Serological detection of an X-associated antigen in the mouse using a cellular radioimmunobinding assay

Journal of Immunological Methods, 52 (1982) 105--110

105

Elsevier Biomedical Press

S E R O L O G I C A L D E T E C T I O N O F AN X - A S S O C I A T E D A N T I G E N IN T H E MOUSE USING A CELLULAR RADIOIMMUNOBINDING ASSAY

JOYCE ANGLIN SHELTON 1 and ELLEN H. GOLDBERG 2 Department o f Microbiology, University of New Mexico School o f Medicine, Albuquerque, NM 87131, U.S.A.

(Received 17 November 1981, accepted 11 January 1982)

Skin grafts between inbred strains of mice that differ with respect to an X-linked histocompatibility locus are generally rejected. Using a cellular radioimmunobinding assay, we have serologically demonstrated the presence of an X-associated antigen on lymphocytes of A/J female mice. Key words: H-X antigen --serological detection

INTRODUCTION E v i d e n c e for t h e e x i s t e n c e o f a h i s t o c o m p a t i b i l i t y locus o n t h e X c h r o m o s o m e (H-X) was first r e p o r t e d b y Bailey ( 1 9 6 3 a , b), w h o f o u n d t h a t w h e n ( C 5 7 B L / 6 ? × B A L B / c ~)F1 males were grafted with tail skin f r o m the p a t e r n a l strain, t h e y w o u l d r e j e c t t h e graft; whereas, a graft f r o m the m a t e r nal strain was n o t rejected. S u b s e q u e n t l y , H i l d e m a n n and C o o p e r ( 1 9 6 7 ) o b s e r v e d t h a t ( C 5 7 B L / 6 ~ × A / J ~)F1 males w o u l d r e j e c t A strain skin grafts, indicating d i f f e r e n c e s at t h e X-linked h i s t o c o m p a t i b i l i t y locus b e t w e e n C 5 7 B L / 6 (B6) a n d A mice. F u r t h e r m o r e , B e r r y m a n and Silvers (1979} grafted various c o m b i n a t i o n s o f F1 h y b r i d males with m a t e r n a l and p a t e r n a l skin and f o u n d t h a t t h e H-X locus is p o l y m o r p h i c , possessing at least 4 alleles. A l t h o u g h H-X is d e t e c t a b l e b y graft r e j e c t i o n , t h e r e has so far b e e n n o conclusive in vitro serological d e m o n s t r a t i o n o f t h e antigen. Using an agar p l a q u e assay, H i l d e m a n n and P i n k e r t o n ( 1 9 6 6 ) d e m o n s t r a t e d t h a t i m m u n i z a t i o n o f ( A / J × B6)F1 males with f e m a l e red b l o o d cells w o u l d elicit a plaquef o r m i n g r e s p o n s e against H-X. H o w e v e r , as t h e a u t h o r s p o i n t o u t , t h e agar

I Present address: Dept. of Biochemistry and Molecular and Cell Biology, Northwestern University, Evanston, IL 60201, U.S.A. 2 To whom all correspondence should be sent. 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press

106 plaque assay requires strict adherence to technical details and conditions in order to achieve reproducibility. Thus, further immunogenetic analysis of the H-X system has been limited by the lack of a simple and reproducible serological assay. In this report, we demonstrate that an X-associated antigen can be detected serologically on lymphocytes, using an 12SI-radioimmunobinding assay. MATERIALS AND METHODS Mice

C57BL/6 (B6) female mice were obtained from the Charles River Breeding Laboratories (Wilmington, MA). A/J males and females were obtained from Jackson Laboratories (Bar Harbor, ME). (B6 ? X A/J d)F~ males and females were bred and maintained in the animal facility at The University of New Mexico. Antiserum

(1) Anti-H-X a antiserum (antiserum against an X-associated antigen found in the A/J strain) was raised by grafting (B6 ? X A/J d)F1 males (XBYA) with (B6 ? X A / J d ) F 1 female (XBXA) tail skin. Grafts measuring i cm 2 were placed on the dorsal side in a prepared graft bed. They were held in place with Vaseline gauze covered with an adhesive strip. Eight weeks after the first graft, the mice were grafted a second time. Animals exhibiting a chronic mosaic rejection p h e n o m e n o n (as described by Bailey, 1963b) were bled via the tail vein 30 days after the second graft. The sera were pooled and frozen at --70°C until used in the assay. As a source of normal mouse serum (NMS), (B6 £ X A/J d)F~ males used to raise anti-H-X a serum were bled via the tail vein prior to grafting, and their sera were pooled. (2) Anti-H-2 a serum was raised in B6 female mice by an initial immunization of 1 X 107 A/J female lymphoid cells given subcutaneously. Four weeks later, a second inoculation of 2.5 X 107 cells were given intraperitoneally (ip), followed by a third immunization (ip) of 5 X 107 cells 2 weeks later. Every 2 weeks thereafter, an inoculum of 1 X l 0 s cells were given ip. Animals were bled via the tail vein at 7, 10, and 14 days after the fifth and subsequent inoculations. Sera were pooled and stored at --70°C until used. (3) 12s I-labeled sheep anti-mouse kappa antibody. Sheep anti-mouse kappa serum was kindly provided by Dr. Noel Warner, The University of New Mexico. The IgG fraction of the antibody was purified by affinity chromatography on a mouse IgG column by Dr. K.S.K. Tung, The University of New Mexico. The antibody was labeled with ~2sI (Amersham) at 0.1 mCi/mg by the chloramine T m e t h o d (Greenwood et al., 1963). Absorption o f H-X a antiserum

Single cell suspensions of spleen from (B6 9 X A/J d)Ft females and

107 (B6 ? × A/J d)F~ males were prepared in phosphate-buffered saline (PBS) and washed 3 times by centrifugation at 200 × g for 10 min. The anti-H-X a serum (diluted 1/16 with PBS) was added to the cells at a 1/4, packed cell v/v, ratio. The cells were suspended in the antiserum and incubated for 30 min at 4°C with gentle agitation every 5 min. The cells were then pelleted by centrifugation at 90 × g for 10 min. The antiserum was removed and either used immediately in the assay or frozen until used.

12SI-radioimrnunobinding assay The radioimmunobinding assay used was a modification of t h a t described by Tsu and Herzenberg (1978). Briefly, single cell suspensions of A/J female or B6 female splenocytes were prepared in PBS. Red blood cells were lysed with ACK (8.29 g/1 NH4C1, 1.0 g/1 KHCO3, 0.037 g/1 EDTA). After washing the remaining cells 3 times with PBS by centrifugation at 200 × g, they were suspended in PBS at a concentration of 7.5 × 106 cells/ml. Fifty microliters of the appropriate cell suspension were added to each well of a soft plastic microtiter plate (Dynatech Laboratories) that had previously been coated with bovine serum albumin (BSA) made 3% with PBS. Fifty microliters of antiserum diluted in PBS (1/16--1/64, at doubling dilutions) were then added to triplicate wells of both A/J and B6 cells. After incubating the plate for 30 min at 4°C, it was washed 3 times with BSA, made 0.2% with PBS, by centrifugation at 200 × g for 10 min. The 12SI-labeled sheep anti-mouse kappa antibody, diluted in 3% BSA, was then added to each well in 50 pl volumes to give 1--2 × 10 s counts per well. After incubating for 30 min at room temperature, the plate was washed 5 times and allowed to dry. The a m o u n t of 12sI bound to the cells in each well was determined and was expressed as a percentage of the total 12SI-labeled sheep anti-mouse kappa a n t i b o d y added to each well according to the following formula: ~2sI cpm/well × 100 = % 12sI bound total 12sI cpm added/well

Statistical analysis The results obtained in each experiment when A/J female cells were reacted with anti-H-X a serum were compared to the results obtained with B6 cells, the specificity control, using 2-way, hierarchical, nested analysis of variances as described by Zar (1974). RESULTS The results of 7 experiments summarized in Table I clearly show that anti-H-X a serum reacts with A strain spleen cells. The maximal response was obtained at 1/16 dilution of antiserum; at higher concentrations (antiserum diluted 1/2--1/8) the a m o u n t of ~2sI b o u n d was always less than that f o u n d at a 1/16 dilution. In each experiment, there was a significant difference

1

1.08 0.49

2

1.40 1.00

1.30 0.90

1.09 0.62

4

1 Antiserum was diluted l/16 at which dilution the greatest percentage 2 Percent lzsI bound. 3 Serum was diluted l/16. Average of 7 experiments.

0 (XBXB)

NJ ‘i’ (XA XA )

B6

3

of “‘1

1.20 0.95

5

bound

1.24 1.01

7

was obtained

1.32 0.93

6

cells

‘.

(see Fig. 1).

5.20 1.10

Anti-H-2 a serum 3

2

spleen

1

serum against A/J and B6 female

Controls

tests with anti-H-Xa

Experiment

of 1251-radioimmunobinding

Strain donating splenocytes

Summary

TABLE

0.76 0.80

NMS 3

109 TABLE 2

A b s o r p t i o n e x p e r i m e n t s s h o w i n g s p e c i f i c i t y o f anti-H-X a serum for the X-associated antigen f o u n d o n A / J female s p l e n o c y t e s . A n t i - H - X a s e r u m I absorbed w i t h s p l e n o c y t e s f r o m

T e s t Cell XAX A

XBXA

_

XB YA

+ 3

Unabsorbed

+

XBXB

2

1 D i l u t e d 1 / 1 6 b e f o r e absorption. 2 _ = negative reaction ( < 0 . 9 % 12s I b o u n d ) . 3 + = positive reaction (/> 1 . 6 % 12sI b o u n d ) .

(P < 0.01) between the values obtained when a 1/16 dilution of anti-H-X a serum was reacted with A/J cells as compared to when it was reacted with B6 cells (the specificity control). In addition, the percent '2sI bound to B6 cells was comparable to the percent bound when NMS was used. The variation among values for triplicate wells was less than 5%. Furthermore, when reacted with A strain cells, the activity of the antiserum decreased with increasing dilution, indicating specificity of the reaction for the X-associated antigen. (A representative experiment is illustrated in Fig. 1 .) To further prove specificity, anti-H-X a serum was absorbed with (B6 9 × A/J d)F, female (XBXA) or (B6 ? X A/J d)F, male (XBYA) spleen cells, and

2 , 0 ~~D

,.-.., Z Z3, 0 a3 t.-i

cr

°

F-Z

j,JcELLs

I,O

W W n

O

,_____~.,~_____,~86CELLS ~

O

0 z

I

1/16

ANTI H - X a

I

l

1/32

1/64

// CONTROLS

SERUM DILUTIONS

Fig. 1. D e t e c t i o n o f an X - a s s o c i a t e d antigen o n s p l e n o c y t e s o f A / J f e m a l e m i c e b y a cellular r a d i o i m m u n o b i n d i n g assay, using serum f r o m ( B 6 ~ x A / J d ) F 1 m a l e s grafted w i t h ( B 6 9 × A / J g)F1 f e m a l e tail skin. B6 cells serve as the s p e c i f i c i t y control.

110 then cells. H-X a cells,

tested in the r a d i o i m m u n o b i n d i n g assay against A/J and B6 female In summary, Table 2 shows t hat female (XBXA) cells remove the antiactivity from the serum; whereas, serum absorbed with male (XBYA) or left unabsorbed, retains its ability to react with A/J cells.

DISCUSSION Because H-X is a weak antigenic system, previous a t t e m p t s t o d e t e c t an a n t i b o d y response to it have n o t been reproducible or reliable. Thus, the i m m u n o g e n e t i c analysis of the H-X system has been limited t o transplantation studies. For this reason, we developed a r a d i o i m m u n o b i n d i n g assay for t h e serological d e t e c t i o n o f an X-associated antigen. Although the reaction is weak, as co mp ar ed to the reaction obtained when anti-H-2 serum is used, it is highly reproducible. In all 7 experiments, the reaction obtained with the anti-H-X a serum was higher against the A strain cells as com pared to the react i o n seen when B6 cells were used as targets. It is necessary to poi nt o u t t h a t although the antiserum was raised by skin grafting across the H-X barrier, t h a t th e a n t i b o d y d e t e c t e d in this assay m ay be directed against ot her X-linked gene products unrelated to the H-X transplantation antigen. However, this assay provides a reliable m e t h o d for t he serological d e t e c t i o n of an X-associated antigen. Its use may, t her e f ore, allow furt her analysis of the genetics o f the H-X antigenic system. ACKNOWLEDGEMENTS This wo r k was s u p p o r t e d by National Institutes of Health Research Grants AI 1 1 5 6 0 and HD 13082. Ellen H. Goldberg is the recipient of Research Career Dev elo p m ent Award AI 00353 from the National Institutes of Health. REFERENCES Bailey, D.W., 1963a, Transplantation 1, 70. Bailey, D.W., 1963b, Science 141,631. Berryman, P.L. and W.K. Silvers, 1979, Immunogenetics 9,363. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89,114. Hildemann, W.H. and R.L. Cooper, 1967, Transplantation 5,707. Hildemann, W.H. and W. Pinkerton, 1966, J. Exp. Med. 124, 885. Tsu, T.T. and L.A. Herzenberg, 1978, in: Selected Methods in Cellular Immunology, eds. B. Mishell and S. Shiigi (Freeman, San Francisco, CA) pp. 373--397. Zar, J.H., 1974, Biostatistical Analysis (Prentice-Hall, Englewood Cliffs, NJ) p. 193.