Systems and methods for testing and screening using compound-binding substrates - Patents.com

Abstract

To provide a system and method for testing and screening using compound-binding substrates [Solution] The present disclosure provides compositions, methods, kits, and systems for detecting antibodies in biological samples. In some embodiments, the methods / systems are particularly useful for detecting antibodies in patients against RBCs treated with pathogen-inactivating compounds. The disclosed compositions, methods, kits, and systems provide, among other uses, a means for detecting antibodies in biological samples, such as screening potential recipients of pathogen-inactivated RBCs for pre-existing cross-linking-reactive antibodies prior to infusion of the treated RBCs.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 438,909, filed December 23, 2016, the contents of which are incorporated by reference herein in their entirety.

[0002] The present disclosure relates to systems and methods for testing and / or screening biological samples using compound-binding substrates, such as red blood cell reagents. [Background technology]

[0003] Transfusion of blood or blood components is a commonly used medical procedure. Blood products are typically tested for compatibility between the donor and recipient prior to transfusion or utilization of the blood product in the recipient.

[0004] Blood group antigens are polymorphic protein or carbohydrate residues on red blood cells. These antigens can stimulate antibody responses in individuals lacking the antigen, and some antibodies (e.g., clinically significant antibodies) can result in hemolytic transfusion reactions or hemolytic disease. Since there are more than 300 blood group antigens (BGAs) present on red blood cells, transfusion recipients are often exposed to such antigens, and immune responses against blood group antigens are observed with conventional red blood cell transfusions. Immune responses are more frequent in patients with multiple exposures to allogeneic red blood cells and / or certain conditions (e.g., SCD, thalassemia).

[0005] Detection of clinically significant blood group alloantibodies (crossmatch test) is an important test in pretransfusion testing. Donors and patients are usually typed for ABO and Rh(D) since these are considered the most important antigens for safe transfusion. Typesetting is generally not performed for minor antigens, such as those of the Rh(C / c and E / e), Kell(K / k), Duffy(Fya, Fyb), Kidd(Jka / Jkb), and MNS(M / N and S / s) systems, although plasma is screened for antibodies to these antigens. If the patient's antibody screen is negative, the units for red blood cell (RBC) transfusion must also be ABO and Rh(D) compatible. If antibodies to clinically significant minor antigens are present, the units should additionally lack the corresponding antigens. Traditionally, antibody screening tests have been performed as solid-phase red blood cell adhesion assays (e.g., Galileo system, Immucor) or in vitro agglutination tests, such as antiglobulin tests (e.g., direct antiglobulin test (DAT), indirect antiglobulin test (IAT)). This involves the use of human RBCs of known specificity, which are tested against a plasma or serum sample. After an incubation period, and most often after the addition of a second antibody solution, the mixture is observed for hemagglutination. More recently, the test has also been performed using microplate and column agglutination technology systems known in the art, which can also be used with some degree of automation. However, current tests are limited in the range of clinically significant antibodies that they can detect.

[0006] The transmission of diseases through blood products and other biological materials remains a serious health problem. Nucleic acid targeting compounds, such as psoralens and psoralen derivatives for photochemical treatment with UVA light, have been introduced into blood products, such as platelets and plasma, to inactivate pathogens prior to transfusion and reduce the risk of transfusion-transmitted infections. Other nucleic acid targeting compounds, such as S-303 (e.g., amstarin), have been used to target RBC S-303 compounds have been developed for pathogen inactivation treatment of infectious diseases. S-303 compounds form covalent crosslinks with contaminating pathogens and with the nucleic acids of white blood cells, blocking their replication and reducing the risk of transfusion-transmitted infections. However, certain amounts of these compounds or their derivatives may react with other nucleophiles of RBC components, including those on the surface of RBCs, which may result in the binding of the compounds or their derivatives (e.g., moieties) to RBCs in certain cases, such as through the formation of surface-bound adducts. Furthermore, even if such pathogen-inactivated RBCs are generally considered to be safe and non-immunogenic, the compounds or their derivatives bound to RBCs may, in some cases, be recognized by pre-existing antibodies or cause undesirable immune responses in certain patients. Summary of the Invention [Means for solving the problem]

[0007] Therefore, a need exists for a method / system for detecting antibodies in patients against RBCs that have been treated with pathogen inactivating compounds.

[0008] The disclosed compositions, methods, kits and systems provide, among other uses, a means for detecting antibodies in biological samples, such as screening potential recipients of pathogen-inactivated RBCs for pre-existing cross-linking reactive antibodies prior to infusion of the treated RBCs. In some embodiments, the compositions, methods, kits and systems are particularly useful for detecting antibodies in patients against RBCs treated with S-303 compounds.

[0009] In one aspect, a kit is provided that contains: (a) a first container containing a first substrate, where a moiety is attached to the surface of the first substrate, the moiety being selected from the group consisting of a compound of any of formulas I, II, III, IV, V, VI, VII, VIII, and IX, and a derivative thereof, or a salt or stereoisomer of any of the foregoing; and (b) a second container containing a second substrate, where the surface of the second substrate lacks an attached moiety. In some embodiments, the moiety is selected from the group consisting of a compound of any of formulas I, II, III, VII, VIII, and IX, and a derivative thereof, or a salt or stereoisomer of any of the foregoing. In some embodiments, the moiety is selected from the group consisting of a compound of any of formulas IV, V, and VI, and a derivative thereof, or a salt or stereoisomer of any of the foregoing. In some embodiments, the moiety is selected from the group consisting of a compound of any of formulas IV(a)-IV(h), and a derivative thereof, or a salt or stereoisomer of any of the foregoing. In some embodiments, the moiety is S-303, a derivative of S-303, or a salt of any of the foregoing. In some embodiments, the moiety is a non-fragile analog of S-303 or a salt thereof. In some embodiments, the moiety bound to the surface of the substrate is present at a loading level of at least about 10,000 moieties per unit of substrate. In some embodiments, the moiety bound to the surface of the substrate is present at a loading level of at least about 50 moieties / μm 2In some embodiments, the compound and the moiety bound to the surface are the same. In some embodiments, the compound and the moiety bound to the surface are different. In some embodiments, each of the first and second substrates comprises a portion of a polymer particle, matrix, or assay plate. In some embodiments, the first substrate comprises red blood cells, and the second substrate comprises red blood cells, and the red blood cells of the first and second substrates are from one or more common donors. In some embodiments, the kit further comprises a third container containing a third substrate, and the moieties at the first level are bound to the surface of the first substrate, and the moieties at the second level are bound to the surface of the third substrate, and the second level is less than the first level. In some embodiments, the first level of the moieties bound to the surface of the first substrate is at least three times higher than the second level of the moieties bound to the surface of the third substrate. In some embodiments, the third substrate comprises a portion of a polymer particle, matrix, or assay plate. In some embodiments, the third substrate comprises red blood cells, and the red blood cells of the first, second, and third substrates are obtained from one or more common donors. , 6, 7, 8, 9, 10, or 11 samples, each of the samples in the first substrate comprising red blood cells obtained from a different blood donor and the second substrate comprising a corresponding sample comprising red blood cells from each of the separate blood donors. In some embodiments, the 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 samples represent multiple blood types. In some embodiments, the first substrate and the second substrate comprise red blood cells obtained from a donor of blood type O. In some embodiments, the first substrate, the second substrate, and the third substrate comprise red blood cells obtained from a donor of blood type O. In some embodiments, the first substrate and the second substrate comprise red blood cells in a buffered suspension medium at about 0.5% to about 5% red blood cells. In some embodiments, the first substrate, the second substrate, and the third substrate comprise red blood cells in a buffered suspension medium at about 0.5% to about 5% red blood cells. In some embodiments, the first container containing the first substrate, the second container containing the second substrate, and / or the kit containing the first container and the second container are suitable for storage at 2-8°C. In some embodiments, the first container containing the first substrate, the second container containing the second substrate, and / or the kit containing the first container and the second container are suitable for storage at room temperature. In some embodiments, the first container containing the first substrate, the second container containing the second substrate, and / or the kit containing the first container and the second container are suitable for storage below -20°C.

[0010] In another aspect, a method of preparing a panel of substrates is provided comprising: a) providing first and second samples, each comprising a substrate; and b) treating the substrate of the first sample with a compound, where treatment with the compound causes a moiety to bind to the substrate surface of the first sample, and the second sample is not treated with the compound, thereby producing a panel comprising a first sample of substrates having a surface-bound moiety and a second sample of substrates lacking a surface-bound moiety, wherein the compound is selected from the group consisting of any of the compounds of Formulae I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, a method of preparing a panel of polymer particles is provided comprising: a) providing first and second samples comprising polymer particles; and b) treating the polymer particles of the first sample with a compound, where treatment with the compound causes moieties to be attached to the particle surface of the first sample, and the second sample is not treated with the compound, thereby producing a panel comprising a first sample of polymer particles having surface-attached moieties and a second sample of polymer particles lacking surface-attached moieties, wherein the compound is selected from the group consisting of any of the compounds of Formulae I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, a method of preparing a panel of red blood cells is provided comprising: a) providing a first and a second sample comprising red blood cells; and b) treating the red blood cells of the first sample with a compound, where treatment with the compound results in binding of a moiety to the surface of the red blood cells of the first sample, and where the second sample is not treated with the compound, thereby producing a panel comprising a first sample of red blood cells having a surface-bound moiety and a second sample of red blood cells lacking a surface-bound moiety, wherein the compound is selected from the group consisting of any of the compounds of Formula I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or a salt or stereoisomer of any of the foregoing.In some embodiments, the compound is selected from the group consisting of any of the compounds of formula I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the compound is selected from the group consisting of any of the compounds of formula IV(a)-IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the compound is a derivative of S-303, or a salt thereof. In some embodiments, the compound is a non-fragile analog of S-303, or a salt thereof. In some embodiments, the surface-bound moiety is a derivative of S-303, or a salt thereof. In some embodiments, the surface-bound moiety is a non-fragile analog of S-303, or a salt thereof. In some embodiments, the moieties bound to the surface of the red blood cells are present at a loading level of at least about 10,000 moieties per red blood cell. In some embodiments, the red blood cells. The moieties bound to the surface of 2In some embodiments, the compound and the surface-binding moiety are the same. In some embodiments, the compound and the surface-binding moiety are different. In some embodiments, the red blood cells of the first and second samples are from the same blood donor. In some embodiments, the red blood cells of the first and second samples are from a donor of blood type O. In some embodiments, the treatment of the substrate of the first sample with the compound, where the treatment with the compound causes the moiety to bind to the substrate surface of the first sample, is performed at 2-8° C. In some embodiments, the treatment of the substrate of the first sample with the compound, where the treatment with the compound causes the moiety to bind to the substrate surface of the first sample, is performed at room temperature. In some embodiments, the method further comprises, after step (b), washing the first and second samples. In some embodiments, the method further comprises, after step (b), resuspending the first and second samples in a buffered suspension medium at about 0.5% to about 5% red blood cells. In some embodiments, the method further comprises, after step (b), adding a cryopreservation agent to the first and second samples and freezing the first and second samples. In some embodiments, the method further comprises, after step (b), storing the first and second samples at refrigerated temperatures (e.g., 2-8° C.). In some embodiments, the method further comprises, after step (b), storing the first and second samples at less than about −20° C. In some embodiments, the method further comprises, after step (b), storing the first and second samples at room temperature. In some embodiments, the method further comprises treating a third sample comprising red blood cells with a compound, wherein treatment of the red blood cells of the third sample with the compound results in a second level of moieties bound to the surface of the red blood cells of the third sample, wherein the first level of moieties is bound to the surface of the red blood cells of the first sample, and the second level is less than the first level. In some embodiments, the method further comprises washing the third sample after treating the third sample with the compound.In some embodiments, the method further comprises adding a cryopreservation agent to the third sample after treating the third sample with the compound, and freezing the third sample.In some embodiments, the method further comprises resuspending the third sample in a buffered suspension medium at about 0.5% to about 5% red blood cells after treating the third sample with the compound. In some embodiments, the method further comprises storing the third sample at refrigerated or room temperature after treating the third sample with the compound.

[0011] In another aspect, a method of testing a sample for the presence of an antibody that binds to a compound is provided comprising the steps of: a) providing a sample comprising serum or plasma; b) contacting the sample with a substrate, wherein a moiety is bound to the substrate surface, the moiety being selected from the group consisting of a compound of any of Formulas I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII, and IX, and derivatives thereof, or a salt or stereoisomer of any of the foregoing; and c) assaying the amount of binding between an antibody from the sample and the surface-binding moiety of the substrate, wherein binding between the antibody and the surface-binding moiety indicates that the antibody binds the compound. In some embodiments, a method of testing a sample for the presence of an antibody that binds a compound comprises the steps of: a) providing a sample comprising serum or plasma; b) contacting the sample with a polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, wherein a moiety is bound to the surface of the polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, the moiety being selected from the group consisting of a compound of any of Formulas I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; and c) assaying the amount of binding between an antibody from the sample and a surface-bound moiety of the polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, wherein binding between the antibody and the surface-bound moiety indicates that the antibody binds the compound. In some embodiments, a method of testing a sample for the presence of an antibody that binds a compound comprises the steps of: a) providing a sample comprising serum or plasma; b) contacting the sample with a moiety that is bound to the surface of the polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, the moiety being selected from the group consisting of a compound of Formula I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; and c) assaying the amount of binding between an antibody from the sample and a surface-bound moiety of the polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, wherein binding between the antibody and the surface-bound moiety indicates that the antibody binds the compound. The method includes contacting red blood cells with a moiety bound to the surface of the red blood cells, the moiety being selected from the group consisting of any of the compounds of formulae I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; and c) assaying the amount of binding between an antibody from the sample and the surface-binding moiety of the red blood cells, the binding between the antibody and the surface-binding moiety indicating that the antibody binds to the compound. In some embodiments, the compound is selected from the group consisting of any of the compounds of formulae I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the compound is selected from the group consisting of any of the compounds of formulae IV(a)-IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the compound is a derivative of S-303, or a salt thereof. In some embodiments, the compound is a non-fragile analog of S-303, or a salt thereof. In some embodiments, the surface-binding moiety is a derivative of S-303, or a salt thereof. In some embodiments, the surface-bound moiety is a non-fragile analog of S-303, or a salt thereof. In some embodiments, the moieties bound to the surface of the red blood cell are present at a loading level of at least about 10,000 moieties per red blood cell. In some embodiments, the moieties bound to the surface of the red blood cell are present at a loading level of at least about 50 moieties / μm. 2In some embodiments, the compound and the surface-binding moiety are the same. In some embodiments, the compound and the surface-binding moiety are different. In some embodiments, step (c) comprises comparing the amount of binding between the antibody from the sample and the surface-binding moiety to a reference, where an increase in the amount of binding compared to the reference indicates the presence of the antibody in the sample. In some embodiments, step (c) comprises assaying the amount of binding between the antibody from the sample and red blood cells lacking the surface-binding moiety. In some embodiments, step (c) further comprises assaying the amount of binding between the antibody from the sample and red blood cells having a lower amount of the surface-binding moiety. In some embodiments, step (b) comprises contacting the sample and the red blood cells with an anti-human globulin, and step (c) comprises assaying the amount of agglutination.

[0012] In another aspect, a method of providing a red blood cell transfusion to a patient is provided, the red blood cell transfusion comprising red blood cells treated with a pathogen inactivating compound, the method comprising: a) providing a sample comprising serum or plasma from the patient; b) contacting the sample with a first substrate, wherein a moiety is bound to the surface of the substrate; c) assaying an amount of binding between an antibody from the sample and the surface-binding moiety of the first substrate compared to a reference, wherein binding between the antibody and the surface-binding moiety indicates that the antibody binds to the red blood cells treated with the pathogen inactivating compound; d) determining whether the amount of binding is higher than the reference; and f) providing the blood transfusion to the patient pursuant to a determination that the amount of binding is not higher than the reference. In some embodiments, the surface-binding moiety is selected from the group consisting of any of the compounds of formula I, II, III, IV, V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the surface-binding moiety is selected from the group consisting of any of the compounds of formula I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the surface-binding moiety is selected from the group consisting of any of the compounds of formula IV, V, and VI, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the surface-binding moiety is selected from the group consisting of any of the compounds of formula IV(a)-IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the surface-binding moiety is S-303, a derivative of S-303, or a salt of any of the foregoing. In some embodiments, the surface-binding moiety is a non-fragile analog of S-303 or a salt thereof. In some embodiments, the compound and the surface-binding moiety are the same. In some embodiments, the compound and the surface-binding moiety are different. In some embodiments, the first substrate comprises a polymer particle, a matrix, or a portion of an assay plate. In some embodiments, the first substrate comprises red blood cells. In some embodiments, the step (c) comprises assaying the amount of binding between the antibodies from the sample and a second substrate lacking surface-binding moieties. In some embodiments, the second substrate is the same as the first substrate. In some embodiments, step (c) further comprises assaying the amount of binding between the antibodies from the sample and a third substrate having a lower amount of surface-binding moieties compared to the first substrate. In some embodiments, the third substrate is the same as the first substrate. In some embodiments, step (b) comprises contacting the sample and substrate(s) with anti-human globulin, and step (c) comprises assaying the amount of agglutination. In some embodiments, the moieties bound to the surface of the substrate(s) are not the same as the pathogen inactivation compound.

[0013] In another aspect, a composition is provided that comprises a human red blood cell, wherein a moiety selected from the group consisting of any of the compounds of Formulae I, II, III, IV(a)-IV(h), VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing, is bound to the surface of the human red blood cell. In some embodiments, the moiety is selected from the group consisting of any of the compounds of Formulae I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. In some embodiments, the moiety is a non-fragile analog of S-303 or a salt thereof. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a graph showing the level of antibody staining of treated red blood cells.

[0015] [Diagram 2] FIG. 2 is a graph showing the level of antibody staining of treated red blood cells.

[0016] [Diagram 3] FIG. 3 is a graph showing the level of antibody staining of treated red blood cells.

[0017] [Figure 4]FIG. 4 is a graph showing the level of antibody staining of treated red blood cells.

[0018] [Diagram 5] FIG. 5 is a graph showing the level of antibody staining of treated red blood cells.

[0019] [Figure 6] FIG. 6 is a graph showing the level of antibody staining of treated red blood cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure provides methods and systems for testing and / or screening biological samples using substrates having compounds bound thereto. The inventors have found that while the compounds (e.g., pathogen inactivation compounds) used according to various embodiments of the present disclosure can be safe and effective for treating (e.g., pathogen inactivation) red blood cells (RBCs), such treatments can potentially cause the compounds to react with other nucleophiles in the RBCs, and in certain cases, can cause the compounds to bind to the RBCs, such as to the surface of the RBCs (e.g., form surface-bound adducts). Such binding can result in antibodies specific for the compound or a portion thereof also binding to the treated RBCs, if such antibodies are present in the patient infused with the treated RBCs. Thus, it is important to test biological samples (e.g., samples from patients) to assess for the presence or absence of antibodies (e.g., clinically significant antibodies) against the compound or portions thereof. The present application contemplates the methods / systems described herein and recognizes their use in testing biological samples for the presence of antibodies against the compounds using compositions and / or kits that include the disclosed compounds bound onto a substrate. According to some embodiments, the methods / systems are particularly suited for pre-transfusion detection of pre-existing antibodies in a patient that react with RBCs that have been treated with a pathogen inactivation compound.

[0021] Provided herein are compositions and kits / systems of compound-bound substrates (e.g., RBCs bound with pathogen inactivating compounds), methods of preparing the compositions, and uses thereof. .

[0022] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a substrate" means one substrate or more than one substrate.

[0023] "Biological sample" or simply "sample", as that term is used herein, refers to a sample, such as, but not necessarily, obtained from an animal (e.g., human), which sample or components thereof can be used in accordance with the methods of the present disclosure to assess the presence, absence and / or levels of antibodies. Such samples include, but are not limited to, any biological sample, such as biological fluids (e.g., blood, serum, plasma, lymph, semen, spit, saliva, sputum, tears, etc.), and any sample obtained from an animal (e.g., human) that can be assayed for the presence or absence of antibodies.

[0024] "Alkyl" as used herein refers to a cyclic, branched, or straight-chain chemical group containing carbon and hydrogen, such as methyl, pentyl, and adamantyl. An alkyl group can be unsubstituted or substituted with one or more substituents, such as halogen, alkoxy, acyloxy, amino, hydroxyl, thiol, carboxy, benzyloxy, phenyl, benzyl, or other functional groups. An alkyl group can be saturated or unsaturated (e.g., containing -C=C- or -CC- subunits) at one or several positions. Typically, an alkyl group will contain 1 to 12 carbon atoms, such as 1 to 10 carbon atoms or 1 to 8 carbon atoms, unless otherwise specified.

[0025] "Heteroalkyl," as used herein, refers to an alkyl chain having one or more N, O, S, or P heteroatoms incorporated into the chain. The heteroatom(s) may bear none, one, or more than one of the substituents described above. "Heteroatom" also includes the heteroatoms N, S, and P in oxidized form. Examples of heteroalkyl groups include, without limitation, methoxy, ethoxy, and other alkoxy groups; ether-containing groups, amide-containing groups, such as polypeptide chains; ring systems, such as piperidinyl, lactams, and lactones; and other groups incorporating heteroatoms into the carbon chain. Typically, heteroalkyl groups will contain 1-12 carbon atoms, e.g., 1-10 carbon atoms or 1-8 carbon atoms, in addition to the heteroatom(s), unless otherwise specified.

[0026] "Aryl" or "Ar" is optionally unsubstituted or includes amino, hydroxyl, C 1~8 It refers to unsaturated aromatic carbocyclic groups having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), substituted with alkyl, alkoxy, halo, thiol, and other substituents.

[0027] A "heteroaryl" group is an unsaturated, aromatic carbocyclic group having a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., acridinyl, indolyl, or benzothienyl) and having at least one heteroatom in at least one of the rings, such as N, O, or S. The ring(s) are optionally unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy, halo, thiol, acyloxy, carboxy, benzyloxy, phenyl, benzyl, and other substituents. composition

[0028] A composition comprising a substrate having a surface-binding moiety, the surface-binding moiety being capable of binding to a compound present in a biological sample, such as a compound used to prepare pathogen-inactivated red blood cells. Provided herein are compositions suitable for binding the antibodies capable of binding to the antibody. Base material

[0029] As will be appreciated by those skilled in the art, the substrate can be any solid support known in the art. The solid support may be composed of any material (e.g., biological material, synthetic material) or matrix suitable for binding the surface-binding moiety. Examples of solid supports include polymers or copolymers (e.g., polyesters, polyethers, polyolefins, polyamides, polysaccharides, polyurethanes, styrene and styrene derivatives, polystyrene, cellulose), plastics, glass, gold and other metals, and the like. Additional examples of solid supports include cells (e.g., RBCs), cell membranes, and other cell-derived materials. The solid support can present the surface-binding moiety in a two-dimensional format (e.g., plates) or a three-dimensional format (e.g., cells, particles, beads, or other spherical or quasi-spherical objects). Additional examples of solid supports include particles (e.g., polymer particles), such as beads and microspheres (e.g., synthetic beads and microspheres, polymeric or copolymeric beads and microspheres). Binders can also be used to provide consistency and structure to the solid support. The substrate can be part of an assay plate. A particular solid support is made of one or more materials to which the biological sample does not bind or does not substantially bind. For example, the solid support may be made of a material to which the antibody does not bind or does not substantially bind (e.g., a material that does not contain the antigen(s) to which the particular antibody binds). In particular, the solid support is made of a material to which the antibody does not bind or does not substantially bind to pathogen-inactivated red blood cells.

[0030] Any solid support for use in the compositions and methods of the present invention may be suitable for covalent or non-covalent binding to surface-binding moieties. In some embodiments, the solid support is inert. In some embodiments, the surface-binding moiety can be covalently or non-covalently bound to the solid support material itself. In other embodiments, the solid support material is functionalized with reactive groups (e.g., amine (-NH2) or ammonium (-NH3+ or -NR3+) functional groups, alcohol functional groups (-OH), carboxyl functional groups (-COOH), isocyanate functional groups (-NCO)) that can interact with or otherwise be covalently or non-covalently bound to surface-binding moieties.

[0031] It may be advantageous for the substrate to be stable at a variety of different temperatures, such as room temperature, refrigerated temperatures, or freezer temperatures. In some embodiments, the substrate is stable at room temperature. In some embodiments, the substrate is stable at refrigerated temperatures (e.g., 1-6°C, 2-8°C, 2-4°C). In some embodiments, the substrate is stable at 0°C or below 0°C (e.g., -20°C). In some embodiments, the substrate is stable at -80°C.

[0032] According to certain embodiments of the present disclosure, the substrate comprises RBCs. For example, RBCs, which are frequently used in immunohematological tests, can be a substrate as provided herein.

[0033] In some embodiments, the RBCs are prepared from human blood. The substrate can include RBCs from donors of any blood type, e.g., O, A, B, or AB. The RBCs may be designed to specifically support the resolution of one or more antibody mixtures. In some embodiments, the RBCs express an antigen selected from the group consisting of antigens C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. ​​In some embodiments, the RBCs express Rh(D). In some embodiments, the RBCs express R1R1, R2R2, R1 wThe RBCs are of an Rh type selected from the group consisting of R1, R0r, and rr. The RBCs can be stored at room temperature (e.g., in a buffered suspension medium with 0.5%-5% RBCs), at refrigerated temperatures (e.g., in a buffered suspension medium with 0.5%-5% RBCs), or they can be frozen (e.g., frozen). Store in a freezing preservative, e.g., glycerolyte, at -80°C.

[0034] The RBCs utilized as substrates in the compositions of the present invention can be obtained from a single donor, or they can be obtained from two or more donors (e.g., a mixture of RBCs from two or more donors). In some embodiments, the RBC substrate comprises RBCs from two, three, four, five, six, seven, eight, nine, ten, or eleven or more individual donors. In some cases, the RBCs from two or more donors have one or more characteristics in common, such as ABO blood type, or the presence or absence of other RBC antigens (e.g., common RBC antigens, clinically relevant RBC antigens). In other cases, the RBCs from two or more donors are heterogeneous with respect to a particular characteristic, such as the presence or absence of ABO blood type or other RBC antigens (e.g., common RBC antigens, clinically relevant RBC antigens). In some embodiments, the RBC substrate comprises RBCs with a mixture of ABO blood types. In some embodiments, the RBC substrate is homogeneous with respect to Rh factors. In other embodiments, the RBC substrate is heterogeneous with respect to Rh factor. In some embodiments, the RBC substrate is homogeneous with respect to any one or more of the following antigens: Cs a , Cs b , Er a , Er b ,Vel,ABTI,Lan,At a , Jr. a , AnWj, Sd a , Batty(By), Biles(Bi), Box(Bx a )Christiansen(Chr a ), HJK, HOFM, JFV, JONES, Jensen(Je a), Katagiri (Kg), Livesay (Li a ), Milne, Oldeide (OI a ), Peters(Pt a ), Rasmussen (RASM), Reid (Re a ), REIT, SARA, Torkildsen(To a ), and Bennett-Goodspeed (Bg). In some embodiments, the RBC substrate is heterogeneous with respect to any one or more of the following antigens: Cs a , Cs b , Er a , Er b ,Vel,ABTI,Lan,At a , Jr. a , AnWj, Sd a , Batty(By), Biles(Bi), Box(Bx a )Christiansen(Chr a ), HJK, HOFM, JFV, JONES, Jensen(Je a ), Katagiri (Kg), Livesay (Li a ), Milne, Oldeide (OI a ), Peters(Pt a ), Rasmussen (RASM), Reid (Re a ), REIT, SARA, Torkildsen(To a ), and Bennett-Goodspeed (Bg).

[0035] RBCs for use as substrate in the compositions and methods described herein can be obtained by any method known in the art.For example, RBCs can be obtained by standard RBC donation techniques (e.g., apheresis, double red collection).Alternatively, RBCs can be obtained from whole blood donations via separation of RBCs from other components of whole blood, for example, via centrifugation or other standard fractionation techniques.In some embodiments, RBCs are sterilized via any method known in the art before or after functionalization with surface-binding moieties.

[0036] Those skilled in the art will recognize that compounds can be bound to a substrate in an array according to some embodiments of the present disclosure. As used herein, the term "array" refers to a generally ordered arrangement of binding compounds on a substrate, such as glass or plastic. Typically, the array can be in the form of a series of regularly spaced regions to which compounds are bound. Such arrays can be described as chips. For example, arrays in multi-well microplates can be scanned by automated equipment. surface bonding part

[0037] According to various embodiments of the present application, the moiety is attached to a substrate surface. In some embodiments, the moiety is a pathogen inactivating compound. In some embodiments, the moiety is a pathogen inactivating compound ( For example, the moiety is a derivative of the pathogen inactivating compound). In some embodiments, the moiety is a compound that has a similar or common moiety to the pathogen inactivating compound. The substrate can be treated with the pathogen inactivating compound such that the compound or a derivative thereof binds to the substrate surface. Alternatively, the substrate can be treated with a moiety that is itself a derivative of the pathogen inactivating compound such that the reactive moiety itself binds to the substrate or further its derivatives bind to the substrate. Alternatively, the substrate can be treated with a compound that has a similar or common moiety to the pathogen inactivating compound such that the similar or common moiety binds to the substrate. In some embodiments, the moiety is a derivative of a compound comprising an acridine, such as an acridine mutagen, a monoalkylating agent, a bisalkylating agent, an acridine mustard (e.g., 6-chloro-9-(3-[(2-chloroethyl)ethylamino]propylamino)-2-methoxyacridine, Sigma #I2888), or an acridine half mustard (e.g., 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride, Sigma #I3636), and the substrate can be treated with the acridine-containing compound or a derivative of the compound.

[0038] Pathogen inactivation compounds are compounds that inactivate one or more nucleic acid-containing agents in red blood cells or other blood-based compositions, which nucleic acid-containing agents can cause disease in humans, other mammals, or vertebrates.Pathogens can be unicellular or multicellular.Examples of pathogens are bacteria, viruses, protozoa, fungi, yeast, mold, and mycoplasma that cause disease in humans, other mammals, or vertebrates.The genetic material of pathogens can be DNA or RNA, and the genetic material can be present as single-stranded or double-stranded nucleic acid.

[0039] Pathogen inactivation compounds have been developed, which usually have electrophilic groups that react with pathogens, more specifically with pathogen nucleic acid.For example, U.S. Patent Nos. 5,691,132; 6,177,441; 6,410,219; 6,143,490; and 6,093,725, the disclosures of which are incorporated herein by reference, describe the use of compounds with nucleic acid targeting moieties and electrophilic moieties that react with nucleic acid to inactivate pathogens.U.S. Patent Nos. 6,093,725 and 6,514,987, the disclosures of which are incorporated herein by reference, describe compounds in which the nucleic acid targeting moiety of the compound is linked to a reactive electrophilic moiety via a hydrolyzable linker.Compounds containing such hydrolyzable linkers can be called fragile compounds. Under certain conditions, the linker of a frangible compound undergoes a hydrolysis reaction, which results in decoupling of the nucleic acid targeting moiety and the electrophilic moiety of the compound. A compound containing a nucleic acid targeting moiety linked to an electrophilic moiety via a non-hydrolyzable linker can be referred to as a non-frangible compound.

[0040] In some embodiments, the pathogen inactivating compound includes compounds that include functional groups that are reactive groups, e.g., electrophilic groups, or that can form, e.g., in situ, reactive groups, e.g., electrophilic groups. In some cases, the pathogen inactivating compound requires photoactivation to become reactive. In some cases, the pathogen inactivating compound does not require photoactivation to become reactive. For example, the functional group may be a mustard group, a mustard group intermediate, a mustard group equivalent, an epoxide, a formaldehyde, or a formaldehyde synthon. Such functional groups may be capable of forming reactive groups in situ, e.g., electrophilic aziridine, aziridinium, thiirane, or thiiranium ions, etc. The mustard group may be a mono- or bis-(haloethyl)amine group or a mono(haloethyl)sulfide group. Mustard equivalents are groups that react by a mechanism similar to mustard, e.g., by forming reactive intermediates, such as aziridinium and aziridine groups or thiirane and thiiranium groups. Examples include aziridine derivatives, mono- or bis-(mesylethyl)amine groups, mono-(mesylethyl)sulfur groups, and the like. Examples of the reactive groups of the pathogen inactivating compound include hydroxylamine, mono- or bis-(tosylethyl)amine and mono-(tosylethyl)sulfide groups. Formaldehyde synthons are any compounds that decompose to formaldehyde, including hydroxylamines, such as hydroxymethylglycine. The reactive groups of the pathogen inactivating compound can react with the nucleic acid of the pathogen, such as nucleophilic groups on the nucleic acid. The reactive groups can also react with the nucleophilic groups of the quencher.

[0041] In some embodiments, the pathogen inactivating compound includes a moiety that allows the compound to target nucleic acids, such as an anchoring moiety. The anchoring moiety can be non-covalently bound to a nucleic acid biopolymer, such as DNA or RNA, and includes moieties also referred to as nucleic acid binding ligands, nucleic acid binding groups, or nucleic acid binding moieties. In certain embodiments, the anchoring moiety is linked to a reactive electrophilic moiety via a hydrolyzable linker.

[0042] In some embodiments, the compound used to treat the substrates provided herein, or the resulting moieties attached to the surface of the substrates provided herein, are non-brittle compounds or derivatives thereof. The non-brittle compounds may be compounds of any of the formulas I, II, or III, as follows:

[0043] Formula I is [ka] wherein n is an integer from 1 to 12, inclusive, and X is CH2, NH, O, or S, or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0044] Formula II is [ka] (wherein n is an integer from 1 to 12, inclusive), or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0045] Formula III is [ka] wherein one or two of R1, R2, R3, R4, and R5 are independently a (2-chloroethyl)amino group or a (2-bromoethyl)amino group, optionally bearing a second 2-ethylhalo group on the amine, attached to the tricyclic ring by a chain of 1 to 9 carbons, which chain optionally contains one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, which chain optionally contains one or more unsaturated bonds or carbonyl groups, and which chain is optionally substituted with one or more lower alkyl groups; The remaining R1, R2, R3, R4, and R5 are independently hydrogen, lower alkyl, lower alkoxy, halogen, -CH2OR6, or -CH2NR7R8, where R6, R7, and R8 are each independently hydrogen or lower alkyl.

[0046] Specific non-brittle compounds are: [ka] [ka] and salts or stereoisomers (including enantiomers and diastereomers) thereof.

[0047] In some embodiments, the compounds used to treat the substrates provided herein, The resulting moiety attached to the surface of the substrate provided herein is a brittle compound or a derivative thereof. The brittle compound may be a compound of any of formulas IV, V, or VI, or a derivative of a compound of any of formulas IV, V, or VI, as follows:

[0048] Formula IV is [ka] wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8 and R9 is -VWXE as defined below, and the remainder of R1, R2, R3, R4, R5, R6, R7, R8 and R9 are independently -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 is selected from the group consisting of Where -R 10 are independently H, -C1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 is heteroalkyl, V is independently -R 11 -, -NH-R 11 -or-N(CH3)-R 11 -, where -R 11 - is, independently, -C 1~8 Alkyl-, -C 1~8 Heteroalkyl-, -aryl-, -heteroaryl-, -C 1~3 Alkyl-Aryl-, -C 1~3 Heteroalkyl-aryl-, -C 1~3 Alkyl-heteroaryl-, -C 1~3 Heteroalkyl-Heteroaryl-, -aryl-C 1~3 Alkyl-, -aryl-C1~3 Heteroalkyl-, -heteroaryl-C 1~3 Alkyl-, -heteroaryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl-, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl- or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 heteroalkyl-, W is independently -C(=O)-O-, -OC(=O)-, -C(=S)-O-, -OC(=S)-, -C(=S)-S-, -SC(=S)-, -C(=O)-S-, -SC(=O)-, -OS(=O)2-O-, -S(=O)2-O-, -OS(=O)2-, -C(=O)-NR 10 -, -NR 10 -C(=O)-, -OP(=O)(-O R 10 )-O-, -P(=O)(-OR 10 )-O-, -OP(=O)(-OR 10 )-and X is independently -R 11 - and E is independently -N(R 12 )2, -N(R 12 )(R 13 ), -SR 12 , and [ka] is selected from the group consisting of Where -R 12 is -CH2CH2-G, where each G is independently -Cl, -Br, -I, -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5, or -OS(=O)2-C6H4-CH3; R 13 independently -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0049] In some embodiments of formula IV, R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. In other embodiments, at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen.

[0050] In some embodiments of Formula IV, R9 is -VWXE. In some embodiments, V is -NH-R 11 In some such embodiments, R 11 -C 1~8 In certain embodiments, V is -NH-R 11 - where R 11 is linear-C 1~8 In some embodiments, V is -N(CH3)-R 11 In some such embodiments, R 11 -C 1~8 In certain embodiments, V is -N(CH3)-R 11 - where R 11 is linear-C 1~8 It is alkyl-.

[0051] In some embodiments of Formula IV, W is -C(=O)-O-. In some embodiments, W is -C(=O)-NR 10 In some embodiments, W is -C(=O)-NH-.

[0052] In some embodiments of Formula IV, X is -R 11 -, where -R 11 -Ha-C 1~8 In some embodiments, X is a straight chain -C 1~8 It is alkyl-.

[0053] In some embodiments of formula IV, E is -N(R 12)2, where R 12 is -CH2CH2-G. In some such embodiments, each G is independently -Cl, -Br, or -I. In some such embodiments, both G moieties are -Cl.

[0054] In some embodiments of formula IV, R9 is -NH-C 1~8 Alkyl-C(=O)-OC 1~8 In some embodiments, R9 is -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, or -NH-(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2. In some such embodiments, each of R1, R2, R3, R4, R5, R6, R7, and R8 is hydrogen.

[0055] In some embodiments of formula IV, R9 is -NH-C 1~8 Alkyl-C(=O)-NH-C 1~8 In some embodiments, R9 is -NH-(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -NH-(CH2)3-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, or -NH-(CH2)-C(=O)-NH-(CH2)3-N(CH2CH2Cl)2. In some such embodiments, each of R1, R2, R3, R4, R5, R6, R7, and R8 is hydrogen.

[0056] Formula V is: [ka] (Wherein, R1, R2, R3, R4, R5, R6, R7, and R8 are independently -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 is selected from the group consisting of Where -R 10 are independently H, -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroa Rukill, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C1~3 is heteroalkyl, R 20 is -H or -CH3, R 21 -R 11 -WXE, Where -R 11 - is, independently, -C 1~8 Alkyl-, -C 1~8 Heteroalkyl-, -aryl-, -heteroaryl-, -C 1~3 Alkyl-Aryl-, -C 1~3 Heteroalkyl-aryl-, -C 1~3 Alkyl-heteroaryl-, -C 1~3 Heteroalkyl-Heteroaryl-, -aryl-C 1~3 Alkyl-, -aryl-C 1~3 Heteroalkyl-, -heteroaryl-C 1~3 Alkyl-, -heteroaryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl-, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl- or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 heteroalkyl-, W is independently -C(=O)-O-, -OC(=O)-, -C(=S)-O-, -OC(=S)-, -C(=S)-S-, -SC(=S)-, -C(=O)-S-, -SC(=O)-, -OS(=O)2-O-, -S(=O)2-O-, -OS(=O)2-, -C(=O)-NR10 -, -NR 10 -C(=O)-, -OP(=O)(-OR 10 )-O-, -P(=O)(-OR 10 )-O-, -OP(=O)(-OR 10 )-and X is independently -R 11 - and E is independently -N(R 12 )2, -N(R 12 )(R 13 ), -SR 12 , and [ka] wherein -R 12 is -CH2CH2-G, where each G is independently -Cl, -Br, -I, -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5, or -OS(=O)2-C6H4-CH3; R 13 is independently -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0057] In some embodiments of Formula V, R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. In other embodiments, at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen. In some embodiments of Formula V, R 20 is H. In some embodiments, R 20 is -CH3.

[0058] In some embodiments of Formula V, R 21 -R 11 In some embodiments, R 11 -C 1~8 alkyl-, e.g., methyl or ethyl. In some embodiments, R 11 is linear-C 1~8 In some embodiments, R 11 is -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or -(CH2)8-.

[0059] In some embodiments of Formula V, W is -C(=O)-O-. In some embodiments, W is -C(=O)-NR 10 In some embodiments, W is -C(=O)-NH-.

[0060] In some embodiments of Formula V, X is -R 11-, where -R 11 -Ha-C 1~8 In some embodiments, X is a linear -C alkyl-. In some embodiments, X is methyl or ethyl. 1~8 In some embodiments, X is -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or -(CH2)8-. In some such embodiments, R 20 is H. In some such embodiments, R 20 is -CH3.

[0061] In some embodiments of Formula V, E is -N(R 12 )2, where R 12 is -CH2CH2-G. In some such embodiments, each G is independently -Cl, -Br, or -I. In some such embodiments, both G moieties are -Cl.

[0062] In some embodiments of Formula V, R 21 -C 1~8 Alkyl-C(=O)-OC 1~8 In some embodiments, R 21 is -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2, or -(CH2)-C(=O)-O-(CH2)-N(CH2CH2Cl)2. In some such embodiments, R 20 is H. In some such embodiments, R 20 is -CH3. In any of the above embodiments, each of R1, R2, R3, R4, R5, R6, R7, and R8 can be hydrogen.

[0063] In some embodiments of Formula V, R 21 -C 1~8 Alkyl-C(=O)-NH-C 1~8 In some embodiments, R 21 is -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2, or -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2Cl)2. In some such embodiments, R 20 is H. In some such embodiments, R 20 is -CH3. In any of the above embodiments, each of R1, R2, R3, R4, R5, R6, R7, and R8 can be hydrogen.

[0064] Formula VI is [ka] (In the formula, R 44 , R 55 , At least one of R3, R4, R5, and R8 is -VWXE, and R 44 , R 55 , the remainder of R3, R4, R5, and R8 are independently -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 is selected from the group consisting of Where -R 10 are independently H, -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 is heteroalkyl, V is independently -R 11 -, -NH-R 11 -or-N(CH3)-R 11 -, where -R 11 - is, independently, -C 1~8 Alkyl-, -C 1~8 Heteroalkyl-, -aryl-, -heteroaryl-, -C 1~3 Alkyl-Aryl-, -C 1~3 Heteroalkyl-aryl-, -C 1~3 Alkyl-heteroaryl-, -C 1~3 Heteroalkyl-Heteroaryl-, -aryl-C 1~3 Alkyl-, -aryl-C 1~3 Heteroalkyl-, -heteroaryl-C 1~3 Alkyl-, -heteroaryl-C 1~3Heteroalkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl-, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl- or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 heteroalkyl-, W is independently -C(=O)-O-, -OC(=O)-, -C(=S)-O-, -OC(=S)-, -C(=S)-S-, -SC(=S)-, -C(=O)-S-, -SC(=O)-, -OS(=O)2-O-, -S(=O)2-O-, -OS(=O)2-, -C(=O)-NR 10 -, -NR 10 -C(=O)-, -OP(=O)(-OR 10 )-O-, -P(=O)(-OR 10 )-O-, -OP(=O)(-OR 10 )-and X is independently -R 11 - and E is independently -N(R 12 )2, -N(R 12 )(R 13 ), -SR 12 , and [ka] wherein -R 12is -CH2CH2-G, where each G is independently -Cl, -Br, -I, -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5, or -OS(=O)2-C6H4-CH3; R 13 is independently -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0065] It should be appreciated that in the above formula IV, the acridine core is the anchoring moiety, the -VWX- group(s) comprise the frangible linker, and the E group(s) are the effector group. Similarly, in the above formula VI, the psoralen core is the anchoring moiety, the -VWX- group(s) comprise the frangible linker, and the E group(s) are the effector group. Formula V is a subset of formula IV.

[0066] In some embodiments, the frangible compound of formula IV is a compound of formulas IV(a)-IV(h), or a salt or stereoisomer (including enantiomers and diastereomers) thereof, where all variables not specifically indicated are the same as for formula IV above:

[0067] Formula IV(a): W is independently -OC(=O)-, -C(=S)-O-, -OC(=S)-, -C(=S)-S-, -SC(=S)-, -C(=O)-S-, -SC(=O)-, -OS(=O)2-O-, -S(=O)2-O-, -OS(=O)2-, -C(=O)-NR 10 -, -NR 10 -C(=O)-, -OP(=O)(-OR 10 )-O-, -P(=O)(-OR 10 )-O-, -OP(=O)(-OR 10 )-is;

[0068] Formula IV(b): V is independently -R 11 - or -N(CH3)-R 11 -, where -R 11 - is, independently, -C 1~8 Alkyl-, -aryl-, -heteroaryl-, -C 1~3 Alkyl-Aryl-, -C 1~3 Heteroalkyl-aryl-, -C 1~3 Alkyl-heteroaryl-, -C 1~3 Heteroalkyl-Heteroaryl-, -aryl-C 1~3 Alkyl-, -aryl-C 1~3 Heteroalkyl-, -heteroaryl-C 1~3 Alkyl-, -heteroaryl-C1~3 Heteroalkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl-, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl- or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl-;

[0069] Formula IV(c):-R 11 - is independently -aryl-, -heteroaryl-, -C 1~3 Alkyl-Aryl-, -C 1~3 Heteroalkyl-aryl-, -C 1~3 Alkyl-heteroaryl-, -C 1~3 Heteroalkyl-Heteroaryl-, -aryl-C 1~3 Alkyl-, -aryl-C 1~3 Heteroalkyl-, -heteroaryl-C 1~3 Alkyl-, -heteroaryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl-, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl-, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl-, -C 1~3Heteroalkyl-aryl-C 1~3 Heteroalkyl-, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl- or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl-;

[0070] Formula IV(d):-R 12 is -CH2CH2-G, where each G is independently -Br, -I, -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5, or -OS(=O)2-C6H4-CH3;

[0071] Formula IV(e): E is independently -N(R 12 )(R 13 ), -SR 12 , and [ka] wherein -R 12 is -CH2CH2-G, where each G is independently -Cl, -Br, -I, -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5, or -OS(=O)2-C6H4-CH3; R 13 is independently -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 is heteroalkyl;

[0072] Formula IV(f): W is independently -C(=S)-O-, -OC(=S)-, -C(=S)-S-, -SC(=S)-, -C(=O)-S-, -SC(=O)-, -OS(=O)2-O-, -S(=O)2-O-, -OS(=O)2-, -C(=O)-NR 10 -, -NR 10 -C(=O)-, -OP(=O)(-OR 10 )-O-, -P(=O)(-OR 10 )-O-, -OP(=O)(-OR 10 )-is;

[0073] Formula IV(g):-R 12 is -CH2CH2-G, where each G is independently -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5, or -OS(=O)2-C6H4-CH3;

[0074] Formula IV(h): At least one of R1, R2, R3, R4, R5, R6, R7, R8 and R9 is -VWXE as defined for formula IV, and at least one of R1, R2, R3, R4, R5, R6, R7, R8 and R9 is - R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R10 , -C(=O)-OR 10 , and -OC(=O)-R 10 and the remainder of R1, R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 and R10 is as defined for formula IV.

[0075] In some embodiments, the compounds used to treat the substrates provided herein, or the resulting moieties attached to the surface of the substrates provided herein, are non-brittle compounds, such as non-brittle analogs of compounds of any of formulas IV, V, or VI. Such non-brittle compounds may be compounds of any of formulas VII, VIII, or IX, as follows:

[0076] Formula VII is [ka] wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8, and R9 is -VXE, where V, X, and E are as defined for formula IV, and the remainder of R1, R2, R3, R4, R5, R6, R7, R8, and R9 are independently -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 is selected from the group consisting of Where -R 10 are independently H, -C 1~8Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0077] In some embodiments of formula VII, R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. In other embodiments, at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen.

[0078] In some embodiments of Formula VII, R9 is -VXE, where V, X, and E are as defined for Formula IV. In some embodiments, V is -NH-R 11 In some such embodiments, R 11 -C 1~8 In certain embodiments, V is -NH-R 11 - where R 11 is linear-C 1~8 In some embodiments, V is -N(CH3)-R 11 In some such embodiments, R 11 -C 1~8 In certain embodiments, V is -N(CH3)-R 11 - where R 11 is linear-C 1~8 It is alkyl-.

[0079] In some embodiments of Formula VII, X is -R 11 -, where -R 11 -Ha-C 1~8 In some embodiments, the moiety -VX- is -NH-C 1~8 In some embodiments, the moiety -VX- is -NH-(CH2)-, -NH-(CH2)2-, -NH-(CH2)3-, -NH-(CH2)4-, -NH-(CH2)5-, -NH-(CH2)6-, -NH-(CH2)7-, or -NH-(CH2)8-. In some embodiments, the moiety -VX- is -N(CH3)-C 1~8 In some embodiments, the moiety -VX- is -N(CH3)-(CH2)-, -N(CH3)-(CH2)2-, -N(CH3)-(CH2)3-, -N(CH3)-(CH2)4-, -N(CH3)-(CH2)5-, -N(CH3)-(CH2)6-, -N(CH3)-(CH2)7-, or -N(CH3)-(CH2)8-.

[0080] In some embodiments of Formula VII, E is -N(R 12)2, where R 12 is -CH2CH2-G. In some such embodiments, each G is independently -Cl, -Br, or -I. In some such embodiments, both G moieties are -Cl.

[0081] In some embodiments of Formula VII, R9 is -NH-C 1~8 It is alkyl-N(CHCHCl). In some embodiments, R is -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), -NH-(CH)-N(CHCHCl), or -NH-(CH)-N(CHCHCl). In some such embodiments, each of R, R, R, R, R, R, R, R, R, R, R, R, and R is hydrogen.

[0082] Formula VIII is [ka] (Wherein, R1, R2, R3, R4, R5, R6, R7, and R8 are independently -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2,- F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 is selected from the group consisting of Where -R 10 are independently H, -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 is heteroalkyl, R 20 is -H or -CH3, R 21 -R 11 -XE, where R 11 , X, and E are as defined for formula V. or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0083] In some embodiments of formula VIII, R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. In other embodiments, at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen.

[0084] In some embodiments of Formula VIII, R 20is H. In some embodiments, R 20 is -CH3.

[0085] In some embodiments of Formula VIII, R 21 -R 11 -XE, where R 11 , X, and E are as defined for formula V. In some embodiments, R 11 -C 1~8 alkyl-, e.g., methyl or ethyl. In some embodiments, R 11 is linear-C 1~8 In some embodiments, R 11 is -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or -(CH2)8-.

[0086] In some embodiments of Formula VIII, X is -R 11 -, where -R 11 -Ha-C 1~8 In some embodiments, the moiety -R 11 -X- is -C 1~8 In some such embodiments, R 20 is H. In some such embodiments, R 20 is -CH3. In some embodiments, the moiety -R 11 -X- is -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or -(CH2)8-. In some such embodiments, R 20 is H. In some such embodiments, R 20 is -CH3.

[0087] In some embodiments of Formula VIII, E is -N(R 12 )2, where R 12is -CH2CH2-G. In some such embodiments, each G is independently -Cl, -Br, or -I. In some such embodiments, both G moieties are -Cl.

[0088] In some embodiments of Formula VIII, R 21 -C 1~8 In some embodiments, R 21 -(CH2)-N(CH2CH2Cl)2 , -(CH2)2-N(CH2CH2Cl)2, -(CH2)3-N(CH2CH2Cl)2, -(CH2)4-N(CH2CH2Cl)2, -(CH2)5-N(CH2CH2Cl)2, -(CH2)6-N(CH2CH2Cl)2, -(CH2)7-N(CH2CH2Cl)2, or -(CH2)8-N(CH2CH2Cl)2. In some such embodiments, R 20 is H. In some such embodiments, R 20 is -CH3. In any of the above embodiments, each of R1, R2, R3, R4, R5, R6, R7, and R8 can be hydrogen.

[0089] Formula IX is [ka] (In the formula, R 44 , R 55 At least one of R3, R4, R5, and R8 is -VXE, where V, X, and E are as defined for formula VI; R 44 , R 55 , the remainder of R3, R4, R5, and R8 are independently -H, -R 10 , -OR 10 , -NO2, -NH2, -NH-R 10 , -N(R 10 )2, -F, -Cl, -Br, -I, -C(=O)-R 10 , -C(=O)-OR 10 , and -OC(=O)-R 10 is selected from the group consisting of Where -R 10 are independently H, -C 1~8 Alkyl, -C 1~8 Heteroalkyl, -aryl, -heteroaryl, -C 1~3 Alkyl-aryl, -C 1~3 Heteroalkyl-aryl, -C 1~3 Alkyl-heteroaryl, -C 1~3 Heteroalkyl-heteroaryl, -aryl-C 1~3 Alkyl, -aryl-C 1~3 Heteroalkyl, -heteroaryl-C 1~3 Alkyl, -heteroaryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Aryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Alkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Alkyl-Aryl-C 1~3 Heteroalkyl, -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Alkyl, -C 1~3 Heteroalkyl-aryl-C 1~3 Heteroalkyl, -C 1~3 Alkyl-Heteroaryl-C 1~3 Heteroalkyl, or -C 1~3 Heteroalkyl-Heteroaryl-C 1~3 Heteroalkyl or a salt or stereoisomer (including enantiomers and diastereomers) thereof.

[0090] A specific example of a pathogen inactivating compound suitable for use in the compositions and methods of the present invention is β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester (also alternatively referred to herein as “S-303”), the structure of which, including its salts, is as follows: [ka]

[0091] In some embodiments, the compound used in the treatment of the substrates provided herein, or the generated moiety attached to the surface of the substrate provided herein, is S-303. In some embodiments, the compound used in the treatment of the substrates provided herein, or the generated moiety attached to the surface of the substrate provided herein, is a derivative of S-303. In some embodiments, the compound used in the treatment of the substrates provided herein, or the generated moiety attached to the surface of the substrate provided herein, is a non-fragile analog of S-303 or a derivative thereof.

[0092] In some embodiments, the compound used in the treatment of the substrates provided herein, or the resulting moiety attached to the surface of the substrates provided herein, is a compound selected from the following compounds, or salts or stereoisomers (including enantiomers and diastereomers) thereof: [ka] [ka] [ka] [ka] [ka]

[0093] In some embodiments of any of the above compounds, the compound is a chloride salt. In some embodiments of any of the above compounds, the compound is a dihydrochloride salt. In some embodiments, the compound is a dihydrochloride salt of S-197. In some embodiments, the compound is a dihydrochloride salt of S-220.

[0094] In some embodiments, the compounds used in the treatment of the substrates provided herein, or the generated moieties attached to the surface of the substrates provided herein, are derivatives (e.g., hydrolyzed derivatives) of any of the aforementioned compounds. In some embodiments, the compounds used in the treatment of the substrates provided herein, or the generated moieties attached to the surface of the substrates provided herein, are non-fragile analogs of any of the aforementioned compounds.

[0095] Derivatives of compounds provided herein can include a nucleic acid targeting moiety, a portion of a nucleic acid targeting moiety, an electrophilic moiety, or a portion of an electrophilic moiety. Derivatives of compounds provided herein can include a) a nucleic acid targeting moiety or a portion of a nucleic acid targeting moiety, and b) an electrophilic moiety or a portion of an electrophilic moiety. Derivatives of compounds provided herein can include only a nucleic acid targeting moiety or a portion of a nucleic acid targeting moiety. Derivatives of compounds provided herein can include only an electrophilic moiety or a portion of an electrophilic moiety. When a compound is non-fragile, its derivatives can include a) a nucleic acid targeting moiety or a portion of a nucleic acid targeting moiety, and b) an electrophilic moiety or a portion of an electrophilic moiety, where a) and b) are linked to each other via a non-hydrolyzable linker. When a compound is fragile, its derivatives can include a) a nucleic acid targeting moiety or a portion of a nucleic acid targeting moiety, and b) an electrophilic moiety or a portion of an electrophilic moiety, where a) and b) are linked to each other via a hydrolyzable linker.When a compound is fragile, its derivatives can include a) a nucleic acid targeting moiety or a portion of a nucleic acid targeting moiety linked to a portion representing the hydrolysis product of the hydrolyzable linker.When a compound is fragile, its derivatives can include an electrophilic moiety or a portion of an electrophilic moiety linked to a portion representing the hydrolysis product of the hydrolyzable linker.The portion representing the hydrolysis product includes, without limitation, alcohols, amines, carboxylic acids, esters, and salts or stereoisomers of any of the foregoing.

[0096] Any of the compounds or derivatives described herein may be the starting material for reacting with a substrate to form any of the compositions provided herein. Alternatively, any of the compounds or derivatives described herein may be bound to a substrate in any of the compositions provided herein. Any of the derivatives provided herein may be formed from the compound of which it is a derivative, or an equivalent molecular structure may be prepared (e.g., synthesized) independently from the compound of which it is a derivative.

[0097] It is understood that a given substrate unit, (e.g., a single red blood cell, a single polystyrene bead) will contain one or more surface-bound moieties. The surface-bound moieties may be uniform across a given substrate unit. Alternatively, the surface-bound moieties may be heterogeneous across a given substrate unit (e.g., a red blood cell). Heterogeneity across a given substrate unit may result from the reaction of the substrate unit with a heterogeneous set of compounds or derivatives. Heterogeneity across a given substrate unit may also result from the reaction of the substrate unit with a homogeneous compound or derivative, where the reaction of the compound or derivative with the substrate results in two or more different reaction products bound to the substrate surface. In some cases, the surface-bound moieties may be uniform across any particular substrate unit, but heterogeneous across a population of substrate units (e.g., red blood cells). This may result from the reaction of different substrate units with different compounds or derivatives, followed by mixing of the resulting substrate units to form a heterogeneous population of surface-bound substrate units (e.g., red blood cells).

[0098] In any of the embodiments described herein, the surface-binding moiety can be present in an amount on the substrate surface, also referred to as the loading level of the substrate. The loading level can be described on a per-unit-substrate basis, for example, on a per-cell basis (e.g., red blood cells), per-bead basis, or per-particle basis (e.g., parts per cell, parts per bead, parts per particle). In some embodiments, the loading level of the substrate may be at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 8,000, at least about 10,000, at least about 12,000, at least about 14,000, at least about 17,000, at least about 20,000, at least about 25,000, at least about 30,000, at least about 35,000, at least about 40,000, or at least about 50,000 parts per unit of substrate (e.g., per red blood cell). In some embodiments, the loading level of the substrate may be no more than about 50,000, no more than about 75,000, or no more than about 100,000 parts per unit of substrate (e.g., per red blood cell). In some embodiments, the loading level of the substrate is between about 1,000 and about 100,000, between about 1,000 and about 75,000, between about 1,000 and about 50,000, between about 1,000 and about 25,000, between about 5,000 and about 50,000, between about 5,000 and about 25,000, between about 5,000 and about 50,000, between about 5,000 and about 25,000, or between about 5,000 and about 50,000. It may be a portion between 0 and about 15,000, between about 8,000 and about 15,000, between about 25,000 and about 50,000, between about 25,000 and about 40,000, between about 25,000 and about 35,000, between about 25,000 and about 30,000, between about 30,000 and about 40,000, or between about 30,000 and about 35,000.In some embodiments, the loading level of the substrate is about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 12,000, about 13,000, or more per unit of substrate (e.g., per red blood cell). , about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 20,000, about 22,000, about 24,000, about 26,000, about 28,000, about 30,000, about, about 32,000, about 35,000, about 40,000, about 45,000, about 50,000, about 60,. 000, about 70,000, about 80,000, about 90,000, or about 100,000 parts.

[0099] The loading level is based on a unit area basis, e.g., on a unit surface area basis (e.g., 1 μm of substrate surface area). 2 In some embodiments, the loading level of the substrate can be described in terms of at least about 5 parts / μm. 2 , at least about 7 parts / μm 2 , at least about 10 parts / μm 2 , at least about 15 parts / μm 2 , at least about 20 parts / μm 2 , at least about 25 parts / μm 2 , at least about 30 parts / μm 2 , at least about 35 parts / μm 2 , at least about 40 parts / μm 2 , at least about 50 parts / μm 2 , at least about 60 parts / μm 2 , at least about 70 parts / μm 2 , at least about 80 parts / μm 2 , at least about 90 parts / μm 2 , at least about 100 parts / μm 2 , at least about 120 parts / μm 2 , at least about 140 parts / μm 2 , at least about 160 parts / μm 2, at least about 180 parts / μm 2 , at least about 200 parts / μm 2 , at least about 225 parts / μm 2 , at least about 250 parts / μm 2 , at least about 275 parts / μm 2 , at least about 300 parts / μm 2 , at least about 350 parts / μm 2 , at least about 400 parts / μm 2 , at least about 450 parts / μm 2 , or at least about 500 parts / μm 2 In some embodiments, the loading level of the substrate may be about 500 parts / μm 2 Below, about 750 parts / μm 2 Less than or equal to 1,000 parts / μm 2 In some embodiments, the loading level of the substrate may be from about 5 to about 1,000 parts / μm 2 Between about 5 and about 750 parts / μm 2 Between about 5 and about 500 pieces / μm 2 Between about 20 and about 500 pieces / μm 2 Between about 20 and about 400 pieces / μm 2 Between about 20 and about 200 pieces / μm 2 Between about 20 and about 100 pieces / μm 2 Between about 30 and about 200 pieces / μm 2 Between about 30 and about 100 pieces / μm 2 Between about 50 and about 100 pieces / μm 2 Between about 100 and about 500 pieces / μm 2 Between about 100 and about 400 pieces / μm 2 Between about 140 and about 400 pieces / μm 2 Between about 140 and about 300 pieces / μm 2 Between about 140 and about 250 pieces / μm 2 Between about 180 and about 300 pieces / μm 2 In some embodiments, the loading level of the substrate may be between about 5 parts / cm 2, about 7 pieces / cm 2 , about 10 pieces / cm 2 , about 15 pieces / cm 2 , about 20 pieces / cm 2 , about 25 pieces / cm 2 , about 30 pieces / cm 2 , about 35 pieces / cm 2 , about 40 pieces / cm 2 , about 45 pieces / cm 2 , about 50 pieces / cm 2 , about 55 pieces / cm 2 , about 60 pieces / cm 2 , about 65 pieces / cm 2 , about 70 pieces / cm 2 , about 75 pieces / cm 2 , about 80 pieces / cm 2 , about 85 pieces / cm 2 , about 90 pieces / cm 2 , about 95 pieces / cm 2 , about 100 pieces / cm 2 , about 110 pieces / cm 2 , about 120 pieces / cm 2 , about 130 pieces / cm 2 , about 140 pieces / cm 2 , about 150 pieces / cm 2 , about 160 pieces / cm 2 , about 170 pieces / cm 2 , about 180 pieces / cm 2 , about 190 pieces / cm 2 , about 200 pieces / cm 2 , about 225 pieces / cm 2 , about 250 pieces / cm 2 , about 275 pieces / cm 2 , about 300 pieces / cm 2 , about 325 pieces / cm 2 , about 350 pieces / cm 2 , about 375 pieces / cm 2 , about 400 pieces / cm 2 , about 425 pieces / cm 2 , about 450 pieces / cm 2 , about 475 pieces / cm2 , about 500 pieces / cm 2 , about 600 pieces / cm 2 , about 700 pieces / cm 2 , about 800 pieces / cm 2 , about 900 pieces / cm 2 , or about 1,000 pieces / cm 2 It may be.

[0100] The loading level may be substantially uniform across a group of substrate units, or it may be non-uniform across a group of substrate units. The loading levels provided herein may represent a quantified average loading level across a group of substrate units. Loading levels may be determined by any method known in the art, such as labeling the compound (e.g., radiolabeled, immunolabeled, chemically labeled, chemiluminescent, fluorescently labeled) prior to addition to the substrate and then removing the label after the compound is combined with the substrate. The loading level can be measured by detecting. Alternatively or additionally, the loading level can be measured by directly or indirectly labeling the compound after adding to the substrate (e.g., immunolabeling, immunofluorescence, chemiluminescence) and then detecting the label. Generally, such labeling is performed separately (e.g., as a control, as a calibration curve) from the compounds used in the kits and methods provided herein, as a comparison or reference point when determining absolute or relative loading levels. Such labeling can be directly quantified using quantitative or semi-quantitative methods, or compared to a reference standard, such as cells, beads or other materials with a known amount of similarly labeled molecules on the surface (e.g., FACS analysis).

[0101] In some embodiments, the composition (e.g., the partially bonded substrate) is stable at room temperature. In some embodiments, the composition is stable at refrigerated temperatures (e.g., 2-8° C.). In some embodiments, the composition is stable at 0° C. and / or below 0° C. (e.g., −20° C.). In some embodiments, the composition is stable at −80° C. The composition may be suitable for storage at room temperature for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 4 months, about 6 months, about 9 months, or about 12 months or longer. The composition may be suitable for storage at refrigerated temperatures for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 4 months, about 6 months, about 9 months, or about 12 months or longer. The composition may be suitable for storage at 0° C. and / or below 0° C. for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 3 months, about 6 months, about 9 months, or about 1 year or longer. The composition may be suitable for storage at -80°C for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 3 months, about 6 months, about 9 months, or about 1 year or longer. The composition may be stored in the presence of a liquid, a buffer, or any suitable solution (e.g., a non-reactive solution). For example, if the substrate is red blood cells, the composition can be stored in red blood cell additive solution, blood bank saline, buffered suspension medium, or other suitable solution. Red blood cell additive solutions and buffered suspension medium are known in the art.When the substrate is red blood cells, the composition can be stored as a suspension of about 0.3% to about 10% RBC, about 0.5% to about 5% RBC, about 0.5% to about 1% RBC, about 1% to about 1.5% RBC, about 1.5% to about 2% RBC, about 2% to about 2.5% RBC, about 2.5% to about 3% RBC, about 3% to about 3.5% RBC, about 3.5% to about 4% RBC, about 4% to about 4.5% RBC, about 4.5% to about 5% RBC, or about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% RBC. When the composition is frozen for storage, a cryopreservation agent can also be added, such as any cryopreservation agent known in the art.

[0102] The stability and suitability for storage of the compositions provided herein (e.g., substrates having moieties attached thereto) may depend on the nature of the surface-attached moiety. For example, a surface-attached moiety lacking a hydrolyzable linker (e.g., a non-brittle compound or derivative thereof, or a hydrolysis product of a brittle compound) may be more stable than a surface-attached moiety containing a hydrolyzable linker (e.g., a brittle compound or derivative thereof). In some embodiments, the compositions provided herein containing a surface-attached moiety lacking a hydrolyzable linker are stable at room temperature. In some embodiments, the compositions are stable at refrigerated temperatures. In some embodiments, the compositions are stable at 0° C. and / or below 0° C. In some embodiments, the compositions are stable at −80° C. The compositions may be stable for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about The composition may be suitable for storage at room temperature for 4 months, about 6 months, about 9 months, or about 12 months or longer. The composition may be suitable for storage at refrigerated temperature for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 4 months, about 6 months, about 9 months, or about 12 months or longer. The composition may be suitable for storage at 0° C. and / or below 0° C. for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 3 months, about 6 months, about 9 months, or about 1 year or longer. The composition may be suitable for storage at -80°C for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 3 months, about 6 months, about 9 months, or about 1 year or longer. In some embodiments, the compositions provided herein containing surface-binding moieties containing hydrolyzable linkers are stable at room temperature. In some embodiments, the compositions are stable at refrigerated temperatures. In some embodiments, the compositions are stable at 0°C and / or below 0°C. In some embodiments, the compositions are stable at -80°C. The composition may be suitable for storage at room temperature for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 4 months, about 6 months, about 9 months, or about 12 months or longer. The composition may be suitable for storage at refrigerated temperatures for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 4 months, about 6 months, about 9 months, or about 12 months or longer.The composition may be suitable for storage at 0° C. and / or below 0° C. for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 2 months, about 3 months, about 6 months, about 9 months, or about 1 year or more. The composition may be suitable for storage at −80° C. for at least about 1 hour, about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, 2 months, about 3 months, about 6 months, 9 months, or about 1 year or more. kit

[0103] Provided herein are various kits that include any of the compositions (e.g., substrates having surface-binding moieties) described herein. In some embodiments, the kits include instructions that describe the use of the substrate and / or substrates having surface-binding moieties to perform any of the methods described herein. The kits may also contain any combination of additional components that may be necessary or useful to perform any of the methods described herein. Exemplary kits are described herein, but other useful kit contents will be apparent to one of skill in the art in light of this disclosure.

[0104] Provided herein are kits for detecting the presence of antibodies in a biological sample according to the methods described herein. In some embodiments, the kits include a first container containing a first substrate, where a moiety is attached to the surface of the first substrate, the moiety being selected from the compounds and derivatives thereof described herein, and a second container containing a second substrate, where the surface of the second substrate lacks an attached moiety. The moiety attached to the surface of the first substrate can be any compound or derivative described herein, such as a pathogen inactivating compound or derivative thereof. In some embodiments, the moiety attached to the surface of the first substrate is S-303 or a derivative thereof. In some embodiments, the moiety attached to the surface of the first substrate is a non-fragile analog of S-303 or a derivative thereof. In some embodiments, the moiety attached to the surface of the first substrate is a compound of formula I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII, and XI. and derivatives thereof, and salts and solvates of any of the foregoing. In some embodiments, the moiety attached to the surface of the first substrate is selected from the group consisting of compounds of formulas I, II, III, VII, VIII, and XI, and derivatives thereof, and salts and solvates of any of the foregoing. In some embodiments, the moiety attached to the surface of the first substrate is selected from the group consisting of compounds of formulas IV, V, and VI, and derivatives thereof, and salts and solvates of any of the foregoing. In some embodiments, the moiety attached to the surface of the first substrate is selected from the group consisting of compounds of formulas IV(a)-IV(h), and derivatives thereof, and salts and solvates of any of the foregoing.

[0105] The first and second substrates can be composed of any suitable substrate material, for example, any substrate material described herein. The first and second substrates can be composed of cells (e.g., red blood cells). Alternatively, the first and second substrates can be composed of cell membranes or cell-derived materials. For example, the first and second substrates can include membrane preparations derived from red blood cells, such as red blood cell ghosts (i.e., membranes of lysed red blood cells). Alternatively, the first and second substrates can be composed of solid supports. As described herein, the solid supports can be composed of materials (e.g., biological materials, synthetic materials) or matrices, including, without limitation, polymers or copolymers (e.g., polyesters, polyethers, polyolefins, polyamides, polysaccharides, polyurethanes, styrene and styrene derivatives, polystyrene, cellulose), plastics, glass, gold and other metals, and the like. The solid supports can present surface-bound moieties in two-dimensional formats (e.g., plates, multi-well plates) or three-dimensional formats (e.g., cells, beads or other spherical or quasi-spherical objects). The substrate can be part of an assay plate. A particular solid support is composed of one or more materials to which the biological sample does not bind or does not substantially bind. For example, the solid support may be composed of a material to which an antibody (e.g., an antibody specific for a compound or a portion thereof) does not bind or does not substantially bind. In particular, the solid support is composed of a material to which an antibody does not bind or does not substantially bind to pathogen-inactivated red blood cells. Usually, the first and second substrates are composed of the same material. In some embodiments, the first and second substrates may comprise red blood cells or a membrane preparation derived from red blood cells immobilized on the surface of a solid support, such as the solid supports described above. The red blood cells or membrane preparations can be immobilized on the surface of the solid support in various ways known in the art, for example, by means of antibodies against non-blood group antigens expressed on the cell surface, by adhesion using poly-L-lysine, organic dyes or lectins (e.g., lectins with specificity for galactosyl moieties).Preferably, the reagents used are compatible with the cell surface and capable of maintaining stable attachment of the cells or membranes to the solid support throughout the assay.

[0106] In some embodiments, both the first and second substrates are red blood cells. The RBCs of the first and second substrates can be obtained from one or more common donors or from different blood donors. In some cases, the RBCs of the first substrate and the RBCs of the second substrate have one or more characteristics in common, such as ABO blood type (e.g., blood type O), Rh type, or the presence or absence of other RBC antigens (e.g., common RBC antigens, clinically significant RBC antigens, clinically relevant RBC antigens). In other cases, the RBCs of the first substrate and the RBCs of the second substrate differ with respect to certain characteristics, such as the presence or absence of ABO blood type or other RBC antigens (e.g., common RBC antigens, clinically relevant RBC antigens). In some embodiments, the first and second substrates exhibit one or more antigens commonly found in biological samples (e.g., whole blood, red blood cells, serum, or plasma). The presence of such an antigen can serve as a control to indicate that the biological sample has been properly contacted with the first and / or second substrate.

[0107] The loading level of surface-bound moieties on the first substrate (e.g., red blood cells) can be any loading level described herein, such as a loading level sufficient to detect antibodies to the compound or portions thereof, etc. In some embodiments, the loading level of the first substrate can be at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 8,000, at least about 10,000, at least about 12,000, at least about 14,000, at least about 17,000, at least about 20,000, at least about 25,000, at least about 30,000, at least about 35,000, at least about 40,000, or at least about 50,000 moieties per unit of substrate (e.g., per red blood cell). In some embodiments, the loading level of the first substrate may be about 50,000 or less, about 75,000 or less, or about 100,000 or less parts per unit of substrate (e.g., per red blood cell). In some embodiments, the loading level of the first substrate may be between about 1,000 and about 100,000, between about 1,000 and about 75,000, between about 1,000 and about 50,000, between about 1,000 and about 25,000, between about 5,000 and about 50,000, between about 5,000 and about 25 ... The number of molecules may be a portion between about 1,000 and about 15,000, between about 8,000 and about 15,000, between about 25,000 and about 50,000, between about 25,000 and about 40,000, between about 25,000 and about 35,000, between about 25,000 and about 30,000, between about 30,000 and about 40,000, or between about 30,000 and about 35,000.In some embodiments, the loading level of the first substrate is about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000, about 16,000, or more per unit of substrate (e.g., per red blood cell). 0, about 17,000, about 18,000, about 20,000, about 22,000, about 24,000, about 26,000, about 28,000, about 30,000, about, about 32,000, about 35,000, about 40,000, about 45,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, or about 100,000 parts. In some embodiments, the loading level of the first substrate is at least about 5 parts / μm. 2 , at least about 7 parts / μm 2 , at least about 10 parts / μm 2 , at least about 15 parts / μm 2 , at least about 20 parts / μm 2 , at least about 25 parts / μm 2 , at least about 30 parts / μm 2 , at least about 35 parts / μm 2 , at least about 40 parts / μm 2 , at least about 50 parts / μm 2 , at least about 60 parts / μm 2 , at least about 70 parts / μm 2 , at least about 80 parts / μm 2 , at least about 90 parts / μm 2 , at least about 100 parts / μm 2 , at least about 120 parts / μm 2 , at least about 140 parts / μm 2 , at least about 160 parts / μm 2 , at least about 180 parts / μm 2 , at least about 200 parts / μm 2 , at least about 225 parts / μm 2 , at least about 250 parts / μm 2, at least about 275 parts / μm 2 , at least about 300 parts / μm 2 , at least about 350 parts / μm 2 , at least about 400 parts / μm 2 , at least about 450 parts / μm 2 , or at least about 500 parts / μm 2 In some embodiments, the loading level of the first substrate may be about 500 parts / μm 2 Below, about 750 parts / μm 2 Less than or equal to 1,000 parts / μm 2 In some embodiments, the loading level of the first substrate may be from about 5 to about 1,000 parts / μm 2 Between about 5 and about 750 parts / μm 2 Between about 5 and about 500 pieces / μm 2 Between about 20 and about 500 pieces / μm 2 Between about 20 and about 400 pieces / μm 2 Between about 20 and about 200 pieces / μm 2 Between about 20 and about 100 pieces / μm 2 Between about 30 and about 200 pieces / μm 2 Between about 30 and about 100 pieces / μm 2 Between about 50 and about 100 pieces / μm 2 Between about 100 and about 500 pieces / μm 2 Between about 100 and about 400 pieces / μm 2 Between about 140 and about 400 pieces / μm 2 Between about 140 and about 300 pieces / μm 2 Between about 140 and about 25 0 pieces / μm 2 Between about 180 and about 300 pieces / μm 2 In some embodiments, the loading level of the first substrate may be between about 5 parts / cm 2 , about 7 pieces / cm 2 , about 10 pieces / cm 2 , about 15 pieces / cm 2 , about 20 pieces / cm 2 , about 25 pieces / cm2 , about 30 pieces / cm 2 , about 35 pieces / cm 2 , about 40 pieces / cm 2 , about 45 pieces / cm 2 , about 50 pieces / cm 2 , about 55 pieces / cm 2 , about 60 pieces / cm 2 , about 65 pieces / cm 2 , about 70 pieces / cm 2 , about 75 pieces / cm 2 , about 80 pieces / cm 2 , about 85 pieces / cm 2 , about 90 pieces / cm 2 , about 95 pieces / cm 2 , about 100 pieces / cm 2 , about 110 pieces / cm 2 , about 120 pieces / cm 2 , about 130 pieces / cm 2 , about 140 pieces / cm 2 , about 150 pieces / cm 2 , about 160 pieces / cm 2 , about 170 pieces / cm 2 , about 180 pieces / cm 2 , about 190 pieces / cm 2 , about 200 pieces / cm 2 , about 225 pieces / cm 2 , about 250 pieces / cm 2 , about 275 pieces / cm 2 , about 300 pieces / cm 2 , about 325 pieces / cm 2 , about 350 pieces / cm 2 , about 375 pieces / cm 2 , about 400 pieces / cm 2 , about 425 pieces / cm 2 , about 450 pieces / cm 2 , about 475 pieces / cm 2 , about 500 pieces / cm 2 , about 600 pieces / cm 2 , about 700 pieces / cm 2 , about 800 pieces / cm 2, about 900 pieces / cm 2 , or about 1,000 pieces / cm 2 It may be. Usually, the loading level of the first substrate is sufficient for the detection of antibodies in biological samples. In particular, the loading level is sufficient to distinguish biological samples from patients who have or are expected to have an immune response (e.g., pre-existing antibodies) against pathogen-inactivated red blood cells (e.g., RBC administered by infusion) from biological samples from patients who do not have or are expected to not have an immune response (e.g., pre-existing antibodies) against pathogen-inactivated red blood cells (e.g., RBC administered by infusion).

[0108] The kit may further include a third container containing a third substrate, where a first level portion is bound to the surface of the first substrate, and a second level portion is bound to the third substrate, and the second level is less than the first level. Those skilled in the art will understand that the second level portion can provide an internal reference for the detection of antibodies to the surface-bound portion at the first level portion. Thus, in some embodiments, the relative loading levels of the first and third substrates are sufficient to distinguish biological samples from patients who have or are expected to have an immune response (e.g., pre-existing antibodies) to pathogen-inactivated red blood cells (e.g., RBC administered by infusion) from biological samples from patients who do not have or are expected to not have an immune response (e.g., pre-existing antibodies) to the infused pathogen-inactivated red blood cells. The loading level of the first substrate may be at least about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 100 times, about 250 times, about 500 times, 1,000 times, about 5,000 times, or about 10,000 times or more higher than the loading level of the third substrate. In some embodiments, the loading level of the first substrate is about 2 to about 500 times, about 2 to about 100 times, about 2 to about 50 times, about 2 to about 20 times, about 2 to about 10 times, about 2 to about 5 times, about 4 to about 100 times, about 4 to about 50 times, about 4 to about 20 times, or about 4 to about 10 times higher than the loading level of the third substrate. In some embodiments, the loading level of the first substrate is at least about 100 parts / μm. 2 , about 110 pieces / μm 2 , about 120 pieces / μm 2 , about 130 pieces / μm 2 , about 140 pieces / μm 2 , about 150 pieces / μm 2 , about 160 pieces / μm 2 , about 180 pieces / μm 2 , about 200 pieces / μm 2 , about 225 parts / μm 2 , about 250 pieces / μm 2 , about 275 parts / μm 2 , about 300 pieces / μm2 , about 325 parts / μm 2 , about 350 pieces / μm 2 , about 400 pieces / μm 2 , about 450 pieces / μm 2 , about 500 pieces / μm 2 and the loading level of the third substrate is about 100 parts / μm 2 Below, about 90 parts / μm 2 Below, about 80 parts / μm 2 Below, about 70 parts / μm 2 Below, about 60 parts / μm 2 Below, about 50 parts / μm 2 Below, about 40 parts / μm 2 Below, about 30 parts / μm 2 In some embodiments, the loading level of the first substrate is about 110 to about 750 parts / μm 2 Between about 110 and about 500 pieces / μm 2 Between about 110 and about 400 pieces / μm 2 Between about 110 and about 300 pieces / μm 2 Between about 140 and about 750 pieces / μm 2 Between about 140 and about 500 pieces / μm 2 Between about 140 and about 400 pieces / μm 2 Between about 140 and about 300 pieces / μm 2 Between about 200 and about 400 pieces / μm 2 Between about 200 and 300 pieces / μm 2 and the loading level of the third substrate is between about 10 and about 100 parts / μm 2 Between about 20 and about 100 pieces / μm 2 Between about 30 and about 100 pieces / μm 2 Between about 40 and about 100 pieces / μm 2 Between about 50 and about 100 pieces / μm 2 In some embodiments, the loading level of the first substrate is between about 110 parts / μm 2 , about 120 pieces / μm 2 , about 130 pieces / μm 2, about 140 pieces / μm 2 , about 150 pieces / μm 2 , about 160 pieces / μm 2 , about 180 pieces / μm 2 , about 200 pieces / μm 2 , about 225 parts / μm 2 , about 250 pieces / μm 2 , about 275 parts / μm 2 , about 300 pieces / μm 2 , about 325 parts / μm 2 , about 350 pieces / μm 2 , about 400 pieces / μm 2 , about 450 pieces / μm 2 , about 500 pieces / μm 2 and the loading level of the third substrate is about 10 parts / μm 2 , about 20 pieces / μm 2 , about 30 pieces / μm 2 , about 40 pieces / μm 2 , about 50 pieces / μm 2 , about 60 pieces / μm 2 , about 70 pieces / μm 2 , about 80 pieces / μm 2 , about 90 pieces / μm 2 , about 100 pieces / μm 2In some embodiments, the loading level of the first substrate is at least about 15,000, about 17,000, about 20,000, about 25,000, about 30,000, about 40,000, or about 50,000 parts (e.g., red blood cells) per unit of substrate, and the loading level of the third substrate is no more than about 14,000, no more than about 12,000, no more than about 10,000, no more than about 8,000, no more than about 6,000, or no more than about 5,000 parts (e.g., red blood cells) per unit of substrate. In some embodiments, the loading level of the first substrate is between about 15,000 to about 75,000 cells (e.g., red blood cells) per unit of substrate, between about 17,000 to about 75,000 cells, between about 20,000 to about 75,000 cells, between about 25,000 to about 75,000 cells, between about 30,000 to about 75,000 cells, between about 40,000 to about 75,000 cells, between about 15,000 to about 50,000 cells, between about 17,000 to about 50,000 cells, between about 20,000 to about 50,000 cells, between about 25,000 to about 50,000 cells, between about 30,000 to about 50,000 cells, between about 40,000 to about 75,000 cells, The loading level of the third substrate is between about 1,000 and about 15,000, between about 3,000 and about 15,000, between about 5,000 and about 15,000, between about 10,000 and about 15,000, between about 1,000 and about 14,000, between about 3,000 and about 14,000, between about 5,000 and about 14,000, between about 7,000 and about 14,000, between about 10,000 and about 14,000, between about 5,000 and about 12,000, and between about 5,000 and about 10,000. In some embodiments, the loading level of the first substrate is about 15,000, about 17,000, about 20,000, about 25,000, about 30,000, about 35,000, about 40,000, about 45,000, about 50,000, about 60,000, or about 75,000 parts (e.g., red blood cells) per unit of substrate, and the loading level of the third substrate is about 1,000, about 3,000, about 5,000, about 7,000, about 10,000, about 12,000, or about 14,000 parts (e.g., red blood cells) per unit of substrate.Loading levels disclosed herein can include absolute amounts, such as those described above, or relative loading levels, such as the loading level of a first substrate compared to a third substrate.

[0109] Typically, the third substrate will be composed of the same material as the first and / or second substrate. In some embodiments, the first, second, and third substrates are the same. If there is heterogeneity in the chemical structure or binding chemistry of the surface-attached moieties on the first and third substrates, the third substrate can have the same or different distribution of surface-attached moieties than the first substrate. In some embodiments, the first, second, and third substrates are comprised of RBCs, and the RBCs in these three substrates are obtained from one or more common donors. In some embodiments, the first, second, and third substrates all display one or more antigens commonly found in biological samples (e.g., whole blood, red blood cells, serum, or plasma).

[0110] In addition to the first panel as described herein, a second panel can be used according to some embodiments of the invention. The RBCs of the second panel can be obtained from one or more common donors. The RBCs of the second panel can be obtained from the same or different donors as the RBCs of the first panel. The surface-binding moieties of the second panel can be the same as or different from the surface-binding moieties of the first panel. The surface-binding moieties of the second panel can be the same as the surface-binding moieties of the first panel, and the loading levels for the two panels can be the same or different. The substrates of the first and second panels can display additional antigens, such as one or more antigens commonly found in biological samples (e.g., whole blood, red blood cells, serum, or plasma). The additional antigens of the first panel can be the same as or different from the additional antigens of the second panel.

[0111] The first, second, and optional third containers of the kits provided herein can be made of any material suitable for storing the first, second, and optional third substrates (which may or may not have surface-binding moieties). The containers can be made of glass, plastic, or any suitable polymeric material. The containers can be sterile and can further provide an environment for maintaining the sterility of the contents of the containers. It is understood that the containers are suitable for storing the contents thereof at an appropriate temperature (e.g., room temperature, refrigerated temperature, freezer temperature, 0°C, -20°C, -80°C).

[0112] The kits provided herein may further contain additive solutions, buffers, or other solutions that can be used to carry out any of the methods provided herein. The additive solutions, buffers, or other solutions may be useful for preserving the substrates of the kits (e.g., substrates containing surface-binding moieties or substrates lacking surface-binding moieties). The additive solutions, buffers, or other solutions may be useful in the process of contacting the substrates of the kits (e.g., substrates containing surface-binding moieties or substrates lacking surface-binding moieties) with one or more biological samples. Depending on the intended use of the additive solutions, buffers, or other solutions, these components may be premixed with the substrates of the kits (e.g., substrates containing surface-binding moieties or substrates lacking surface-binding moieties) or they may be contained separately in the kit.

[0113] The kits provided herein may further contain additional components (e.g., reagents) for use in visualizing or otherwise determining the presence or absence of antibodies bound to the surface-bound moieties during or after contacting the biological sample. This may include anti-human globulin or other materials that induce agglutination, as well as any other components useful in visualization methods, such as the IAT or DAT. In certain embodiments, the kits provided herein may contain materials suitable for use as controls (e.g., standards).

[0114] As will be readily appreciated by those of skill in the art, systems that include the above-described kits are also provided, for example systems that further include automated equipment that can be used in accordance with the methods described herein. Preparation method

[0115] Provided herein are methods for preparing a panel of substrates (e.g., polymer particles, RBCs). In some embodiments, the methods include preparing a first and a second panel, each panel comprising a substrate. The method includes providing a sample and treating the substrate of the first sample with a compound or derivative provided herein. In some embodiments, the method includes providing a first and a second sample comprising polymer particles (e.g., beads, microspheres) and treating the polymer particles of the first sample with a compound or derivative provided herein. In some embodiments, the method includes providing a first and a second sample comprising RBCs and treating the RBCs of the first sample with a compound or derivative provided herein. In some embodiments, the RBCs of the first and second samples are from the same blood donor. The treatment step may further include additional reagents, such as acids, bases, suitable catalysts, or any other reagents that may facilitate the reaction of the compound or derivative with the substrate. A suitable solvent (e.g., water) or buffer (e.g., RBC additive solution, blood bank saline) may be utilized in the treatment step. The treatment step may result in covalent or non-covalent attachment of moieties to the surface of the substrate (e.g., polymer particles, RBCs) of the first sample. In some embodiments, the generated surface-bound moieties have the same or substantially the same chemical structure as the compound or derivative used to treat the substrate (e.g., polymer particles, RBCs) of the first sample. In some embodiments, the generated surface-bound moieties have a substantially different chemical structure than the compound or derivative used to treat the substrate (e.g., polymer particles, RBCs) of the first sample. For example, the generated surface-bound moieties may be some type of hydrolysis product of the reaction. Since the second sample is not treated with the compound, this, together with the first sample, generates a panel including a first sample of substrate (e.g., polymer particles, RBCs) with surface-bound moieties and a second sample of substrate (e.g., polymer particles, RBCs) lacking surface-bound moieties.

[0116] In some embodiments, the method for preparing a panel of substrates (e.g., polymer particles, RBCs) may further include one or more steps of reacting the first and second samples with one or more additional antigens, such as one or more antigens commonly found in biological samples (e.g., whole blood, red blood cells, serum, or plasma). The reaction with the one or more additional antigens may be performed before, in parallel with, or after treatment with the compound or derivative. Solvents and additional reagents may be used as necessary in the reaction with the one or more additional antigens.

[0117] The method for preparing a panel of substrates (e.g., polymer particles, RBCs) may further include one or more steps of washing the first and second samples following the step of treating the substrates (e.g., polymer particles, RBCs) with a compound or derivative. The method may also further include a quenching step to facilitate removal of excess free compound or derivative from the substrate treatment mixture. In some embodiments, the quencher may be glutathione. The quenching step may be performed before, in parallel with, or after the washing step. In certain embodiments, the method further includes suspending the first and second samples (e.g., polymer particles, RBCs) in a buffered suspension medium. In certain embodiments, the method further includes adding a cryopreservation agent to the first and second samples after washing.

[0118] In some embodiments, the second sample is treated similarly or identically to the first sample, except that the compound or derivative is omitted from the processing step. This can serve to match the characteristics of the first and second samples so that the second sample provides a suitable control.

[0119] The method includes treating a third sample comprising a substrate (e.g., polymer particles, RBCs) with a compound or derivative in the same manner as the first sample, such that treatment with the compound or derivative results in a lower level of moieties bound to the surface of the substrate (e.g., polymer particles, RBCs) in the third sample compared to the level of moieties bound to the surface of the substrate (e.g., polymer particles, RBCs) in the first sample. The method may further include treating the substrate with a compound or derivative. Thus, the first level of moieties is bound to the surface of the substrate (e.g., polymer particles, RBCs) of the first sample, and the second level is less than the first level. In some embodiments, this can be achieved by treating the substrate (e.g., polymer particles, RBCs) of the third sample with a different amount of the compound or derivative than the substrate (e.g., polymer particles, RBCs) of the first sample. In some embodiments, this can be achieved by instead treating the substrate (e.g., polymer particles, RBCs) of the third sample with the compound or derivative for a shorter length of time than the treatment of the RBCs of the first sample. In yet another alternative, the third sample is treated with a smaller amount of the compound or derivative than that used to treat the first sample, and this treatment is performed on the third sample for a shorter length of time than the treatment of the first sample. Additionally, any of the additional preparation steps described herein for the first sample may also be performed on the third sample.

[0120] In some embodiments, the third sample reacts with one or more additional antigens, such as one or more antigens commonly found in biological samples (e.g., whole blood, red blood cells, or plasma). The reaction with one or more additional antigens can be performed before, in parallel with, or after treatment with the compound or derivative. In the reaction with one or more additional antigens, solvents and additional reagents can be used as necessary.

[0121] In some embodiments, the third sample is subjected to one or more steps of washing steps following the step of treating the substrate (e.g., polymer particles, RBCs) with the compound or derivative. The method can also further include a quenching step that facilitates the removal of excess free compound or derivative from the treatment mixture. The quenching step can be performed before, in parallel with, or after the washing step. In certain embodiments, the method further includes a step of suspending the third sample (e.g., polymer particles, RBCs) in a buffered suspension medium. In certain embodiments, the method further includes a step of adding a cryopreservation agent to the third sample after washing.

[0122] In some embodiments, the first, second, and third samples are treated equally, except that the amount of compound or derivative used in the processing step is less for the third sample compared to the first sample, and for the second sample, the compound or derivative is omitted from the processing step.

[0123] The identity and loading level of the surface-bound moiety on the substrate can be assessed by any suitable method known in the art. The loading level can be determined, for example, by labeling the compound (e.g., radiolabeling, immunolabeling, chemical labeling, fluorescent labeling, chemiluminescence) before adding to the substrate, and then detecting the label after the compound is combined with the substrate. Alternatively or in addition, the loading level can be measured by directly or indirectly labeling the compound (e.g., immunolabeling, immunofluorescence, chemiluminescence) after adding to the substrate, and then detecting the label. Generally, such labeling is performed separately (e.g., as a control, as a calibration curve) from the compounds used in the kits and methods provided herein, as a comparison or reference point in determining absolute or relative loading levels. Such labeling can be directly quantified using quantitative or semi-quantitative methods, or compared to a reference standard, such as cells, beads or other materials with a known amount of similarly labeled molecules on the surface (e.g., FACS analysis). The substrate can be monitored for the presence of at least a certain level of a particular generated surface-bound moiety. The substrate can be monitored for the presence of any generated surface-bound moieties of at least a certain loading level.

[0124] The first, second, and optional third samples can be sterilized according to any method known in the art. Sterilization can occur before or after treatment with the compound or derivative.

[0125] The panel of substrates (e.g., polymer particles, RBCs) including the first, second, and optional third samples can be packaged for storage. Any suitable storage containers (e.g., boxes, bags, bottles, tubes) known in the art can be used. In some embodiments, the storage containers are sterilized. In some embodiments, refrigeration is required. For example, if the moieties bound to the substrate contain hydrolyzable linkers as described above, the samples in the panel can be frozen for storage. In other embodiments, the samples in the panel do not need to be frozen. How to use

[0126] Provided herein are methods for testing biological samples using the kits described herein. In various embodiments, the substrate in the kits described herein comprises a polymer particle, a matrix, a portion of an assay plate, or a cell, such as an RBC. How to test the sample

[0127] Provided herein is a method of testing a sample for the presence of an antibody that binds a compound, the method comprising: a) providing a sample comprising serum or plasma; b) contacting the sample with a substrate, wherein a moiety is bound to the surface of the substrate, the moiety being selected from the group consisting of any of the compounds of Formulae I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; and c) assaying the amount of binding between the antibody from the sample and the surface-bound moiety of the substrate, wherein binding between the antibody and the surface-bound moiety indicates that the antibody binds to the compound.

[0128] In some embodiments, a method of testing a sample for the presence of an antibody that binds a compound includes the steps of: a) providing a sample comprising serum or plasma; b) contacting the sample with a polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, wherein a moiety is bound to a surface of the polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, the moiety being selected from the group consisting of a compound of any of Formulas I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; and c) assaying the amount of binding between an antibody from the sample and the surface-bound moiety of the polymeric particle (e.g., bead, microsphere), matrix, or portion of an assay plate, wherein binding between the antibody and the surface-bound moiety indicates that the antibody binds to the compound.

[0129] In some embodiments, a method of testing a sample for the presence of an antibody that binds a compound includes the steps of: a) providing a sample comprising serum or plasma; b) contacting the sample with red blood cells, wherein a moiety is bound to the surface of the red blood cells, the moiety being selected from the group consisting of a compound of any of Formulas I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or a salt or stereoisomer of any of the foregoing; and c) assaying the amount of binding between an antibody from the sample and the surface-binding moiety of the red blood cells, wherein binding between the antibody and the surface-binding moiety indicates that the antibody binds to the compound.

[0130] Provided herein are methods for testing a sample for the presence of an antibody that binds a compound. In some embodiments, the method includes providing a sample comprising serum or plasma, contacting the sample with a substrate (e.g., RBCs) where a moiety is bound to a surface of the substrate, and subsequently assaying the amount of binding between antibodies from the sample and the surface-bound moieties of the substrate.

[0131] In some embodiments, the assaying step comprises comparing the amount of binding between the antibody from the sample and the surface-binding moiety to a reference, where an increase in the amount of binding compared to the reference indicates the presence of the antibody in the sample. In some embodiments, the reference is the same substrate as the substrate with the surface-binding moiety, except that the surface-binding moiety is absent.

[0132] In some embodiments, the assaying step includes assaying the amount of binding between the antibody from the sample and a substrate lacking surface-binding moieties (e.g., RBCs). The method may further include assaying the amount of binding between the antibody from the sample and a substrate (e.g., RBCs) having a lower amount of surface-binding moieties compared to the sample.

[0133] In some embodiments, assaying the amount of binding between antibodies from the sample and the substrate (e.g., RBCs) comprises contacting the substrate to which the sample has been added with anti-human globulin, and assaying comprises assaying for the amount of agglutination. Conventional methods for measurement can be used, such as solid-phase red blood cell adhesion assays (e.g., Galileo System, Immucor), or IATs and DATs (e.g., gel cards, ID-Card DiaScreen gel cards, Bio-Rad).

[0134] In some embodiments, the test method provided herein is used to determine whether a subject is expected to have an immune response to infusion of pathogen-inactivated red blood cells. Exemplary pathogen-inactivated red blood cell compositions that can be used for infusion into a subject are described in US6,270,952, US2003 / 0113704, US7,655,392, and WO2009 / 126786, the contents of each of which are incorporated herein by reference. In some embodiments, the surface-bound moiety on the substrate used in the method is the same as the pathogen-inactivating compound used to treat the red blood cells for infusion, or a derivative of such a pathogen-inactivating compound. In other embodiments, the surface-bound antigen on the substrate used in the method is an analog of the pathogen-inactivating compound used to treat the red blood cells for infusion, or a derivative of such an analog. For example, if the pathogen-inactivating compound used to treat the red blood cells for infusion is a fragile compound (e.g., S-303), a non-fragile analog of the pathogen-inactivating compound can be used as the surface-bound moiety on the substrate. The non-fragile analogue may exhibit a similar epitope for antibody binding as the pathogen inactivating compound, and may exhibit a similar binding affinity for antibodies as the pathogen inactivating compound itself. The advantage of using a non-fragile analogue as a surface-binding moiety may be that a substrate containing a non-fragile surface-binding moiety may be more stable than a substrate containing a fragile surface-binding moiety. Thus, a substrate containing a non-fragile surface-binding moiety may be suitable for storage at room temperature or at refrigerated temperatures, whereas a substrate containing a corresponding fragile surface-binding moiety may require freezer storage to avoid degradation of the surface-binding moiety (e.g., via hydrolysis of the fragile linker).

[0135] In some embodiments, the sample is from a subject who needs to infuse red blood cells.For example, the sample can be a serum sample, a plasma sample, or other blood sample from a subject who needs to infuse red blood cells.In some embodiments, the method of testing a sample provided herein comprises a first step of obtaining a sample from a subject.The sample can be obtained from a subject by any method known in the art, such as standard blood collection from an arm or finger.

[0136] The method can be carried out using any of the compositions or kits provided herein. If the composition or kit is stored in a freezer, the composition or kit is removed from the freezer and thawed before contacting the kit or substrate of the composition (e.g., red blood cells) with the sample. The kit can be thawed in the refrigerator or at room temperature. How to Give a Blood Transfusion

[0137] Provided herein is a method of providing a blood transfusion to a subject in need thereof, the transfusion comprising RBCs treated with a pathogen inactivation compound (e.g., a pathogen inactivation compound described herein). The method includes providing a sample (e.g., a serum sample or a plasma sample) from the subject, contacting the sample with a substrate (e.g., polymer particles, RBCs), where a moiety is bound to the surface of the substrate, assaying the amount of binding between an antibody from the sample and the surface-bound moiety of the sample compared to a reference to determine whether the amount of binding between the antibody and the surface-bound moiety is higher than the reference, and providing an infusion of the pathogen-inactivated red blood cells to the subject upon determining that the amount of binding is not higher than the reference. The surface-bound moiety may be the same as the pathogen inactivation compound used to treat the red blood cells for infusion. Alternatively, the surface-bound moiety may be a derivative (e.g., a hydrolyzed derivative) of the pathogen inactivation compound used to treat the red blood cells for infusion. In yet another alternative, the surface-bound moiety may be an analog of the pathogen inactivation compound used to treat the red blood cells for infusion. For example, if the pathogen inactivating compound used to treat red blood cells for infusion contains a frangible linker (e.g., S-303), the surface-binding moiety may be a non-frangible analog thereof or a derivative of such a non-frangible analog.

[0138] In some aspects, the method includes providing a sample (e.g., a serum sample or a plasma sample) from a subject, contacting the sample with a substrate (e.g., polymer particles, RBCs), where the moiety is bound to the surface of the substrate, assaying the amount of binding between an antibody from the sample and the surface-bound moiety of the sample compared to a reference, determining whether the amount of binding between the antibody and the surface-bound moiety is higher than the reference, and providing a drip of non-pathogen-inactivated red blood cells to the subject following a determination that the amount of binding is higher than the reference. The surface-bound moiety may be the same as the pathogen inactivation compound used to treat the red blood cells for infusion. Alternatively, the surface-bound moiety may be a derivative (e.g., a hydrolyzed derivative) of the pathogen inactivation compound used to treat the red blood cells for infusion. In yet another alternative, the surface-bound moiety may be an analog of the pathogen inactivation compound used to treat the red blood cells for infusion. For example, if the pathogen inactivating compound used to treat red blood cells for infusion contains a frangible linker (e.g., S-303), the surface-binding moiety may be a non-frangible analog thereof or a derivative of such a non-frangible analog.

[0139] In some embodiments, the method includes providing a sample comprising serum or plasma, contacting the sample with a substrate (e.g., polymer particles, RBCs) where a moiety is bound to the surface of the substrate, and subsequently assaying the amount of binding between antibodies from the sample and the surface-bound moieties of the substrate.

[0140] In some embodiments, the assaying step comprises comparing the amount of binding between the antibody from the sample and the surface-binding moiety to a reference, where an increase in the amount of binding compared to the reference indicates the presence of the antibody in the sample. In some embodiments, the reference is the same substrate as the substrate with the surface-binding moiety, except that the surface-binding moiety is absent.

[0141] In some embodiments, the assaying step includes assaying the amount of binding between the antibody from the sample and a substrate lacking surface-binding moieties (e.g., polymer particles, RBCs). The method may further include assaying the amount of binding between the antibody from the sample and a substrate (e.g., polymer particles, RBCs) having a lower amount of surface-binding moieties compared to the sample.

[0142] In some embodiments, assaying the amount of binding between antibodies from a sample and a substrate (e.g., polymer particles, RBCs) comprises contacting the substrate to which the sample has been added with an anti-human globulin, and assaying comprises assaying for the amount of agglutination. Conventional methods for measurement, such as the IAT and DAT, can be used.

[0143] In some embodiments, the determination that the amount of antibody binding to the surface-binding moiety of the substrate is not higher than the reference is made using a substrate lacking surface-binding moiety as a reference. In some embodiments, the determination that the amount of antibody binding to the surface-binding moiety of the substrate is not higher than the reference is made using a substrate with a lower loading level of surface-binding moiety as a reference. It may be advantageous to have a reference substrate that differs from the test substrate only in the presence or absence of surface-binding moiety or only in terms of the loading level of surface-binding moiety.

[0144] A sample from a subject can be collected and tested for binding to the surface-binding portion of an antibody immediately prior to the intended infusion of red blood cells, or within 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, or 24 hours, 48 ​​hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks or more prior to the intended infusion of red blood cells. If a subject receives more than one infusion of red blood cells within a period of time, a sample from the subject can be tested according to the methods described herein prior to each infusion of red blood cells (e.g., pathogen-inactivated red blood cells), or only prior to the first infusion of red blood cells (e.g., pathogen-inactivated red blood cells), or on a periodic basis as determined by a medical professional. EXAMPLES

[0145] The present invention is further illustrated by the following non-limiting examples. Example 1 Preparation of red blood cell panel Red blood cell panel with S-303

[0146] S-303 treated and control reagent RBCs were prepared to include: 1) control RBCs with no bound adducts from S-303, 2) RBCs treated with S-303 and glutathione (GSH) quencher under conditions that produce high levels of bound adducts from S-303, and 3) RBCs treated with S-303 and glutathione (GSH) quencher under conditions that produce low levels of bound adducts from S-303. More specifically, RBC units were obtained from human donors by standard blood collection techniques, and each RBC unit was divided into three matched, smaller (sub) 100 mL unit samples, which were then used to prepare the reagent cell panel under the three different treatment conditions. To generate high levels of binding adducts, one of three matched RBC units was mixed with a processing solution having about 0.2 mM S-303 and about 2 mM GSH (see, e.g., U.S. Patent Nos. 7,655,392 and 8,900,805), incubated overnight at room temperature, washed with 0.9% NaCl, and frozen in glycerolyte at -80°C. To generate low levels of binding adducts, one of three matched RBC units was mixed with a processing solution having about 0.2 mM S-303 and about 20 mM GSH, incubated overnight at room temperature, washed with 0.9% NaCl, and frozen in glycerolyte at -80°C. Control RBCs were not subjected to the S-303 / GSH treatment, washed with 0.9% NaCl, and frozen in glycerolyte at -80°C.

[0147] A panel of reagents was evaluated to determine whether RBC reagents with higher levels of surface adducts would provide greater sensitivity in antibody detection compared to RBC reagents with lower levels of surface adducts. A previously characterized monoclonal antibody (generated from mice immunized with KLH-conjugated S-197, a non-fragile analog of S-303) was titrated and various mAb dilutions were used in a gel card assay (e.g., ID-MTS™ Ortho Diagnostics, Raritan, NJ). Anti-acridine S-197 mAb titrations tested against RBCs with low S-303 adducts yielded an average titer (the reciprocal of the highest dilution) of 8,000. In contrast, anti-acridine S-197 mAb dilutions tested against RBCs with high S-303 adducts yielded an average titer of 32,000. These data indicate that high adduct RBCs provide higher antibody detection against the acridine moiety of S-303 RBCs. Bond stability of non-brittle analog compounds

[0148] Experiments were performed to evaluate the binding to RBC substrates by compound S-220, a non-fragile analog of S-303. The experiments also compared the stability of the surface-bound portion of the S-220 compound to that of similarly bound fragile S-303. More specifically, glutathione (GSH) quencher and S-220 compound were added to human RBC substrates at concentrations of 2.0 mM and 0.2 mM, respectively, and then incubated overnight at 37° C. or 4° C. For comparison, GSH quencher and fragile S-303 compound were similarly added to human RBC substrates at concentrations of 2.0 mM and 0.2 mM, respectively, and incubated overnight at 37° C. or 4° C. Both non-fragile S-220 and fragile S-303 compounds contain an acridine moiety as part of their structure. After incubation, RBCs treated with GSH / S-220 and GSH / S-303 were stained with either mouse monoclonal (mAb) or rabbit polyclonal (polyAb) anti-acridine antibodies (also at 4°C or 37°C) and samples were analyzed for antibody binding by flow cytometry using standard methods (quantified as MFI). For immunization, compound S-197 was used to generate both mAb and polyAb anti-acridine antibodies.

[0149] As shown in Figures 1-6, both non-friable S-220 treated RBCs and friable S-303 treated RBCs are able to achieve similar binding levels. Furthermore, although some differences were observed between monoclonal and polyclonal antibody binding to treated RBCs, data for both antibodies showed that S-303 binding to RBCs was not as stable as S-220 binding to RBCs at 37°C, suggesting the benefit of using non-friable compounds in the methods and kits provided herein. Erythrocyte Panel

[0150] Red blood cells (RBCs) are obtained from type O donors by standard blood collection techniques. The cells express one or more of the antigens C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. ​​Each unit of RBCs (approximately 350 mL) is divided into three matched, smaller units (e.g., subunits) of approximately equal volume (designated subunits A, B, and C). Subunit A is treated with a non-fragile analog of S-303 (e.g., S-220, S-197) and 2 mM glutathione, followed by washing with 0.9% NaCl, to generate "high" levels of surface-bound adducts. Subunit B from the same donor unit is treated with the same non-fragile analog of S-303 and 20 mM glutathione, followed by washing with 0.9% NaCl, to generate "low" levels of surface-bound adducts. Other levels of surface-bound adducts can be achieved by varying the non-fragile analog and / or glutathione concentration. Subunit C is not treated with the non-fragile analog of S-303 or glutathione. Subunit C is washed with 0.9% NaCl. After the processing steps described herein, each of the subunits is suspended in a buffered suspension medium (e.g., 3% RBC) and transferred to one or more storage containers. Optionally, glycerolyte is added to each of subunits A, B, and C, and the subunits are then stored at -80°C. Subunits A, B, and C are Form the first panel.

[0151] The second panel is prepared similarly to the first panel, except that the RBCs of subunits A, B, and C are each from a different type O donor. Example 2 Testing of patient serum samples

[0152] One, two, or three first panels of Example 1 are thawed to room temperature. Each first panel contains subunits A, B, and C from a single type O donor. Serum from the patient and anti-human globulin are added to each of the first panel's respective subunits A, B, and C. Each first panel's subunits are monitored for agglutination and assigned a score of 0, 1, 2, or 3 (0 representing no agglutination, 1, 2, and 3 representing increasingly more agglutination).

[0153] If the antibody score for each of the first panel subunits A, B, and C is 0 (i.e., no agglutination is observed), the sample is considered "non-reactive" and the patient may be administered an infusion of RBCs treated with S-303 as a pathogen-inactivating compound.

[0154] If an antibody score of ≥ 1 is observed for any of the RBCs in the first panel, the patient sample undergoes additional testing with a second panel, as described in Example 1. If all three first panel scores are ≥ 1 for either "high" or "low" levels of surface-bound adducts (i.e., subunit A or subunit B) and the corresponding untreated control RBCs (i.e., subunit C) score 0, the sample is classified as "initial reactive." If the second panel scores show the same pattern of reactivity as any of the first panel, the sample is classified as "confirmed reactive." The patient receives an infusion of RBCs not treated with S-303, if necessary.

[0155] If one or two, but not all three, of the first panels have an antibody score of ≥1 against either "high" or "low" levels of surface-bound adducts (i.e., subunit A or subunit B), and the corresponding untreated reagent control RBCs show a score of 0 in all of the first panels, the sample is classified as "non-reactive." If the second panel results show no agglutination from RBCs with surface-bound adducts, the sample is classified as "non-reactive" and the patient can be administered an infusion of RBCs treated with S-303 as a pathogen-inactivating compound.

[0156] If the results of the three first panels show three out of three, two out of three, or one out of three reactivity to both RBCs bearing surface-bound adducts and corresponding control untreated RBCs, the sample is considered to have "putatively alloreactive" antibodies to endogenous red blood cell antigens. The present invention provides, for example, the following items. (Item 1) (a) a first container containing a first substrate, wherein a moiety is attached to a surface of the first substrate, the moiety being selected from the group consisting of a compound of any of Formulas I, II, III, IV, V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; (b) a second container containing a second substrate, the surface of the second substrate being devoid of binding moieties; and Kit including: (Item 2) 2. The kit according to item 1, wherein the moiety is selected from the group consisting of any of the compounds of formula I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 3) 2. The kit according to item 1, wherein the moiety is selected from the group consisting of a compound of formula VIII and its derivatives, or a salt or stereoisomer of any of the foregoing. (Item 4) The kit according to item 3, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. (Item 5) R 20 5. The kit according to item 3 or 4, wherein (Item 6) R 21 Ga-C 1~8 6. The kit according to any one of items 3 to 5, wherein the alkyl group is alkyl-N(CH2CH2Cl)2. (Item 7) 2. The kit according to item 1, wherein the moiety is selected from the group consisting of any of the compounds of formulae IV, V, and VI, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 8) 8. The kit according to item 7, wherein the moiety is selected from the group consisting of any compound of formula IV(a) to IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 9) 2. The kit of item 1, wherein the moiety is S-303, a derivative of S-303, or a salt of any of the foregoing. (Item 10) The kit according to item 1, wherein the moiety is a non-fragile analog of S-303 or a salt thereof. 11. The kit according to item 10, wherein the non-fragile analogue of S-303 is S-197 or S-220. (Item 12) 12. The kit of any one of items 1 to 11, wherein the moieties bound to the surface of the substrate are present at a loading level of at least about 10,000 moieties per unit of substrate. (Item 13) The moieties attached to the surface of the substrate have a density of at least about 50 moieties / μm 2 12. The kit according to any one of items 1 to 11, wherein the antibody is present at a loading level of (Item 14) 14. The kit of any one of items 1 to 13, wherein each of the first and second substrates comprises a portion of a polymer particle, a matrix, or an assay plate. (Item 15) 14. The kit according to any one of items 1 to 13, wherein the first substrate comprises red blood cells and the second substrate comprises red blood cells, the red blood cells of the first and second substrates being obtained from one or more common donors. (Item 16) 16. The kit of any one of items 1 to 15, further comprising a third container containing a third substrate, wherein the moieties at a first level are attached to a surface of the first substrate and the moieties at a second level are attached to a surface of the third substrate, the second level being less than the first level. (Item 17) 17. The kit of claim 16, wherein the first level of the moiety bound to the surface of the first substrate is at least three times higher than the second level of the moiety bound to the surface of the third substrate. (Item 18) 18. The kit of claim 16 or 17, wherein the third substrate comprises a polymer particle, matrix, or a portion of an assay plate. (Item 19) 18. The kit of claim 16 or 17, wherein the third substrate comprises red blood cells, and the red blood cells of the first, second, and third substrates are obtained from one or more common donors. (Item 20) 20. The kit of claim 15 or 19, wherein the first substrate comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 samples, each of the samples in the first substrate comprising red blood cells from a different blood donor, and the second substrate comprises corresponding samples comprising red blood cells from each of the separate blood donors. (Item 21) 21. The kit of item 20, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 samples represent multiple blood types. (Item 22) 1. A method for preparing a panel of red blood cells, comprising: a) providing a first and a second sample comprising red blood cells; b) treating the red blood cells of the first sample with a compound, the treatment with the compound resulting in binding of moieties to the surface of the red blood cells of the first sample; wherein the second sample is not treated with the compound, thereby having surface-binding moieties. a panel is generated comprising a first sample of red blood cells that contain the surface-bound moiety and a second sample of red blood cells that lack the surface-bound moiety; The method of claim 1, wherein the compound is selected from the group consisting of any of the compounds of formula I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 23) 23. The method of claim 22, wherein the compound is selected from the group consisting of any of the compounds of formula I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 24) 23. The method of claim 22, wherein the compound is selected from the group consisting of a compound of formula VIII and its derivatives, or a salt or stereoisomer of any of the foregoing. (Item 25) 25. The method of claim 24, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. (Item 26) R 20 The method according to item 24 or 25, wherein (Item 27) R 21 Ga-C 1~8 27. The method according to any one of items 24 to 26, wherein the alkyl group is alkyl-N(CH2CH2Cl)2. (Item 28) 23. The method of claim 22, wherein the compound is selected from the group consisting of any of the compounds of formulae IV(a) to IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 29) 23. The method of claim 22, wherein the compound is a derivative of S-303 or a salt thereof. (Item 30) 23. The method of claim 22, wherein the compound is a non-fragile analog of S-303, or a salt thereof. (Item 31) 23. The method of claim 22, wherein the surface-binding moiety is a derivative of S-303, or a salt thereof. (Item 32) 23. The method of claim 22, wherein the surface-binding moiety is a non-fragile analog of S-303, or a salt thereof. (Item 33) 33. The method of any one of items 22 to 32, wherein the moieties bound to the surface of the red blood cell are present at a loading level of at least about 10,000 moieties per red blood cell. (Item 34) The moieties bound to the surface of the red blood cell are at least about 50 moieties / μm 2 33. The method according to any one of items 22 to 32, wherein the .alpha.-tocopherol is present at a loading level of. (Item 35) 35. The method according to any one of items 22 to 34, wherein the red blood cells of the first and second samples are obtained from the same blood donor. (Item 36) After step (b), Washing the first and second samples 36. The method according to any one of items 22 to 35, further comprising: (Item 37) After step (b), adding a cryopreservation agent to the first and second samples; freezing the first and second samples; 37. The method according to any one of items 22 to 36, further comprising: (Item 38) 37. The method of any one of items 22 to 36, further comprising the step of storing the first and second samples at refrigerated temperatures after step (b). (Item 39) 38. The method of any one of items 22 to 37, comprising after step (b) storing the first and second samples at less than about -20°C. (Item 40) 40. The method of any one of items 22 to 39, further comprising the step of treating a third sample containing red blood cells with the compound, wherein treatment of the red blood cells of the third sample with the compound results in a second level of the moieties bound to the surface of the red blood cells of the third sample, wherein a first level of the moieties is bound to the surface of the red blood cells of the first sample, and the second level is less than the first level. (Item 41) After treating the third sample with the compound, Washing the third sample. 41. The method of claim 40, further comprising: (Item 42) After treating the third sample with the compound, adding a cryopreservation agent to the third sample; freezing the third sample. The method according to claim 40 or 41, further comprising: (Item 43) 42. The method of claim 40 or 41, further comprising storing the third sample at refrigerated temperatures after treating the third sample with the compound. (Item 44) 1. A method of testing a sample for the presence of an antibody that binds to a compound, comprising: a) providing a sample comprising serum or plasma; b) contacting the sample with red blood cells, wherein a moiety is attached to the surface of the red blood cells, the moiety being selected from the group consisting of any of the compounds of formula I, II, III, IV(a)-IV(h), VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing; c) assaying the amount of binding between an antibody from the sample and a surface binding moiety of the red blood cells, wherein binding between the antibody and the surface binding moiety indicates that the antibody binds to the compound; A method comprising: (Item 45) 45. The method of claim 44, wherein the compound is selected from the group consisting of any of the compounds of formula I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 46) 45. The method of claim 44, wherein the compound is selected from the group consisting of a compound of formula VIII and its derivatives, or a salt or stereoisomer of any of the foregoing. (Item 47) Item 47. The method of item 46, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. (Item 48) R 20 The method according to item 46 or 47, wherein (Item 49) R 21 Ga-C 1~8 49. The method according to any one of items 46 to 48, wherein the alkyl group is alkyl-N(CH2CH2Cl)2. (Item 50) Item 45. The method of item 44, wherein the compound is selected from the group consisting of any of the compounds of formulae IV(a) to IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 51) 45. The method of claim 44, wherein the compound is a derivative of S-303 or a salt thereof. (Item 52) 45. The method of claim 44, wherein the compound is a non-fragile analog of S-303, or a salt thereof. (Item 53) 53. The method of claim 52, wherein the non-fragile analog of S-303 is S-197 or S-220. (Item 54) 45. The method of claim 44, wherein the surface-binding moiety is a derivative of S-303, or a salt thereof. (Item 55) 45. The method of claim 44, wherein the surface-binding moiety is a non-fragile analog of S-303, or a salt thereof. (Item 56) 56. The method of any one of items 44 to 55, wherein the moieties bound to the surface of the red blood cell are present at a loading level of at least about 10,000 moieties per red blood cell. (Item 57) The moieties bound to the surface of the red blood cell are at least about 50 moieties / μm 2 56. The method according to any one of items 44 to 55, wherein the .alpha.-tocopherol is present at a loading level of. (Item 58) 58. The method of any one of items 44 to 57, wherein the compound and the surface-binding moiety are the same. (Item 59) 58. The method of any one of items 44 to 57, wherein the compound and the surface-binding moiety are different. (Item 60) 60. The method of any one of items 44 to 59, wherein step (c) comprises comparing the amount of binding between an antibody from the sample and the surface-binding moiety with a reference, wherein an increase in the amount of binding compared to the reference indicates the presence of the antibody in the sample. (Item 61) 61. The method of claim 60, wherein step (c) comprises assaying the amount of binding between antibodies from the sample and red blood cells lacking the surface-binding moiety. (Item 62) 61. The method of claim 60, wherein step (c) further comprises assaying the amount of binding between antibodies from the sample and red blood cells having a lower amount of the surface-binding moieties. (Item 63) 63. The method of any one of items 44 to 62, wherein step (b) comprises contacting the sample and the red blood cells with an anti-human globulin, and step (c) comprises assaying the amount of agglutination. (Item 64) 1. A method of providing a red blood cell transfusion to a patient, the red blood cell transfusion comprising administering to a patient a pathogen inactivating compound. and treating the red blood cells, the method comprising: a) providing a sample comprising serum or plasma from said patient; b) contacting the sample with a first substrate, wherein a moiety is bound to a surface of the substrate; c) assaying the amount of binding between an antibody from the sample and a surface-binding moiety of the first substrate relative to a reference, where binding between the antibody and the surface-binding moiety indicates that the antibody binds to red blood cells treated with the pathogen inactivating compound; and d) determining whether the amount of binding is higher than the reference. f) providing a blood transfusion to the patient pursuant to a determination that the amount of binding is not higher than the reference. A method comprising: (Item 65) Item 65. The method of item 64, wherein the surface-binding moiety is selected from the group consisting of any compound of formula I, II, III, IV, V, VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 66) Item 65. The method of item 64, wherein the surface-binding moiety is selected from the group consisting of a compound of any of formulas I, II, III, VII, VIII, and IX, and a derivative thereof, or a salt or stereoisomer of any of the foregoing. (Item 67) Item 65. The method of item 64, wherein the surface-binding moiety is selected from the group consisting of a compound of formula VIII and its derivatives, or a salt or stereoisomer of any of the foregoing. (Item 68) Item 68. The method of item 67, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. (Item 69) R 20 The method according to item 67 or 68, wherein (Item 70) R 21 Ga-C 1~8 70. The method according to any one of items 67 to 69, wherein the alkyl group is alkyl-N(CH2CH2Cl)2. (Item 71) Item 65. The method of item 64, wherein the surface-binding moiety is selected from the group consisting of any of the compounds of formulae IV, V, and VI, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 72) 72. The method of claim 71, wherein the surface-binding moiety is selected from the group consisting of any compound of formulae IV(a) to IV(h), and derivatives thereof, or salts or stereoisomers of any of the foregoing: (Item 73) 65. The method of claim 64, wherein the surface-binding moiety is S-303, a derivative of S-303, or a salt of any of the foregoing. (Item 74) 65. The method of claim 64, wherein the surface-binding moiety is a non-fragile analog of S-303 or a salt thereof. (Item 75) 75. The method of claim 74, wherein the non-fragile analog of S-303 is S-197 or S-220. (Item 76) 76. The method of any one of items 64 to 75, wherein the compound and the surface-binding moiety are the same. (Item 77) 76. The method of any one of items 64 to 75, wherein the compound and the surface-binding moiety are different. (Item 78) 78. The method of any one of items 64 to 77, wherein the first substrate comprises a polymer particle, a matrix, or a portion of an assay plate. (Item 79) 78. The method of any one of items 64 to 77, wherein the first substrate comprises red blood cells. (Item 80) 80. The method of any one of items 64 to 79, wherein step (c) comprises assaying the amount of binding between antibodies from the sample and a second substrate lacking the surface-binding moieties. (Item 81) Item 81. The method of item 80, wherein the second substrate is the same as the first substrate. (Item 82) 81. The method of any one of items 64 to 80, wherein step (c) further comprises assaying the amount of binding between antibodies from the sample and a third substrate having a lower amount of the surface-binding moiety compared to the first substrate. (Item 83) Item 83. The method of item 82, wherein the third substrate is the same as the first substrate. (Item 84) 84. The method of any one of items 64 to 83, wherein step (b) comprises contacting the sample and the substrate(s) with an anti-human globulin, and step (c) comprises assaying the amount of agglutination. (Item 85) 85. The method of any one of items 64 to 84, wherein the moiety bound to the surface of the substrate(s) is not the same as the pathogen inactivating compound. (Item 86) A composition comprising a human red blood cell, wherein a moiety selected from the group consisting of any of the compounds of Formulas I, II, III, IV(a)-IV(h), VI, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing, is bound to the surface of the human red blood cell. (Item 87) 87. The composition of claim 86, wherein the moiety is selected from the group consisting of any compound of formula I, II, III, VII, VIII, and IX, and derivatives thereof, or salts or stereoisomers of any of the foregoing. (Item 88) 87. The composition according to item 86, wherein the moiety is selected from the group consisting of a compound of formula VIII and its derivatives, or a salt or stereoisomer of any of the foregoing. (Item 89) Item 89. The composition according to item 88, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each hydrogen. (Item 90) R 20 90. The composition according to item 88 or 89, wherein is H. (Item 91) R 21 Ga-C 1~891. The composition of any one of items 88 to 90, wherein the alkyl group is alkyl-N(CH2CH2Cl)2. (Item 92) 87. The composition of claim 86, wherein the moiety is a non-fragile analog of S-303 or a salt thereof. (Item 93) 93. The composition of claim 92, wherein the non-fragile analog of S-303 is S-197 or S-220.

Claims

1. 1. A method for testing a sample obtained from a patient in need of a red blood cell transfusion for the presence of antibodies that bind to a pathogen inactivating compound, wherein the pathogen inactivating compound is β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester, the method comprising: a) contacting the sample comprising serum or plasma with red blood cells, wherein the surface-bound moieties bind to the surface of the red blood cells, the surface-bound moieties bind to a compound of formula VII: 【Chemical 22】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ is -H; R ９ is -V-X-E, where V is -NH-R １１ - and -R １１ - is -C １-８ alkyl-; X is -R １１ - and E is -N(R １２ ) ２ where -R １２ is -CH ２ CH ２ -G, each G being independently -Cl, -Br, or -I, or a salt or stereoisomer of the foregoing compound; b) assaying the amount of binding between an antibody from the sample and a surface binding moiety of the red blood cells, where binding between the antibody and the surface binding moiety indicates that the antibody binds to the pathogen inactivating compound; A method comprising:

2. The compound of formula VII, or a salt or stereoisomer of the aforementioned compound, is a compound of formula VIII: 【Chemistry 23】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ is -H; R ２０ is H, and R ２１ Ga-C １～８ Alkyl-N(CH ２ CH ２ Cl) ２ 2. The method of claim 1, wherein the compound is a salt or stereoisomer of the foregoing compound.

3. The compound is S-197 【Chemistry 24】 or S-220 【Chemistry 25】 or a salt thereof.

4. 4. The method of claim 1, wherein the moieties bound to the surface of the red blood cells are present at a loading level of at least 10,000 moieties per red blood cell.

5. The moieties bound to the surface of the red blood cell are at least 50 moieties / μm ２ The method of any one of claims 1 to 3, wherein the cation is present at a loading level of

6. 6. The method of claim 1, wherein step (b) comprises comparing the amount of binding between an antibody from the sample and the surface-binding moiety with a reference, wherein an increase in the amount of binding compared to the reference indicates the presence of the antibody in the sample.

7. 7. The method of claim 6, wherein step (b) comprises assaying the amount of binding between antibodies from the sample and red blood cells lacking the surface-binding moiety.

8. 7. The method of claim 6, wherein step (b) further comprises assaying the amount of binding between antibodies from the sample and red blood cells having a lower amount of the surface-bound moieties.

9. 9. The method of claim 1, wherein step (a) comprises contacting the sample and the red blood cells with an anti-human globulin, and step (b) comprises assaying the amount of agglutination.

10. 1. A composition comprising a first substrate for use in a method of providing a red blood cell transfusion to a patient in need thereof, wherein a moiety is attached to a surface of the substrate, the red blood cell transfusion comprising red blood cells treated with a pathogen inactivating compound, the pathogen inactivating compound being β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester, the method comprising: a) contacting a sample comprising serum or plasma obtained from the patient in need of a red blood cell transfusion with the first substrate; b) assaying the amount of binding between an antibody from the sample and a surface-binding moiety of the first substrate relative to a reference, wherein binding between the antibody and the surface-binding moiety indicates that the antibody binds to red blood cells treated with the pathogen inactivating compound, and the surface-binding moiety binds the first substrate to a compound of formula VII: 【Chemistry 26】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ is -H; R ９ is -V-X-E, where V is -NH-R １１ - and -R １１ - is -C １-８ alkyl-; X is -R １１ - and E is -N(R １２ ) ２ where -R １２ is -CH ２ CH ２ -G, each G being independently -Cl, -Br, or -I, or a salt or stereoisomer of the foregoing compound; c) determining whether the amount of binding is higher than the reference; d) providing said red blood cell transfusion to said patient pursuant to a determination that said amount of binding is not higher than said reference; A composition comprising:

11. The compound of formula VII, or a salt or stereoisomer of the aforementioned compound, is a compound of formula VIII: 【Chemical 27】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ is -H; R ２０ is H, and R ２１ Ga-C １～８ Alkyl-N(CH ２ CH ２ Cl) ２ 11. The composition of claim 10, wherein the compound is a salt or stereoisomer of the foregoing compound.

12. The compound is S-197 【Chemistry 28】 or S-220 【Chemical Formula 29】 or a salt thereof.

13. The composition of claim 10 , wherein the first substrate comprises a polymeric particle, a matrix, or a portion of an assay plate.

14. The composition of claim 10 , wherein the first substrate comprises red blood cells.

15. 15. The composition of any one of claims 10 to 14, wherein step (b) comprises assaying the amount of binding between antibodies from the sample and a second substrate lacking the surface-binding moiety.

16. The composition of claim 15, wherein the second substrate is the same as the first substrate.

17. 16. The composition of any one of claims 10 to 15, wherein step (b) further comprises assaying the amount of binding between antibodies from the sample and a third substrate having a lower amount of the surface-binding moieties compared to the first substrate.

18. 20. The composition of claim 17, wherein the third substrate is the same as the first substrate.

19. 19. The composition of any one of claims 10 to 18, wherein step (a) comprises contacting the sample and the substrate(s) with an anti-human globulin, and step (b) comprises assaying the amount of agglutination.

Images