US 20150293O86A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0293.086 A1 MESSMER et al. (43) Pub. Date: Oct. 15, 2015 (54) LATERAL FLOW IMMUNOASSAY Publication Classification (71) Applicant: ABREOS BIOSCIENCES, INC., San (51) Int. Cl. Diego, CA (US) GOIN33/543 (2006.01) GOIN33/68 (2006.01) (72) Inventors: Bradley Todd MESSMER, San Diego, GOIN33/94 (2006.01) CA (US); Peter HABERZ, San Diego, GOIN33/558 (2006.01) CA (US) (52) U.S. Cl. CPC ........ G0IN33/54386 (2013.01); G0IN33/558 (21) Appl. No.: 14/686,578 (2013.01); G0IN33/6854 (2013.01); G0IN 33/94 (2013.01) (22) Filed: Apr. 14, 2015 (57) ABSTRACT Disclosed is a test device for determining the presence or Related U.S. Application Data integrity of a biologic comprising a sample pad for receiving the biologic, a conjugate pad, and a test membrane compris (60) Provisional application No. 61/979,123, filed on Apr. ing at least one test line comprising an immobilized mime 14, 2014. tope, and methods of using the same. Caitro line 3 Wicking Pad oitris: i.e : C:- Qigg Conjugate Paci a Kagga alg3 Fc nanoA Sampie Pad Contro: itse 2 test ine catbids finsatose peptive Flow & 2. --- i wog of active Á: wirging as active A: wroig Aix wigg. A {{ A, ikey 33 g(3 { A, xey is a g3 c{3}{338c: six c:33:33.8d 38.33 to attit.xy 88 artiaoriy assay faiec Patent Application Publication Oct. 15, 2015 Sheet 1 of 6 US 2015/029.308.6 A1 FGURE 1 Cottro tie 3 Wicking Pad Cartoire 3. & EigG Certigate Pad &Kaga cigg FC and As Sarpia Pad Corirai :38 2. Best & catalis time toge peptide Fow - c w i SS 8.338; 8.3: ... ', wog 8* is active A: wrog (; ; ;8&tive YA &oig A. weig A. 3 Ai, key 3.3 g3 to Ab, iikey harai ig3 coatiated Air coatiated & 8 at:38 y 83;{i}{3xy assay faiei assay failed Patent Application Publication Oct. 15, 2015 Sheet 2 of 6 US 2015/029.308.6 A1 3 *33 88 *33 Ritaxia gins Patent Application Publication Oct. 15, 2015 Sheet 3 of 6 US 2015/029.308.6 A1 FGURE 3 ::::::::::::::::::::8: Patent Application Publication Oct. 15, 2015 Sheet 4 of 6 US 2015/029.308.6 A1 FIGURE 4 Patent Application Publication Oct. 15, 2015 Sheet 5 of 6 US 2015/029.3086 A FIGURE 5 Test line 8 83. 8 :8 Patent Application Publication Oct. 15, 2015 Sheet 6 of 6 US 2015/029.3086 A FIGURE 6 Test line 3 Test line 2 US 2015/0293O86 A1 Oct. 15, 2015 LATERAL FLOW IMMUNOASSAY bilized mimetope, (b) determining whether at least one test line undergoes a color change, (c) determining, based on the RELATED APPLICATIONS color change, the activity of the biologic. 0001. The present application claims the benefit of U.S. 0009 Disclosed herein is a kit comprising (a) a test device Provisional Application Ser. No. 61/979,123, filed on Apr. 14, for determining the integrity of a biologic, the test device 2014, by Bradley T. MESSMER et al. and entitled “LAT comprising a sample receiving pad, a conjugatepad, and a test ERAL FLOW IMMUNOASSAY, the entire disclosure of membrane, the test membrane further comprising at least one which is incorporated herein by reference, including the test line and at least one control line, and (b) instructions for drawings. use thereof. BRIEF DESCRIPTION OF THE DRAWINGS GOVERNMENT RIGHTS 0010 FIG. 1 illustrates one embodiment of the lateral flow 0002 The subject matter disclosed herein was made with assay disclosed herein. The top panel illustrates a schematic government support under grants 1R41CA192697-01 and of the prototype. A drop of the biologic sample is placed on a 1R43CA183241-01 awarded by the National Institutes of sample pad and is wicked through the device. After binding Health Small Business Innovation Research (NIH-SBIR). the secondary detection antibody, conjugated to gold, the The government has certain rights in the disclosed subject sample encounters the peptide, anti-light chain, and anti matter. secondary capture agents. The bottom panel illustrates pos sible outcome matrix for an antibody that is known to possess FIELD OF THE INVENTION a kappa light chain. 0003. The present invention is in the field of test devices, 0011 FIG. 2 (A) illustrates one embodiment of the lateral and more specifically in the field of lateral flow test devices. flow assay validating that rituximab binding mimetope pep tide function in immunoassays and competes with the native BACKGROUND OF THE DISCLOSURE antigen (CD20). FIG. 2 (A) is a standard curve for rituximab by peptide based ELISA. Biotinylated peptides are bound 0004 Biologics are valuable therapeutics for treating can onto neutravidin coated ELISA plates. Rituximab is diluted in cers and other diseases. As they are among the most expensive TBST. Each value shows the mean (S.D.) of triplicates. The pharmaceuticals produced and increasingly prescribed, there solid line indicates the mean of the buffer control and the have been a growing number of incidents involving counter dashed line represents the mean +10 times the SD of the feit biologics, such as counterfeit antibodies. These econom buffer control. ics create a considerable incentive for counterfeit or other (0012 FIG. 2 (B) illustrates peptide inhibition of Chronic deceptions. The problem of counterfeit medications is grow Lymphocytic Leukemia (CLL) cell staining. Fluorescently ing rapidly. The Center for Medicines in the Public Interest labeled rituximab is incubated with primary CLL cells and estimates that the worldwide trade for counterfeit medicines evaluated by flow cytometry (solid line). Weak staining for exceeds S75 billion. This number is S25 billion more than the CD20 was observed because CLL cells have low levels of illicit drug trade. Therefore, there is a concern that organized CD20. When peptide RTX-10 was added at a large molar crime will become more active in this area. excess (dashed lines), the cell labeling was largely abrogated. 0005. Furthermore, biologics are sensitive to environmen Control peptides had no effect (not shown). The shaded his tal conditions and improper storage or handling at any point in togram represents CLL cells incubated with fluorescently the Supply chain. Improper handling of a biologic. Such as an labeled normal human IgG. antibody, could reduce or inactivate their ability to bind the 0013 FIG.3 illustrates one embodiment of the lateral flow target antigen and mitigate their therapeutic effect. At present, immunoassay design and development path. The flow chart there are no routine safeguards in place at the time of admin istration to ensure that a patient is receiving the correct, active shows the design process for a lateral flow immunoassay. therapy. 0014 FIG. 4 illustrates one embodiment of mimetope LFA for bevacizumab, trastuzumab, and rituximab. The 0006 Thus, a need remains to develop simple, rapid sample comprising an antibody flows through a conjugatepad assays that Verifies both the presence and integrity of appro loaded with goat anti-human IgG attached to colloidal gold, priate antibody in the sample and that are amenable for use at then onto a membrane striped with the appropriate mimetope any point in the Supply chain, including bedside. Such an peptide, and finally an assay control. A false positive on one assay will provide patients and their physicians an extra anti-lambda test line is shown by an asterisk. degree of confidence that the correct, active therapeutic is (0015 FIG.5 illustrates that the lateral flow assay disclosed administered. herein has wide dynamic range. Samples tested were Ritux imab at 0.0 mg/ml (negative control), 0.01 mg/ml, 0.1 mg/ml. SUMMARY OF THE INVENTION 1.0 mg/ml, and 10 mg/ml (vial concentration). With increas 0007 Disclosed herein is a test device for determining the ing Rituximab concentration, the test line gets more intense. presence or integrity of a biologic comprising a sample pad 0016 FIG. 6 further illustrates that the lateral flow assay for receiving the biologic, a conjugate pad, and a test mem disclosed herein has a wide dynamic range. FIG. 6 shows one brane comprising at least one test line comprising an immo embodiment of the sandwich lateral flow assay with two test bilized mimetope. lines. The test lines comprise peptide mimetope striped at two 0008 Disclosed herein is a method of determining the different concentrations on the membrane. Samples tested are integrity of a biologic comprising (a) contacting the biologic Rituximab at 0.0 mg/ml (negative control), 0.01 mg/ml, 0.1 with a test device, wherein the test device comprises a sample mg/ml, 1.0 mg/ml, and 10 mg/ml (vial concentration). With pad for receiving the biologic, a conjugate pad, a test mem increasing Rituximab concentration, the lower test line gets brane comprising at least one test line comprising an immo less intense whereas the upper test line gets more intense. US 2015/029.3086 A1 Oct. 15, 2015 DETAILED DESCRIPTION OF THE (IgA1 and IgA2), Igl), IgE. IgM, and IgG (IgG1, IgG3 and EMBODIMENTS IgG4) etc. In some embodiments, the antibody is polyclonal 0017. The terminology used herein is for the purpose of or monoclonal. In some embodiments, the antibody is from describing particular embodiments only and is not intended to any origin, such as mouse or human, including a chimeric be limiting of the invention. As used herein, the singular antibody thereof. In some embodiments, the antibody is forms “a,” “an,” and “the' are intended to include the plural humanized. forms as well, unless the context clearly indicates otherwise. (0023 Examples of antibodies include, but are not limited Furthermore, to the extent that the terms “including.” “includes,” “having.” “has.” “with.” or variants thereof are to, bevacizumab, trastuzumab, rituximab, abciximab, adali used in either the detailed description and/or the claims, such mumab, alemtuzumab, basiliximab, belimumab, brentux terms are intended to be inclusive in a manner similar to the imab vedotin, canakinumab, cetuximab, certolizumab pegol, term “comprising.” daclizumab, denosumab, eculizumab, efalizumab, gemtu 0018.