III. Inactivation by Heat of the Isoagglutinins in the Blood of Cattle with Reactivation by the Addition of Fresh Cow Serum
Authors: Little Ralph B. 
Published: December 2022

Discussion and Summary
In this as in the preceding studies on the isoagglutinins of bovines, the agglutinable substance has been called an isoagglutinin. This agglutinative substance has been recognized by other workers and given the same name. The repeated examinations of a large number of individual bloods revealed certain phenomena not encountered in the grouping of the blood of man. The characteristic mass agglutination of the corpuscles by bovine sera and the rapid deterioration of the agglutinins together with reactivation suggested that this agglutinin differed considerably from the agglutinative substance present in human blood. With the use of a method already described (1) it has been possible to divide the blood of bovines into certain groups. The agglutination reactions limited to one group were characteristic and occurred regularly.
However, when the sera were heated at 60°C. for one hour the agglutinative substance was inactivated. A temperature of 56°C. for one hour did not completely inactivate all sera. With certain bloods at this temperature a weak agglutination was frequently demonstrated. When Group I serum was heated at 60°C. for one hour one component of agglutination was destroyed. The agglutination reaction probably depends on the coöperation of several components all of which are necessary to complete the phenomenon. If it is assumed that A represents one component which is thermostable, and B the other component which is thermolabile, with the addition of the normal unheated serum component B was again restored to the heated sera. The agglutination which followed the addition of the reactivating serum with few exceptions was as complete as before the serum was heated. In each reactivation experiment the unheated sera were examined with agglutinable corpuscles to determine the normal agglutination for each blood used. When reactivating sera failed to agglutinate or partially clumped, the same fresh suspensions were generally completely agglutinated when agglutinating serum heated was included in the combination. The reaction then was generally complete, whereas the reactivating sera when used alone only weakly clumped or failed altogether to agglutinate the corpuscles.
When unheated fresh serum of Subgroup IIIa was stored in the refrigerator for one hour with agglutinable corpuscles and then centrifuged clumping did not take place. When heated Group I serum was added later to a suspension of these same cells which had been washed three times in normal saline the reaction was also negative. If the same operation was reversed and the corpuscles were first treated with the heated serum the agglutinative substance was absorbed. The addition of fresh serum to washed cells so treated resulted in a complete agglutination. These observations suggest that the reactivating blood contained a normal concentration of component B but an insufficient amount of component A.
It has been frequently mentioned that in one group of cattle the agglutination reactions varied at different periods. The protocols of some of these cows have been submitted, so that it suffices here to mention that with each serum the agglutinable corpuscles at some period had been clumped one or more times. It has also been shown (1, 3) that in the blood of certain cattle under natural conditions the isoagglutinins become depleted. With other individuals (Cow No. 7) the agglutination may be strongly positive after negative examinations.
So far as this work has progressed the capacity to reactivate was confined to one small group of bloods. In each examination Group II sera failed to reactivate even when the quantity of serum was increased from 0.5 to 1 or 2 cc.
The most logical explanation for this peculiar phenomenon is to consider that potent Group I serum (heated) was reactivated by the addition of a masked Group I serum in which the agglutinative properties had become reduced, for in typing blood samples it was often difficult to decide whether to place them into Group I or III. If after repeated examinations it was found that negative occurred more frequently than positive agglutinations the cattle were placed in Group III. There exists a close similarity in agglutinative properties of the blood of Subgroup IIIa and Group I. Thus the sera of Group IV would occasionally agglutinate the corpuscles of Groups I and III. In later examinations it was found that the Group III bloods agglutinated by these sera were confined to this special type, namely Subgroup IIIa.
The fact that Subgroup IIIa sera occasionally agglutinate washed corpuscles more vigorously than corpuscles prepared from the clot indicated that some inhibitory substance was removed by washing. Group I sera, however, showed no preference ofthis kind. The cells prepared from the clot were agglutinated as completely as were the washed cells. The serum of one cow (no. 32) in each recent examination has failed to agglutinate any corpuscle suspension regardless of the method of preparation. Heated sera were always reactivated as well with this serum as with any other. It may be significant here to mention that when two different reactivating sera were mixed in equal amounts with agglutinable corpuscles, the reaction was no more marked than when each serum was used separately.