Design of a lateral flow diagnostic assay requires optimization of a number of critical parameters. Among these are: antibody pairing, buffer formulation, conjugate rehydration, and flow characteristics. During the development of a 510(k) cleared pregnancy test, Scripps Laboratories evaluated a number of commercially available “dipstick” assays and determined that many of them had obvious shortcomings.
The selection of appropriate antibodies is critical during the development of an immunoassay. The “appropriate” choice is a balancing act dependent on a number of factors, including specificity, relative affinity, stability, and the ultimate requirements of the assay system. It is often possible for an antibody pair to be perfectly suitable for one application but marginal or unsatisfactory for another. A key obstacle in a lateral flow pregnancy test is the affinity of the antibody pair for hCG and the potential cross-reactivity with other related hormones, primarily luteinizing hormone (LH). Both hCG and LH are dimeric proteins composed of an α- and a β-subunit. In the case of these two hormones, the α-subunit is essentially identical, whereas the primary biofunctional and immunological differences are due to the β-subunit. Antibody pairing for a pregnancy test falls into three general categories: α-hCG / β-hCG specific, β-hCG / β-hCG specific, and whole hCG / β-hCG specific. The first two options can be accomplished using monoclonal or polyclonal pairs, while the third option requires a polyclonal antibody against whole molecule hCG.
In general, monoclonal antibody pairing results in greater specificity but with a potential trade-off in lower sensitivity. Conversely, polyclonal antibodies generally result in greater cross-reactivity with the related hormones LH, FSH, and TSH. A number of commercially available assays add an unlabeled “scavenger” antibody specific for LH to prevent or minimize cross-reactivity. In the pregnancy strip developed by Scripps Laboratories, all these options were evaluated. In the final assay format a monoclonal antibody specific for β-hCG (catalog number MC097, part number 90000) was paired with a polyclonal antibody also specific for β-hCG (catalog number GC099, part number 90062). To ensure consistent antibody quality, Scripps Laboratories controls the purification process for both these antibodies. The monoclonal antibody is purified using a combination of ion-exhange chromatography, protein A purification, and gel filtration. The polyclonal antibody is purified using immunoaffinity chromatography and gel filtration. Internal testing indicates that this antibody pair has no cross-reactivity with LH at > 2,000 mIU/ml. We have tested some commercial hCG assays based on an α-hCG / β-hCG configuration and found reactivity when the LH level approaches 800 to 1,000 mIU/ml, even using a scavenger antibody.
Incomplete resuspension/rehydration of the colloidal gold conjugate is a problem for a number of commercially available tests, even after incubation periods beyond the maximum read time for the assay. Colloidal gold that remains in the conjugate pad indicates poor conjugate preparation or inappropriate buffers / manufacturing conditions used for diluting and drying the conjugate. If the antibody is still attached to the colloidal gold particles and is functional, hCG present in the sample will bind but will not enter the test zone of the membrane. This compromises the sensitivity of the assay at the lower limit specification, typically 20 - 25 mIU/ml in urine. If the antibody dissociates from the aggregated gold particle and remains functional it will bind hCG as it flows through the system and also decrease sensitivity of the assay as it forms a sandwich with the immobilized antibody. If the aggregation phenomenon increases over time the effective shelf-life of the assay will decrease.
Another issue with incomplete rehydration of the conjugate is its effect on the flow characteristics through the membrane. Channeling (streaking) can result in a spotted read zone rather than a contiguous line. This can lead to inaccurate test interpretation. Additionally, conjugate that does not resuspend quickly will result in a buffer front that precedes the conjugate through the assay zone. This can increase the prozone effect, the phenomenon where samples with high levels of hCG result in a decreased signal intensity. In samples with high levels of hCG, not all the analyte is captured at the colloidal gold interface. As the buffer front passes through the immobilized antibody it is bound, effectively preventing the colloidal gold conjugate from reacting.
To improve the resuspension and stability of the conjugate, Scripps developed a proprietary buffer formulation (catalog number B0221, part number 90092). This buffer is intended for the resuspension and dilution of the colloidal gold conjugate prior to drying on the conjugate membrane. It prevents aggregate formation and improves stability of the dried conjugate. Real-time studies indicate test stability in excess of 27 months at room temperature. More importantly, all conjugate is released from the pad throughout the product shelf-life. The buffer contains a blocking agent so the membrane does not need to be blocked prior to assembling the test. Components in the buffer also promote rapid and complete resuspension of the conjugate. This results in conjugate flowing through the test zone with the buffer front, minimizing the prozone effect.
Custom formulations can be provided on request.