T-lymphocyte rosette inhibition titering of antisera by direct microassay

Journal of Immunological Methods, 30 (1979) 329--338 © Elsevier/North-Holland Biomedical Press

329

T - L Y M P H O C Y T E R O S E T T E I N H I B I T I O N T I T E R I N G O F A N T I S E R A BY DIRECT MICROASSAY 1

CHARLES BIEBER and EDWARD B. STINSON

Department of Cardiovascular Surgery, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 20 March 1979, accepted 29 June 1979)

A 60-sample micro-rosette inhibition assay for determining the 100% rosette inhibitory titers of heterologous antisera is described. The assay is performed in histocompatibility trays, under oil, using frozen-thawed thymocytes or peripheral blood lymphocytes as the rosette forming lymphoid cells. Antisera dilutions are incubated with lymphocytes for an appropriate period, either with or without added complement; SRBC are inoculated into each well, and rosettes formed by centrifugation of the trays at 200 x g. Following centrifugation, glutaraldehyde is added to each well to fix rosettes to the well bottom and the plates inverted to allow unbound SRBC to fall away. One hundred percent rosette inhibition is determined by low power microscopic examination of the inverted wells. Highly reproducible in-assay (-+4% average standard error) and betweenassay (-+9%) inhibition values are obtained which correlated well (r = 0.99) with values calculated by more conventional methodology.

INTRODUCTION Methods for determining the rosette inhibitory titer (RIT) of anti-lymphoc y t e g l o b u l i n s ( A L G ) {Bach e t al., 1 9 6 9 ; N e l s o n e t al., 1 9 7 5 ) have l i m i t e d s a m p l e c a p a c i t y a n d , in o u r e x p e r i e n c e , have b e e n p o o r l y r e p r o d u c i b l e . T h i s r e p o r t d e s c r i b e s a m i c r o - r o s e t t e i n h i b i t i o n t i t e r i n g assay ( m i c r o - R I T A ) w h i c h has high s a m p l e c a p a c i t y ( 6 0 - - 6 0 0 ) a n d a r e p r o d u c i b l e assay e n d p o i n t t h a t is read directly. The major modifications that distinguish micro-RITA from o t h e r a s s a y s in c u r r e n t use are: (1) m i n i a t u r i z a t i o n , (2) use o f r e c o n s t i t u t e d f r o z e n t h y m o c y t e s as t h e r o s e t t i n g cells, (3) ' f i x i n g ' t h e r o s e t t e s f o r m e d b y t h e a d d i t i o n o f g l u t a r a l d e h y d e , a n d (4) d i r e c t d e t e r m i n a t i o n o f t h e 100% r o s e t t e i n h i b i t o r y d o s e ( R I D ) o f A L G as t h e assay r e s u l t , r a t h e r t h a n calc u l a t i o n o f t h e 25% R I T .

1 This work was supported by Public Health Service Research Grant RR70 from the General Clinical Research Centers, Division of Research Resources, and by the National Heart and Lung Institute Research Grant HL-13108.

330 MATERIALS AND METHODS

Chemicals and reagents Media 199 and fetal calf serum (FCS) were obtained from Gibco, Grand Island, NY. Sheep erythrocytes (SRBC) were obtained from Microbiological Associates, Bethesda, MD. Guinea pig serum (GPS) was drawn fresh from animals obtained from Simonsen Laboratories, Gilroy, CA. Glutaraldehyde and dimethyl sulfoxide (DMSO) were purchased from J.T. Baker, Phillipsburg, NJ. Mineral oil was purchased from Squibb, Inc., New York, NY. Instruments Sixty-well 3034 microtest plates (Becton-Dickenson Company, Oxnard, CA) were used for rosette assay. A Nikon model S-Ke binocular microscope fitted with Nikon 10X and Zeiss 40 X 0.65 NA objectives was used for assay evaluation. An International Model HNS tabletop centrifuge was used for density gradient separations. Assay plates were centrifuged in an International Model K centrifuge fitted with an IEC 4 place rotor. Assay plate carriers were obtained from Cooke Engineering Company, Alexandria, VA. T h y m o c y t e s and peripheral blood lymphocytes were frozen in 2 ml polypropylene screw-cap freezing vials (Linde division Union Carbide, San Francisco, CA). Cells were frozen in a Cryo-Med biofreezer (Cryo-Med, Mt. Clemens, MI). Reagents were dispensed through a Hamilton 50 or 250 pl syringe fitted with a repeating dispenser. Human lymphoid cells SRBC-rosette forming cells were obtained from either peripheral blood or from subtotal t h y m e c t o m y specimens. Peripheral blood was collected in heparin and sedimented over Ficoll-Paque gradients (TM Pharmacia Fine Chemicals, Piscataway, NJ) according to the m e t h o d of Boyum (1968). Eluted lymphocytes were washed twice in Hanks' balanced salt solution (HBSS) and reconstituted at 5 X 106 per ml in 20% FCS-199 media for assay use. To collect t h y m o c y t e s , tissue fragments were dissected free of investing connective tissue and minced with new No. 10 surgical blades over a No. 40 mesh stainless steel grid into HBSS at room temperature. T h y m o c y t e s were resuspended in 20% FCS-199 at 1 X 107 per ml for freezing or 5 X 106 per ml for assay use. Thymocyte freezing T h y m o c y t e ~uspensions were made in 20% FCS-199. All suspensions were brought to 7.5% DMSO by the slow dropwise addition of DMSO immediately prior to freezing. 1 X 107 cells in 1 ml of media were frozen in a biological freezer adjusted to give a freezing rate of 1 ° C/min. Vials containing t h y m o c y t e s were thawed in a 37°C water bath until one crystal of ice remained and then transferred to a 17 mm X 100 mm polystyrene tube containing 15 ml of 20% FCS-199. This tube was then centrifuged at 200 X g for

331 10 min and the cell pellet resuspended in 20% FCS-199 at a concentration of 5 X 106 cells per ml. Viability of lymphoid cells was assessed b y trypan blue dye exclusion and percent of cells capable of forming SRBC rosettes by the m e t h o d of Jondal (1972).

Sheep erythrocytes SRBC were prepared fresh weekly. Cells were washed three times in HBSS (Ca 2÷ and Mg 2÷ free), resuspended to 5 X l 0 s cells per ml, and stored at 4°C until used.

Complement Guinea pigs were used as the complement source. Animals were bled by heart puncture and the serum absorbed by the addition of a 50% volume of SRBC for 1 h at r o o m temperature. Absorbed GPS was dispensed into 0.2 ml aliquots and stored frozen a t - - 7 0 ° C. Heat inactivation of GPS (complement control) was produced at 60°C for 60 min.

An tisera Antisera were raised in New Zealand rabbits to a variety of cell types by two-pulse immunization 28 days apart, followed by bleeding 7, 8, and 9 days following the second immunization. Primary immunization was with 109 cells given subcutaneously and boosting by intravenous administration of the same cell dose. Use of adjuvant varied as indicated in Table 2. All ALG were purified to an approximately 80% gamma-globulin content by the combination of ammonium sulfate precipitation and dialysis-precipitation of euglobulins (Harboe and Ingild, 1973). Gamma-globulin content was determined by the product of the total protein and the percent gamma-globulin purity as determined by gel electrophoresis. ALG of equine origin were provided b y Dr. Paul Satoh of the Upjohn Company, Kalamazoo, MI. Ten dilutions of ALG diluted in HBSS were run in any given assay plate. ALG doses containing 150, 120, 100, 75, 50, 40, 35, 30, 25, and 20 pg/ml were routinely assayed. For suspected high titered ALG, doses containing 20, 15, 10, 7.5, 5, 4, 3, 2, 1, and 0.5 pg/ml were assayed.

Assay procedure RITA was performed in 60-well histocompatibility plates under mineral oil using microsyringes fitted with repeating dispensers to distrib-ute the reagents. Six replicates of each ALG concentration were assayed, three with complement and three without. Each oil-filled well was inoculated with 5 pl of an ALG sample, 1/al of the 5 X 106 cells per ml l y m p h o c y t e suspension and 1 pl of complement or complement control. Following a 16-h incubation at 4 ° C, 1 pl of the SRBC suspension was added to each plate well and the plates immediately centrifuged for 5 min at 200 X g. (Careful centering of the assay plates in the centrifuge carriers was necessary to orientate the broad plane of the plates parallel to the rotor axis during centrifugation. This

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insured an even distribution of cells across the well.) At the completion of centrifugation, 1 pl of 0.5% glutaraldehyde was inoculated into each well in order to prevent SRBC-lymphocyte rosette disruption, and to bind the l y m p h o c y t e s to the well b o t t o m . Five minutes later the plates were inverted, to allow u n b o u n d SRBC to fall away, transferred to a microscope stage and the central two-thirds of individual wells examined. For determining the 100% rosette inhibitory endpoint of an ALG dilution the wells were examined under low power magnification and the last rosettefree well defined (Fig. 1). The logarithms of replicate 100% RID values were used to give an estimate of 100% inhibitory means (Gaddum, 1945). The difference between the log means of standard versus test ALG preparations yielded the log p o t e n c y ratio. Taking the antilog of this latter value provided the relative potency of a test ALG with respect to a standard preparation. Analysis of the t~listribution of standard and test log means provided confidence limits for the log p o t e n c y (Armitage, 1973). To define a measured effect of an ALG in assay, the ratio of rosettes/200 l y m p h o c y t e s for all replicates of each ALG concentration was determined by examination of wells under high power magnification (40X, NA 0.65 objective). A dose-response curve which plotted percent rosettes as a function of ALG concentration was drawn and parallel line methods used for comparing standard and test ALG values (Gaddum, 1953). RESULTS

Determination of optimal SRBC/thymocyte ratio for micro-RITA Figure 2 illustrates the effect of S R B C / t h y m o c y t e ratio on the formation of t h y m o c y t e rosettes in assays performed in the absence of ALG. A ratio of

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335 100 was selected f o r r o u t i n e use since it gave highly r e p r o d u c i b l e r o s e t t e p e r c e n t a g e values (87% + 3% S.E.M.) w i t h o u t seriously a f f e c t i n g light transmission.

Inhibition o f rosette formation by ALG: effect of incubation time Figure 3 illustrates t h e r e l a t i o n o f assay i n c u b a t i o n t i m e to t h e A L G d o s e n e c e s s a r y f o r 1 0 0 % r o s e t t e i n h i b i t i o n . As s t e a d y s t a t e i n h i b i t i o n values were n o t a c h i e v e d b y 6 h o f i n c u b a t i o n at 4°C, o v e r n i g h t (24 h) i n c u b a t i o n s at 4°C w e r e r o u t i n e l y p e r f o r m e d . T h e s a m e e n d p o i n t c o u l d be a c h i e v e d b y 6-h i n c u b a t i o n at r o o m t e m p e r a t u r e w i t h n o sacrifice o f r e p r o d u c i b i l i t y . I n c u b a t i o n s o f 2 h or less at e i t h e r r o o m t e m p e r a t u r e or 4°C generally r e s u l t e d in b o t h high in-assay (replicate) and b e t w e e n - a s s a y v a r i a t i o n .

Comparison of reconstituted frozen thymocytes versus fresh peripheral blood lymphocytes (PBL) in micro-RITA T h y m o c y t e s r e c o n s t i t u t e d f r o m f r o z e n a l i q u o t s a n d fresh P B L o b t a i n e d f r o m d e n s i t y gradients c o u l d be used i n t e r c h a n g e a b l y in t h e m i c r o - R I T A assay w i t h o u t significantly a f f e c t i n g 1 0 0 % R I D o f an A L G (Fig. 4).

Comparison o f ALG rosette inhibition titers obtained independently at two institutions Six e q u i n e A L G s selected t o p r o v i d e a b r o a d r a n g e o f R I T w e r e p r o v i d e d b y t h e U p j o h n C o m p a n y a n d a s s a y e d b y m i c r o - R I T A as d e s c r i b e d . T h e coeff i c i e n t o f c o r r e l a t i o n b e t w e e n t h e U p j o h n 50% R I D values a n d S t a n f o r d 1 0 0 % R I D values was 0 . 9 9 f o r m i c r o - R I T A r u n in t h e p r e s e n c e o f c o m p l e m e n t GPS a n d 0 . 9 4 f o r t h o s e r u n in its a b s e n c e (p < 0 . 0 0 1 a n d 0 . 0 0 3 , respectively) ( T a b l e 1). A m o d i f i c a t i o n o f t h e m e t h o d o f J o n d a l et al. ( 1 9 7 2 ) was used f o r R I T A b y t h e U p j o h n C o m p a n y a n d did n o t include t h e use o f c o m p l e m e n t ; assay e n d p o i n t c o n s i s t e d o f 50% r o s e t t e i n h i b i t i o n . TABLE 1 COMPARISON OF ROSETTE INHIBITION VALUES OF 6 ALG SAMPLES ASSAYED BY THE UPJOHN COMPANY AND STANFORD ALG

17901 (-2) 17903 (-5) 17904 (-5) 17900 (-5)A 16138-8 16128-16A Correlation with Upjohn Company RID

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RID o f various an tisera assayed by micro-RITA The 100% RID and p o t e n c y relative to a house standard were determined f o r a variety o f antisera. The results of these assays are presented in Table 2. Standard errors of replicate values were generally less than 4%, f r e q u e n t l y zero and in no instance greater than 13%. One A L G was assayed in triplicate on five different occasions to establish between-assay reproducibility. The mean 100% RID f or this one ALG, +1 S.E., was 4.03 -+ 0.56 pg/ml in the presence o f c o m p l e m e n t and 8.3 + 2.9 pg/ml in the absence of c o m p l e m e n t . The p o t e n c y of additional ALG samples was measured against this standard by the direct assay m e t h o d . Ninety-five percent confidence limits for the p o t e n c y values in every instance were less than 12%. DISCUSSION The micro-RITA described in this study was designed for use as a direct assay in which increasing doses o f A L G were i nt roduced until a critical event (100% rosette inhibition) could be reproducibly observed. Other rosette inhibition titering assays in current use are of the measured effect t y p e (i.e. t h e y require generation and comparison of dose-response data from b o t h standard and test samples). Each data poi nt in a measured effect RITA must be obtained by visually counting rosettes. The time required for this task affects the assay in two ways. First, average rosette inhibition for each dose assayed is p o o r l y estimated because few replicates can be run. Secondly, in-assay variation is increased by the additional incubation time required by sequential counting of samples. This latter effect probably results from incomplete equilibrium between l y m p h o c y t e s , l y m p h o c y t e rosette inhibitors and l y m p h o c y t e - S R B C binding forces, and, in practice, is difficult to cont rol by assay design. Both o f the above deficiencies are eliminated by the m e t h o d o l o g y presented here. A micro-RITA conveniently provides up to 600 data points per assay and thus the triplicate means of ten dilutions from nine different u nk n o wn s and a standard with and w i t h o u t added c o m p l e m e n t can be determined. Use of micro-RITA to quantitate the dose-response relationship o f a rosette inhibitor requires visual counting of rosettes. However, sample reactions are stopped by the addition of glutaraldehyde, and variation due to sequential counting is, therefore, n o t apparent. Rosette inhibitory A L G doses causing less than 50% inhibition are the most variable in micro-RITA e m p l o y e d as a measured effect assay; doses which cause greater than 50% rosette inhibition have less than 3% standard error. We have used the micro-RITA primarily as a direct assay requiring only the comparison of the average 100% RID of standard and test samples. Perh)rmed in this m a n n e r the m i cr o- R I T A yields t he 100% RID of an ALG in pg o f gamma-globulin per ml with less than 4% in-assay and 9% betweenassay standard error. P o t e n c y values of A L G relative to a house standard have average 95% confidence limits of +8.7% and in every instance less than 12%,,

338 One h u n d r ed per c e nt RID values obtained by micro-RITA for several A LG preparations correlate well with determinations made by anot her institution (Upjohn C o m p a n y ) , utilizing a different technique. This correlative study n o t only provides validation of the micro-RITA, but also demonstrates an advantage in t ha t RID values could be measured by micro-RITA in b o t h the presence and absence of c o m p l e m e n t . Differences between those t wo values are generally parallel for di f f e r ent antisera prepared in the same fashion against identical antigens in one species. Comparison of RID values o f antisera prepared against different antigens (e.g. t h y m o c y t e s versus thym o c y t e memb ra ne ) , however, show wide discrepancies between results obtained with and w i t h o u t c o m p l e m e n t . Antisera made against t h y m o c y t e membranes and live t h y m o c y t e s give 100% RID values of 7.5 and 4.0 pg/ml, respectively, in the presence of c o m p l e m e n t and 150 against 10 pg/ml in its absence. This discrepant finding suggests t hat rosette inhibitory values observed in the absence of c o m p l e m e n t may provide a measure of direct inhibition o f presumptive sheep e r y t h r o c y t e r e c e p t o r sites (Boldt and Armstrong, 1976; Owen and Fanger, 1974), whereas those obtained in the presence of c o m p l e m e n t may be additionally influenced by A L G - c o m p l e m e n t complexed to antigens adjacent to the putative receptors. Thus the micro-RITA, which was initially developed for monitoring rosette inhibitory titers of ALG prepared for administration to transplant recipients, might also be a useful t e c h n iq u e for the de t e c t i on of antibodies directed against T - l y m p h o c y t e specific antigens. To this end the assay is currently being used to m o n i t o r the a n t i b o d y c o n t e n t in culture supernatants of hybrid cells created between mouse m y e l o m a cell lines and splenic l y m p h o c y t e s taken from normal mice i mmu n ized with h u m a n t h y m o c y t e s (Kohler and Milstein, 1975). REFERENCES Armitage, P., 1973, Statistical Methods in Medical Research (Blackwell Scientific, Oxford) p. 442. Bach, J.F., I. Dormont, M. Dardenne and H. Balner, 1969, Transplantation 8, 265. Boldt, D.H. and J.P. Armstrong, 1976, J. Clin. Invest. 57, 1068. Boyum, A., 1968, J. Clin. Lab. Invest. 21 (Suppl.) 97, 1. Gaddum, J.H., 1945, Nature 156,463. Gaddum, J.H., 1953, Pharm. Rev. 5, 87. Harboe, N. and A. Ingild, 1973, in: A Manual of Quantitative Immunoelectrophoresis, eds. N. Axelsen, J. Knoll and B. Weeke (Universitetsforlaget, Oslo) p. 162. Jondal, M., G. Holm and H. Wigzell, 1972, J. Exp. Med. 136,207. Kohler, G. and C. Milstein, 1975, Nature 256,495. Nelson, J.W., A.J. Parcells, A.B. Cosimi and R.E. Essler, 1975, Transplantation 20, 3. Owen, F.L. and M.W. Fanger, 1974, J. Immunol. 113, 1138.