The development of reliable immunologic compositions useful as reagents in immunoassays to determine the presence or absence of antigens or antibodies in a test fluid has and continues to be a sought after goal. One approach which has received a great deal of attention has been the development of reagents for hemagglutination and hemagglutination inhibition systems. These systems employ red blood cells also known as erythrocytes which, for use, are joined with antigenic or antibody substances so as to provide an indicator system which can be used to detect the homologous antibody or the antigenic substance itself.
An early development in hemagglutination inhibition or passive hemagglutination tests was described by Wide and Gemzell, Acta Endocr. 35(1960) 261. They established that the red blood cells (Rbc) coated by or attached to the antigen must be stabilized to be useful hemagglutination inhibition tests, otherwise the red blood cells would need to be fresh daily. While Wide et al. stabilized the erythrocytes before attachment of the antigen, others have stabilized following attachment of the antigen.
Stabilization of Rbc was originally described by Boyden using formaldehyde, J. Exp. Med., 93, 107 (1951) and the concept was extended by Wide, Acta Endocr., supp. 70, 41(1962), when following pretreatment of formaldehyde treated RBC with tannic acid, it was shown that hCG could be absorbed onto the cell surface of the Rbc.
Ling, Brit. J. Haemat., 7, 229(1961), also showed the pyruvic aldehyde could beneficially replace formalin and that pretreatment with tannic acid was not necessary for antigen attachment. However for stabilization, Ling decreed that 48 hours at +4.degree. was necessary. But, the coated erythrocytes are unstable and are also time consuming to prepare.
One approach to improving stability and also of obtaining erythrocytes with higher hemagglutination titers were disclosed in U.S. Pat. No. 3,714,345; 3,715,427 and 3,925,541. There the erythrocytes were treated sequentially with pyruvic aldehyde and then formaldehyde for periods in excess of 12 hours for treatment prior to coating the thus stabilized erythrocyte with an antigen or antibody. The double aldehyde treated erythrocytes were in some cases further subjected to a lengthy freeze-thaw cycle at very low temperatures.
Another approach to obtain antigen sensitized erythrocytes was through the use of bivalent reagents as coupling agents between antigens and erythrocytes, e.g., bis diazotized benzidines illustrated in U.S. Pat. No. 3,236,732; various diols and quinones described in U.S. Pat. No. 3,322,634; toluene-2,4-diisocynates described in Immunochemistry 1:43(1964); etc. Since even the coupling coated erythrocytes are still readily susceptible to decomposition, formaldehyde treatment was usually employed prior to coating with antigen or simultaneously as described in U.S. Pat. No. 3,987,159 where the coupling agent employed was glutaraldehyde. In still a different approach described in U.S. Pat. No. 3,991,175, glutaraldehyde was also utilized as a coupling agent but gelatin was used to stabilize the composition.
The various compositions and methods already noted and numerous others suffer individually from various disadvantages such as instability, lack of reproducibility, false positive results, low hemagglutination titres, cost or time of preparation, and others.
It has now been found that the compositions and methods of this invention demonstrated in part by use in hemagglutination tests, provide a superior grade of stabilized, sensitized cells. The composition of this invention have higher hemagglutination titres, are stable for long periods of time, and give reproducible patterns and results. Another advantage of the herein disclosed invention is the stability of results obtained. For example, in utilizing a commercially available reagent the hemagglutination patterns change with time. Thus, in a pregnancy test, a negative result, i.e., complete hemagglutination within 2 hours, will change over 24 hours to show a quasiinhibition pattern; results recorded from 2-24 hours at room temperature will therefore indicate a significant number of false positive results. However, utilizing the composition and methods of this invention, results are obtained within 2 hours of testing and will not change for about 11 days thereafter or more. Other advantages will become apparent as the description of the invention unfolds.