Field of the Invention and Related Art Statement
The present invention relates to a method of effecting an immunological analysis.
Due to recent progress in the medical field it has become possible to analyze very small amounts of biological substances, and this contributes to early stage diagnosis for various diseases. For instance, malignant tumors can be diagnosed at an early stage by measuring alpha-fetoprotein and carcinoembryonic antigen. Further, immunological diseases can be diagnosed by measuring abnormal secretion of hormones such as insulin and thyroxine and immunoglobulin. Moreover, it has also become possible to effect monitoring during the course of medical treatment. Further analysis for low molecular hapten (incomplete antigen) such as medicines, contributes to plan a dose schedule of medicines.
Most of these biological substances have been analyzed by an immunological method utilizing the antigen-antibody reaction. There have been proposed various immunological analyzing methods. For instance, antigen or antibody is fixed on fine particles made of glass or synthetic resin and is reacted with a sample antibody or antigen, while labeling antibody or antigen labeled with a highly sensitive marker such as radio isotope, fluorescent substance, luminescent substance and enzyme is used to produce antigen-antibody complex. Then the antigen-antibody complex is detected to quantify the sample antigen or antibody.
FIG. 1 shows a reaction process of known enzyme immunological analysis utilizing an enzyme labeling reagent. On an outer surface of an insoluble carrier 1 there is fixed antibody 1A which is specifically reacted with antigen contained in a sample to be analyzed. If sample antibody instead of antigen is to be measured, antigen which is specifically reacted with the sample antibody has to be fixed onto the insoluble carrier 1. At first, the antibody 1A on the carrier 1 is reacted with antigen 2 contained in the sample. Then free antigen which has not been bound with the antibody 1A on the carrier 1 is separated from antigen 1A bound on the carrier 1 by means of washing. This separating process is generally called B-F separation. After that, the carrier 1 is reacted with a labeling reagent 3 comprising antibody 3A labeled with enzyme 3B, said antibody 3A being specifically reacted with the sample antigen 2. Next, the B-F separation is carried out again to separate free labeling reagent from labeling reagent bound with the sample antigen 2 reacted with the antibody 1A fixed on the carrier 1. Further, a color reagent is added to effect a color reaction under the existence of the enzyme 3B of the labeling reagent 3. Finally the reaction liquid is colorimetered to detect an enzyme activity. In this manner the sample substance 2 is quantified at a high sensitivity. The above mentioned immunological analyzing method has become widely practiced.
In the known immunological analyzing method using a labeling reagent containing antibody or antigen, after the antibody or antigen fixed on the carrier has been reacted with the sample antigen or antibody, the first B-F separation is performed, and then after the sample antigen or antibody has been reacted with the labeling reagent, the second B-F separation is carried out. Therefore, the analysis requires a larger number of steps and the analyzing process is liable to be complicated. Moreover, it is necessary to use various kinds of reagents and thus the running cost becomes expensive. It should be noted that an analyzer for effecting the known analysis becomes also complicated and expensive.
Further, since the substance to be analyzed is detected indirectly by using the labeling reagent as intermediate substance, the analysis is liable to be affected by slight change in external condition during the analyzing process. For instance, if the washing process is not carried out correctly, there might be produced measuring errors.
The immunological analysis has been also used to identify or determine a blood type. For instance, in German Patent No. 3,246,873, there is disclosed a method of determining the blood type by photoelectrically detecting a particle agglutination pattern. In this known method, blood cells are first separated by centrifuge, and then separated blood cells are dispersed in a saline solution to form a blood cell solution having a concentration of 2 to 5%. The blood cell solution is delivered into a reaction vessel having a conical bottom and a given antiserum is delivered into the reaction vessel. A particle pattern formed on the conical bottom is uniformly illuminated and is photoelectrically detected. A photoelectric output signal is processed to judge agglutinated or non-agglutinated particle pattern to determine the blood type.
In the above mentioned known blood type determining method, there are required various processes such as the centrifugal separation of blood cells, preparation of the blood cell suspension and the delivery of the antiserum. Therefore, the analyzing process is very cumbersome. Moreover, in order to form the particle pattern the reaction vessel has to remain still for a long time period. Further, in order to detect the particle pattern, there must be arranged correctly a light source, imaging lens, a plurality of light receiving elements, etc. Thus, the whole analyzing apparatus is liable to be large in size, complicated in construction and expensive in cost.