US 201302.52261A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0252261 A1 Opperman et al. (43) Pub. Date: Sep. 26, 2013 (54) COMPOSITIONS AND METHODS FORN Publication Classification VTRO DAGNOSTIC TESTS INCLUDING SULFONCACID COMPOUND (51) Int. Cl. GOIN 2L/78 (2006.01) (71) Applicant: SURMODICS, INC., Eden Prairie, MN (52) U.S. Cl. (US) CPC ...................................... G0IN 21/78 (2013.01) USPC ............ 435/7.72:435/25; 435/288.3: 435/28 (72) Inventors: Gary Opperman, St. Louis Park, MN (57) ABSTRACT (US); Wendy Nelson, Woodbury, MN The invention provides compositions, kits, and methods for (US) performing colorimetric analysis. A Substrate is reacted to generate a chromogenic reaction product, and a reaction stop (21) Appl. No.: 13/849,056 reagent that is a Sulfonic acid is added to stop and Stabilize the reaction product. The absorbance properties of the chromoge (22) Filed: Mar. 22, 2013 nic reaction product can be maintained over significantly longer periods of time of that of conventional reagents and Related U.S. Application Data methods. The Sulfonic acid can be used in assays such as (60) Provisional application No. 61/614,602, filed on Mar. ELISAs in order to provide a more accurate and safer detec 23, 2012. tion of analytes in a biological sample. Patent Application Publication Sep. 26, 2013 Sheet 1 of 5 US 2013/0252261 A1 Figure 1 Signal Stability at 300 pg/well Level of HRP s y C o O - - - - - O.5 MH2SO4 C 1Mphosphoric O.25 MHC 0.2 M sulfamic acid (invention) - - 0.15 Mmethanesulfonic acid (invention) 20 40 60 80 100 120 140 Time (minutes) Patent Application Publication Sep. 26, 2013 Sheet 2 of 5 US 2013/0252261 A1 Figure 2 Signal Stability at 40 pg/well Level of HRP C C e o e - - - - - 0.5 MH2SO4 1Mphosphoric O.25 MHC 0.2 M sulfamic acid (invention) - - 0.15 Mmethanesulfonic acid (invention) 8O 1OO 12O 140 160 18O Time (minutes) Patent Application Publication Sep. 26, 2013 Sheet 3 of 5 US 2013/0252261 A1 0.5 M Sulfuric Acid - Time after addition of stop 4. 10 minutes 3.5 - - - - -30 minutes ................................... 1. 60 minutes 3 - - - - - 2 hours - - - - - Patent Application Publication Sep. 26, 2013 Sheet 4 of 5 US 2013/0252261 A1 Figure 4 0.15 M Methanesulfonic Acid - Time points after stop addition 10 minutes - - - -30 minutes - - - 60 minutes - - - - - 2 hours S e V w C O 1 5 t C C 1 - 1OO Mouse lgG (pg/ml) Patent Application Publication Sep. 26, 2013 Sheet 5 of 5 US 2013/0252261 A1 Figure 5 0.2M Sulfamic Acid - Time points after stop addition 10 minutes - - - - 30 minutes - - - - 60 minutes - - - - - 2 hours S 4 hours vs. 8 hours w k o a. Mouse IgG (pg/ml) US 2013/0252261 A1 Sep. 26, 2013 COMPOSITIONS AND METHODS FOR IN binding reactions. However, the use of these acids has prob VTRO DAGNOSTIC TESTS INCLUDING lems associated with it, namely corrosivity, short signal dura SULFONCACID COMPOUND tion, and toxicity. 0007. The investigator has discovered there is a need to CROSS-REFERENCE TO RELATED provide a non-corrosive stop reagent and a stop reagent that APPLICATION allows for an extended dynamic range and a longer, more stable signal time than previous acids have provided. 0001. The present non-provisional Application claims the benefit of commonly owned provisional Application having SUMMARY Ser. No. 61/614,602, filed on Mar. 23, 2012, entitled COM POSITIONS AND METHODS FOR INVITRO DIAGNOS 0008 Generally, the invention provides compositions, TIC TESTS INCLUDING SULFONIC ACID COM kits, and methods for determination of an analyte in a sample POUND, which Application is incorporated herein by which uses a chromogenic Substrate detectable by colorimet reference in its entirety. ric analysis, and a stop reagent for stopping and stabilizing a reaction composition comprising a chromogenic reaction FIELD product. The stop reagent is a Sulfonic acid. 0009 Experimental studies associated with the invention 0002 The invention relates to compositions and methods have unexpectedly found that the Sulfonic acid stop reagents for in vitro diagnostic tests and in vitro colorimetric tests. stabilize absorbance properties of the chromogenic reaction product over significantly longer periods of times as com BACKGROUND pared to assays using conventional stop reagents. In addition, the stop reagents of the invention allowed for colorimetric 0003 Research and diagnostic procedures require rapid, analysis, especially at high analyte levels, to be performed accurate and qualitative and/or quantitative determinations of over an extended dynamic range. Advantageously, the stop Substances (“analytes') that are present in biological reagents of the invention do not have the corrosivity charac samples, such as biological tissues or fluids, at low concen teristics of sulfuric and hydrochloric acids, which make the trations. For example, the presence of drugs, narcotics, hor components and compositions of the kits and methods of the mones, steroids, polypeptides, prostaglandins or infectious invention more amenable to handling. organisms in blood, urine, saliva, dental plaque, gingival 0010. Accordingly, one aspect of the invention provides a crevicular fluid, and other biological specimens is desirably kit with components for performing a colorimetric assay. The determined in an accurate and rapid fashion for Suitable diag kit comprises a chromogenic Substrate capable of forming a nosis or treatment. chromogenic reaction product detectable using colorimetric 0004. In many cases, an analyte is identified in a sample analysis, and a compound that stops and Stabilizes the chro using a compound that specifically recognizes the chemical mogenic reaction product, the compound being a Sulfonic features of the analyte. Often, monoclonal antibodies specific acid. The kit can optionally include other components like for one or more chemical epitopes on an analyte are used. The one or more redox compounds, such as peroxidases, peroxi complex formed between the antibody and analyte can be dase substrates, oxidases and oxidase Substrates, which can detected by a variety of known methods. The most commonly be used to convert the chromogenic Substrate into the chro used methods employ a signal generating moiety of some mogenic reaction product. Another optional component is an type which is either already attached to the antibody, or analyte binding member, such as an antibody, capable of becomes attached to the antibody through further reaction. specific recognition of an analyte in a biological sample. For example, in the formation of a complex of biotin with Other optional components in the kit include vessels, such as avidin, the complex may be detected using a label on either multiwell plates, in which the colorimetric analysis can be the avidin or biotin molecule. Such a label can be a radioiso carried out and the chromogenic reaction product read using tope or an enzyme conjugated with the avidin or biotin. Alter spectrophotometric equipment. natively, the avidin-biotin complex might be detected by fur 0011. The invention also provides a composition compris ther reaction with a labeled molecule which is specific to ing the Sulfonic acid and chromogenic reaction product either or both parts of the complex. It is commonly known to detectable using colorimetric analysis. The chromogenic do the same with antigens and their corresponding antibodies. reaction product can be stabilized in the presence of the 0005. In diagnostic tests designed to be rapid and easy to Sulfonic acid. The composition can optionally include other use with moderate training in a doctors office or clinic, the components that can be used to generate the chromogenic specific binding ligand of interest (such as an antigen from an reaction product such as a peroxidase, peroxidase Substrate, infectious agent) is often detected using colorimetric, fluo oxidase, and oxidase substrate. rescent or chemiluminescent signals resulting from reaction 0012. The method further provides methods for perform of the enzyme label with its corresponding substrate. ing a colorimetric analysis. The method includes, in the least, 0006. There is a need to produce the signal quickly and a step of adding the Sulfonic acid to a composition containing intensely if the ligand is present. This is commonly done the chromogenic reaction product. The addition of the Sul using a colorimetric detection reagent. Upon addition of the fonic acid stabilizes the chromogenic reaction product which detection reagent a colored product is produced. In many can then be colorimetrically analyzed using equipment Such types of assay, the generation of color is not limited. In order as a spectrophotometer, or alternatively by visualization. The to optimally quantitate the result, a stop reagent is employed method can optionally include one or more steps upstream of to stop the formation of color and hold it at a stable level to the step of adding the compound with the Sulfonic acid group. allow for accurate quantitation. Acids such as Sulfuric acid For example, the method can optionally include step(s) of and hydrochloric acid can be used to stop the production of providing a biological sample having an analyte, contacting detectable signal when peroxidase is used as a label in specific the analyte with an analyte-binding compound, recognizing US 2013/0252261 A1 Sep. 26, 2013 the analyte/analyte-binding compound complex with one or out, the stop reagent is added to the reaction composition to more components, such as a peroxidase, peroxidase Sub maintain favorable color characteristics (e.g., as measured by strate, oxidase, and oxidase Substrate that promote conversion optical absorbance) or signal intensity over extended periods of a Substrate, and formation of the reaction product detect of time. By comparison, more rapid changes in color charac able using colorimetric analysis. teristics (e.g., as measured by optical absorbance) are observed when known stop reagents such as Sulfuric acid (as BRIEF DESCRIPTION OF THE DRAWINGS the only stop reagent in the composition) are used.