Immunologic studies using antigen-coated charcoalApplication to rheumatoid factor RF.код для вставкиСкачать
Immunologic Studies Using Antigen-Coated Charcoal : Application to Rheumatoid Factor (RF) By CHESTERW. GOTTLIEB,KAM-SENG LAU AND VICTORHERBERT a number of sensitive assays for antibodies have been developed using “passive agglutination”.1-2This consists in the suspension of camer particles in a solution, coating of the carrier particle with adsorbed antigen, and subsequent visible agglutination of these carrier particles in the presence of antibody to their adsorbed antigen. The carrier particles included coll0dion,3-~barium stearate,5 erythrocytes ( especially after treatment with tannic acid) (Boyden),6 b e n t ~ n i t e ,and ~ latex.8 Charcoal particles had been used to adsorb horse serum antigen,l schistosomal antigen: and VDRL antigen,1° but these reports did not identify the type of charcoal used, nor did they indicate that there is an almost infinite variety of charcoals. Many charcoals and carbon blacks are unsuitable to serve as carrier particles by reason of size, high density, hydrophobic character requiring an anionic dispersing agent and an alkaline pH to stay in aqueous dispersion, non-uniformity, etc. For these reasons, other workers have been unable to use charcoal successfully in agglutination reactions. Our preliminary studies indicated that charcoals with maximum particle size greater than 60 microns were not suitable VER THE YEARS From the Department of Hematology, The Mount Sinai Hospital and Medical School, New York, N e w York 10029 This work was suppwted b y Research Grants AM 09564 and AM 09062 from the National Institute of Arthritis and Metabolic Diseases of the United States Public Health Service, and the Albert A. List, Frederick Machlin and Anna Ruth Lowenberg Funds. CHESTERW. GOITLIEB, M.D.: Fomwly, Research Assistant, The Department of Hematology. Current address: captain, M.C., Clinical Investi- as carrier particles in immunologic reactions. This report presents the results of studies using a charcoal of smaller than 60micron maximum size. The simplicity of the protocol here reported suggests its wide applicability throughout the range of agglutination reactions currently employing more complex carrier particles. A preliminary report of these findings has been published in abstract form.ll MATERIALS AND METHODS Serum. All sera were stored at -20 C. Serum from 18 patients with rheumatoid arthritis, supplied by Doctor Irwin Oreskes, had been evaluated by him for rheumatoid factor ( R F ) using the latex fixation technic? Antigen. Lyophilized gamma globulin was purchased from Squibb & Sons, 745 Fifth Avenue, New York. Charcoal. Norit A neutral pharmaceutical grade decolorizing carbon purchased from Amend Drug and Chemical Co., New York, was used. This charcoal has a maximum particle size of 53 p Antigen-coated charcoal. The procedure for coating charcoal particles is almost instantaneous. A desired volume of a standard charcoal suspension (generally, 2.5 gm per cent) is mixed with an equal volume of the desired concentration of the antigen to be adsorbed in a flask with constant swirling for one minute. The product is antigencoated charcoal with a ratio of antigen to charcoal by weight which is dependent on the respective gations Branch, Medical Research Laboratory, USA Edgewood Arsenal, Edgewood Arsenal, Mayland 21010. KAM-SENGLAU, M.D.: Formerly, Fellow of the World Health Organization at The Mount Sinai Hospital. Current address: Head, Clinical Laboratories, Faculty of Medicine, The University of Malaya, Kuala Lumpur, Malaysia. VICTORHERBERT,M.D.: Recipient of Career Scientist Award 1-435 from the Health Research Council of the City of N e w York. Professor of Medicine, Mount Sinai School of Medicine. 199 A R T H R ~ I AND S RHEUMATISM, VOL. 10, No. 3 (June 1967) 200 GOTTLIEB, LAW AND HERBERT determined as shown in Table 1. From the Table it can be seen that uncoated charcoal sediments rapidly in buffered13 saline and slowly in the diluted normal and rheumatoid sera (due both to coating of the charcoal by the serum proteins and to retention of excess serum protein in the suspending medium). The sedimentation rate is equally retarded in buffered saline and in dilute normal human serum with a gamma globulin-to-charcoal ratio by weight between 1:1 and 1:40. When the ratio of gamma globulin to charcoal is reduced to 1:80, the sedimentation rate in buffered saline is comparable to the sedimentation rate for uncoated charcoal; in diluted normal human serum the sedimentation rate is increased, but is less than uncoated charcoal. At the 1:80 ratio the gamma globulin coat is insufficient to retard sedimentation, when in buffered saline, but may be sufficient to block additional coating of the charcoal by the protein in normal human serum. That normal human serum of itself is insufficient to retard sedimentation is supported by the observation that the addition of exogenous albumin, polyvinylpyrrolidone (PVP ) or fibrinogen did not further retard the sedimentation of 1:80 gamma globulin-coated charcoal in normal human serum. Using low titer ( 1:160) and high titer ( 1:2560) serum containing RF, sedimentation clearly more rapid than from normal serum was evident at gamma globulin-to-charcoal ratios between 1 : l O and 1:40 (best at 1:40). At a ratio of 1:80, serum containing low or high titers of RF noticeably inRESULTS creased the rate of sedimentation compared One-half ml. of gamma globulin-coated to normal human serum, such that it apNorit A neutral pharmaceutical grade char- proached the rate seen in buffered saline. Table 2 demonstrates the similar results coal, which contained 12.5 mg of charcoal in buffered13 saline to which was added 0.5 of testing 18 sera from patients with rheuml of a 1 : l O dilution of unknown serum, matoid arthritis for RF by the coated charresulted in sedimentation of the charcoal to coal and latex fixation methods. The test the bottom third of the flocculation tube was performed using a 2.5 gm per cent within 15 minutes in the presence of RF, suspension of Norit A, coated 1:40 with but not in the absence of RF. This ratio of gamma globulin (see above). The coated gamma globulin to charcoal by weight was charcoal results were checked for repro- concentrations of materials added and the adsorptive capacity of the charcoal for the coating material. When Ilm-labeled albumin was added to an equal weight of charcoal (Norit A ) , the charcoal adsorbed approximately 10 per cent of its weight of the albumin.= Once the coating material has been adsorbed by charcoal it remains firmly bound, since only a negligible quantity can be removed by repeated washing.= The charcoal and coating material were prepared in physiologic saline or buffered saline. The final suspension was stored at 5 C. It may be stored for two weeks in the case of gamma globulin-coated charcoal. Since the coating process is so simple, it is recommended that it be carried out on the day of assay. Sedimentation and Flocculation. Uncoated charcoal particles are hydrophobic and settle rapidly and completely, forming a pellet at the bottom of a test tube, with a clear supernatant. Sedimentation is complete within 3 minutes when the concentration of charcoal is 10 to 12.5 mg per ml of saline diluent (excellent for easy visualization). Charcoal which has been saturated by the addition of equal weights of albumin, globulins, fibrinogen, hemoglobin, Ficoll ( a polymerized alcohol, Pharmacia Laboratories, Inc. ), dextrans, or polyvinylpyrrolidone (PVP),” and then washed and resuspended in saline, remains dispersed for hours without significant settling. Using standard flocculation tubes (10x 75 mm), 0.5 ml of a 1:10 dilution of serum suspected of containing R F is added to 0.5 ml of a suspension of 2.5 gm per cent charcoal coated with the appropriate concentration of gamma globulin. The rate of sedimentation is compared with that of a control (normal) serum. The degree of sedimentation was graded from 0 to 4’; 0, no sedimentation; 1+, clearing of the upper 4s of the coated charcoal suspension; 2+, clearing of the upper 2/3 of the coated charcoal suspension; 3+, definite pellet formation at the bottom of the tube; 4+,clear supernatant with pellet completely at the bottom of the tube. 1:5 1:10 1 :2u 1 :40 1 :80 3 I) I, 4f rr n II " I, It I, It n fI If fI It I, I+ n 0 It I5 min. 4' huffcrcd saline) 1 :1 1 :2 Time 3 rnin. Reaction saline Buffered Time ff I, It I, tf 4' w It c It fl ,I I# n If n n I, 15 min. rf 4+ W 2* 3+ 1+ W " 0 Time n If I, n n If I, 10 min. b It I1 ,I If If n 15 inin. Titer 1 :I60 Reaction ~ H W fI PI 4+ 3' 14 1' 0 -- I, 5 min. r, It fI It " n ff fI It 10 w n n 15 rnin. Titer 1 :2560 Reaction Time - Serum containing R F ch arcool ratio for redimentation and flocculation of RF sera II f 1+ , 0 Reaction NHS of best gamma globulin: 0 :1 (uncoated charcoal) 1 :1 (rcconstituted in glohutin: Gamma -~~~ Table I.-Deberrnination charcoal - * k ! ! 8 1 2$2 3 202 GOTTLIEB, LAU AND HERBERT Table 2 . 4 o m p a r i s o n of coated charcoal and latex fixation method in 18 sera from patients with rheumatoid arthritis Latex Fixation Coated Charcoal (time, minutes) Reaction Titer 5 10 15 - 0 0 1' 0 2' 3' 2' Specimen Control 396 RA 10210 " 10559 " 10608 " 10609 " 10611 " 10678 " 10750 " 10759 " 10771 " 10802 " 10807 " 10810 " 10838 " 10868 " 10869 " 10891 " 10899 " 10900 ____ Neg Pos I, n n I, 320 2560 640 320 640 n 5120 I, 2560 5120 n R 640 n 1280 1280 5120 160 0 ,, N ,I Neg R n I, ff I, r, ,, 'I ducibility by making 4 separate runs, each in duplicate (8 observations for each sample at each time period). The experimental variation was not greater than 1+ for any serum; the control normal and RF-negative rheumatoid sera never exceeded I+ (i.e. 1/3 sedimented) at 15 minutes, whereas the RF positive sera were never less than 2+ (i.e. 2/3 sedimented) at 15 minutes. The addition of 2 drops of 1:40 gamma globulin-coated charcoal and 1:10 diluted serum, respectively, to a slide, resulted in easily discernible coarsening of the charcoal particles (and clearing of the buffered saline suspension) in the presence of serum containing RF but not in its absence, suggesting the usefulness of the procedure as a rapid slide test. The test was not enhanced by prior incubation of the reactants for periods up to one hour, nor by incubation at 37 C. during the procedure. Buffered saline was superior to physiological saline with respect to clarity of the endpoint. DISCUSSION Charcoals and carbon blacks are capable 0 0 0 2+ 2' 3+ 1' 0 0 0 0 3' 0 0 0 0 0 0 '1 1+ 3' 3' 4' 2' 3' 1' '1 2+ 3+ 1+ 0 0 0 1' 0 - 3+ 3' 4' 3' 3' 2' 2+ 3' 4' 3' '1 '1 1+ 1' 1' of adsorbing a large variety of protein, carbohydrate and lipid substances. Particles of charcoal may, therefore, be coated with a variety of such substances and should be sedimented (and flocculated) under appropriate conditions by antibody to these substances. Thus, in the present communication, charcoal sensitized with gamma globulin can differentiate, in a rapid sedimentation procedure, sera containing RF from RF-negative sera. Similarly, hog intrinsic factor (IF)-coated CAL" charcoal or Carbolac carbon black t in buffered saline may be sedimented by antibody to hog IF (made in rabbits).'l ACKNOWLEDGMENT We are indebted to Doctor Hugo Pollack, Mrs. Katherine Kellett and Mr. John Farrelly for technical assistance. *CAL PULV (5746) charcoal supplied by Basil Louros, Pittsburgh Activated Carbon Co., Trenton, New Jersey. More than 90 per cent of this charcoal has a particle size of 44 p or less. +Supplied by F. A. Heckman, Cabot Corporation, Cambridge, Mass. Carbolac has an 8 to 10 mp particle size and a surface area of approximately 1000 M Z / g m . IMMUXOLOGIC STUDIES USING ANTIGEN-COATED CHARCOAL 203 SUMMARY Appropriate charcoals or carbon blacks may be used as carrier particles for antigens or antibodies in a manner akin to latex, bentonite or erythrocytes. In the present report, charcoal was substituted for latex in a test for rheumatoid factor. Charcoal and carbon black may have wide applicability in immunologic sedimentation, flocculation, or agglutination procedures for recognition of antibody or antigen. They are stable, inexpensive, require no preparation or special storage prior to use, and almost instantly adsorb a wide variety of antigens or antibodies. SUMMARIOIN INTERLINCUA Particulas d e varie generes de carbon pote esser usate como vectores de antigeno o de anticorpore in un maniera simile a latex, bentonita, o erythrocytos. In le studios hic reportate, carbon esseva usate in loco d e latex in un test pro factor rheumatoide. I1 es possibile que carbones ha un extense applicabilitate in procedimentos de sedimentation, flocculation, o agglutination immunologic pro le recognition de anticorpore o antigeno. Iste materiales es stabile e incostose e require nulle preparation e nulle special mesuras de preservation ante lor uso. 1110s absorbe quasi instantaneemente un extense varietate de antigenos o anticorpores. 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