PRELIMINARY REPORT ON A SUBSTANCE WHICH
INHIBITS ANTI-Rh SERUM*
BETTINA B. CARTER, M.S.
From the Institute of Pathology, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania
Witebsky and Klendshoj, 8 Hallauer,6 Landsteiner, 7 Goebel5 and others have
isolated blood group specific substances of carbohydrate nature from various
sources, i.e., gastric juice, red blood cells, saliva and commercial peptone.
Belkin and Wiener1 have demonstrated the property Rh in the stromata of red
blood cells and have used the stromata as antigen. Gallagher and Pillischer4
have used stromata from group O, Rh-positive cells to immunize guinea pigs
to the Rh factor. Calvin and co-workers2 have obtained from stromata material
which they consider to be a lipoprotein, called elinin, which has Rh specificity.
All of these workers with Rh used physical methods for separating the active
substance.
It is possible by chemical means to obtain a substance which completely
inhibits high-titered standard human anti-Rh serum. This substance is produced as follows: pooled, washed, packed, group 0, Rh-positive cells are shaken
at 4 C. with an equal volume of distilled water in order to lake the cells. Five
volumes of 95 per cent alcohol are added to the laked cells in order to precipitate
the protein. The flask containing the material is rotated for thirty minutes
and allowed to stand at 4 C. over-night. The red, powdery precipitate is separated from the alcohol by filtration and allowed to dry at 4 C ; 5 volumes of
anesthesia ether are added to this powder. The flask is rotated for thirty
minutes and kept at 4 C. Extraction continues for eight days, with thirty
minutes of rotation daily. At the end of this period, the red powder is separated from the ether by filtration and discarded. The ether filtrate is evaporated with the aid of an electric fan. One six-inch Petri plate is filled with the
filtrate and refilled as soon as evaporation will allow. Since the amount of
inhibiting substance obtained is small, it is desirable to make all evaporations
in the same container.
Complete evaporation of the ether leaves a white to cream colored lipid
material. A volume of 800 cc. packed cells yields approximately 300 mg. of
the substance. The material is soluble in 95 per cent alcohol, acetone, xylol
and chloroform. It seems to be closely associated with the blood cholesterol
since it gives positive Liebermann-Burchard and Salkowski's tests. A 1:5
dilution of an anti-Rh standard human serum with a titer of 4 plus in a 1:600
dilution is completely inhibited when this substance is present in a concentration of 0.25 per cent. Some lots have yielded material which would inhibit in
a concentration of 0.0125 per cent. This substance does not inhibit hightitered A and B serums.
* Received for publication, April 28,1947.
646
ANTI-Rr INHIBITING
SUBSTANCE
647
The method used for testing inhibition is as follows: 50 mg. of the substance
is dissolved in 5 ml. 95 per cent alcohol by shaking gently in a water bath at
56 C. One ml. of this alcoholic solution is mixed rapidly with 1 ml. of 0.85 per
cent saline by pouring the mixture back and forth from one vial to another.
Equal volumes of this suspension and the Rh serum, are combined. The usual
practice is to allow the mixture of serum and suspension to incubate at 37 C.
for fifteen minutes, but tests show that the inhibition takes place at once. Controls are set up by combining equal volumes of 50 per cent alcohol in saline and
the same anti-Rh serum. This mixture is incubated simultaneously with the
combination of inhibitor and serum. The inhibited serum and the control
serum are tested against a series of group 0 , Rh-positive cells, as shown below
in the usual method used for determining Rh sensitization.
Cells
Serum with inhibitor..
Control serum
ORh+ ORh+ ORh+ ORh+ ORh+ ORh+ ORh+ ORh+ ORh+ ORh-
+-H-+
++++ ++++ ++++ ++++ ++++ ++++ ++++
The cells used are 2 per cent suspensions, each from a different donor. The
serum, before combining with inhibitor or with 50 per cent alcohol in the control, is diluted 1:5. The inhibited serum and the control serum are tested
by adding two drops of the serum for each test to two drops of cell suspension
in saline. The tests are incubated for thirty minutes at 37 C.
In order to be sure that the inhibition was not merely a physical adsorption
of antibody on lipid particles, the suspension of alcoholic inhibitor in saline was
centrifuged. The supernatant fluid and the particles resuspended in saline
were each used in equal volumes with the anti-Rh serum to see if either alone
would inhibit. The supernatant fluid inhibited the serum; the resuspended
particles did not. Also the inhibition tests as outlined above were set up using
cholesterol (Eimer and Amend, C.P.) in place of inhibitor in the same concentration. No inhibition took place.
Twenty-four guinea pigs were inoculated intraperitoneally and two rabbits
were inoculated intravenously with a 10 per cent suspension of this lipid material. Twenty-four pigs were inoculated with 10 per cent suspensions of human
group O, Rh-positive red blood cells as controls. Inoculations were made intraperitoneally and the methods previously described elsewhere,3 of injecting on
alternate days for a total of six inoculations, were followed for both red blood
cells and lipid suspension. Each of the control animals produced agglutinins
for group O, Rh-positive cells; the lowest titer was 1:160. None of the animals inoculated with inhibitor substance produced antibodies to human red
blood cells. However, when equal volumes of 10 per cent lipid suspension in
50 per cent alcohol and 10 per cent egg albumen (Eimer and Amend, soluble
scales) in saline were injected intraperitoneally in the same manner, five out
of the twenty-four guinea pigs inoculated developed agglutinins demonstrable
in 1:10 dilution for group O, Rh-positive cells. In each of the 72 animals.
inoculations of 2 ml. of the appropriate material were made on alternate days
648
CARTER
until a total of six injections had been given. Bleedings were made from the
heart one week after the final inoculation. Each of these pigs was tested before
the series of inoculations was begun to make sure that the normal blood contained no agglutinins for group O, Rh-positive cells. None of the normal
pigs showed agglutinins.
As a third check on the specific nature of this lipid inhibitor, tests were carried
out to determine whether a suspension of this substance plus human standard
anti-Rh serum would fix complement. Extensive experimentation was necessary to determine the antigen dilution range, but finally it proved possible to
demonstrate complement fixation with the Rh inhibitor as antigen. In the
lot under investigation, 0.5 ml. Rh inhibitor at a 1:2000 dilution with human
anti-Rh serum, 0.05 ml. in 0.5 ml. dose, will fix two full units of complement to
produce a 4 plus reaction. The complement-fixation tests were set up exactly
as in a quantitative Kolmer procedure for the serologic test of syphilis, using
carefully titrated lyophilized complement and high-titered amboceptor. Serum
controls were negative and, as an additional control, normal human serum was
set up in place of the anti-Rh serum in an identical quantitative procedure and
tested simultaneously. This control was invariably negative.
Calvin and Behrendt 2 found their elinin fraction to be thermolabile, but
mentioned an ether-soluble material obtainable only occasionally which was
thermostable. The activity of our ether-soluble fraction is resistant to heating
at 56 C. It is probable that the presence of protein renders the elinin susceptible to heat. When the protein is absent, the inhibitor substance resists
inactivation.
It would seem logical to assume that the ether-soluble inhibitor of Rh antibody is hapten. Apparently, in itself, it is incapable of stimulating the production of antibody, but in company with a protein "carrier", Rh antibody is
produced. The possibility of making use of Rh hapten in the desensitization
of Rh sensitized mothers or in the prevention of sensitization is alluring to many
investigators. Work of this type is under way now and will be reported at a
later date.
In the process of obtaining inhibitor from red blood cells, it is desirable to
save the initial alcoholic filtrate. Some of the inhibitor may be obtained from
this by concentration under vacuum.
SUMMARY
A fraction has been separated from group O, Rh-positive cells which is nonantigenic in experimental animals, but which is antigenic when injected simultaneously with a protein carrier. This substance, which is lipid in nature,
specifically inhibits the agglutinins present in anti-Rh serum. Also it will
fix complement in conjunction with human anti-Rh serum. It resists inactivation by heat and is probably Rh hapten in impure form.
REFERENCES
1. B B L K I N , R U T H B . , AND W I E N E R , A L E X A N D E R S.: D e m o n s t r a t i o n of t h e properties, A,
B , M, N , and R h in red-cell s t r o m a t a .
1944.
Proc. Soo. Exper. Biol, and Med., 56: 214-217,
ANTI-Rr INHIBITING SUBSTANCE
649
2. C A L V I N , M E L V I N , E V A N S , R O B E R T S., B E H R E N D T , V E K A , AND C A L V I N , G E N E V I E V E :
Rh
antigen and h a p t e n . N a t u r e of antigen a n d i t s isolation from erythrocyte stroma.
Proc. Soc. Exper. Biol, and Med., 61: 416-419, 1946.
3. CARTER, BETTINA B . : T h e production of R h antiserum in guinea pigs through inoculation with human red blood cells. Am. J . Clin. P a t h . , 15: 278-279, 1945.
4. GALLAGHER, F E E D W., AND P I L L I S C H E E , G E O E G E P . : E x p e r i m e n t a l production of a n t i - R h
sera by t h e use of human erythrocyte s t r o m a t a , Science, 105: 344-345, 1947.
5. GOEBEL, WALTHER F . : T h e isolation of t h e blood group A specific substance from commercial peptone. J . Exper. Med., 68: 221-227, 1938.
6. HALLAUER, C : Weitere Versuche zur Isolierung wasserloslicher Gruppenstoffe aus
menschlichen E r y t h r o z y t e n . Ztschr. f. Immunitatsforsch. u. exper. T h e r a p . , 83: 114123, 1934.
7. LANDSTEINER, K . : Note on t h e group specific substance of horse saliva. Science, 7 6 :
351,1932.
8. W I T E B S K Y , E R N E S T , AND K L E N D S H O J , N I E L S C.: T h e isolation of an O specific substance
from gastric juice of secretors and carbohydrate-like substances from gastric juice
of non-secretors. J . Exper. Med., 73: 655-667, 1941.