Evaluation of human and murine monoclonal anti-rhésus antibodies

Revue Fran~aise de Transfusion T o m e XXXI. - N ° 2. - 1 9 8 8

et I m m u n o - h 6 m a t o l o g i e

175

Evaluation of human and murine monoclonal anti-rhesus antibodies b y L. M a n n e s s i e r * a n d H . B r o l y * * * Laboratoire d'Immunologie Erythrocytaire ** Laboratoire des Anticorps Monoclonaux C.KT.S. Lille 21 rue Camille Gu6rin 59012 Lille Cedex, France

Thirty monoclonal anti-Rhesus antibodies were tested : 21 anti-D, 4 anti-E, 4 anti-e and 1 anti-c, ragarding their specificity, avidity, intensity, titer and score. Their type, mode and mean were determined by flow cytometry assay. Then, we tried to determine if the anti-D could adequately detect Du samples and also studied their behaviour with 13 partial and 2 depleted D antigens.

MATERIALS AND METHODS H e m a g g l u t i n a t i o n tests in tube

Immediate spin method (LS.) The red blood cells (RBC) are washed 3 times and diluted to 3 % in saline. 2 drops of 3 % suspension and 2 drops of supernatant or its dilutions are mixed in saline in a tube. Then, the tube is centifuged at 1 00 rpm for 1 minute. Button of cells is gently shaken and results are read by eye. The titer is the reciprocal of the highest dilution at which the antibody produces a positive reaction. The score is a numerical value to each positive reaction in the liIration ; the value is based on the strength of agglutination in each t~be [t]. A 3 plus reaction scored 10, a 2 plus 8, a 1 plus 5 and a weak reaction 2.

Room temperature incubation method (RTI) The method is the same as I.S. except that the incubation period is 60 minutes at room temperature.

176

~VNESSIER L.

37 C incubation method (37-1) The method is the same as RTI except that incubation is at 37 C instead of room temperature.

Indirect antiglobulin test (IA T) 2 drops of supernatant or its dilutions in saline and 2 drops of 3 % cell suspension previously washed 3 times in saline are mixed in a tube. The contents of the tube are gently mixed and incubated for 45 minutes at 37 C. Then, cells are washed 3 times in saline and 1 drop of antiglobulin serum (CRTS Lille) is added. The tube is centrifugated at 1 000 rpm for 1 minute. The results are recorded as previously described.

Papain 3 7 C- Two stage method (P-3 7) 1 volume of washed packed red cells and 1 volume of 1 % papain solution (CRTS Lille) are put in a tube. The tube is incubated for 7 minutes at 37 C. Then, the RBC are washed 3 times and diluted to 4 % in saline. A drop of supernatant is added to a drop of 4 % suspension. The tube is gently shaken and the result noted.

BromeIin 3 7 C - Two stage method (B-3 7) 1 volume of washed packed red cells and 1,5 volume of 0,25 % bromelin solution (CRTS Lille) are put in a tube. The tube is incubated for 15 minutes at 37 C. Then, the method is the same as P-37 except that the incubation is 60 minutes and the tube is centrifuged at 1 800 rpm for 1 minute.

AB serum 3 7 C method (AB-3 7) Prior to the test, 1 volume of supernatant is diluted with 1 volume of 30 % bovine albumin solution. 2 drops of reagent or its dilutions in AB serum and 2 drops of 3 % cell suspension in AB serum are put in a tube. Then, the tube is incubated at 37 C for 60 minutes and centrifuged at 1 800 rpm for 1 minute.

Slide t e s t s

Slide room temperature (S-RT) One drop of supernatant is mixed with one drop of 10 % cell suspension in saline. The slide is then rocked for 3 minutes and the results are recorded as the ~ in tube method ~.

Slide 3 7 C (S-3 7) The method is the same as S-RT except that the slide is prewarmed at 37 C.

Slide albumin 3 7 C (S-A-3 7) Prior to the test, 1 volume of supernatant is diluted with 1 volume of 30 % bovine albumin solution. Then, 1 drop of this reagent is mixed with 1 drop of 40 % cell suspension in plasma. The slide is then rocked for 3 minutes and the results recorded.

HUMAN AND MURINE MONOCLONAL ANTI-RH

177

Flow citometry assay In the first stage, washed RBC are sensitized with supernatant antibodies. 50 Ill of supernatant is added to 50 Ill of 1% cell suspension. The contents of the tube are gently mixed and incubated at 37 C for 30 minutes. Then, the cells are washed 3 times. If during the procedure, some clumps appear, they are systematically broken on a Vortex. In a second stage, 100 Ill of a 1% solution in PBS pH 7,2 of one of these fluorescent antibodies : - FITC conj-goat F(ab') 2 anti-human IgM(Mu ch-sp) TAGO ; - FITC conj-goat F(ab') 2 anti-human IgG TAGO ; is added. The reagents will bind specifically on the anti-D which is complexed to the D antigen of the red cells. After incubation at room temperature for 30 minutes, the RBC are washed 3 times in PBS pH 7,2 and the clumps broken on a Vortex. Assay is performed on a flow cytometer FACSTAR (Becton-Dickinson) equipped with a laser Argon which emits at 488 nm. Only the free RBC are counted up to 10 000 per sample. Then, the results are compared according to their mode and mean on a log plot.

RESULTS Anti-D Specificity Serological tests were performed by standard hemagglutination rections : RT, 37-I, IAT, P-37 and B-37 methods with 20 RBC of different Rhesus phenotypes and also with one LW negative and one Dash phenotype. Whatever the supernatants were, they were specific with the D antigen.

Avidity and intensity Avidity is the value based on the appearance time of the first agglutinates. Intensity is the value based on the agglutination strength after 3 minutes. Serological tests were performed on slide at room temperature or 37 C with a pool of 3 DCcee phenotypes RBC. Table I shows the results.

Titer and score Titrations were performed by IS, RTI, 37-I, IAT and AB-37 with the same RBC as mentionned above. Table II shows the results.

Flow cytometry assay RBC sensitized by the anti-D were the same'as mentioned above. Type as well as results of mode and mean were reported in Table IL Generally the IgM anti-D react in saline. Their titer and score were not enhanced by use of AB serum or antiglobulin serum. As for the IgG anti-D, they only react with the last two reagents except one (13 W 3) which was able to react in saline and its reaction strength on slide was greatly improved at 37 C on the opposite for IgM anti-D.

178

MANNESSIER I~

TABLE I

Avidity and intensity of human monoclonal anti-D.

S

-

RT

S

-

S-A-37

37

NUMBER

INT.

AVID

INT.

AVID

INT.

AVID

6

w

1

+++

8"

+++

5"

NT

NT

6

w

3

-

-

+/-

1 '35"

++

15"

6

w

4

+++

5"

+++

3"

NT

NT

6

w

5

++

25"

++

20"

NT

NT

6

w

6

+/-

2 '40"

NT

NT

6

w

7

NT

NT

+++

10"

NT

NT

++

13"

NT

NT

6 w 8

-

i0

w

1

i0

w

2

10

w

3

10

w

4

10w

-

+/-

+/-

1 '4 5 "

2'20"

+/-

2 '30"

++

25"

5

-

-

+/-

3'

+++

15"

13

w

3

+

16"

+++

9"

++

i0"

16

w

5

-

-

+/-

2 '30"

+++

10"

16

w

6

+

23"

++

20"

NT

NT

23

w

3

-

-

NT

NT

24

w

4

+++

5"

+++

4 "

NT

NT

26

%4 5

++

12"

++

8 "

NT

NT

26

w

6

+/-

2 '3 5 "

+/-

i'

+++

5"

26

w

7

-

-

+/-

1 '40"

NT

NT

26

w

8

-

-

+/-

1 '40"

++

9"

Evaluation/or lY testing A sample was considered to be a Du if it was negative for polyclonal reagent on slide at 37 C and positive when confirmed by the IAT. 57 samples were tested by IS, RTI, 37-I, S-RT with the supernatants 6 W l, 6 W 4 , 6 W 5 , 16W6, 24W4, 26 W 5, 13 W 3 and by S-A-37 and IAT with the following supernatants : 6 W 3, 6 W 8 , 10W2, 10W4, 10W5, 1 6 W 5 , 2 6 W 6 , 2 6 W S a n d 13W3.

179

HUMAN AND MURINE MONOCLONAL ANTI.RH

TABU II Titer and score ( ) according to hemagglutination reactions. Type, mean and mode obtained by flow cytometry assay.

Z

H

~

~

H

~

H

H

H

A

E~

E~

E~

~

H

H

H

H

H

H

H

H

H

~

"~

H

H

A

~

v

H

E~

v

E~

E~

~

E~

v

~

v

v

v

I

~

I

~D

z

~

C0

r-~

E~ Z

~r ~

~ ~

~ Z

E~ Z

E~ Z

E~ Z

~ Z

~

~

~

~

~

~

~

0

0

~

E~ Z

~

~

E~ Z

0

~

v

v

A

A

~o

A 0% P-

A L~

E~ Z

c0

E~ ~

~

E~ Z

~ ~

"~ ~

E~ Z

E~ Z

E~ Z

2

~

~

~

~

~

~

~

~

~

180

MANNESSZF~ L.

Results recorded in Table I I I showed that the supernatant 24 W 4 was the more sensitive to detect D" by slide test. Furthermore, by RT-I it was able to detect all the D" tested. As for the IgG anti-D used by IAT, the following supernatants : 10 W 4, 10 W 5, 10 W 2, 26 W 6 and 6 W 8 possessed the best medium intensity. The titer of these antibodies was 512 by IAT except the 10 W 4 which was weaker.

TABLE IIl Evaluation of monoclonal anti-D for D u testing. The m e d i u m reaction strength is expressed as : +++ scored 3, ++ 2, + 1, + 0,5. MI/S is the ratio of m e d i u m intensity a n d anti-D score by ]AT.

ANTI-D

Total

Du

Total positive reactions ...........................................

Tested

RT-I 37-I S-RT ................................

I-S ....................... 6 w

1

57

34

42

31

12

6 w

4

51

33

34

33

16

6w

5

51

17

25

13

5

I16 w

6

57

13

17

18

2

4

57

55

57

52

38

5

35

23

29

29

8

3

23

7

5

3

0

i 124 w

I 126 w

I LI3 w

[ iA N T I - D

i l

Total D u tested

[Total positive Reactions ........ S-A-37 IAT

Medium

Intensity

MI/SxI00

.............................

I. . . . . .

I 3

41

2

41

1,5

2,0

6 w

8

35

2

35

1,9

2,2

ll0w I Ii0 w

2

57

2

57

2,1

2,4

4

57

3

57

2,2

3,1

5

57

3

57

2,1

2,8

5

57

2

57

1,3

1,6

6

45

4

45

1,9

2,3

8

57

3

57

1,1

1,7

3

35

0

35

1,6

2,2

i 6 w

l I

i

I il0 w

I i16 w

J 126 w

t 126 w

I LI3 w

i

HUMAN AND MURINE MONOCLONAL ANTI-RH

181

Evaluation [or partial and depleted D antigens All antibodies were tested by RT-I, 37-I, IAT, P-37 with 13 partial and 2 depleted D antigens. As for slide tests, S-RT has been used for IgM anti-D and S-A-37 for IgG. Using the classification of ~PPETFand SANCER [3] partial D antigen characteristics are : KHA: D I I I a THO: DI[Ia JEF: D I V a OBA: D V c STR: DVI BOU : Uncharacterized, positive with 100 % of polyclonal anti-D GUE : Uncharacterized, positive with 60 % of polyclonal anti-D LOU : Uncharacterized, positive with 20 % of polyclonal anti-D DEL : Uncharacterized, positive with 35 % of polyclonal anti-D VER : Uncharacterized, positive with 60 % of polyclonal anti-D SIO : Uncharacterized, positive with 20 % of polyclonal anti-D BER : Probably D VI FOU : Uncharacterized, positive with 75 % of polyclonal anti-D Among the 2 depleted D antigens, PER is uncharacterized and PAU possesses the R°Har phenotype which was first reported by GmLES and al. [4]. Results of the tests are recorded in Table IV.

L W negative and Dash phenotypes Supernatants were tested with rhesus positive and LW (a-) RBC. Results were the same as those obtained with Rhesus positive and LW positive RBC. Supernatants were also tested with Dash RBC which possess an enhanced D antigen [2] bu the results were not significant.

Anti-E

Specificity Serological tests were performed by the same methods as anti-D with 20 selected RBC. Antibody supernatants 16 W 7 and 16 W 8 were specific with the E antigen whatever method was used. On the other hand, the antibody 26 W 10 was not sprcific by enzymatic methods and 26 W 15 by B-37.

Titer, score, avidity and intensity All the tests were performed with a pool of 3 ddccEe and 3 DCCEe RBC. According to the type and behaviour of the antibody, the methods used were S-RT or S-A-37 for avidity and intensity and IS, RT-I, IAT, P-37 or AB-37 for titer and score. Results are recorded in Table V.

Flow cytometry assay Assay was the same as for anti-D except that the phenotype was DCcEe. Results are recorded in Table VI.

18 2

~V~SS~F~ L TABLE I V

Evaluation of monoclonal anti-D for partial and depleted D antigens. C o l u m n 1 : RT-L - 2 : 37-I. - 3. : IAT. - 4 : P - 3 Z - 5 : S - R T o r ~-A-37. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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MANNESS1ER L

TABLE V Titer, Score, Avidity andlmensityofthe 4anfi-Eand 1 anfi-c. l ANTIBODY R E D C E L L TYPE] T E S T .......................... i. . . . .

AVIDITY

I 16 W 7

ddccEe

IP-37 fIAT IAB-37 iS-A-37

25"

I DCCEe

IP-37 [IAT IAB-37

I 16 W 8

dccEe

iIS iRT-I IS-RT

8"

I DCCEe

iIS iRT-I

I 26 W 10

dccEe

fIAT IAB-37 IS-A-37

INTENSITY

.....................

30"

l [

TITER

SCORE

J.................

+++ +++ +++ +

128 32 2 NT

65 41 18 NT

+++ +++ ++

64 32 2

53 41 i0

+++ +++ +++

32 256 NT

53 68 NT

+++ +++

32 64

50 63

+++ +++ ++

256 64 NT

76 61 NT

256 8

71 29

8

28

I

26 W 15

DCCEe

fIAT 1AB-37

+++ ++

dccEe

iP-37

++

I DCCEe

IP-37

DCcee

fIAT IAB-37 iS-A-37

++

I 26 W 9

25"

+++ +++ ++

8

28

256 16 NT

71 30 NT

Test with a weakened E antigen Supernatants were tested with a weakened E antigen. The only anti-E able to react on slide is the 16 W 8 which is an excellent antibody.

Antic

Specificity Serological tests were performed by the same methods as for anti-D with 20 selected RBC. The antibody 26 W 9 was specific by RT-I, 37 - I and IAT with the c antigen but by enzymatic methods, false positive reactions were observed.

Titer, score, avidity and intensity Serological tests were performed by AB - 37, IAT and S-A-37 with a pool of 3 DCcee RBC. Results are recorded in Table V.

Flow cytometry assay Assay was the same as for the anti-D and the results are recorded in Table l/Z.

185

H UMAN AND M URINE M ONOCLONAL ANTI-RIft TABLE V I Flow cytometry assay of anti-E a n d anti-c : determination of type, m o d e a n d m e a n .

ANTIBODY LABELLED - ANTIBODY MODE AVERAGE TYPE .......................................................................... 16 w 7

anti-IgG anti-IgM-Mu

3,5 1,0

16 w 8

anti-IgG anti-IgM-Mu

26 w i0

4,6 2,3

IgG

1,0 53

2,3 49

IgM

anti-IgG anti-IgM-Mu

3,5 1,0

8,5 2,2

IgG

26 w 15

anti-IgG anti-IgM-Mu

1,0 1,0

2,2 2,2

undetermined

26 w 9

anti-IgG anti-IgM-Mu

9,0 1,0

9,5 2,2

IgG

.

Antbe F o u r s u p e r n a t a n t s : 19 W 6, 25 W 1, 25 W 2 a n d 29 W 1 were tested. The results s h o w e d t h a t these antibodies did n o t h a v e the specificity anti-e since t h e RBC of following p h e n o t y p e s were agglutinated : D-/D-- a n d D c-/D c-.

Anti-G and anti-Rh The a b s e n c e of i n f o r m a t i v e RBC for these specificities h a s p r e v e n t e d us testing these s u p e r n a t a n t s .

BIBLIOGRAPHY [1] ROUGER Ph., SALMON Ch. - La pratique des Groupes et Groupages Erythrocytaires. Masson, 1981. [2] SAL~ONCh., CARTRONJ.P., ROUGER Ph. - The Human Blood Groups. Masson, USA, 1984. [3] TWPETrP. and SANGERR. -- Further Observations on Subdivisions of the Rh antigen D. Arzd. Lab., 1977, 23, 476-480. [4] GILESC., CROSSLANDJ.D., HAGGAS W.K. and LONGSTERG. - An Rh Gene Complex which results in a ~ new ~ antigen detectable by a specific antibody, anti-Rh 3 t Vox Sang, 1971, 21, 289-01.