o n t a i n i n g increasing c o n c e n t r a t i o n s o f T4 were t r e a t e d w i t h o n e v o l u m e o f 1.3 M 8a n i l i n o - l - n a p h t h a l e n e s u l f o n i e acid in '/5 m M s o d i u m veronal b u f f e r , pH 8.6, a n d m i x e d w i t h T 4 - D e x t r a n c o n j u g a t e a n d particles c o a t e d w i t h r a b b i t anti-T4 i m m u n o g l o b u l i n s .
43 DISCUSSION In the present paper, we have shown that by counting latex particles with a device designed for counting blood cells, it was possible to measure agglutination accurately and, hence, to develop a new immunoassay of general applicability. The versatility of the system has been illustrated by its use for the determination of antibodies such as human IgM rheumatoid factor and rabbit anti-BSA IgG, antigens such as HPL and a l-fetoprotein, or haptens such as DNP-lysine and T4. Counting the free particles rather than detecting the agglutinates by eye increased the sensitivity by a factor of 20, as indicated by the test performed on rheumatoid sera. C l q can agglutinate IgG-coated latex (Ewald and Schubart, 1966) but n o t at the ionic strength and pH of the buffer system used (Sobel et al., 1975). The heat-resistant agglutinating activity of some sera from apparently healthy blood donors was presumably due to the presence of low levels of rheumatoid factor. Interference by rheumatoid factor can easily be avoided by reduction with DTT. IgG antibodies and sero-negative rheumatoid sera were capable of agglutinating latex in the PACIA system, but the activity of the rheumatoid sera appeared only with human IgG-coated latex. This suggests that IgG rheumatoid factor is not liable to interfere in the immunoassay performed with goat antibody-coated latex. When applied to antigens dissolved in buffer, the threshold of sensitivity of the PACIA system reached about 10 pg/1. However, serum contains factors inhibiting agglutination. One of them is thermolabile and might correspond to complement factors, but simple heating did not suffice to suppress the interference by serum. By increasing the density of antibodies of the particles their agglutinability is improved but remains still lower than in buffer. Since the interference of serum is not identical for all sera, it is necessary to neutralize this effect by diluting all samples at least 10 times, which results in a practical sensitivity of about 100 pg/1 for the determination of serum proteins. Such a sensitivity makes the PACIA system suitable for the immunoassay of many plasma proteins, including IgE. Happens, even in the presence of serum concerning, 10 ttg/1 of T4 has been detected. Such a sensitivity opens a large field of application for the determination of many physiological haptens and drugs, such as digoxin, morphine, d i p h e n y l h y d a n t o i n or gentamycin. Except for determining haptens, the PACIA system has been completely automated using a continuous flow system performing 20 or 30 assays per hour. A u t o m a t i o n for hapten assay is in progress and will later be described in detail. Extensive studies on precision, accuracy and correlations with other techniques are now being performed for the determination of HPL, IgE, a~-fetoprotein, digoxin and T4. From preliminary results, we can report that the coefficient of variation of 10 repeated determinations of HPL in serum was
44 less t h a n 2%. A s w e h a v e s e e n , a p o s s i b l e c a u s e o f e r r o r is t h e p r e s e n c e o f rheumatoid factor which may cause the agglutination of latex coated with goat or rabbit immunoglobulins. However, this difficulty can be easily overcome by treating the serum samples with 0.005 M DTT for 5 min. In conclusion, preliminary experiments performed with the PACIA system suggest that this technique compares well with radioimmunoassay, especially in t e r m s o f t i m e a n d f a c i l i t y . P A C I A r e q u i r e s r e l a t i v e l y s m a l l a m o u n t s o f a n t i b o d i e s a n d , in t h e c a s e o f p r o t e i n s , n o p u r i f i e d a n t i g e n is n e e d e d . REFERENCES Allington, M.J., 1971, Scand. J. Haematol. Suppl. 13, 115. Blume, P. and L.J. Greenberg, 1975, Clin. Chem. 21, 1234. Cambiaso, C.L., A. Goffinet, J.-P. Vaerman and J.F. Heremans, 1975, Immunochemistry 12, 273. Cambiaso, C.L., H.A. Riccomi, P.L. Masson and J.F. Heremans, 1974, J. Immunol. Methods 5,293. Carvajal, C., J. Di Bella and M. Stinger, 1976, Am. J. Clin. Pathol. 65, 547. Chopra, I.J., 1972, J. Clin. Endocrinol. Metab. 34,938. Christian, C.L., R.M. Bryan and D.L. Larson, 1958, Proc. Soc. Exp. Biol. Med. 98, 820. Cohen, R.J. and G.B. Benedek, 1975, Immunochemistry 12,349. De~elid, G., N. De~elid, N. Muid and B. Pende, 1971, Eur. J. Biochem. 20, 553. Eisen, H.N., 1964, in: Methods in Medical Research, vol. 10, ed. H.N. Eisen (Year Book Medical Publishers, Inc., Chicago) p. 122. Ewald, R.W. and A.F. Schubart, 1966, J. Immunol. 97, 100. Fleck, L. and Z. Evenchik, 1962, Nature 194, 548. Gharib, H., R.J. Ryan, W.E. Mayberry and T. Hockert, 1971, J. Clin. Endocrinol. Metab. 33, 509. Helmet, R. arid J.L. Abruzzo, 1972, Immunochemistry 9 , 9 2 1 . Hoign6, R., M. Jaeger and R. Gautier-Hunter, 1959, Acta Allergol. XIII, 364. Horwitz, C.A., H. Polesky, P. Odenbrett, M. Gronli, A. Horowitz, R. Diamond and P.C.J. Ward, 1971, Am. J. Obstet. Gynecol. 111, 808. Leek, A.E., C.T. Ruoss, M.J. Kitau and T. Chard, 1975, Brit. J. Obstet. Gynaecol. 82,669. Letchworth, A.T., R.J. Boardman, C. Bristow, J. Landon and T. Chard, 1971, J. Obstet. Gynaecol. Brit. Common. 78, 542. Porath, J., K. Aspberg, H. Drevin and R. Ax6n, 1973, J. Chromatogr. 86, 53. Robbins, J.L., G.A. Hill, B.N. Carle, J.H. Carlquist and C. Marcus, 1962, Proc. Soc. Exp. Biol. Med. 109,321. Schmidt, W.A.K., M. Klein and H. Brade, 1975, Arch. Virol. 48, 99. Schultze, H.E. and J.F. Heremans, 1966, in: Molecular Biology of Human Proteins, Vol. 1 (Elsevier, Amsterdam) p. 262. Singer, J.M. and C.M. Plotz, 1956, Am. J. Med. 21, 888. Singer, J.M., C.M. Plotz, E. Pader and S.K. Alster, 1957, Am. J. Clin. Pathol. 28, 1957. Sobel, A.T., V.A. Bokisch and H.J. Mfiller-Eberhard, 1975, J. Exp. Med. 142, 139.