A simple procedure for determining Lewis phenotypes in human saliva

Journal of Immunological Methods,

62 (1983) 73-78

73

Elsevier JIM 2703

A Simple Procedure for Determining Lewis Phenotypes in Human Saliva Zenon Steplewski *, Meenhard Herlyn, Magdelena Blaszczyk and

Hilary Koprowski The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104 U.S.A.

(Received 1 February 1983, accepted 18 February 1983) We have selected 4 murine monoclonal antibodies that specifically bind to Lewis type blood group haptens to establish an assay in which antigens present in human saliva are bound to polystyrene beads and then detected by monoclonal antibodies in radioimmunoassay or in peroxidase-antiperoxidase immunoassay. Two of these antibodies detect both the Lea and Le b determinants (Le~b), 1 antibody detects only Le ~, and 1 only Le b. All Lewis phenotypes (Lea-b-, Le a + b - , Le a - b + and Le~+b+) are easily detectable in this assay. Of the 60 individuals tested, 1 w a s Le a + b + , 4 Lea b , 12 Le a + b - and 43 Le a-b+.

Key words:

Lewis phenotypes -- Le ~ --

Le ~

Introduction Most of the m e m b e r s of the large p a n e l of m u r i n e m o n o c l o n a l anti-gastrointestinal cancer antibodies available (Koprowski et al., 1979; Steplewski a n d Koprowski, 1982) are directed against glycolipids expressed by h u m a n t u m o r cells ( M a g n a n i et al., 1981; Blaszczyk et al., 1982; Brockhaus et al., 1982). Some of the m o n o c l o n a l a n t i b o d i e s directed against the c a r b o h y d r a t e sequences of glycolipids have a p p a r e n t t u m o r specificities (Koprowski et al., 1981; M a g n a n i et al., 1981, 1982; Brockhaus et al., 1982; Pukel et al., 1982). I m m u n i z a t i o n of mice with colon c a r c i n o m a cell lines SW1116 a n d SW1222 (Leibovitz et al., 1976) has yielded h y b r i d o m a s that secrete m o n o c l o n a l a n t i b o d i e s directed against Lewis b l o o d group haptens: Le b (Brockhaus et al., 1981), Le a, a n d against d e t e r m i n a n t s c o m m o n to L e a a n d Le b antigens (Blaszczyk et al., 1983). We have used these m o n o c l o n a l a n t i b o d i e s to establish a simple assay in which it becomes possible to determine Lewis p h e n o t y p e by testing h u m a n saliva. * To whom correspondence should be addressed: Zenon Steplewski, The Wistar Institute, 36th Street at Spruce, Philadelphia, PA 19104, U.S.A. Abbreviations: BSA, bovine serum albumin; DAB, diaminobenzidine; PAP, peroxidase-antiperoxidase; GICA, gastrointestinal cancer-associated antigen; PBS, phosphate-buffered saline; RIA, radioimmunoassay. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.

74 Materials and Methods

Monoclonal antibodies Monoclonal antibodies are secreted by hybridomas established after fusion of splenocytes of mice immunized by injection of SW 1116 and SW 1222 colon carcinoma cells (Leibovitz et al., 1976) with mouse myeloma P3X63 Ag8 variant 653 (Kearney et al., 1979). Antibody (IgM) from hybridoma 1116NS-10 c1.17 (10-17) is directed against the Le b antigen of the human Lewis blood group system (Brockhaus et al., 1981). The recently developed monoclonal antibody 151-6-A7-9 (A7-9), IgG3 isotype, recognizes specifically lacto-N-fucopentaose II structure - - an Le ~ active terminal sequence (Blaszczyk et al., 1983). Two other monoclonal antibodies, 151-5G3-5 (G3-5) and 151-5-G2-12 (G2-12), bind to both Lea and Le b glycolipids of the Lewis blood group system and recognize both lacto-N-difucopentaose II (Le a) and lacto-N-difucohexaose I (Le b) structures (Blaszczyk et al., 1983).

Samples Saliva samples (1 ml) collected from 60 individuals of both sexes and ranging in age from 25 to 75 years, were inactivated by heating for 30 min at 85°C in a water bath. Samples were diluted 1:4 in phosphate-buffered saline (PBS) and stored at - 2 0 ° C until assayed.

Assay procedures Saliva samples were diluted 1:20 or as specified (Table II). Polystyrene beads (Precision Plastic Ball Co., Chicago, IL) were incubated with saliva samples (200 ~l/bead), and the coated beads were washed in 1% gelatin in 0.01 M sodium borate, p H 8.0 (1 h at room temperature), and placed in reaction trays. Two hundred microliters of hybridoma-derived tissue culture supernatants containing monoclonal antibodies 10-17 (for Le b), A7-9 (for Le a) and G2-12 and G3-5 (for Le ~b) were added to the beads and incubated for 1 h at room temperature. After washing 3 times in PBS containing 0.5% bovine serum albumin (BSA), the binding of the monoclonal antibody was measured by radioimmunoassay (RIA) or detected in an enzyme-linked immunoabsorbent assay using a peroxidase-antiperoxidase (PAP) system (biotin-avidin immunoperoxidase kit obtained from Vectastain ABC unit, Vector Labs, Burlingame, CA). In R1A, beads preincubated with antibody were then incubated with 125I-labeled affinity-purified rabbit anti-mouse F(ab')2 immunoglobulin (1 × 105 c p m / b e a d ) for 30 min at room temperature, washed 3 times with 0.5% BSA in PBS and counted. For the PAP assay, beads were incubated with monoclonal antibodies, exposed to biotinylated anti-mouse IgG for 30 min at room temperature, washed 3 times with PBS, exposed to avidin DH-biotinylated horseradish peroxidase H (ABC reagent) for 30 min at room temperature and again washed 3 times with PBS. Finally the peroxidase substrate (5 mg diaminobenzidine (DAB), 10/~1 of 30% H202, 100 ~1 of 1 M imidazole in 10 ml of 0.1 M Tris buffer, pH 7.6) was added. Incubation for 5-10 min results in clear dark brown staining of positive beads.

75 Results and Discussion

Fig. 1 shows the typical pattern of reactivity of saliva samples from 4 different individuals with 4 monoclonal antibodies included in the PAP assay. Saliva of CP bound all 4 antibodies, thus this individual's Lewis blood group could be described as Le (a+b+). The saliva of individual 24 did not react with any of the 4 antibodies and could be diagnosed as Le (a-b-). The saliva sample JL reacted with antibodies G3-5, G2-12 (Le ab) and thus represents a Lewis-positive phenotype. Because it bound antibody A7-9 but not 10-17, it is diagnosed as Le (a+b-). Lastly, the saliva sample of individual IS bound both Le ~b antibodies and antibody 10-17, but not A7-9 and is thus of the Le (a-b+) phenotype. A simultaneous assay performed on the same set of samples in RIA (Table I) confirmed the results obtained by PAP assays. Either method can, therefore, be used. The antigens of the Lewis blood group are very well represented in the saliva of Lewis-positive individuals. As shown in Table II, saliva samples of Le ( a + b ) and Le t~-b+) individuals could be diluted up to 100,000 with clearly positive binding of the 4 antibodies. Saliva of the Le (~-b-) individual did not bind these antibodies at any of the dilutions tested. Of the 60 individuals tested (Table III), we found one who expressed Lewis blood group phenotype Le (a+b+), 4 with the Le ( a - b - ) phenotype, 12 with Le (a+b-) and 43 with the Le ta-b+) phenotype. The Le " antigen contains the terminal sugar sequence: G a l f l l - 3 G l c N A c .... 4

I Fuccd

f-

CP

24

JL

IS

~'

Le~

,~

Lea

p~ Le b

Le ab Lo

Cont

Fig. 1. A panel of 4 different individuals, representing 4 different Lewis blood group phenotypes. Saliva samples were incubated with 4 monoclonal antibodies: G3-5 (Le ab); A7-9 (Le a ); 10-17 (Le b), G2-12 (Le ab) and P3 (control supernatant), and binding detected by a PAP assay using the avidin-biotin system.

76 TABLE I B I N D I N G OF A N T I - L E W I S A N T I B O D I E S TO SALIVA SAMPLES AS D E T E C T E D IN RIA ~ Binding (cpm) of saliva samples

Antibo dy Code

Specificity

CP

24

JL

IS

G3-5 A7-9 10-17 G2-12 P3X63 Ag8

Le ab Le" Le b Le ab Control

9860 10780 4490 10580 452

480 33 320 180 426

9530 10790 690 10060 453

12010 350 I 1090 12710 481

a Input 100,000 cpm per bead. Values represent cpm per single bead from 2 consecutive assays under the same conditions.

TABLE II LEVELS OF Le A N T I G E N S IN SALIVA SAMPLES AS D E F I N E D BY M O N O C L O N A L A N TIB O D IES D I R E C T E D A G A I N S T Lea (A7-9) A N D Le b (10-17) HA P TEN S Saliva samples

Binding of antibodies (cpm) " at saliva dilutions

Code

Phenotype

20

50

100

1000

10000

100000

49 93 44

Le ~a+b-) Le (a-b+) Lena 8-7

10800 12350 550

10000 11810 600

9500 8750 430

4300 5300 420

1800 1980 440

400 900 480

a Input 100,000 c p m p e r bead.

TABL E III D I S T R I B U T I O N OF LEWIS PHENOTYPES IN T H E SALIVA SAMPLES F R O M 60 I N D I V I D U A L S Le a+b+ reacted with all 4 antibodies. Le a - b - did not react with any of the 4 antibodies. Le ~+b- did not react with antibody 10-17. Le a b+ did not react with antibody A7-9. N u m b e r of individuals typed as

Total

Lea+b+

Lea-b-

Le a+b

Lea-b+

1(1.6) *

4(6.6)

12(20.0)

43(71.6)

60

* In parentheses: % of total.

a n d Le b a n t i g e n c o n t a i n s the same terminal sequence with an additional fucosyl residue: Gal/~I-3GlcNAc .... 2 4

I

I

Fucal

Fucal

77 The Lewis blood group terminal sugars occur in the glycolipids and glycoproteins (Watkins, 1980). Approximately 75% of the population expresses the Le ~a-h÷) phenotype, whereas about 20% of the population is unable to synthesize the Le b terminal sugar sequence due to the lack of the fucosyltransferase required for the Fucal-2 Gal linkage (Shen et al., 1968) and thus expresses the Le la÷b )phenotype. Finally, about 5% of the population (Race and Sanger, 1975) lacks both the Fuccd-2 Gal enzyme and the fucosyltransferase responsible for the formation of Fuccd-4 GlcNAc linkage (Grollman et al., 1969). The Le ~-h-~ individuals are probably also unable to synthesize the gastrointestinal cancer-associated antigen (GICA), a tumor-associated antigen containing sialylated lacto-N-fucopentaose II (sialylated Lea), since this monosialoganglioside (Magnani et al., 1981) has a carbohydrate structure (Magnani et al., 1982): NeuNAcc~2-3Gal/31-3GlcNAc/31-3Gal/3Glc .... 4 Fucal and requires the synthesis of Lewis terminal sugar sequences for its expression. We have postulated (Koprowski et al., 1982) a relationship between the distribution of Lena-~-), Lena+ b-) and Le ~a- b+) phenotypes in patients with gastric and pancreatic cancer as compared with their spouses and siblings. The simple assay presented here for the analysis of Lewis phenotypes, in conjunction with the serum assay for GICA, should facilitate testing of this hypothesis.

Acknowledgements Supported by Grants CA 10815, CA 21124 and CA 25874 from The National Cancer Institute, Grant RR-05540 from the Division of Research Resources and funds from W.W. Smith Foundation.

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