US2454752A - Production of antibodies - Google Patents

Description

Patented Nov. 23, 1948 2,454,752 PRODUCTION OF ANTIBODIES? Arthur F. Coca, ;radell. N. J assignor. by mesne assignments. to American Cyanamid Company, New York, N, Y.,, a corporation of Maine No Drawing.

This invention relatesto. the production of a t odi s, more particularly to -the pr p t of blood serum containing agglutinins. of value n th identification and. classification of blood groups and blood types.

It has now been firmly established that the blood of higher animals; contains certain antigenic substances and: antibodies, such as, specifically, agg-lutinogens and agglutini-ns; Early w-ork demonstrated the fact: that human blood contained two distinct hema-agglutinogens which were called A and B: and two hema-agglutinins which were. called anti-A and anti-B. Corresponding agglutinogens and agglutinins do not cot-exist in the blood. of. one. individual. It has been possible, therefore, to.- group: the blood of human beings into; one of four classes, namely, group- A, which blood: contains the, agglutinogen A and the agglutinin anti-B; group 13, containing agglutirrogen B and agglutinin. anti-A; group AB, which blood contains agglutinogens A and B but no, agglutinins; and group 0., whi-ch contains no agglutinogens but agglutim'ns anti-A and anti- B. The agglutinogens are found in; the erythros cytes; andtissue cells: whereas the agglutinins are found in theserum...

When the blood of. a donor of group Ais-introduced. into the blood stream of an individual of group B" or 0, during a, blood transfusion the agglutinin anti-A of the groupB, or 0, blood will agglutinate or. hemolyze the erythrocytes of. the donor blood: and a serious accident may' occur. Similarly, hemolysis may occur: in the-event that the donor hasblood or group B and the recipient has bloodof group A 017-0, Since the blood serum of; blood of group Q contains bQth-s agglutinins anti-A and anti B itwould: appear that blood of group. 0- could not beused; as donor blood in transfusions to; patients of" groups A-, B, or AB. However; as. thevolumeot blood; donated. is relatively scall and as? the recipientuhasa substantial proportion of. the: substance; A on; B, in his; body tissueszin. addition, to-that. in. his; b1ood';,neutraliization of the anti-A or anti-B takes place in most cases without seriousd-ifliculty.

Although the relative distribution. of the agglutinins anti-A, and anti-B.- varies somewhat. with racial. groups. it. hasv been. found that. the blood of. approximately 42% of human beings in the United States contains; the: agglutinin anti-B, 10.9% of; human beings have' blood containingthe ag lutinin anti-A, and; approximately 45%. have blood with both anti -Al and, anti-13.. It isapparent, therefore, that, it. is ot considerable-practical importance to be: able toldetmmine ra idly and Application December 20, 1944, Serial No. 569,112.

11. Claims. (Cl..167'78) accurately the blood groups of individuals so that blood transfusions may be given when needed without unnecessary delay and without danger of untoward reactions.

Other antigenic factors, such as M and N, have been reported but. as these antigens do not have the ability to cause isoimmunization they are not considered of significance in blood transfusions. They are important, however, in blood typing in determining parentage in medico-legal cases. A group of miscellaneous, unidentified antigens has been: designated as group X, but this group, too, is usually disregarded. in grouping blood for transfusions.

Withirr thevpast few years another important antigenic substance has been discovered in human blood This factor was originally discovered in: the bloodof the Rhesus monkey and has been designated: at the Rh factor. This antigenic substance has the abiilty to cause isoimmunization and is, therefore, an important factor to be taken intoconsideration when blood transfusions are to. be made. Approximately 87% of the individuals in this country have been found to have the Rh" factor in their blood whereas the remaining 13% are Rh negative. Since the Rh antigen is an agglutinogen, it will. give rise to the production of agglutinins and there is the grave possibility that those individuals who have Rh negative blood may become isoimmunized against the 'Rhfactor and may, during the course of a subsequent. blood transfusion from a Rh positive donor, suffer a reaction which may be fatal.

It has been suspected for sometime and now seems to be fairly well established that most cases oil erythroblastosis fetalis are caused by isoimmunization. For example, the mother may boot the lit-h negative type whereas the fetus is, through heredity, Rh positive. Because of some defect in the placenta, someof the fetal blood may enter the blood stream, of the mother, giving rise to anti-Rh;agglutininswhich then pass back into the fetus to cause hemolysis of its red; blood cells.

The mothers blood in such circumstances may contain antirRh agglutinins, and in the event that she requires ablood transfusion and if the blood donor is Rh positive, a severe accident may It, is obviously necessary, therefore, to provide-a rapid and effective method of determining the'bloodtypes Rh and anti-Rh.

Methods ofgrouping blood of the A, B, AB and 0 types havebeen developed and used for some time. Unfortunately, the production of typing serum isnot arr-easy matter and the product has m t been of? the potency desired. The typing anti-Rhserum and blood be placed in a warm water bath at 37 C. for about an hour, then centrifuged, resuspended if necessary and the results determined with the aid of a hand lens.

It isquite apparent that this delicate test is not I suitable for general use. 7

In the production of the testing sera it is necessary to obtain a supply of serum containing agglutinins of the proper type, anti-A, anti-B, anti- Rh, or the like, but free from undesirable agglu.

tinins which might interfere with the test. For example, grouping serum for testing for blood of Group A should contain the agglutinin anti A, but none of anti-B, anti-Rh or anti-X. To obtain such serum it is first necessary to obtain a supply of blood with the proper agglutinin in it. This may be accomplished by injecting the appropriate antigen into the blood stream of a warm-blooded animal and, after agglutinins have been formed, bleeding the animal.

To obtain blood serum containing anti-A it has been the practice to inject human A cells into one of the higher animals, preferably a rabbit. During the course of a week or two the rabbit develops the agglutinin anti-A and its blood may be withdrawn and the serum used in the production of blood typing substance. Unfortunately, human erythrocytes also contain other antigenic substances which give rise to the production of corresponding agglutinins in the rabbits blood stream. These antigens, or agglutinogens, are known collectively as factor X and it happens that the X antigens are more potent than the A antigens in stimulating the production of antibodies. As a result, the relatively strong antigen X competes unfavorably with the antigen A in the immunization and the rabbit blood contains a relatively high proportion of the agglutinin anti-X and less than could be desired of anti-A. In most animals, and in fact in most of the rabbits used in this method of producing agglutinins, the yield of anti-A, and also anti-B and anti-Rh, is too small to be of any use. When the rabbit blood contains a suitably high potency of anti-A, it may be used only after removing the anti-X agglutinin. This is usually accomplished by a process called absorption, or neutralization, in which the anti-X factor in the rabbit serum is absorbed by the X factor in blood cells and are thus removed from the fluid. It has usually been considered necessary to first dilute the serum with ten to twenty volumes of saline and then mix ten to twenty volumes of blood of group O with each volume of the immunized rabbit serum. As will be seen, the production of blood grouping sera requires the use of excessive amounts of blood and usually fails to give a product of the potency desired,

I have discovered that blood grouping sera containing agglutinins in much higher potency than those previously thought possible can be obtained by the process to be'described and claimed herein which depends upon the principle of blocking out undesirable antigens from the blood material used in creating agglutinins of the desired type. For example, to produce blood grouping serum containin a relatively high agglutination power for blood of any desired group I employ in the immunization blood cells in which the undesirable, antigenic substances which might interfere are rendered impotent by neutralization or otherwise to produce antibodies.

As pointed out before, it is the usual practice to inject rabbits with blood containing the agglutinogen A, or B, with the expectation that the agglutinin anti-A, or anti-B, will be produced.

This method does not take into account the disturbing influence of the more potent agglutinogen X which simultaneously causes the production of anti-X in the rabbit blood to the detriment of the production of anti-A, or anti-B. To overcome this detrimental action, I first block out or inactivate the antigenic substance or substances collectively known as X. This is done by treatment of the A blood with a suitable blocking serum containing suflicient anti-X to neutralize the factor X. The resulting blood cells with factor X relatively inactive but containing factor A, or B as the case may be, may then be injected into rabbits or other suitable warm-blooded anifuels with the result that a much higher production of anti-A, or B, is obtained in the blood stream of the immunized animal. Blood from the so immunized animal may then be recovered and processed to obtain serum having a much greater potency of anti-A, or anti-B, but containing greatly reduced amounts of other interfering agglutinins.

To obtain suitable blockin sera for use in the process of the present invention, I inject rabbits or other warm-blooded animals with washed erythrocytes of human blood of group 0. After the lapse of a suitable period of time the agglutinin anti-X is developed in the blood stream of the animal and may be withdrawn and processed to obtain serum with a relatively high concentration of anti-X. When this serum is mixed with normal blood containing erythrocytes having the agglutinogens A or B or X, the agglutinogen X is neutralized, leaving the agglutinogens A or B in practically undiminished potency.

When preparing blood sera for testing for the Rh factor, in accordance with the present invention, the same general principle described above is applicable. It is necessary, however, to make provision also for blocking out the agglutinogens A and B. Accordingly, the blocking serum that is produced will contain the agglutinins anti-A, anti-B; and anti-X so that the blood substance used in injecting animals to bring about the formation of anti-Rh contains no other important agglutinogen other than the factor Rh.

In order that my invention may be better understood, the following examples are given which illustrate in detail a preferred process of obtaining blood grouping sera for the groups A, B and Rh. It will be understood, of course, that these examples are given primarily by way of illustration and are not intended to limit'my invention to the exact procedure and details described.

Example 1 The following example is directed to the production of serum containing a high potency of the agglutinin anti-A. As a preliminary step, suitable blood blocking serum is first prepared. This is ,done by obtaining a supply of human blood cells of group O which cells contain the agglutinogen X but do not contain the agglutinogens Aand B. The erythrocytes are washed twice with an equal volume of sterile, physiological saline. Healthy rabbits are then givenlan ensure intravenous injection of cc. of the washed cells in 1 cc. of saline solution. After intervals of three or four days the rabbits are again injected with the washed cells as before. These injections are repeated for a total of three to five times. Six or seven days after the last injection the animal is bled and the serum recovered. The resulting blood serum will be found to contain a relatively high concentration of the agglutinin anti-X but with none of the agglutinins anti-A or anti-B. The filtered serum may be frozen and stored for some time without preservatives until ready ior use.

Antigenic substance for the production of anti-A is pre ared by obtaining a supply of erythrocytes of group A. For ordinary blood grouping Work pooled group A blood is preierred as it contains both of the sub groups A1 and A2. When preparing blood grouping reagents specific to groups A1, A2, etc. blood from these groups is used. The sterile erythrocytes are washed twice with an equal volume of sterile saline in a centrifuge. To the Washed cells is added an equal volume of the blocking serum prepared as described above and two volumes of saline. Ordinarily it will be found that this amount of blocking serum saturates the X antigens which may be present in the group A cells. ()bviously, more or less of the blocking serum may be used, depending upon the relative amounts of factor X in the group A cells and the amount of anti-X in the blocking serum. As the A cells do not absorb all of the anti-X, it is usually necessary to add more than the theoretical amount of the anti-X than the antigen X present would indicate.

Healthy rabbits are then given an intravenous injection with 2 cc. of the antigenic A blood substance just described as a first sensitizing dose. After about five days the rabbits are again given a subcutaneous injection of 2 cc. of the mixture. Two hours later they are given a 2 cc. intravenous injection of the mixture. The subcutaneous and intravenous injections are repeated three times at three to five dayintervals.

The animal is then bled 50 cc. one week after the last injection.

Occasionally some rabbits will not produce antibodies in the desired manner and such animals are eliminated from further use. The remaining rabbits may be given subsequent injections with subsequent bleedings until they have lost their value for such production.

The resulting blood will be found to contain a relatively high potency of the agglutinin anti-A and relatively low potencies of anti-X or anti-B. If these latter aggiutinins are found to be presout, they may be removed from the undiluted serum by absorption with three or four volumes of a mixture of B and 0 cells by treatment at room temperature with constant but slight agitation for about fifteen minutes. The blood serum containing anti-A may then be recovered and assayed for potency by methods known to those skilled in the art.

After determination of the potencyof theserum it is ready for. use. It is usually. advisable to adjust the titer of the serum to a fixed value so, that comparable results may be. obtained when using the product for testing for group A. This may be accomplished by diluting the serum with saline to a given potency. When it is desired to prepare the product in the form of a powder for storage and use, sucrose, sodium chloride, or some other inert diluent which has no deleterious died on. th a glut nat on o blee m be d 6 eltherj before or after the product is dried. Dyestuffs may also be incorporated in the mixture to serve as a means of identification apart from the label on the product. The usual method of drying is to first freeze the product and then evaporate the Water therefrom while maintaining the product in a frozen state in the manner lgnown to those in the art. The dried product .may then be pulverized and is ready for use.

same 2 Ezcample 3 This example describes the production of anti- Rh serum. Goats are used as the immunizing animal since they have been found to be particularly suitable for the production of anti-Rh serum of high otency.

As in the preceding examples, preliminary steps involve the production of an immunizing substance in which the antigenic material of the desired type predominates-in this case the agglutinogenic substance Rh. This anti-genie substance is obtained by treating the blood cells of the Rhesus monkey with agglutinins which neutralize the antigenic substances other than the factor Rh which are found in the erythrocytes of the Rhesus monkey. As this kind of blood contains the agglutinins A, B and X, the blocking serum will have the agglutinins anti-A, anti- B, and anti-X.

Suitable blocking serum for the production of anti-Rh is prepared by obtaining a quantity of human erythrocytes which are positive for factors A and B but preferably negative for factor Rh. These blood cells are Washed three times with sterile, normal saline and are then resuspended in saline, each 1 cc. of cell sediment being suspended in 4 cc. of the saline solution. The suspension is then placed in a water bath for forty minutes at 5660 C., then removed, and

5. cc. of distilled water'for each cc. of the cell suspension is added. After holding at room temperature for hour, 2.06 cc. of 10% sodium chloride is added for each cc. of the cell suspension to give a product containing 1% of sodium chloride. The suspension is then centrifuged, the. supernatant liquid removed and the sediment, which is stroma, is recovered.

Healthy goats are then given a subcutaneous injection of 10, co. in each hind leg of an immunizing substance containing 40 cc. of the stroma described above containing approximately, equal parts of B-Rh negative cells and A-Rh negative cells, 160 cc. of mineral oil, 40 cc. of Falba base (lanolin derivative) and a very small amount of a phenol mercuroborate preservative. The injection is repeated after ten days. After another ten days the goat is given a subcutaneous injection of 3 cc. of the stroma in 3 cc. of saline solution. Four. hours later the animal is given an intravenous injection of 1.5 cc. of the stroma in 3- cc. of saline. After three days the sub cutaneous and intravenous injections arev repcated. Seven days after the last injection thev animal may be bled to obtain a suitable blocking serum.

Rhesus monkey cells are washed three times with sterile, physiological saline solution and then saturated with the blocking serum just described.

Enough of the blocking serum is added so that the supernatant liquid contains a small excess of anti-A, anti-B, and anti-X. The mixture of blocking serum and monkey cells is then allowed to stand in an ice box overnight after which the supernatant liquid is removed and the cells are frozen. On thawing, the mass becomes fluid and easily injectable. Each goat is then injected five times at ten day intervals intramuscularly with about 63 cc. of a mixture of cc. of the saturated monkey cells, 25 cc.-of aluminum cream, and 13 cc. of saline.

Following the immunization, the animal is bled and the serum recovered. Serums having a high potency of agglutinins effective against the Rh factor may be used directly in blood typing.

moving traces of anti-A, anti-B and anti-X by treatment with normal animal blood containing factors A, B and X as described in Example 1. The purified serum may be diluted and used in blood typing or may be first dried for more convenient handling. If the product is dried, it is usually desired to dilute it with an inert solid diluent which does not effect the agglutination test as in Example 1.

To use this more potent Rh agglutinin in typing blood it is merely necessary to prepare a mixture of two drops to the blood to be tested with onehalf cc. of saline and place one drop of the cell suspension on a glass slide with an equal drop of the typing serum just described. The two drops are mixed and spread out on the slide. Within five minutes a strong clumping action will be easily observed with the naked eye .if the blood being tested is Rh positive. Obviously this is a considerable improvement over the Rh test now' in use.

Blood typing reagents specific to types M and N are prepared exactly as in the preparation of the grouping reagents A and B with the exception, of course, that OM or ON cells are used in immunizing the rabbits, the X factor being blocked out in the same way with anti-X.

I claim:

1. The method of producing blood serum containing a high titer of a specific hemagglutinin, the steps of which comprise obtaining blood substance having hemagglutinogenic properties, mixing therewith hemagglutinins effective against the hemagglutinogenic substances other than those of the desired specific hemagglutinin whereby the undesired hemagglutinogenic substances are rendered substantially impotent to stimulate the production of antibodies, introducing into the blood stream of a higher animal an effective amount of the thus treated hemagglutinogenic substance, allowing the formation of corresponding hemagglutins to take place and thereafter removing at least part of the blood of said animal to obtain blood serum containing the desired hemagglutinin.

2. The method of producing blood serum containing the agglutinin anti-A the steps which comprise mixing with a blood substance having the agglutinogenic substances A and X a blood substance containing anti-X in sufficient amounts to substantially neutralize the agglutinogenic factor X present in said blood substance whereby the agglutinogenic substances X comprise mixing with a blood substance having containing anti-A.

I 3. The method of producing blood serum containing the agglutinin anti-A the steps which the agglutinogenic substances A' and X a blood substance containing anti-X in sufficient amounts to substantially neutralize the agglutinogenic factor X present in said blood sub- U stance whereby the agglutinogenic substances X and thereafter removing at least part of the blood of said animal to obtain blood serum containing anti-A.

4. The method of producing blood serum containing the agglutinin anti-B the steps which comprise mixing with a blood substance having the agglutinogenic substances B and X a blood substance containing anti-X in sufiicient amounts to substantially neutralize the agglutinogenic factor X present in said blood substance whereby the agglutinogenic substances X are rendered substantially impotent to stimulate the production of antibodies, introducing into the blood stream of a higher animal an effective amount of the thus treated agglutinogenic substance, a1- lowing the formation of anti-B to take place and thereafter removing at least part of the blood of said animal to obtain blood serum containing anti-B.

5. The method of producing blood serum containing the agglutinin anti-B the steps which comprise mixing with a blood substance having the agglutinogenic substances B and X a blood substance containing anti-X insufficient amounts to substantially neutralize the agglutinogenic factor X present in said blood substance whereby the agglutinogenic substances X are rendered substantially impotent to stimulate the production of antibodies, introducing into the blood stream of a rabbit an effective amount of the thus treated agglutinogenic substance, al-

lowing the formation of anti-B to take place and thereafter removing at least part of the blood of said animal to obtain blood serum containing anti-B.

6. The method of producing blood serum containing the agglutinin anti-A the steps which comprise introducing into the blood stream of a rabbit erythrocytes of blood of group O and after a suitable concentration of agglutinin anti-X has been formed therein removing at least part of said blood, mixing a sufiicient quantity of the anti-X substance thus obtained with erythrocytes of group A to neutralize the antigenic substance A contained therein, introducing into the blood stream of a rabbit an amount of the thus treated erythrocytes of group A sufficient to produce an immunizing effect, allowing the formation of anti-A to take place in the blood stream of the rabbit and thereafter removing at least part of the blood of said rabbit to obtain blood serum having a relatively high titer of anti-A.

taining the agglutinin anti-B the steps which comprise introducing into the blood stream of a rabbit erythrocytes of blood of group O and after a suitable concentration of agglutinin anti-X has been formed therein removing at least part of said blood, mixing a sufficient quantity of the anti-X substance thus obtained with erythrocytes of group B to neutralize the antigenic substance X contained therein, introducing into the blood stream of a, rabbit an amount of the thus treated erythrocytes of group B suflicient to produce an immunizing effect, allowing the formation of anti-B to take place in the blood stream of the rabbit and thereafter removing at least part of the blood of said rabbit to obtain blood serum having a relatively high titer or anti-B.

8. The method of producing blood serum containing the agglutim'n anti-Rh the steps which comprise introducing into the blood stream of a higher animal the agglutinogenic substances A, B, and X and, after a suitable concentration of anti-A, anti-B, and anti-X has been formed in the blood stream of the animal, removing at least part of said blood, mixing a sufficient quantity of the antibodies thus obtained with Rh erythrocytes to neutralize the antigenic substances A, B and X therein, introducing into the blood stream of a higher animal an amount of the thus treated Rh erythrocytes sufficient to produce an immunizing effect, allowing the formation of anti-Rh to take place in the blood stream of the animal and thereafter removing at least part of the blood of the said animal to obtain a blood serum having a relatively high titer of anti-Rh.

9. The method of producing blood serum containing a high titer of the agglutinin anti-Rh the steps which comprise preparing a blocking serum containing the agglutinogens A, B and X, mixing said blocking serum with erythrocytes of the Rhesus monkey to neutralize the antigenic substances A, B, and X therein, introducing into the blood stream of a goat an amount of the thus treated Rhesus erythrocytes in which the antigenic substances A, B and X have been rendered substantially impotent to stimulate the production of antibodies, allowing the formation of anti- Rh to take place in the blood stream of the goat and thereafter removing at least part of the blood of the goat to obtain blood serum having a relatively high titer of anti-Rh.

10. The method which comprises injecting into a warm blooded animal agglutinogens of group X and after agglutinins of group anti-X have been formed withdrawing at least part of the blood of said animal, mixing the thus produced anti-X with normal erythrocytes to neutralize the agglutinogens of group X therein and injectin the resulting agglutinogenic substance into a warm blooded animal whereby a high titer of agglutinins is produced in the blood serum of the animal.

11. The method which comprises injecting into a warm blooded animal agglutinogens of the group X, A, and B and after agglutinins of the group anti-X, anti-A, and anti-B have been formed withdrawing at least part of the blood of said animal, mixing the thus produced anti-X, anti- A, and anti-B with Rh erythrocytes to neutralize the agglutinogens X, A, and B therein and injecting the resulting agglutinogenic substance into a. goat whereby a high titer of Rh agglutinins is produced in the blood serum of the goat.

ARTHUR F. COCA.

REFERENCES CITED The following references are of record in the file of this patent:

Morgan, An Artificial Antigen with Blood- Group A Specificity, in The British Journ. of Experimental Pathology, vol, XXIV, No. 2, April 1943, pages 41-49.

Potent Typing Sera article by Witebsky et al. in Proc. Soc. Exptl. Biol. 8: Med, March 1944, pages 167 to 170*.