1. Field of the Invention
The present invention relates to immunochromatography and more particularly to sequential solid phase immunoassays and a system used in performing the immunoassays.
2. Description of Related Art
The use of lateral flow immunochromatography along with colloidal dye conjugates is well known in the field of rapid diagnostics. Various diagnostic kits have been developed and marketed based on these methods. In principle, a specific member of a combining pair (an antigen or an antibody, for example) is immobilized onto a specific capture zone in one of many different kinds of membranes, such as nitrocellulose. As a fluid sample containing the complementary member of the combining pair (complementary antigen or antibody) passes through the capture zone, it is captured by the immobilized member. Another biological reagent, which also specifically binds to the captured member, is conjugated to one of a number of colored particles such as colloidal gold and is used to visualize the captured member. Frequently the colored particle conjugate is in dry form and strategically placed between the capture zone and the sample application zone and in the path of sample flow, so that the fluid sample can reconstitute the conjugate and react with it before reaching the capture zone.
For the detection of antigens, this represents a rather straight-forward method in which the capture zone contains immobilized antibodies (A) specific for the antigen and the conjugate contains specific antibodies (B). At the capture zone, the antigen is essentially sandwiched between two antibodies; one immobilized on the membrane and the other on the colored conjugate. This sandwich is easily detected visually or by a reader.
The prior art provides three different general approaches to antibody detection. In the double antigen sandwich method, the antigen is immobilized onto the membrane, and is also conjugated onto the colored particles. The specific antibody in the biological specimen to be detected essentially cross links with the two antigens. The advantages of this method are that it is rapid, sensitive and can be done in one single step, without the need for wash steps. The chief disadvantages of this method are that: 1) it is sometimes difficult to conjugate antigens onto particles as each antigen may have a unique set of conditions in which it needs to be conjugated to retain immunological reactivity and avoid steric hindrance; 2) it is not possible to distinguish between IgG and IgM antibodies, which may be very important in clinical situations (to distinguish between primary and secondary infections); and 3) when the antibody/antigen ratio falls outside of the range of effective formation of immobilized antigen-antibody-conjugated antigen cross-links, the test renders a false negative resulting in decreased sensitivity due to the prozone effect.
In an antibody capture method, specific anti-IgG or anti-IgM (or a combination of both) are immobilized onto the membrane at the capture zone. Antigen is conjugated onto the colored particle. Specific antibodies contained in the biological specimen react and bind with the antigen-colored particle conjugate and as it passes through the capture zone, become captured by the antibodies. The advantages of this method include the fact that IgG and IgM antibodies can be distinguished, and the reaction can be completed as a one step reaction without wash steps. The chief disadvantages are: 1) that conjugation of the antigen could present a challenge as mentioned above with regard to the double antigen method; and 2) that when the specific titer is low (in other words, the specific antibodies' concentrations are low) and the overall IgG or IgM antibody levels are high, the sensitivity may be decreased due to a low ratio of specific to nonspecific antibody levels and to the finite number of capture sites in the capture zone proportionately more occupied by extraneous antibodies. This is because the ratio of specific to nonspecific antibody level is low, and the capture sites of the capture zone which are finite may be proportionately more occupied by extraneous antibodies. This is particularly so in the case of IgG.
In a second antibody method, the antigen is immobilized onto the capture zone of the membrane. The specific antibody contained in the fluid sample is captured by the antigen as the specimen passes through the capture zone. A secondary antibody (specific for IgG or IgM or both) conjugated onto the colored particle is then applied, and as it passes through the capture zone, binds to the captured specific antibodies, thereby being visualized. Using this method, IgG and IgM can be distinguished by the use of different conjugates which are specific for one or the other antibody. Another advantage is that antigens do not need to be conjugated to the colored particles; a universal set of secondary antibodies (anti-IgG or IgM) can be used for many different assays, thus simplifying manufacturing in a commercial setting. When done in a sequential manner, first with the addition of the sample, followed by a wash step to wash away extraneous non-specific antibodies, and then the addition of the conjugate, the method is not subject to being overwhelmed by excessive extraneous antibodies. The chief disadvantage is that one or more wash steps need to be implemented to avoid the exhaustion of the conjugate. If there is too much non-specific antibody in the specimen, it will bind to and exhaust the conjugate, leaving an insufficient amount to bind to the specific antibodies and causing low sensitivity.
U.S. Pat. No. 6,818,455 to May et al. entitled “Capillary Immunoassay and Device Therefor Comprising Mobilizable Particulate Labelled Reagents” discloses an analytical device having an immobilized labeled reagent adjacent a lower end of a test strip. A sample to be tested is added behind the immobilized labeled reagent and reconstitutes the labeled reagent which then moves toward a reaction zone. The disclosed immunoassay suffers the disadvantages mentioned above related to antibody tests and low sensitivity due to the prozone effect related to antigen tests.
US Patent Application Publication No. 2004/0235189 to Lu entitled “Reversed Chromatographic Immunoassay” discloses a chromatographic immunoassay test strip comprising a solid support having a pre-dried immobilized ligand/tracer. In use, a specimen is added ahead of the immobilized ligand B/tracer and a buffer is used to dissolve the ligand B/tracer, facilitate the movement of the sample along the strip, and allow it to react with a binder (ligand A) immobilized in a test region. The buffer may also clean up the strip of bound material outside of the test region. The disclosed immunoassay suffers the disadvantages of requiring a separate buffer and reduced sensitivity due to the relatively limited pre-dried immobilized ligand B/tracer which is insufficient to achieve sensitive detection.
What is needed then is a sequential solid phase immunoassay and system thereof that overcomes the limitations of the prior art. What is further needed is a sequential solid phase immunoassay that utilizes the secondary antibody method for the detection of antibodies in a membrane-based test. What is also needed is a sequential solid phase immunoassay that utilizes stabilized liquid secondary antibody conjugates of sufficient quantity as visualization reagents. What is further needed is a sequential solid phase immunoassay that provides for a fluid specimen added to a fluid specimen application zone distal from a capture zone having an immobilized antigen. What is also needed is a sequential solid phase immunoassay that provides for application of an excess secondary antibody conjugate in the fluid specimen application zone or distal of the fluid specimen application zone, after the fluid specimen is added such that the conjugate flows in the same direction as the fluid specimen and behind the fluid specimen. What is further needed is a sequential solid phase immunoassay that provides a sequential reaction to thereby provide better sensitivity at both a low end and a high end of analyte concentration. What is further needed is a sequential solid phase immunoassay wherein the liquid secondary antibody conjugate serves to wash excess non-specific antibodies and continuously supplies sufficient reactive conjugate material throughout the entire test run time, such that extraneous non-specific antibodies do not overwhelm the system.
USPTO Backgrounds
USPTO Backgrounds