EA044190B1 - SYSTEMS AND METHODS FOR TESTING AND SCREENING USING COMPOUND-RELATED SUBSTRATES - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No.

62/438909, filed December 23, 2016, the contents of which are hereby incorporated by reference in their entirety.

Field of technology

The invention relates to systems and methods for testing and/or screening biological samples using compound-bound substrates, such as reagent red blood cells.

State of the art

Transfusion of blood or blood components is common in medical practice. Typically, blood products are tested for compatibility between the donor and recipient before transfusion or use of the blood product on the recipient.

Blood group antigens are polymorphic protein or carbohydrate residues on red blood cells. These antigens can stimulate a humoral response in individuals who lack them, and some antibodies (eg, clinically significant antibodies) can lead to a transfusion hemolytic reaction or hemolytic disease. There are more than 300 blood group antigens (BGAs) on red blood cells, so transfusion recipients are often exposed to such antigens and, in the case of conventional red blood cell transfusions, immune responses to them are observed. Immune responses occur more frequently in patients with repeated exposure to allogeneic red blood cells and/or certain conditions (eg, SCD, thalassemia).

Detection of clinically significant blood group alloantibodies (donor-recipient blood compatibility test) is the main test in pre-transfusion testing.

Donors and patients are typically ABO and Rh(D) typed because... they are considered the most important antigens for safe transfusion. As a rule, typing is not performed for minor antigens, such as antigens of the Rh (C/c and E/e), Kell (K/k), Duffy (Fya, Fyb), Kidd (Jka/Jkb) and MNS (M/) systems. N and S/s), and the plasma is screened for antibodies against these antigens. If the patient's antibody screening results are negative, red blood cell transfusion units must also be ABO- and Rh(D)-compatible. If an antibody against a clinically significant minor antigen is present, the units must additionally lack the corresponding antigen. A common antibody screening test is a solid-phase red cell adhesion assay (e.g., Galileo System, Immucor) or an in vitro agglutination test such as an antiglobulin test (e.g., direct antiglobulin test (DAT), indirect antiglobulin test (IAT)) . These involve the use of human red blood cells of known specificity, tested against plasma or serum samples. After an incubation period and, most often, the addition of secondary antibody solutions, the mixture is examined for hemagglutination. Recently, this test has also been carried out using microplate and column agglutination technology systems known in the art, which can also be used with some degree of automation. However, the existing test is limited by the range of clinically relevant antibodies that can be detected with it.

Disease transmission through blood products and other biological materials remains a major public health problem. To inactivate pathogens before transfusion and reduce the risk of transfusion infections, blood products such as platelets and plasma include nucleic acid-targeting compounds such as psoralens and psoralen derivatives for UVA photochemical treatment. Other nucleic acid-targeting compounds have been developed to treat red blood cells to inactivate pathogens, such as S-303 (eg amustalin). Compound S-303 forms covalent cross-links with contaminating pathogen and leukocyte nucleic acids to block replication and reduce the risk of transfusion-borne infections. However, some of these compounds or their derivatives may potentially react with other nucleophiles in components of red blood cells, including nucleophiles on their surface, which in particular cases may result in the binding of the compound or its derivatives (e.g., residues) to red blood cells, for example, through the formation of bound surface of adducts. Even though such pathogen-inactivated RBCs are generally considered safe and non-immunogenic, the compounds or derivatives thereof bound to RBCs may in some cases be recognized by pre-existing antibodies or induce undesirable immune responses in some patients.

Thus, there is a need for methods/systems for detecting antibodies against red blood cells treated with pathogen-inactivating compounds in patients.

The essence of the invention

The compositions, methods, kits and systems of the present invention, among other uses, provide a means for detecting antibodies in a biological sample, for example, for screening potential recipients of pathogen-inactivated red blood cells for pre-existing cross-antibodies prior to infusion of the treated red blood cells. In some embodiments, the compositions, methods, kits, and systems are particularly suitable for detecting antibodies in patients

- 1 044190 against erythrocytes treated with compound S-303.

In one aspect, the invention relates to a kit comprising: (a) a first container containing a first substrate, wherein a functional moiety is associated with a surface of the first substrate, and wherein the functional moiety is selected from the group consisting of compounds of any of formulas I, II, III, IV, V, VI, VII, VIII and IX and their derivatives or salts or stereoisomers of any of the above compounds; and (b) a second container containing a second substrate, wherein there is no associated functional moiety on the surface of the second substrate. In some embodiments, the functional moiety is selected from the group consisting of compounds of any of formulas I, II, III, VII, VIII and IX and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the functional moiety is selected from the group consisting of compounds of any of formulas IV, V and VI and derivatives thereof, or a salt or stereoisomer of any of the above compounds. In some embodiments, the functional moiety is selected from the group consisting of compounds of any of formulas IV(a)-IV(h) and derivatives thereof, or a salt or stereoisomer of any of the above compounds. In some embodiments, the functional moiety is S-303, a derivative of S-303, or a salt of any of the above compounds. In some embodiments, the functional moiety is a refractory analogue of S-303 or a salt thereof. In some embodiments, the functional moiety associated with the surface of the substrate is present at a loading level of at least about 10,000 functional moieties per unit of substrate. In some embodiments, the functional moiety associated with the surface of the substrate is present at a loading level of at least about 50 functional moieties/μm ² . In some embodiments, the compound and the surface-associated functional moiety are the same. In some embodiments, the compound and the functional moiety associated with the surface are different. In some embodiments, the first and second substrates each comprise a polymer particle, matrix, or assay plate portion. In some embodiments, the first substrate contains red blood cells, where the second substrate contains red blood cells, and where the red blood cells from the first and second substrates are obtained from one or more common donors. In some embodiments, the kit further comprises a third container containing a third substrate, wherein the first level of functional moiety is associated with a surface of the first substrate, wherein the second level of functional moiety is associated with the surface of a third substrate, and where the second level is smaller than the first level. In some embodiments, the first level of functional moiety associated with the surface of the first substrate is at least 3 times the second level of functional moiety associated with the surface of the third substrate. In some embodiments, the third substrate comprises a polymer particle, matrix, or assay plate portion. In some embodiments, the third substrate comprises red blood cells, and the red blood cells from the first, second, and third substrates are obtained from one or more common donors. In some embodiments, the first substrate contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 samples, where each of the first substrate samples contains red blood cells obtained from a different blood donor, and where the second substrate contains a corresponding sample , containing red blood cells from each of the different blood donors. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 samples correspond to multiple blood types. In some embodiments, the first substrate and the second substrate comprise red blood cells obtained from a blood type O donor. In some embodiments, the first substrate, second substrate, and third substrate comprise red blood cells obtained from a blood type O donor. In some embodiments, the first substrate and the second substrate contains red blood cells in a buffered suspension medium at an amount of from about 0.5% to about 5% red blood cells. In some embodiments, the first substrate, second and third substrate contain red blood cells in a buffered suspension medium at an amount of from about 0.5% to about 5% red blood cells. In some embodiments, a first container containing a first substrate, a second container containing a second substrate, and/or a set containing a first container and a second container are suitable for storage at 2-8°C. In some embodiments, a first container containing a first substrate, a second container containing a second substrate, and/or a set containing a first container and a 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 set containing the first container and the second container are suitable for storage at temperatures less than -20°C.

In another aspect, the invention relates to a method for producing a panel of substrates, comprising: a) producing a first and a second sample, each containing a substrate; and b) treating the substrate from the first sample with the compound, wherein treatment with the compound results in binding of the functional moiety to the surface of the substrate from the first sample; wherein the second sample is not treated with the compound, thereby providing a panel comprising a first substrate sample with a surface-bound functional moiety and a second substrate sample lacking the surface-bound functional moiety, and wherein the compound is selected from the group consisting of compounds

- 2 044190 any of formulas I, II, III, IV(a)-IV(h), VII, VIII and IX and their derivatives or salts or stereoisomers of any of the above compounds. In some embodiments, the invention relates to a method for producing a panel of polymer particles, comprising: a) producing a first and second sample containing polymer particles; and b) treating the polymer particles from the first sample with a compound, wherein the treatment with the compound causes the functional moiety to bind to the surface of the particles from the first sample; wherein the second sample is not treated with the compound, thereby providing a panel containing a first sample of polymer particles with a surface-bound functional moiety and a second sample of polymer particles lacking a surface-bound functional moiety, and wherein the compound is selected from the group consisting of compounds of any from formulas I, II, III, IV(a)-IV(h), VII, VIII and IX and their derivatives or a salt or stereoisomer of any of the above compounds. In some embodiments, the invention relates to a method for obtaining a panel of red blood cells, comprising: a) obtaining first and second samples containing red blood cells; and b) treating the red blood cells from the first sample with the compound, wherein the treatment with the compound causes the functional moiety to bind to the surface of the red blood cells from the first sample; wherein the second sample is not treated with the compound, thereby obtaining a panel containing a first sample of red blood cells with a surface-bound functional moiety and a second sample of red blood cells lacking a surface-bound functional moiety, and wherein the compound is selected from the group consisting of compounds of any of the formulas I, II, III, IV(a)-IV(h), VII, VIII and IX and their derivatives or salts or stereoisomers of any of the above compounds. In some embodiments, the compound is selected from the group consisting of compounds of any of formulas I, II, III, VII, VIII and IX and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the compound is selected from the group consisting of compounds of any of formulas IV(a)-IV(h) and derivatives thereof, or a salt or stereoisomer of any of the above compounds. In some embodiments, the compound is a derivative of S-303 or a salt thereof. In some embodiments, the compound is a refractory analogue of S-303 or a salt thereof. In some embodiments, the surface-associated functional moiety is a derivative of S-303 or a salt thereof. In some embodiments, the surface-bound functional moiety is a refractory analogue of S-303 or a salt thereof. In some embodiments, the functional moiety associated with the surface of the red blood cells is present at a loading level of at least about 10,000 functional moieties per red blood cell. In some embodiments, the functional moiety associated with the surface of the red blood cells is present at a loading level of at least about 50 functional moieties/μm ² . In some embodiments, the compound and the surface-associated functional moiety are the same. In some embodiments, the compound and the surface-associated functional moiety are different. In some embodiments, the red blood cells from the first and second samples are obtained from the same blood donor. In some embodiments, the red blood cells from the first and second samples are obtained from a blood type O donor. In some embodiments, treatment of the substrate from the first sample with a compound, wherein treatment with the compound causes the functional moiety to bind to the surface of the substrate from the first sample, is carried out at 2-8°C WITH. In some embodiments, treatment of the substrate from the first sample with the compound, wherein treatment with the compound results in binding of the functional moiety to the surface of the substrate from the first sample, is carried out at room temperature. In some embodiments, the method after step (b) further includes washing the first and second samples. In some embodiments, the method after step (b) further includes resuspending the first and second samples in a buffered suspension medium at an amount of from about 0.5% to about 5% red blood cells. In some embodiments, the method after step (b) further includes adding a cryopreservative to the first and second samples and freezing the first and second samples. In some embodiments, the method after step (b) further includes storing the first and second samples at a refrigeration temperature (eg, 2-8°C). In some embodiments, the method after step (b) includes storing the first and second samples at a temperature less than about -20°C. In some embodiments, the method after step (b) includes storing the first and second samples at room temperature. In some embodiments, the method further includes treating a third sample containing red blood cells with a compound, wherein treating the red blood cells from the third sample with the compound results in a second level of functional moiety associated with the surface of the red blood cells from the third sample, wherein the first level of functional moiety is associated with the surface of the red blood cells from the first sample, and where the second level is less than the first level. In some embodiments, after treating the third sample with the compound, the method further includes washing the third sample. In some embodiments, after treating the third sample with the compound, the method further includes adding a cryopreservative to the third sample and freezing the third sample. In some embodiments, after treating the third sample with the compound, the method further includes resuspending the third sample in a buffered suspension medium at an amount of from about 0.5% to about 5% red blood cells. In some embodiments, after treating the third sample with the compound, the method further includes storing the third sample at a refrigerated temperature or at room temperature.

In another aspect, the invention relates to a method of testing a sample for the presence of an antibody that binds to a compound, comprising: a) obtaining a sample containing serum or plasma; b) bringing the sample into contact with the substrate, where the functional moiety is associated with the surface of the substrate; and wherein the functional moiety is selected from the group consisting of compounds 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 above compounds; and c) analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety from the substrate, wherein binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to the compound. In some embodiments, a method of testing a sample for the presence of an antibody that binds to a compound includes: a) obtaining a sample containing serum or plasma; b) bringing the sample into contact with a polymer particle (eg, bead, microsphere), matrix, or assay plate portion, wherein the functional moiety is bound to the surface of the polymer particle (eg, bead, microsphere), matrix, or assay plate portion; and wherein the functional moiety is selected from the group consisting of compounds 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 above compounds; c) analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety on a polymer particle (e.g., beads, microspheres), matrix, or assay plate portion, where binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to connection. In some embodiments, a method of testing a sample for the presence of an antibody that binds to a compound includes: a) obtaining a sample containing serum or plasma; b) bringing the sample into contact with red blood cells, where the functional fragment is associated with the surface of the red blood cells; and wherein the functional moiety is selected from the group consisting of compounds 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 above compounds; and c) analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety on the red blood cells, where binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to the compound. In some embodiments, the compound is selected from the group consisting of compounds of any of formulas I, II, III, VII, VIII and IX and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the compound is selected from the group consisting of compounds of any of formulas IV(a)-IV(h) and derivatives thereof, or a salt or stereoisomer of any of the above compounds. In some embodiments, the compound is a derivative of S-303 or a salt thereof. In some embodiments, the compound is a refractory analogue of S-303 or a salt thereof. In some embodiments, the surface-associated functional moiety is a derivative of S-303 or a salt thereof. In some embodiments, the surface-bound functional moiety is a refractory analogue of S-303 or a salt thereof. In some embodiments, the functional moiety associated with the surface of the red blood cells is present at a loading level of at least about 10,000 functional moieties per red blood cell. In some embodiments, the functional moiety associated with the surface of the red blood cells is present at a loading level of at least about 50 functional moieties/μm ² . In some embodiments, the compound and the surface-associated functional moiety are the same. In some embodiments, the compound and the surface-associated functional moiety are different. In some embodiments, step (c) involves comparing the degree of binding between the antibody from the sample and the surface-bound functional moiety to a reference, and wherein the increased degree of binding relative to the reference is indicative of the presence of the antibody in the sample. In some embodiments, step (c) includes analyzing the extent of binding between the antibody from the sample and red blood cells that lack the surface-bound functional moiety. In some embodiments, step (c) further includes analyzing the extent of binding between the antibody from the sample and red blood cells containing a reduced amount of surface bound functional moiety. In some embodiments, step (b) includes contacting the sample and red blood cells with antiglobulin serum, and where step (c) includes analyzing the degree of agglutination.

In another aspect, the invention relates to a method of performing a transfusion of red blood cells into a patient, wherein the red blood cell transfusion includes red blood cells treated with a pathogen-inactivating compound, comprising: a) obtaining a sample containing serum or plasma of the patient; b) bringing the sample into contact with a first substrate, wherein the functional moiety is bound to the surface of the substrate; c) analyzing the degree of binding between the antibody from the sample and the surface-bound functional moiety from the first substrate compared to a reference, wherein the binding between the antibody and the surface-bound functional moiety indicates that the antibody

- 4 044190 binds to red blood cells treated with a pathogen-inactivating compound; d) determining whether the degree of binding exceeds the standard; and f) if it is determined that the degree of binding does not exceed the standard, administering a blood transfusion to the patient. In some embodiments, the surface-associated functional moiety is selected from the group consisting of compounds of any of formulas I, II, III, IV, V, VI, VII, VIII and IX and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the surface-associated functional moiety is selected from the group consisting of compounds of any of formulas I, II, III, VII, VIII and IX and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the surface-associated functional moiety is selected from the group consisting of compounds of any of formulas IV, V and VI, and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the surface-associated functional moiety is selected from the group consisting of compounds of any of formulas IV(a)-IV(h) and derivatives thereof, or a salt or stereoisomer of any of the above compounds. In some embodiments, the surface-associated functional moiety is S-303, a derivative of S-303, or a salt of any of the above compounds. In some embodiments, the surface-bound functional moiety is a refractory analogue of S-303 or a salt thereof. In some embodiments, the compound and the surface-associated functional moiety are the same. In some embodiments, the compound and the surface-associated functional moiety are different. In some embodiments, the first substrate comprises a polymer particle, matrix, or assay plate portion. In some embodiments, the first substrate comprises red blood cells. In some embodiments, step (c) includes analyzing the extent of binding between the antibody from the sample and a second substrate that lacks a surface-bound functional moiety. In some embodiments, the second substrate is the same as the first substrate. In some embodiments, step (c) further includes analyzing the extent of binding between the antibody from the sample and a third substrate containing a smaller amount of surface-bound functional moiety compared to the first substrate. In some embodiments, the third substrate is the same as the first substrate. In some embodiments, step (b) includes contacting the sample and substrates with antiglobulin serum, and where step (c) includes analyzing the degree of agglutination. In some embodiments, the functional moiety associated with the surface of the substrates is different from the pathogen-inactivating compound.

In another aspect, the invention relates to a composition containing human red blood cells, wherein the functional moiety is selected from the group consisting of compounds of any 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 above compounds, bound to the surface of human erythrocytes. In some embodiments, the functional moiety is selected from the group consisting of compounds of any of formulas I, II, III, VII, VIII and IX and derivatives thereof or a salt or stereoisomer of any of the above compounds. In some embodiments, the functional moiety is a refractory analogue of S-303 or a salt thereof.

Brief description of the figures

Fig. 1 is a graph of antibody staining levels of treated red blood cells. Fig. 2 is a graph of antibody staining levels of treated red blood cells. Fig. 3 is a graph of antibody staining levels of treated red blood cells. Fig. 4 is a graph of antibody staining levels of treated red blood cells. Fig. 5 is a graph of antibody staining levels of treated red blood cells. Fig. 6 is a graph of antibody staining levels of treated red blood cells.

Detailed description

The present invention relates to methods and systems for testing and/or screening biological samples using a substrate containing a related compound. The present inventors have discovered that although a compound (eg, a pathogen-inactivating compound) used in various embodiments of the present invention may be safe and effective for treating red blood cells (eg, inactivating pathogens), such treatment may potentially result in the compound will react with other nucleophiles in the red blood cells, which in particular cases may result in the compound binding to the red blood cells, such as to the surface of the red blood cells (eg, the formation of surface-bound adducts). Such binding may also result in the formation of antibodies specific for the compound or portion thereof also binding to the treated red blood cells if such antibodies are present in the patient to whom the treated red blood cells are transfused. Thus, it is important to test a biological sample (eg, a sample from a patient) to assess the presence or absence of antibodies (eg, clinically significant antibodies) against a compound or part thereof. The present application includes the methods/systems presented herein and the use of these methods/systems in testing a biological sample for the presence of an antibody against a compound using compositions and/or kits containing the compounds of the present invention coupled to substrates. In some embodiments, the methods/systems are particularly useful for detecting pre-existing antibodies reacting with red blood cells treated with a pathogen-inactivating compound in patients prior to transfusion.

The present invention relates to compositions of compound-bound substrates (eg, red blood cells bound to a pathogen-inactivating compound), kits/systems, methods for preparing the compositions, and uses thereof.

As used herein, singular terms are used to refer to one or more (ie, at least one) grammatical entities. By way of example, substrate means one substrate or multiple substrates.

As used herein, the term biological sample or simply sample means a sample, such as a sample, optionally obtained from an animal (eg, a human), wherein the sample or component thereof can be used to assess the presence, absence and/or level of an antibody by the methods of the invention. Such a sample includes, by way of non-limiting examples, any biological sample, such as, for example, a biological fluid (e.g., blood, serum, plasma, lymph, semen, sputum, saliva, mucus, tears, etc.) and any sample derived from an animal (such as a human) that can be analyzed for the presence or absence of an antibody.

As used herein, the term alkyl refers to a cyclic, branched or straight-chain chemical group containing carbon and hydrogen, such as methyl, pentyl and adamantyl. Alkyl groups may be unsubstituted or substituted with one or more substituents, for example, halogen, alkoxy, acyloxy, amino, hydroxyl, thiol, carboxy, benzyloxy, phenyl, benzyl or other functional group. Alkyl groups may be saturated or unsaturated (eg, containing -C=C- or -C-C- subunits) at one or more positions. Typically, unless otherwise stated, alkyl groups will contain from 1 to 12 carbon atoms, such as from 1 to 10 carbon atoms or from 1 to 8 carbon atoms.

As used herein, the term heteroalkyl refers to an alkyl chain with one or more N, O, S or P heteroatoms embedded in the chain. Heteroatoms may or may not bear one or more of the substituents described above. Heteroatoms also include oxidized forms of N, S, and P heteroatoms. Non-limiting examples of heteroalkyl groups include methoxy, ethoxy, and other alkoxy groups; ether-containing groups, amide-containing groups such as polypeptide chains; ring systems such as piperidinyl, lactam and lactone; and other groups including heteroatoms in the carbon chain. Typically, unless otherwise indicated, heteroalkyl groups will contain, in addition to heteroatoms, from 1 to 12 carbon atoms, for example, from 1 to 10 carbon atoms or from 1 to 8 carbon atoms.

The term aryl or Ar refers to an unsaturated aromatic carbocyclic group containing a single ring (eg, phenyl) or multiple fused rings (eg, naphthyl or anthryl), optionally unsubstituted or substituted by an amino group, hydroxyl, C ₁ . ₈ -alkyl, alkoxy group, halogen, thiol and other substituents.

Heteroaryl groups are unsaturated aromatic carbocyclic groups containing one ring (for example, pyridyl or furyl) or multiple fused rings (for example, acridinyl, indolyl or benzothienyl) and at least one heteroatom, such as N, O or S, at least one of the rings. The rings are optionally unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy, halogen, thiol, acyloxy, carboxy, benzyloxy, phenyl, benzyl and other substituents.

Compositions.

The present invention relates to compositions containing a substrate containing a surface-bound functional moiety, wherein the surface-bound functional moiety is suitable for binding antibodies against a compound, such as, for example, a compound used in the production of pathogen-inactivated red blood cells that may be present in a biological sample .

Substrate.

As will be appreciated by one skilled in the art, the substrate can be any solid support known in the art. The solid support may consist of any material (eg, biological, synthetic) or matrix suitable for binding a surface-bound functional moiety. Examples of solid supports include polymers or copolymers (eg, polyesters, polyethers, polyolefins, polyamides, polysaccharides, polyurethanes, styrene and styrene derivatives, polystyrene, cellulose), plastic, glass, gold and other metals, etc. Additional examples of solid supports include cells (eg, red blood cells), cell membranes, and other cell-derived material. The solid support may expose the surface-associated functional moiety in a two-dimensional format (eg, a tablet) or a three-dimensional format (eg, a cell, particle, bead, or other spherical or quasi-spherical object). Additional examples of solid supports include particles (eg, polymer particles),

- 6 044190 such as, for example, beads and microspheres (for example, synthetic beads and microspheres, beads and microspheres made of polymers or copolymers). Bonding agents can also be used to impart integrity and structure to solid substrates. The substrate may be part of an assay plate. Particular solid supports are composed of one or more materials to which the biological sample will not or substantially will not bind. For example, the solid support may be composed of material to which antibodies do not bind or substantially not bind (eg, material that does not contain antigens to which specific antibodies bind). In particular, the solid support consists of a material to which antibodies against red blood cells with inactivated pathogens do not bind or substantially do not bind.

Any solid support for use in the compositions and methods of the invention may be suitable for covalently or non-covalently attaching a surface-bound functional moiety. In some embodiments, the solid support is inert. In some embodiments, the surface-bound functional moiety may be covalently or non-covalently linked to the solid support material itself. In other embodiments, the solid support material is functionalized with a reactive group (e.g., amine (-NH2) or ammonium (-NH3+ or -NR3+) functional group, alcohol functional group (-OH), carboxyl functional group (-COOH), isocyanate functional group (-NCO)), which can interact with or otherwise covalently or non-covalently bind to a surface-bound functional moiety.

It may be advantageous for the substrate to be stable at various temperatures, such as room temperature, refrigeration temperature, or freezing temperature. In some embodiments, the substrate is stable at room temperature. In some embodiments, the substrate is stable at refrigeration temperatures (eg, 1-6°C, 2-8°C, 2-4°C). In some embodiments, the substrate is stable at a temperature of 0°C or less (eg, -20°C). In some embodiments, the substrate is stable at -80°C.

In some embodiments of the present invention, the substrate contains red blood cells. For example, red blood cells, often used in immunohematological tests, can be a substrate, as presented herein.

In some embodiments, the red blood cells are obtained from human blood. The substrate may include red blood cells from any blood group, for example, from type O, type A, type B, or type AB donors. Red blood cells may be specifically designed to support resolution of one or more antibody mixtures. In some embodiments, the red blood cells express antigens selected from the group consisting of C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb antigens. In some embodiments, the red blood cells express Rh(D). In some embodiments, the red blood cells are of a Rh type selected from the group consisting of R1R1, R2R2, ^R1wR1 , _R0r , and rr. Red blood cells can be stored at room temperature (eg, in buffered suspension medium at 0.5-5% red blood cells), refrigerated temperature (eg, in buffered suspension medium at 0.5-5% red blood cells), or they can be frozen (eg, in a cryopreservative such as glycerolite) and store at -80°C.

The red blood cells used as substrates in the compositions of the invention can be obtained from a single donor or from two or more donors (eg, a mixture of red blood cells obtained from two or more donors). In some embodiments, the red blood cell substrate comprises red blood cells obtained from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more separate donors. In some cases, red blood cells from two or more donors share one or more properties, such as ABO blood type or the presence or absence of other red blood cell antigens (eg, common red cell antigens, clinically relevant red blood cell antigens). In other cases, red blood cells from two or more donors are heterogeneous with respect to specific properties, such as ABO blood group or the presence or absence of other red blood cell antigens (eg, common red blood cell antigens, clinically relevant red blood cell antigens). In some embodiments, the red blood cell substrate includes red blood cells having a mixture of ABO blood groups. In some embodiments, the erythrocyte substrate is homogeneous with respect to the Rh factor. In other embodiments, the erythrocyte substrate is heterogeneous with respect to the Rh factor. In some embodiments, the erythrocyte substrate is homogeneous with respect to any one or more of the following antigens: Csa, Cs ^b , Era, Er ^b , Vel. ABTI, Lan, Ata, Jra, AnWj, Sda, Batty (By), Biles (Bi), Box (Bx ^a ) Christiansen (Chra), HJK, HOFM, JFV, JONES, Jensen (Je ^a ), Katagiri (Kg) , Livesay (Lia), Milne, Oldeide (OI ^a ), Peters (Pt ^a ), Rasmussen (RASM), Reid (Rea), REIT, SARA, Torkildsen (To ^a ) and Bennett-Goodspeed (Bg). In some embodiments, the erythrocyte substrate is heterogeneous with respect to any one or more of the following antigens: Csa, Cs ^b , Era, Er ^b , Vel. ABTI, Lan, Ata, Jra, AnWj, Sda, Batty (By), Biles (Bi), Box (Bx ^a ) Christiansen (Chra), 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).

Red blood cells for use as a substrate in the compositions and methods presented in

- 7 044190 of the present description, can be obtained by any method known in this field. For example, red blood cells can be obtained using standard red blood cell donation methods (eg, apheresis, double-dose red blood cell donation). Alternatively, red blood cells can be obtained from whole blood donations by separating the red blood cells and other components of the whole blood, for example, by centrifugation or other standard fractionation method. In some embodiments, the red blood cells are sterilized by any method known in the art before or after functionalization using a surface-bound functional moiety.

One skilled in the art will appreciate that in some embodiments of the present invention, the compound may be coupled to a substrate in an array. As used herein, the term array refers to a generally ordered arrangement of interconnected compounds on a substrate such as glass or plastic. Typically, an array may be in the form of a series of evenly spaced and demarcated areas to which connections are associated. Such arrays can be described as a chip. For example, arrays in multiwell microplates can be scanned using automated equipment.

A functional fragment associated with a surface.

In various embodiments of the present invention, the functional moiety is associated with the surface of the substrate. In some embodiments, the functional moiety is a pathogen-inactivating compound. In some embodiments, the functional moiety is a derivative of (eg, a portion of) a pathogen-inactivating compound. In some embodiments, the functional moiety is connected to a portion similar to or common to the pathogen-inactivating compound. The substrate can be treated with a pathogenic activating compound such that the compound or derivative thereof binds to the surface of the substrate. Alternatively, the substrate can be treated with a functional moiety that is itself a derivative of the pathogen-inactivating compound such that the reactive functional moiety itself binds to the substrate or a further derivative thereof binds to the substrate. Alternatively, the substrate can be treated with a connection to a moiety similar or common to the pathogen-inactivating compound such that the similar or common moiety binds to the substrate. In some embodiments, the functional moiety is a derivative of an acridine-containing compound, such as, for example, an acridine mutagen, a monoalkylator, a bis-alkylator, acridine mustard (e.g., 6-chloro-9-(3-[(2chloroethyl)ethylamino]propylamino)-2 -methoxyacridine, Sigma #12888) or acridine hemi-mustard (e.g., 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride, Sigma #13636), and the substrate can be treated with a compound or derivative of a compound containing acridine

Pathogen-inactivating compounds are compounds that inactivate one or more nucleic acid-containing pathogens in red blood cells or other blood-based compositions where the nucleic acid-containing pathogens are capable of causing disease in humans, other mammals, or vertebrates. The pathogen can be unicellular or multicellular. Examples of pathogens are bacteria, viruses, protozoa, fungi, yeasts, molds, and mycoplasmas that cause disease in humans, other mammals, or vertebrates. The genetic material of the pathogen may be DNA or RNA, and the genetic material may be present in the form of single-stranded or double-stranded nucleic acid.

Pathogen-inactivating compounds have been developed, typically containing electrophilic groups that react with pathogens, more specifically with pathogen nucleic acids. For example, US Pat. Nos. 5,691,132, 6,177,441, 6,410,219, 6,143,490, and 6,093,725, the disclosures of which are hereby incorporated by reference, describe the use of compounds containing a nucleic acid-reactive component as well as an electrophilic component that reacts with nucleic acid to inactivate the pathogen. US Pat. Nos. 6,093,725 and 6,514,987, the disclosures of which are therefore incorporated herein by reference, describe compounds wherein a nucleic acid-targeting moiety is coupled to a reactive electrophilic moiety via a hydrolyzable linker. Compounds containing such hydrolyzable linkers may be referred to as cleavable compounds. Under particular conditions, the linker from a readily cleavable compound will undergo hydrolysis, thereby cleaving the nucleic acid-targeting component from the electrophilic component of the compound. Compounds containing a nucleic acid-targeting moiety linked to an electrophilic moiety via a non-hydrolyzable linker may be referred to as refractory compounds.

In some embodiments, pathogen-inactivating compounds include compounds containing a functional group that forms or is capable of forming and has formed, for example in situ, a reactive group, such as an electrophilic group. In some cases, a pathogen-inactivating compound requires photoactivation to become reactive. In some cases, a pathogen-inactivating compound does not require photoactivation to be reactive. For example, the functional group may be mustard gas

- 8 044190 group, mustard group intermediate, mustard group equivalent, epoxide, formaldehyde or formaldehyde synthon. Such functional groups may form an in situ reactive group such as an electrophilic aziridine, aziridinium, thiirane or thiiranium ion. The mustard group may be a mono- or bis-(haloethyl)amino group or a mono(haloethyl)sulfide group. A mustard equivalent is a group that reacts through a mechanism similar to mustard gases, for example, forming reactive intermediates such as aziridinium and aziridine groups or thiirane and thiiranium groups. Examples include aziridine derivatives, mono or bis(mesylethyl)amino groups, mono(mesylethyl)sulfide groups, mono or bis(tosylethyl)amino groups, and mono(tosylethyl)sulfide groups. A formaldehyde synthon is any compound that decomposes to formaldehyde, including hydroxylamine such as hydroxymethylglycine. The reactive moiety of the pathogen-inactivating compound can react with nucleic acids of pathogens, for example, with nucleophilic groups on the nucleic acid. The reactive group can also react with the nucleophilic group of the quencher.

In some embodiments, the pathogen-inactivating compounds include a component that directs the compound to nucleic acids, such as an anchor moiety. The anchor portion contains a functional moiety capable of non-covalently binding to a nucleic acid biopolymer such as DNA or RNA, and is also referred to as a nucleic acid binding ligand, nucleic acid binding moiety, or nucleic acid binding functional moiety. In certain embodiments, the anchor moiety is coupled to the reactive electrophilic moiety via a hydrolyzable linker.

In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a refractory compound or a derivative thereof. The refractory compounds may be any of the following formulas I, II or III.

Formula I is:

Formula (I) wherein n is an integer 1-12, inclusive, and X is CH ₂ , NH, O or S, or a salt or stereoisomer thereof (including enantiomers and diastereomers).

Formula II is:

Formula (II) where n is an integer 1-12, inclusive, or a salt or stereoisomer thereof (including enantiomers and diastereomers).

- 9 044190

Formula III is:

Formula (III) 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 with a second 2-ethylhalogen group on the amine attached to the tricyclic ring by a chain of one to nine carbon atoms, wherein the chain optionally contains one or more heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur atoms, wherein the chain optionally contains one or more unsaturated bonds or carbonyl groups, and wherein the chain is optionally substituted with one or more lower alkyl groups; and the remainder of R1, R2, R3, R4 and R5 are independently hydrogen, lower alkyl, lower alkoxy, halogen, CH2OR6 or -CH2NR7R8, wherein each of R6, R7 and R8 is independently hydrogen or lower alkyl.

Specific refractory compounds include the following:

and their salts or stereoisomers (including enantiomers and diastereomers).

In some embodiments, the compound treated with a substrate provided herein, or the resulting functional moiety bound to the surface of a substrate provided herein, is a readily cleavable compound or a derivative thereof. The readily cleavable compound may be a compound of any of formulas IV, V or VI or a derivative of a compound of any of the following formulas IV, V or VI, as follows.

- 10 044190

Formula IV is:

wherein at least one of R _b R2, R ₃ , R ₄ , R ₅ , R ₆ , R7, R ₈ and R ₉ is -VWXE, as defined below, and the rest of R _b R2, R ₃ , R ₄ , R ₅ , R ₆ , R7, R ₈ and R ₉ are independently selected from the group consisting of -H, -R10, -O-Rio, -NO2, -NH2, -NH-R10, N(R _w )2, -F, -Cl, -Br, -I, -C(=O) _-RW , -C(=0)-0-Rio and -OC(=0)-Rio, where -R ₁₀ independently represents 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 -heteroαlkyl-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 ₁₃ -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;

V is independently -R11-, -NH-R _n - or -N(CH ₃ )-R ₁₁ -, where -R11- is independently -C _1-8 -alkyl-, -C _1-8 -heteroalkyl- , -aryl-, -heteroaryl-, -C _1-3 -alkyl-aryl-, -C _1-3 -heteroalkylaryl-, -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 -heteroalkylaryl-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 -heteroal kil-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 independently represents -C(=O)-O-, -O-C(=O)-, C(=S)-O-, -O-C(=S)-, -C(=S)-S-, - S-C(=S)-, -C(=O)-S-, -S-C(=O)-, -O-S(=O)2-O-, -S(=O)2-O-, -O-S( =O)2-, -C(=O)-NRio-, -NRio-C(=O)-, -O-P(=o) (-ORio)0-, -P(=O) (-ORio)- O-, -O-P(=O) (-ORio)-;

X independently represents -R11-; And

O -CHSN ₂

E is independently selected from the group consisting of -N(R ₁₂ ) ₂ , -N(R ₁₂ )(R ₁₃ ), -SR ₁₂ and ' where -R ₁₂ is -CH2CH2-G, wherein each G is independently - Cl, -Br, -I, -OS(=O)2-CH ₃ , -0-S (=O)2-CH2-C6H5, or -0-S (=O)2-C6H4-CH3; and where R ₁₃ independently represents -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 ₁₃ -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 thereof (including enantiomers and diastereomers).

In some embodiments of Formula IV, R1, _R2 , _R3 , R4, _R5 , _R6 , R7, and _R8 are each hydrogen. In other embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of R1, R2, _R3 , R4, _R5 , R6, R7, _R8 represent hydrogen.

In some embodiments of Formula IV, R ₉ is -VWXE. In some embodiments, V is -NH-R11-. In some such embodiments, R11 is -C _1-8 -alkyl-, such as methyl or ethyl. In specific embodiments, V is -NH-R11-, wherein R11 is linear -C _1-8 -alkyl-. In some embodiments, V is -N(CH ₃ )-R ₁₁ -. In some such embodiments, R11 is -C _1-8 -alkyl-, such as methyl or ethyl. In specific embodiments, V is -N(CH ₃ )-R ₁₁ -, where R11 is linear -C _1-8 -alkyl-.

In some embodiments of Formula IV, W is -C(=O)-O-. In some embodiments, W is -C(=O)-NR _1o -. In some embodiments, W is -C(=O)-NH-.

In some embodiments of Formula IV, X is -R11-, where -R11- is -C _1-8 -alkyl. In some embodiments, X is methyl. In some embodiments, X is ethyl. In some embodiments, X is linear -C _1-8 -alkyl-.

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 functional moieties of G are -Cl.

In some embodiments of Formula IV, R ₉ is -NH-C ₁ . ₈ -αalkyl-C(=O)OC ₁ . ₈ -alkyl-N(CH ₂ CH ₂ Cl) ₂ . In some embodiments, R ₉ is -NH-(CH ₂ )C(=O)-O-(CH2)-N(CH2CH2C1)2, -NH-(CH2)2-C(=O)-O-( CH2)-N(CH2CH2C1)2, -NIROM-G ΟΜΜΟΗ.··N(CH2CH2Cl)2, -NH-(CH2)2-C(=O)-O-(CH2)2-N(CH2CH2C1)2, -NIRON.-GOMROM-NO or

-NH-(CH ₂ )-C(=O)-O-(CH ₂ ) ₃ -N(CH ₂ CH ₂ C1) ₂ . In some such embodiments, R1, R2, _R3 , _R4 , _R5 , R6, R7, and _R8 are each hydrogen.

In some embodiments of Formula IV, R ₉ is -NH-C _1-8 -αalkyl-C(=O)NH-C ₁ . ₈ -alkyl-N(CH ₂ CH ₂ Cl) ₂ . In some embodiments, R ₉ is -NH-(CH ₂ )C(=O)NH-(CH2)-N(CH2CH2C1)2, -NH-(CH2)2-C(=O)-NH-(CH2 )-N(CH2CH2C1)2, -NH-(CH2)-C(=O)-NH(CH2)2-N(CH2CH2Cl)2, -NH-(CH2)2-C(=O)-NH-( CH2)2-N(CH2CH2C1)2, -MH-(CH2)3-C(=O)-MH-(CH2)N(CH ₂ CH ₂ Cl) ₂ or -NH-(CH ₂ )-C(= O)-NH-(CH ₂ ) ₃ -N(CH ₂ CH ₂ C1) ₂ . In some such embodiments, R1, R2, _R3 , R4, _R5 , _R6 , R7, and _R8 are each hydrogen.

Formula V is:

(V) where R1, R2, _R3 , _R4 , _R5 , _R6 , R7 and _R8 are independently selected from the group consisting of -H, _-R1o , -O-Rw, -NO2, -NH2, - NH-Rio, N(Rio)2, -F, -Cl, -Br, -I, -C(=0)-Rio, -C(=0)-0-Rio and -0-C(=0) -Rio, where -R ₁₀ independently represents 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 ₁₃ -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, -C1. 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;

R ₂₀ represents -H or -CH ₃ ; And

R ₂₁ is -R11-WXE, where -R _n - independently represents -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-arylC _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-ary-C _1-3 -heteroalkyl-, -C _1-3 alkyl-heteroaryl-C ₁ _ ₃ -heteroalkyl- or - C 1 _ ₃ -heteroalkyl-heteroaryl-C 1 _-3 -heteroalkyl-;

W independently represents -C(=O)-O-, -O-C(=O)-, C(=S)-O-, -O-C(=S)-, -C(=S)-S- , -S-C(=S)-, -C(=O)-S-, -S-C(=O)-, -O-S(=O)2-O-, -S(=O)2-O-, - O-S(=O)2-, -C(=O)-NR10-, -NR10-C(=O)-, -0-P(=0)(-0Rio)О-, -P(=O) ( -ORio)-O-, -O-P(=O) (-ORio)-;

X independently represents -R11-; And

O -CHSN ₂

E is independently selected from the group consisting of -N(R ₁₂ ) ₂ , N(R ₁₂ )(R ₁₃ ), -SR ₁₂ and/where -R ₁₂ is -CH2CH2-G, wherein each G is independently -Cl , -Br, -I, -OS(=O)2-CH ₃ , -OS(=O)2-CH2-C6H5 or -OS^b-CH-CHj;

and where R ₁₃ independently represents -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-

- 12 044190

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 thereof (including enantiomers and diastereomers).

In some embodiments of Formula V, R1, R2, _R3 , _R4 , R5, _R6 , _R7 , and R8 are each hydrogen. In other embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of R1, _R2 , _R3 , R4, R5 , R6, _R7 , _R8 represent hydrogen. In some embodiments of Formula V, R20 is H. In some embodiments, R20 is -CH ₃ .

In some embodiments of the formula, VR ₂₁ is -R11-WXE. In some embodiments, R11 is -C _1-8 -alkyl-, such as methyl or ethyl. In some embodiments, R11 is linear -C _1-8 -alkyl-. In some embodiments, R11 is -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, (CH ₂ ) ₅ -, -(CH ₂ ) ₆ -, -(CH ₂ ) ₇ - or ^-(CH 2)8 ^- .

In some embodiments of the formula, VW is -C(=O)-O-. In some embodiments, W is -C(=0)-NR ₁₀ -. In some embodiments, W is -C(=O)-NH-.

In some embodiments of formula VX is -R11-, where -R11- is -C _1-8 -alkyl-. In some embodiments, X is methyl or ethyl. In some embodiments, X is linear -C _1-8 -alkyl-. In some embodiments, X is -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, (CH ₂ ) ₅ -, -(CH ₂ ) ₆ -, -(CH ₂ ) ₇ or -(CH ₂ ) ₈ -. In some such embodiments, R ₂₀ is H. In some such embodiments, R ₂₀ is -CH ₃ .

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

In some embodiments of formula VR ₂₁ is -C _1-8 alkyl-C(=O)-OC _1-8 alkyl-N(CH ₂ CH ₂ Cl) ₂ . In some embodiments, R ₂₁ is -(CH ₂ )-C(=O)O(CH ₂ )-N(CH ₂ CH ₂ C1) ₂ , -(CH ₂ ) ₂ -C(=O)O-( CH ₂ )-N(CH ₂ CH ₂ C1) ₂ , -(CH ₂ )-C(=O)-O-(CH ₂ ) ₂ -N(CH ₂ CH ₂ C1) ₂ , -(CH2)2- C(=O)-O-(CH2)2-N(CH2CH2C1)2, -(CH2)3-C(=O)-O-(CH2ZHCH2CH2C1)2 or -< _CH2 )-C(=O)- O-(CH ₂ )3N(CH ₂ CH ₂ Cl) ₂ . In some such embodiments, R ₂₀ is H. In some such embodiments, R ₂₀ is -CH ₃ . In any of the above embodiments, each of R1, _R2 , _R3 , R4, R5, R6, _R7 and _R8 may be hydrogen.

In some embodiments of Formula V, R21 is -C _1-8 alkyl-C(=O)-NH-C _1-8 alkyl-N(CH ₂ CH ₂ Cl) ₂ . In some embodiments, R ₂₁ is -(CH2)-C(=O)-NH-(CH2)-N(CH2CH2C1)2, -(CH2)2-C(=O)-NH-(CH2)-N (CH2CH2C1)2, -(CH ₂ )-C(=O)-NH(CH2)2-N(CH2CH2Cl)2, -(CH2)2-C(=O)-NH-(CH2)2-N( CH2CH2C1)2, -(ch2)3-c(=0)-nh-(ch2)-n(ch2CH2C1)2 or

-(CH ₂ )-C(=O)-NH-(CH ₂ ) ₃ -N(CH ₂ CH ₂ C1) ₂ . In some such embodiments, R ₂₀ is H. In some such embodiments, R ₂₀ is -CH ₃ . In any of the above embodiments, each of R1, _R2 , _R3 , _R4 , R5, _R6 , _R7 and _R8 may be hydrogen.

Formula VI is:

R _B (VI) where at least one of R ₄₄ , R ₅₅ , R ₃ , R ₄ . R5 and _R8 are -VWXE, and the others of _R44 , _R55 , _R3 , _R4 , R5 and _R8 are independently selected from the group consisting of -H, _-R1o , _-OR10 , _-NO2 . -NH ₂ , -NHR10, -N(Rio)2, -F, -Cl, -Br, -I, -C(=O)-R10, -C(=O)-OR _W and -OC(=O )-R _w where -R ₁₀ independently represents 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 ₁₃ -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 - 13 044190 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;

V is independently -R11-, -NH-R11- or -N(CH ₃ )-R _n -, where -R _n - is independently -C ₁ . ₈ -alkyl-, -C ₁ . ₈ -heteroalkyl-, -aryl-, -heteroaryl-, -C1. 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-arylC _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 independently represents -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-, -NR10-C(=O)-, -OP(=O)(-OR _w )O-, -P(=O)(-OR10) -O-, -OP(=O)(-OR10)-;

X independently represents -R11-; And

O -CH CH ₂

E is independently selected from the group consisting of -N(R ₁₂ ) ₂ , N(R ₁₂ )(R _i3 ), -SR ₁₂ and ' 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;

and where R ₁₃ independently represents -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 ₁₃ -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 thereof (including enantiomers and diastereomers).

It will be understood that in Formula IV above, the acridine core is the anchor functional moiety, the -V-W-X- groups contain a cleavable linker, and the E groups are effector groups. Likewise, in Formula VI above, the psoralen core is the anchor functional moiety, the -V-W-X- groups contain a cleavable linker, and the E groups are the effector groups. Formula V is a subtype of formula IV.

In some embodiments, the readily cleavable compound of formula IV is a compound of formula IV(a)-IV(h) or a salt or stereoisomer thereof (including enantiomers and diastereomers), wherein all group variables not specifically listed are the same as those above formula IV:

Formula IV(a): W independently represents -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-, -NR10-C(=O)-, -oP(=O)(-OR10)-O-, -P(=O)(-OR10)-O- , -0-P(=0)(-ORio) Formula IV(b): V is independently -R11-, or -N(CH ₃ )-R11-, where -R11- is independently -C ₁ . ₈ -αlkyl-, -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-, heteroarylC _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 -heteroalkylheteroaryl-C _1-3 -alkyl-, -C _1-3 -heteroalkyl-aryl-C _1-3 -heteroalkyl -, -C _1-3 -alkyl-heteroaryl-C _1-3 -heteroal kyl- or -C _1-3 -heteroalkyl-heteroaryl-C _1-3 -heteroalkyl-;

Formula IV(c): -R11- independently represents -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-;

Formula IV (d): -R ₁₂ is -CH2CH2-G, where each G is independently -Br, -I, -OS(=O)2-CH3, OS(=O)2-CH2-C6H ₅ or -OS(=O)2-C6H4-CH3;

O -CH-tH ₂

Formula IV(e): E is independently selected from the group consisting of -N(R ₁₂ )(R ₁₃ ), -SR ₁₂ and ' where -R ₁₂ is -CH2CH2-G, wherein each G is independently -Cl, -Br, -I, -OS(=O) ₂ -CH ₃ , -OS(=O) ₂ -CH ₂ -C ₆ H ₅ or -OS(=O) ₂ -C ₆ H ₄ -CH ₃ ; and where R ₁₃ independently represents -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- 14 044190 heteroaryl-C _{1- 3 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 -heteroalkylheteroaryl-C _1-3 -heteroalkyl;

Formula IV(f): W independently represents -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-, -NR1o-C(=O)-, -OP(=O)(-OR _w )O-, -P(=O)(-OR10)-O-, -OP(=O)( -OR10)-;

Formula IV(g): -R ₁₂ is -CH ₂ CH ₂ -G, where each G is independently -OS(=O)2-CH ₃ , -OS(=O)2-CH2-C6H ₅ or - OS (=O)2-C6H4-CH3;

Formula IV(h): at least one of R1, _R2 , _R3 , R4, _R5 , R6, R7, _R8 and _R9 is -V-WX-E as defined for Formula IV, at least At least one of R1, _R2 , _R3 , R4, _R5 , _R6 , R7, _R8 and _R9 is selected from the group consisting of _-R10 , _-OR10 , _-NO2 , _-NH2 , -NH -R ₁₀ , -N(R ₁₀ ) ₂ , -F, -Cl, -Br, -I, -C(=O)-R ₁₀ , -C(=O)-OR ₁₀ and -OC(=O) -Ri ₀ , and the rest of R1, R ₂ , R ₃ , R4, R ₅ , R ₆ , R7, R ₈ and R ₉ are independently selected from the group consisting of -H, -R ₁₀ , -OR ₁₀ , -NO ₂ , -NH ₂ , -NH-R ₁₀ , -N(R ₁₀ ) ₂ , -F, -Cl, -Br, -I, -C(=O)-R ₁₀ , -C(=O)-OR ₁₀ and -OC(=O)-R ₁₀ , where R ₁₀ is as defined for formula IV.

In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a refractory compound, such as a refractory analogue of a compound of any of formulas IV, V, or VI. Such a refractory compound may be any of the following formulas VII, VIII or IX. Formula VII is:

(VII) 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 rest of R1, _R2 , _R3 , _R4 , _R5 , _R6 , R7, Rs and _R9 are independently selected from the group consisting of -H, _-R10 , _-OR10 , _-NO2 , -NH ₂ , -NH-R ₁₀ , -N(R ₁₀ ) ₂ , -F, -Cl, -Br, -I, -C(=O)-R _W , -C(=O)-O- R10 and -OC(=O)-R _W , where -R ₁₀ independently represents 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 ₁₃ -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 thereof (including enantiomers and diastereomers).

In some embodiments of Formula VII, R1, _R2 , _R3 , R4, _R5 , _R6 , R7, and _R8 are each hydrogen. In other embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of R1, _R2 , _R3 , R4, R ₅ , R6, R7, _R8 represent hydrogen.

In some embodiments of Formula VII, R ₉ is -VXE, wherein V, X, and E are as defined for Formula IV. In some embodiments, V is -NH-R11-. In some such embodiments, R11 is -C _1-8 -alkyl-, such as methyl or ethyl. In specific embodiments, V is -NH-R11-, wherein R11 is linear -C _1-8 alkyl-. In some embodiments, V is N(CH ₃ )-R ₁₁ -. In some such embodiments, R11 is -C _1-8 -alkyl-, such as methyl or ethyl. In specific embodiments, V is -N(CH ₃ )-R ₁₁ -, where R11 is linear -C _1-8 -alkyl-.

In some embodiments of Formula VII, X is -R11-, where -R11- is -C _1-8 -alkyl-. In some embodiments, the functional moiety -VX- is -NH-C _1-8 -alkyl-. In some embodiments, the functional moiety -V-X is -NH-(CH ₂ )-, -NH-(CH ₂ ) ₂ -, -NH-(CH ₂ ) ₃ -, -NH-(CH ₂ ) 4-, -NH-(CH ₂ ) ₅ -, -NH-(CH ₂ ) ₆ -, -NH-(CH ₂ ) ₇ - or -NH-(CH ₂ ) ₈ -. In some embodiments, the functional moiety -V-X is -N(CH ₃ )-C _1-8 -alkyl-. In some embodiments, the functional moiety -VX- is -N(CH ₃ )-(CH ₂ )-, -N(CH ₃ )-(CH ₂ ) ₂ -, -N(CH ₃ )-(CH ₂ ) ₃ -, -N(CH ₃ )-(CH ₂ ) ₄ -, ^-N(CH 3 ^)-(CH 2 ⁾ 5 ^- , -^(6^3)-(6^2)6-, ^-N(CH 3 ^)-(C H2 ⁾ 7 ^{- or} -^6^3)-(6^2)8 ^- .

- 15 044190

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

In some embodiments of Formula VII, R ₉ is -NH-C ₁ . ₈ -alkylN(CH2CH2Cl)2. In some embodiments, R ₉ is -NH-(CH ₂ )-N(CH2CH ₂ Cl)2, -NH-(CH ₂ ) ₂ -N(CH ₂ CH ₂ Cl) ₂ , -NH-(CH ₂ ) ₃ -N (CH ₂ CH ₂ Cl) ₂ , -NH-(CH ₂ ) ₄ -N(CH ₂ CH ₂ Cl) ₂ , -NH-(CH ₂ ) ₅ N(CH ₂ CH ₂ Cl) ₂ , - NH-(CH ₂ ) ₆ -N(CH ₂ CH ₂ Cl) ₂ , -NH-(CH ₂ )7-N(CH ₂ CH ₂ Cl) ₂ or -NH-(CH ₂ ) ₈ -N(CH ₂ CH ₂ Cl) ₂ . In some such embodiments, R1, R2, _R3 , R4, _R5 , R6, R7, and _R8 are each hydrogen.

Formula VIII is:

(VIII) where R1, R2, _R3 , R4, _R5 , _R6 , _R7 and _R8 are independently selected from the group consisting of -H, _-R10 , _-OR10 , -NO2, -NH2, -NH -R10, N(R _w )2, -F, -Cl, -Br, -I, -C(=O)-Rio, -C(=O)-O-Rio and -OC(=O)-R _W , where _{-R 10} independently represents 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;

R ₂₀ represents -H or -CH ₃ ; And

R ₂₁ represents -R ₁₁ -XE, where R ₁₁ , X and E are as defined for formula V; or a salt or stereoisomer thereof (including enantiomers and diastereomers).

In some embodiments of Formula VIII, _R1 , R2, _R3 , R4, _R5 , _R6 , R7, and _R8 are each hydrogen. In other embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of _R1 , _R2 , _R3 , R4, _R5 , _R6 , R7, _R8 represent hydrogen.

In some embodiments of Formula VIII, R ₂₀ is H. In some embodiments, R ₂₀ is -CH ₃ .

In some embodiments of Formula VIII, R ₂₁ is -R ₁₁ -XE, where R ₁₁ , X and E are as defined for Formula V. In some embodiments, R ₁₁ is -C _1-8 -alkyl-, for example , methyl or ethyl. In some embodiments, R _n is linear -C _1-8 -alkyl-. In some embodiments, Rn is (CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ )3-, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -, -(CH ₂ ) 6-, -(CH ₂ ) ₇ - or - (CH ₂ )8-.

In some embodiments of Formula VIII, X is -R ₁₁ -, where -R ₁₁ - is -C _1-8 -alkyl-. In some embodiments, the functional moiety -R ₁₁ -X is -C _1-8 -alkyl-. In some such embodiments, R ₂₀ is H. In some such embodiments, R ₂₀ is -CH ₃ . In some embodiments, the functional moiety -R ₁₁ -X- is -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -, -(CH ₂ ) ₆ -, -(CH ₂ ) ₇ - or -(CH ₂ ) ₈ -. In some such embodiments, R ₂₀ is H. In some such embodiments, R ₂₀ is -CH ₃ .

In some embodiments of Formula VIII, E is -N(R ₁₂ ) ₂ , wherein R ₁₂ is -CH ₂ CH ₂ -G. In some such embodiments, each G is independently -Cl, -Br, or -I. In some such embodiments, both functional moieties of G are -Cl.

In some embodiments of Formula VIII, R21 is -C1-8-alkylN(CH ₂ CH ₂ Cl) ₂ . In some embodiments, R ₂₁ is - (CH ₂ )-N(CH ₂ CH ₂ Cl) ₂ , -(CH2)2-N(CH2CH2Cl)2, -(CH2)3-N(CH2CH2Cl)2, -( CH2)4-N(CH2CH2Cl)2, -(CH2)5-N(CH2CH2Cl)2, -(CH2)6N(CH ₂ CH ₂ Cl) ₂ , -(CH ₂ ) ₇ -N(CH ₂ CH ₂ Cl ) ₂ or -(CH ₂ ) ₈ -N(CH ₂ CH ₂ Cl) ₂ . In some such embodiments, R ₂₀ is H. In some such embodiments, R ₂₀ is - 16 044190

CH ₃ . In any of the above embodiments, each of R1, R2, _R3 , R4, _R5 , _R6 , _R7 and R8 may be hydrogen.

Formula IX is:

wherein at least one of R4 ₄ , R ₅₅ , R ₃ , R4, R ₅ and R8 is -VXE, where V, X and E are as defined for formula VI;

and the rest of R44, _R55 , _R3 , R4, _R5 and R8 are independently selected from the group consisting of -H, _-R10 , -OR10, -NO2, -NH2, -NH-R10, N( _Rw ) 2, -F, -Cl, -Br, -I, -C(=O)-R _w , -C(=O)-OR _w and -OC(=O)-R _w , where -R _w independently represents is H, -C ₁ _ ₈ -alkyl, -C ₁ _ ₈ -heteroalkyl, -aryl, -heteroaryl, -C ₁ _ ₃ -alkyl-aryl, -C1. 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, -C1- 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, -C1- 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 thereof (including enantiomers and diastereomers).

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

In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is S-303. In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a derivative of S-303. In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a refractory analogue of S-303 or a derivative thereof.

In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a compound selected from the following compounds, or a salt or stereoisomer (including enantiomers and diastereomers) thereof:

- 17 044190

- 18 044190

- 19 044190

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

In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a derivative (eg, a hydrolyzed derivative) of any of the above compounds. In some embodiments, the compound treated with a substrate provided herein or the resulting functional moiety bound to the surface of a substrate provided herein is a refractory analogue of any of the above compounds.

A derivative of a compound provided herein may include a nucleic acid functional moiety, a portion of a nucleic acid functional moiety, an electrophilic functional moiety, or a portion of an electrophilic functional moiety. A derivative of a compound provided herein may include a) a nucleic acid functional fragment or a portion of a nucleic acid functional fragment, and b) an electrophilic functional fragment or a portion of an electrophilic functional fragment. A derivative of a compound provided herein may include only a nucleic acid-acting functional fragment or a portion of a nucleic acid-acting functional fragment. A derivative of a compound provided herein may include only an electrophilic functional moiety or a portion of an electrophilic functional moiety. If the compound is difficult to cleave, its derivative may include a) an affecting

- 20 044190 on nucleic acids, a functional fragment or part of a functional fragment affecting nucleic acids, and b) an electrophilic functional fragment or part of an electrophilic functional fragment, where a) and b) are linked to each other by means of a non-hydrolysable linker. If the compound is readily cleavable, its derivative may comprise a) a nucleic acid functional moiety or part of a nucleic acid functional moiety and b) an electrophilic functional moiety or part of an electrophilic functional moiety, wherein a) and b) are linked to each other by a hydrolyzable linker . If the compound is readily cleavable, its derivative may comprise a) a nucleic acid-acting functional moiety or a portion of a nucleic acid-acting functional moiety linked to a functional moiety that is the hydrolysis product of a hydrolyzable linker. If the compound is readily cleavable, its derivative may include an electrophilic functional moiety or a portion of an electrophilic functional moiety linked to a functional moiety that is the hydrolysis product of the hydrolyzable linker. Functional hydrolysis product moieties include, but are not limited to, alcohols, amines, carboxylic acids, esters, and salts or stereoisomers of any of the above compounds.

Any of the compounds or derivatives provided herein can be used as starting material for reaction with a substrate to produce any of the compositions provided herein. Alternatively, any of the compounds or derivatives provided herein may be associated with a substrate in any of the compositions provided herein. Any derivative provided herein may be prepared from the compound of which it is a derivative, or an equivalent molecular structure may be obtained (eg, synthesized) independently of the compound of which it is a derivative.

It should be understood that said substrate unit (eg, one red blood cell, one polystyrene bead) will contain one or more surface-bound functional moieties. The surface-bound functional moieties may be homogeneous relative to the specified substrate unit. Alternatively, the surface-associated functional moieties may be heterogeneous relative to the specified substrate unit (eg, red blood cell). Heterogeneity relative to a specified substrate unit may result from the reaction of a substrate unit with a heterogeneous set of compounds or derivatives. Heterogeneity with respect to a specified substrate unit can also result from the reaction of a substrate unit with a homogeneous compound or derivative, where the reaction of the compound or derivative with the substrate results in the formation of two or more different reaction products associated with the surface of the substrate. In some cases, surface-associated functional moieties may be homogeneous with respect to any particular substrate unit, but heterogeneous with respect to a group of substrate units (eg, 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 produce a heterogeneous group of surface-bound substrate units (eg, red blood cells).

In any of the embodiments presented herein, the surface-bound functional moiety may be present on the surface of the substrate in an amount also referred to as the substrate loading level. The loading level can be described on a per unit basis, such as per cell (eg, red blood cell), per bead, or per particle (eg, functional fragments per cell, functional fragments per bead, functional fragments per particle). In some embodiments, the substrate loading level may be at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 8000 , 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, according to at least about 40,000 or at least about 50,000 functional fragments per unit substrate (eg, per red blood cell). In some embodiments, the substrate loading level may be no more than about 50,000, about 75,000, or about 100,000 functional fragments per unit of substrate (eg, per red blood cell). In some embodiments, the substrate loading level may be from about 1,000 to about 100,000, from about 1,000 to about 75,000, from about 1,000 to about 50,000, from about 1,000 to about 25,000, from about 5,000 to about 50,000, from about 5,000 to about 25,000 , from about 5,000 to about 15,000, from about 8,000 to about 15,000, from about 25,000 to about 50,000, from about 25,000 to about 40,000, from about 25,000 to about 35,000, from about 25,000 to about 30,000, from about 30,000 to about 40,000, or from about 30,000 to about 35,000

- 21 044190 functional fragments per unit of substrate (for example, per erythrocyte). In some embodiments, the substrate loading level may be about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 13000, about 14000 , approximately 15000, approximately 16000, approximately 17000, approximately 18000, approximately 20000, approximately 22000, approximately 24000, approximately 26000, approximately 28000, approximately 30000, approximately 32000, approximately 35000, approximately 40000, approximately 45000, approximately 5000 0, approximately 60000 , about 70,000, about 80,000, about 90,000, or about 100,000 functional fragments per unit substrate (eg, per red blood cell).

The loading level can be described in terms of per unit area, for example, per unit surface area (eg, functional fragments per µm ² surface area of the substrate). In some embodiments, the substrate loading level may be at least about 5, at least about 7, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30 , at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, according to at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 functional fragments/μm ² . In some embodiments, the substrate loading level may be no more than about 500, about 750, or about 1000 functional fragments/μm ² . In some embodiments, the substrate loading level may be from about 5 to about 1000, from about 5 to about 750, from about 5 to about 500, from about 20 to about 500, from about 20 to about 400, from about 20 to about 200 , from about 20 to about 100, from about 30 to about 200, from about 30 to about 100, from about 50 to about 100, from about 100 to about 500, from about 100 to about 400, from about 140 to about 400, from about 140 to about 300, from about 140 to about 250, or from about 180 to about 300 functional fragments/μm ² . In some embodiments, the substrate loading level may be about 5, about 7, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65 , approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, approximately 500, approximately 600, approximately 700, approximately 800, approximately 900, or approximately 1000 functional fragments/cm ² .

The loading level may be substantially homogeneous with respect to a group of substrate units or may be heterogeneous with respect to a group of substrate units. The loading levels presented herein may represent a weighted average loading level relative to a group of substrate units. The loading level can be measured by any method known in the art, for example, by labeling (eg, reactive labeling, immunolabeling, chemical labeling, chemiluminescent labeling, fluorescent labeling) of the compound before addition to the substrate and then detecting the label after combining the compound with the substrate. Alternatively or additionally, the loading level can be measured by direct or indirect labeling (eg, immunolabeling, immunofluorescent labeling, chemiluminescent labeling) of the compound after addition to the substrate and then detecting the label.

Typically, such labeling is performed separately (eg, as a control, as a standard curve) from the compound used in the kits and methods presented herein, as a comparator or reference in determining absolute loading levels or relative loading levels. Such labeling can be directly quantified using a quantitative or semi-quantitative method, or can be compared to a standard such as

- 22 044190 for example, cells, beads or other materials with a known amount of a similarly labeled molecule on the surface (for example, FACS analysis).

In some embodiments, the composition (eg, the substrate associated with the functional moiety) is stable at room temperature. In some embodiments, the composition is stable at refrigeration temperatures (eg, 2-8°C). In some embodiments, the composition is stable at temperatures of 0°C and/or less than 0°C (eg, -20°C). In some embodiments, the composition is stable at -80°C. The composition may be suitable for storage 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 more at room temperature. The composition may be suitable for storage 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 more at refrigeration temperature.

The composition may be suitable for storage 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, approximately 4 weeks, approximately 5 weeks, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, or approximately 1 year or more at 0°C and/or less than 0°C. The composition may be suitable for storage 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 at -80°C. The composition can be stored in the presence of a liquid, buffer or any suitable solution (eg, a chemically inactive solution). For example, if the substrate is red blood cells, the composition can be stored in a red blood cell supplement solution, blood bank saline, buffered suspension medium, or other suitable solution. Red blood cell additive solutions and buffered suspension media are known in the art. If the substrate is red blood cells, the compositions can be stored as a suspension of about 0.3% to about 10% red blood cells, about 0.5% to about 5% red blood cells, about 0.5% to about 1% red blood cells, from about 1% to about 1.5% of red blood cells, from about 1.5% to about 2% of red blood cells, from about 2% to about 2.5% of red blood cells, from about 2.5% to about 3% of red blood cells, from about 3% up to about 3.5% of erythrocytes, from about 3.5% to about 4% of erythrocytes, from about 4% to about 4.5% of erythrocytes, from about 4.5% to about 5% of erythrocytes 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% red blood cells. If the composition will be frozen for storage, a cryopreservative may also be added, such as, for example, any cryopreservative known in the art.

The stability of the compositions (eg, functional moiety-bound substrates) provided herein and their suitability for storage may depend on the nature of the surface-bound functional moiety. For example, a surface-bound functional moiety that lacks a hydrolyzable linker (e.g., a refractory compound or derivative thereof, or a hydrolysis product of an easily cleavable compound) may be more stable than a surface-bound functional moiety containing a hydrolyzable linker (e.g., a highly cleavable compound or its derivative). In some embodiments, a composition provided herein containing a surface-bound functional moiety that lacks a hydrolyzable linker is stable at room temperature. In some embodiments, the composition is stable at refrigeration temperatures. In some embodiments, the composition is stable at temperatures of 0°C and/or less than 0°C. In some embodiments, the composition is stable at -80°C. The composition may be suitable for storage 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 more at room temperature. The composition may be suitable for storage 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 more at refrigeration temperature. The composition may be suitable for storage 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 at a temperature of 0°C and/or less than 0°C. The composition may be suitable for storage 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 at -80°C. In some embodiments, a composition provided herein containing a surface-bound functional moiety containing a hydrolyzable linker is stable at room temperature. In some embodiments, the composition is stable at refrigeration temperatures. In some embodiments, the composition is stable at temperatures of 0°C and/or less than 0°C. In some embodiments, the composition is stable at -80°C. The composition may be suitable for storage 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 more at room temperature. The composition may be suitable for storage 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 more at refrigeration temperature. The composition may be suitable for storage 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, approximately 4 weeks, approximately 5 weeks, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, or approximately 1 year or more at 0°C and/or less than 0°C. The composition may be suitable for storage 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 at -80°C.

Sets.

The present invention relates to various kits containing any of the compositions (eg, substrates containing surface-associated functional moieties) presented herein. In some embodiments, the kit contains instructions that describe the use of substrates and/or substrates containing surface-associated functional moieties to perform any of the methods presented herein. The kits may also contain any combination of additional components that may be necessary or useful to carry out any of the methods presented herein. Although the present description provides examples of kits, the contents of other kits that can be used will be apparent to those skilled in the art in light of the present description.

The present invention relates to a kit for detecting the presence of an antibody in a biological sample by the methods presented in the present description. In some embodiments, the kit includes a first container containing a first substrate, wherein a functional moiety is associated with a surface of the first substrate, and wherein the functional moiety is selected from compounds and derivatives thereof provided herein; and a second container containing a second substrate, wherein there is no associated functional moiety on the surface of the second substrate. The functional moiety associated with the surface of the first substrate may be any compound or derivative provided herein, such as a pathogen-inactivating compound or derivative thereof. In some embodiments, the functional moiety associated with the surface of the first substrate is S-303 or a derivative thereof. In some embodiments, the functional moiety associated with the surface of the first substrate is difficult to cleave

- 24 044190 splicable analogue of S-303 or its derivative. In some embodiments, the functional moiety associated with the surface of the first substrate is selected from the group consisting of compounds of formula I, II, III, IV, IV(a)-IV(h), V, VI, VII, VIII and XI and thereof derivatives and salts and solvates of any of the above compounds. In some embodiments, the functional moiety associated with the surface of the first substrate is selected from the group consisting of compounds of formula I, II, III, VII, VIII and XI and their derivatives and salts and solvates of any of the above compounds. In some embodiments, the functional moiety associated with the surface of the first substrate is selected from the group consisting of compounds of formula IV, V and VI and their derivatives and salts and solvates of any of the above compounds. In some embodiments, the functional moiety associated with the surface of the first substrate is selected from the group consisting of compounds of formula IV(a)-IV(h) and derivatives thereof and salts and solvates of any of the above compounds.

The first and second substrates may consist of any suitable substrate material, such as any substrate material provided herein. The first and second substrates may consist of cells (eg, red blood cells). Alternatively, the first and second substrates may consist of cell membranes or cell-derived material. For example, the first and second substrates may comprise membrane preparations derived from red blood cells, such as red blood cell ghosts (ie, lysed red blood cell membranes). Alternatively, the first and second substrates may consist of a solid support. As provided herein, the solid support may consist of a material (e.g., biological, synthetic) or matrix including, but not limited to, polymers or copolymers (e.g., polyesters, polyethers, polyolefins, polyamides, polysaccharides, polyurethanes, styrene and styrene derivatives, polystyrenes, celluloses), plastic, glass, gold and other metals, etc. The solid support may expose the surface-associated functional moiety in a two-dimensional format (eg, plate, multiwell plate) or three-dimensional format (eg, cell, bead, or other spherical or quasi-spherical object). The substrate may be part of an assay plate. Particular solid supports are composed of one or more materials to which the biological sample will not or substantially does not bind. For example, the solid support may consist of a material to which antibodies (eg, antibodies specific for a compound or portions thereof) do not bind or substantially do not bind. In particular, the solid support consists of a material to which antibodies against pathogen-inactivated red blood cells do not bind or substantially do not bind. Typically, the first and second substrates will be composed of the same material. In some embodiments, the first and second substrates may comprise red blood cells or membrane preparations derived therefrom immobilized on the surface of a solid support, such as, for example, the above solid support. Red blood cells or membrane preparations can be immobilized on the surface of a solid support by various methods known in the art, for example, using an antibody against a non-blood group antigen expressed on the cell surface or by adhesion using poly-L-lysine, organic dyes or lectins (eg, lectins having specificity for galactosyl functional moieties). Preferably, the reagent used is compatible with the cell surface and can stably maintain cell or membrane binding to the solid support throughout the assay.

In some embodiments, the first and second substrates are red blood cells. Red blood cells from the first and second substrates can be obtained from one or more common donors or different blood donors. In some cases, the red blood cells from the first substrate and the red blood cells from the second substrate share one or more properties, such as ABO blood type (eg, blood type O), Rh type, or the presence or absence of other red blood cell antigens (eg, common red blood cell antigens, clinically significant erythrocyte antigens). In other cases, the red blood cells from the first substrate and the red blood cells from the second substrate differ in specific properties, such as ABO blood type or the presence or absence of other red blood cell antigens (eg, common red blood cell antigens, clinically relevant red blood cell antigens). In some embodiments, the first and second substrates have one or more antigens typically found in a biological sample (eg, whole blood, red blood cells, serum, or plasma). The presence of such an antigen can serve as a control indicating that the biological sample is appropriately in contact with the first and/or second substrate.

The loading level of the surface-bound functional moiety on the first substrate (eg, red blood cells) may be any loading level provided herein, such as, for example, a loading level sufficient to detect antibodies against the compound or functional moieties thereof. In some embodiments, the first substrate loading level may be at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 8000, at least about 10000, at least about 12000, at least about 14000, at least about 17000, at least about 20000, according to

- 25 044190 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 functional fragments per unit substrate (eg, per red blood cell). In some embodiments, the loading level of the first substrate may be no more than about 50,000, about 75,000, or about 100,000 functional fragments per unit of substrate (eg, per red blood cell). In some embodiments, the first substrate loading level may be from about 1,000 to about 100,000, from about 1,000 to about 75,000, from about 1,000 to about 50,000, from about 1,000 to about 25,000, from about 5,000 to about 50,000, from about 5,000 to about 25,000, from about 5,000 to about 15,000, from about 8,000 to about 15,000, from about 25,000 to about 50,000, from about 25,000 to about 40,000, from about 25,000 to about 35,000, from about 25,000 to about 30,000, from about 30,000 to about 4,000 0 or from about 30,000 to about 35,000 functional fragments per unit substrate (eg, per red blood cell). In some embodiments, the first substrate load level may be about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 13000, about 14000, approximately 15000, approximately 16000, approximately 17000, approximately 18000, approximately 20000, approximately 22000, approximately 24000, approximately 26000, approximately 28000, approximately 30000, approximately 32000, approximately 35000, approximately 40000, approximately 45000, approximately 50000, approx. 60,000, approximately 70,000, approximately 80,000, approximately 90,000, or approximately 100,000 functional fragments per unit substrate (eg, per red blood cell). In some embodiments, the first substrate loading level may be at least about 5, at least about 7, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 225, at least about 250, at least about 275, at least at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 functional fragments/μm ² . In some embodiments, the loading level of the first substrate may be no more than about 500, about 750, or about 1000 functional fragments/μm ² . In some embodiments, the first substrate loading level may be from about 5 to about 1000, from about 5 to about 750, from about 5 to about 500, from about 20 to about 500, from about 20 to about 400, from about 2 to about 200, about 20 to about 100, about 30 to about 200, about 30 to about 100, about 50 to about 100, about 100 to about 500, about 100 to about 400, about 140 to about 400 , from about 140 to about 300, from about 140 to about 250, or from about 180 to about 300 functional fragments/μm ² . In some embodiments, the first substrate loading level may be about 5, about 7, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, approximately 500, approximately 600, approximately 700, approximately 800, approximately 900 , or approximately 1000 functional fragments/cm ² . Typically, the loading level of the first substrate will be sufficient to detect antibodies in a biological sample. In particular, the loading level will be sufficient to distinguish between a biological sample from a patient that has or is expected to have an immune response (eg, pre-existing antibodies) to a pathogen-inactivated red blood cell (eg, red blood cells given by infusion) and a biological specimen from a patient who does not have, or is not expected to have, immune

- 26 044190 response (for example, pre-existing antibodies) to red blood cells with inactivated pathogens (for example, red blood cells administered by infusion).

The kit may further include a third container containing a third substrate, wherein the first level of functional fragment is associated with a surface of the first substrate, the second level of functional fragment is associated with the third substrate, and the second level is smaller than the first level. One skilled in the art will appreciate that the second level of functional moiety may provide an internal reference for detecting an antibody against a surface-bound functional moiety in the first level of functional moiety. Thus, in some embodiments, the relative loading levels of the first and third substrates will be sufficient to distinguish a biological sample from a patient that has or is expected to have an immune response (e.g., pre-existing antibodies) to pathogen-inactivated red blood cells (e.g., red blood cells infused) and a biological sample from a patient who does not have, or is not expected to have, an immune response (eg, pre-existing antibodies) to 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, 1000 times, about 5000 times, or about 10,000 times or more the loading level of the third substrate. In some embodiments, the first substrate loading level is about 2 times to about 500 times, about 2 times to about 100 times, about 2 times to about 50 times, about 2 times to about 20 times, about 2 times to about 20 times 10 times, about 2 times to about 5 times, about 4 times to about 100 times, about 4 times to about 50 times, about 4 times to about 20 times, or about 4 times to about 10 times the load level third substrate. In some embodiments, the first substrate loading level is at least about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 180, about 200, about 225, about 250, about 275, about 300 , about 325, about 350, about 400, about 450, about 500 functional fragments/μm ² , and the third substrate loading level is no more than about 100, about 90, about 80, about 70, about 60, about 50, about 40, approximately 3 0 functional fragments/µm ² . In some embodiments, the first substrate loading level is from about 110 to about 750, from about 110 to about 500, from about 110 to about 400, from about 110 to about 300, from about 140 to about 750, from about 140 to about 500 , from about 140 to about 400, from about 140 to about 300, from about 200 to about 400, from about 200 to about 300 functional fragments/μm ² , and the third substrate loading level is from about 10 to about 100, from about 20 to about 100, from about 30 to about 100, from about 40 to about 100, from about 50 to about 100 functional fragments/μm ² . In some embodiments, the first substrate loading level is about 110, about 120, about 130, about 140, about 150, about 160, about 180, about 200, about 225, about 250, about 275, about 300, about 325, about 350 , about 400, about 450, about 500 functional fragments/μm ² , and the loading level of the third substrate is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 functional fragments/µm ² . In 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 functional fragments per substrate unit (e.g., red blood cell), and the loading level of the third substrate is not more than about 14,000, about 12,000, about 10,000, about 8,000, about 6,000, or about 5,000 functional fragments per unit substrate (eg, red blood cell). In some embodiments, the first substrate load level is from about 15,000 to about 75,000, from about 17,000 to about 75,000, from about

20,000 to approximately 75,000, from approximately 25,000 to approximately 75,000, from approximately

30,000 to approximately 75,000, from approximately 40,000 to approximately 75,000, from approximately

15,000 to approximately 50,000, from approximately 17,000 to approximately 50,000, from approximately

20,000 to approximately 50,000, from approximately 25,000 to approximately 50,000, from approximately

- 27 044190

30,000 to about 50,000, about 40,000 to about 50,000 functional fragments per substrate unit (e.g., red blood cell), and the third substrate loading level is from about 1,000 to about 15,000, from about 3,000 to about 15,000, from about 5,000 to about 15,000, from from about 10,000 to about 15,000, from about 1000 to about 14,000, from about 3,000 to about 14,000, from about 5,000 to about 14,000, from about 7,000 to about 14,000, from about 10,000 to about 14,000, from about 5,000 to about 12,000, from about 5,000 to approximately 10,000 functional fragments per unit substrate (eg, red blood cell). In some embodiments, the first substrate loading level 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, about 75,000 functional fragments per unit substrate (e.g., red blood cell) and the loading level of the third substrate is about 1000, about 3000, about 5000, about 7000, about 10000, about 12000, about 14000 functional fragments per unit substrate (eg, red blood cell). Loading levels as presented herein may include absolute amounts, such as those set forth above, or relative loading levels, such as the loading level of a first substrate relative to a third substrate.

Typically, the third substrate will be composed of the same material as the first and/or second substrates. In some embodiments, the first, second, and third substrates are the same. In the event that there is heterogeneity in the chemical structure or binding pattern of the surface-bound functional moiety on the first and third substrates, the third substrate may have the same or a different distribution of surface-bound functional moieties compared to the first substrate. In some embodiments, the first, second, and third substrates consist of red blood cells, and the red blood cells from these three substrates are obtained from one or more common donors. In some embodiments, the first, second, and third substrates all have one or more antigens typically found in a biological sample (eg, whole blood, red blood cells, serum, or plasma).

In addition to the primary panel presented herein, a secondary panel may be used in some embodiments of the present invention. Red blood cells from the secondary panel can be obtained from one or more common donors. Red blood cells from a secondary panel can be obtained from the same or different donors than red blood cells from a primary panel. The surface-associated functional fragments from the second panel may be the same as or different from the surface-associated functional fragments from the first panel. The surface-associated functional fragments from the secondary panel may be the same as the surface-associated functional fragments from the first panel, and the loading levels for the two panels may be the same or different. The substrates from the first and second panels may have additional antigens, such as one or more antigens typically found in a biological sample (eg, whole blood, red blood cells, serum or plasma). The additional antigens from the first panel may be the same as or different from the additional antigens from the second panel.

The first, second, and optional third containers of the kits provided herein can be made from any material suitable for storing the first, second, and optional third substrates, which may or may not contain surface-associated functional moieties. Containers can be made of glass, plastic or any suitable polymeric material. The containers may be sterile and may further include a medium to maintain the sterility of the contents of the containers. It should be understood that the containers will be suitable for storing their contents at an appropriate temperature (eg, room temperature, refrigeration temperature, freezing temperature, 0°C, -20°C, 80°C).

The kits provided herein may additionally contain additive solutions, buffers, or other solutions that can be used to perform any of the methods presented herein. Additive solutions, buffers, or other solutions can be used to store the substrates (eg, substrates containing surface-bound functional moieties or substrates not containing surface-bound functional moieties) from the kits. Additive solutions, buffers, or other solutions can be used in bringing substrates (eg, substrates containing surface-associated functional moieties or substrates not containing surface-associated functional moieties) from arrays 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 (e.g., substrates containing surface-associated functional moieties or substrates not containing surface-associated functional moieties) from the kit or may be included in the kit apart.

The kits presented herein may additionally contain additional

- 28 044190 components (eg, reagents) for use in imaging or otherwise determining the presence or absence of antibodies associated with surface-bound functional moieties, before or after contact with a biological sample. These may include antiglobulin serum or other agglutination material, as well as any other components that can be used in imaging modalities such as IAT or DAT. In certain embodiments, the kits provided herein may contain materials suitable for use as controls (eg, standards).

The invention also relates to systems containing the above kits, as will be understood by one skilled in the art. For example, the system further includes automated equipment that can be used in the methods presented herein.

Methods of obtaining.

The present invention relates to methods for producing a panel of substrates (eg, polymer particles, red blood cells). In some embodiments, the method includes obtaining a first and a second sample, each containing a substrate, and treating the substrate from the first sample with a compound or derivative provided herein. In some embodiments, the method includes obtaining first and second samples containing polymer particles (eg, beads, microspheres) and treating the polymer particles from the first sample with a compound or derivative provided herein. In some embodiments, the method includes obtaining first and second samples containing red blood cells, and treating the red blood cells from the first sample with a compound or derivative provided herein. In some embodiments, the red blood cells from the first and second samples are obtained from the same blood donor. The treatment step may further include additional reagents, such as an acid, a base, a suitable catalyst, or any other reagent that can facilitate the reaction of the compound or derivative with the substrate. During the processing step, suitable solvents (eg water) or buffers (eg red blood cell additive solutions, blood bank saline) can be used. The processing step may result in covalent or non-covalent binding of the functional moiety to the surface of the substrate (eg, polymer particles, red blood cells) from the first sample. In some embodiments, the resulting surface-bound functional moiety has the same or substantially the same chemical structure as the compound or derivative used to treat the substrate (eg, polymer particles, red blood cells) from the first sample. In some embodiments, the resulting surface-bound functional moiety has a substantially different chemical structure than the compound or derivative used to treat the substrate (eg, polymer particles, red blood cells) from the first sample. For example, the resulting surface-bound functional moiety may be a type of hydrolyzed reaction product. Because the second sample is not treated with the compound and, together with the first sample, constitutes a panel including a first sample of substrate (e.g., polymer particles, red blood cells) with a functional moiety bound to the surface and a second sample of substrate (e.g., polymer particles, red blood cells) in which there is no bound with a functional fragment surface.

In some embodiments, a method for producing a panel of substrates (e.g., polymer particles, red blood cells) may further include one or more steps of reacting a first and a second sample with one or more additional antigens, such as one or more antigens typically found in a biological sample ( e.g. whole blood, red blood cells, serum or plasma). The reaction with one or more additional antigens can be carried out before, during or after treatment with the compound or derivative. In the reaction with one or more additional antigens, solvents and additional reagents may be used as necessary.

The method of producing a panel of substrates (eg, polymer particles, red blood cells) may further include one or more steps of washing the first and second samples after the step of treating the substrate (eg, polymer particles, red blood cells) 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 can be performed before, during or after the washing step. In certain embodiments, the method further includes the step of suspending the first and second samples (eg, polymer particles, red blood cells) in a buffered suspension medium. In certain embodiments, the method further includes the step of adding a cryopreservative to the first and second samples after washing.

In some embodiments, the second sample is processed similarly or identically to the first sample, except that the compound or derivative is eliminated in the processing step. This can be used to optimize the performance of the first and second samples such that the second sample is a suitable control.

The method may further include treating a third sample containing a substrate (eg, polymer particles, red blood cells) with a compound or derivative in a manner similar to the treatment of permeate.

- 29 044190 of the third sample such that treatment with the compound or derivative results in a lower level of functional moiety associated with the surface of the substrate (e.g., polymer particles, red blood cells) from the third sample relative to the level of functional moiety associated with the surface of the substrate (e.g. polymer particles, red blood cells) from the first sample. Thus, the first level of functional fragment is associated with the surface of the substrate (eg, polymer particles, red blood cells) from the first sample, and the second level is smaller than the first level. In some embodiments, this may be achieved by treating the substrate (eg, polymer particles, red blood cells) from the third sample with a different amount of the compound or derivative than the substrate (eg, polymer particles, red blood cells) from the first sample. In some embodiments, this may alternatively be achieved by treating the substrate (eg, polymer particles, red blood cells) from the third sample with the compound or derivative for a shorter period of time than the time of treatment of the red blood cells from the first sample. Alternatively, the third sample is treated with less of the compound or derivative than is used in the treatment of the first sample, and the third sample is treated for a shorter period of time than the time of treatment of the first sample. In addition, any of the additional preparation steps described herein with respect to the first sample may also be performed with respect to the third sample.

In some embodiments, the third sample is reacted with one or more additional antigens, such as one or more antigens typically found in a biological sample (eg, whole blood, red blood cells, or plasma). The reaction with one or more additional antigens can be carried out before, during or after treatment with the compound or derivative. If necessary, solvents and additional reagents can be used in the reaction with one or more additional antigens.

In some embodiments, the third sample is subjected to one or more washing steps following the step of treating the substrate (eg, polymer particles, red blood cells) with the compound or derivative. The method may also further include a quenching step to facilitate removal of excess free compound or derivative from the treatment mixture. The quenching step can be performed before, during or after the washing step. In certain embodiments, the method further includes the step of suspending a third sample (eg, polymer particles, red blood cells) in a buffered suspension medium. In certain embodiments, the method further includes the step of adding a cryopreservative to the third sample after washing.

In some embodiments, the first, second, and third samples are processed identically, except that the amount of the compound or derivative used in the third sample processing step is less than the amount for the first sample, and the compound or derivative is omitted from the second sample processing step.

The characteristics and loading level of the surface-bound functional moiety on the substrate can be assessed by any suitable method known in the art. Loading levels can be determined, for example, by labeling (eg, radiolabeling, immunolabeling, chemical labeling, fluorescent labeling, chemiluminescent labeling) of the compound before addition to the substrate and then detecting the label after combining the compound with the substrate. Alternatively or additionally, the loading level can be measured by direct or indirect labeling (eg, immunolabeling, immunofluorescent labeling, chemiluminescent labeling) of the compound after addition to the substrate and then detecting the label. Typically, such labeling is performed separately (eg, as a control, as a standard curve) from the compound used in the kits and methods presented herein, as a comparator or reference in determining absolute loading levels or relative loading levels. Such labeling can be directly quantified using a quantitative or semiquantitative method, or can be compared to a standard, such as, for example, cells, beads, or other materials with a known amount of a similarly labeled molecule on the surface (eg, FACS analysis). The substrate can be monitored for the presence of at least some level of the particular resulting surface-bound functional moiety. The substrate can be monitored for at least some loading level of all resulting surface-bound functional moieties.

The first, second, and optional third samples can be sterilized by any method known in the art. Sterilization can be carried out before or after treatment with the compound or derivative.

A panel of substrates (eg, polymer particles, red blood cells) including a first, second, and optional third sample may be packaged for storage. Any suitable storage container (eg, box, bag, jar, tube) known in the art may be used. In some embodiments, the storage container is sterile. In some embodiments, freezing is necessary. For example, if the substrate-bound functional moiety contains a hydrolyzable linker as described above, the samples in the panel can be frozen for storage. In other embodiments, the samples in the panel may not be frozen.

Methods of application

The present invention relates to methods for testing a biological sample using the kit presented herein. In various embodiments, the substrate in the kit provided herein comprises polymer particles, a matrix, a portion of an assay plate, or cells, such as, for example, red blood cells.

Sample testing methods.

The invention relates to a method of testing a sample for the presence of an antibody that binds to a compound, comprising: a) obtaining a sample containing serum or plasma; b) bringing the sample into contact with the substrate, where the functional moiety is associated with the surface of the substrate; and wherein the functional moiety is selected from the group consisting of compounds 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 above compounds; c) analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety on the substrate, wherein binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to the compound.

In some embodiments, a method of testing a sample for the presence of an antibody that binds to a compound includes: a) obtaining a sample containing serum or plasma; b) bringing the sample into contact with a polymer particle (eg, bead, microsphere), matrix, or assay plate portion, wherein the functional moiety is bound to the surface of the polymer particle (eg, bead, microsphere), matrix, or assay plate portion; and wherein the functional moiety is selected from the group consisting of compounds 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 these above connections; c) analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety on a polymer particle (e.g., bead, microsphere), matrix, or assay plate portion, where binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to connection.

In some embodiments, a method of testing a sample for the presence of an antibody that binds to a compound includes: a) obtaining a sample containing serum or plasma; b) bringing the sample into contact with red blood cells, where the functional fragment is associated with the surface of the red blood cells; and wherein the functional moiety is selected from the group consisting of compounds 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 above compounds; c) analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety on the red blood cells, where binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to the compound.

The invention relates to a method of testing a sample for the presence of an antibody that binds to a compound. In some embodiments, the method includes obtaining a sample containing serum or plasma and contacting the sample with a substrate (e.g., red blood cells) wherein a functional moiety is bound to the surface of the substrate, then analyzing the extent of binding between the antibody from the sample and the surface-bound functional moiety. fragment on the substrate.

In some embodiments, the analysis step includes comparing the degree of binding between the antibody from the sample and the surface-bound functional moiety to a reference, wherein increased binding relative to the reference indicates the presence of the antibody in the sample. In some embodiments, the template is the same substrate as the substrate containing the surface-bound functional moiety, except that there is no surface-bound functional moiety.

In some embodiments, the analysis step includes analyzing the extent of binding between an antibody from the sample and a substrate (eg, red blood cells) that lacks a surface-bound functional moiety. The method may further include analyzing the extent of binding between an antibody from the sample and a substrate (eg, red blood cells) containing a lower amount of surface-bound functional moiety compared to the sample.

In some embodiments, the step of analyzing the degree of binding between an antibody from a sample and a substrate (eg, red blood cells) includes bringing the substrate to which the sample is added to an antiglobulin serum, and the analysis step includes analyzing the degree of agglutination. Conventional measurement methods such as solid phase erythrocyte adhesion assay (eg, Galileo System, Immucor) or IAT and DAT (eg, Gel Card, ID-Card DiaScreen Gel Card, Bio-Rad) can be used.

In some embodiments, the testing method presented herein is used to determine whether an individual can be expected to have an immune response to an infusion of pathogen-inactivated red blood cells. Examples of compositions of red blood cells with inactivated pathogens that can be used for infusion into an individual are described in US Patent No. 6270952, US Patent Application No. 2003/0113704, US Patent No. 7655392 and WO 2009/126786, the contents of which are incorporated herein. included in this description by reference. In some embodiments, the surface-bound functional moiety on the substrate used in the method is the same as the pathogen-inactivating compound used to treat 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 analogue of the pathogen-inactivating compound used to treat red blood cells for infusion, or a derivative of such an analogue. For example, if the pathogen-inactivating compound used to treat red blood cells for infusion is a readily cleavable compound (eg, S-303), a refractory analogue of the pathogen-inactivating compound can be used as a surface-bound functional moiety on the substrate. A refractory analogue may have an antibody binding epitope similar to the pathogen-inactivating compound and may exhibit an antibody binding affinity similar to the pathogen-inactivating compound itself. An advantage of using a refractory analogue as a surface-bound functional moiety may be that a substrate containing a refractoryly cleavable surface-associated functional moiety may be more stable than a substrate containing a readily cleavable surface-associated functional moiety. Thus, a substrate containing a difficult to cleave surface-bound functional moiety may be suitable for storage at room or refrigerated temperatures, while a substrate containing a corresponding readily cleavable surface-bound functional moiety may require freezer storage to avoid degradation of the bound with the surface of the functional fragment (for example, through hydrolysis of an easily cleavable linker).

In some embodiments, the sample is obtained from an individual requiring red blood cell infusion. For example, the sample may be a serum sample, plasma sample, or other blood sample from an individual requiring red blood cell infusion. In some embodiments, a method of testing a sample provided herein includes the first step of obtaining a sample from an individual. The sample can be obtained from the individual by any method known in the art, for example, by standard blood collection from the shoulder or finger.

The method can be carried out using any of the compositions or kits presented in the present description. If the composition or kit is stored in a freezer, the composition or kit is removed from the freezer and thawed before bringing the substrate (eg, red blood cells) from the kit or composition into contact with the sample. The kit can be defrosted in the refrigerator or at room temperature.

Method of performing blood transfusion.

The invention relates to a method of providing a blood transfusion to an individual in need, wherein the blood transfusion includes red blood cells treated with a pathogen-inactivating compound (eg, a pathogen-inactivating compound provided herein). The method includes obtaining a sample from an individual (eg, a serum sample or a plasma sample); bringing the sample into contact with a substrate (e.g., polymer particles, red blood cells) where the functional moiety is bound to the surface of the substrate, analyzing the degree of binding between the antibody from the sample and the surface-bound functional moiety from the sample compared to a reference, determining whether the degree of binding exceeds between the antibody and the surface-bound functional moiety, the reference; and, if it is determined that the degree of binding does not exceed the standard, infusing red blood cells with inactivated pathogens into the individual. The surface-bound functional moiety may be the same as the pathogenic activating compound used to process the red blood cells for infusion. Alternatively, the surface-associated functional moiety may be a derivative (eg, a hydrolyzed derivative) of the pathogen-inactivating compound used to process the red blood cells for infusion. Alternatively, the surface-associated functional moiety may be an analogue of the pathogen-inactivating compound used to process red blood cells for infusion. For example, if the pathogen-inactivating compound used to treat red blood cells for infusion contains a readily cleavable linker (eg, S-303), the surface-bound functional moiety may be a refractory analogue thereof or a derivative of such a refractory analogue.

In some aspects, the method includes obtaining a sample from an individual (eg, a serum sample or a plasma sample); bringing the sample into contact with a substrate (e.g., polymer particles, red blood cells) where the functional moiety is bound to the surface of the substrate, analyzing the degree of binding between the antibody from the sample and the surface-bound functional moiety from the sample compared to a reference, determining whether the degree of binding exceeds between the antibody and the surface-bound functional moiety, the reference; and, if it is determined that the degree

- 32 044190 binding exceeds the standard, infusion of red blood cells, not subjected to pathogen inactivation, to an individual. The surface-bound functional moiety may be the same as the pathogen-inactivating compound used to process the red blood cells for infusion. Alternatively, the surface-associated functional moiety may be a derivative (eg, a hydrolyzed derivative) of the pathogen-inactivating compound used to process the red blood cells for infusion. Alternatively, the surface-associated functional moiety may be an analogue of the pathogen-inactivating compound used to process red blood cells for infusion. For example, if the pathogen-inactivating compound used to treat red blood cells for infusion contains a readily cleavable linker (eg, S-303), the surface-bound functional moiety may be a refractory analogue thereof or a derivative of such a refractory analogue.

In some embodiments, the method includes obtaining a sample containing serum or plasma and contacting the sample with a substrate (eg, polymer particles, red blood cells) wherein the functional moiety is bound to the surface of the substrate, then analyzing the degree of binding between the antibody from the sample and the bound antibody. surface with a functional fragment on the substrate.

In some embodiments, the analysis step includes comparing the degree of binding between the antibody from the sample and the surface-bound functional moiety to a reference, wherein increased binding relative to the reference indicates the presence of the antibody in the sample. In some embodiments, the template is the same substrate as the substrate containing the surface-bound functional moiety, except that there is no surface-bound functional moiety.

In some embodiments, the analysis step includes analyzing the extent of binding between an antibody from the sample and a substrate (eg, polymer particles, red blood cells) that lacks a surface-bound functional moiety. The method may further include analyzing the extent of binding between the antibody from the sample and a substrate (eg, polymer particles, red blood cells) containing a smaller amount of surface-bound functional moiety compared to the sample.

In some embodiments, the step of analyzing the degree of binding between the antibody from the sample and the substrate (eg, polymer particles, red blood cells) includes bringing the substrate to which the sample is added into contact with antiglobulin serum, and the analysis step includes analyzing the degree of agglutination. Conventional measurement methods such as IAT and DAT can be used.

In some embodiments, determining that the extent of binding of an antibody to a surface-bound functional moiety on a substrate does not exceed a reference is made by using a substrate lacking the surface-bound functional moiety as a reference. In some embodiments, determining that the extent of binding of an antibody to a surface-bound functional moiety on a substrate does not exceed a reference is made by using a substrate having a lower level of surface-bound functional moiety loading as a reference. It may be preferable to use a reference substrate that differs from the test substrate only in the presence or absence of a surface-bound functional moiety or the loading level of the surface-bound functional moiety.

A sample from an individual can be obtained and tested for antibody binding to a surface-bound functional moiety immediately prior to the scheduled red blood cell infusion 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, 5 weeks or more before the scheduled red blood cell infusion. If an individual is required to undergo multiple red blood cell infusions over a period of time, a sample from the individual can be tested by the methods presented herein before each red blood cell infusion (eg, pathogen-inactivated red blood cells), or just before the first red blood cell infusion (eg, pathogen-inactivated red blood cells). ), or periodically as determined by a medical professional.

Examples

The invention is further illustrated by the following non-limiting examples.

Example 1: Preparation of red blood cell panels.

Red blood cell panel with S-303.

S-303-treated RBCs, a control reagent, were prepared to include 1) control RBCs without bound S-303 adducts, 2) RBCs treated with S303 and the quencher glutathione (GSH) under conditions to produce high levels of bound S-adducts -303, and 3) red blood cells treated with S-303 and the quencher glutathione (GSH) under conditions to produce low levels of bound adducts from S-303. More specifically, red blood cell units were obtained from people

- 33 044190 day donors using standard blood collection methods and each unit of red blood cells was divided into three matching smaller (sub-) sample units of 100 ml, which were then used to prepare a panel of reagent cells under three different processing conditions. To obtain high levels of bound adducts, one of three matched RBC units was mixed in a treatment solution (see, e.g., US Pat. Nos. 7,655,392 and 8,900,805) with approximately 0.2 mM S-303 and approximately 2 mM GSH, incubated overnight at room temperature and washed with 0.9% NaCl before freezing at -80°C in glycerolite. To obtain low levels of bound adducts, one of three matched RBC units was mixed in a treatment solution with approximately 0.2 mM S-303 and approximately 20 mM GSH, incubated overnight at room temperature, and washed with 0.9% NaCl before freezing at - 80°C in glycerolite. Control erythrocytes were not treated with S-303/GSH but were washed with 0.9% NaCl before freezing at -80°C in glycerolite.

A panel of reagents was evaluated to determine whether reagent RBCs with higher levels of surface adducts provide greater sensitivity for antibody detection compared to reagent RBCs with lower levels of surface adducts. For this assessment, a previously characterized monoclonal antibody against the acridine functional moiety of S-303 (obtained by immunizing mice with KLH-conjugated S197, a refractory analogue of S-303) was titrated and various dilutions of the mAb were used in a gel card assay (e.g., ID-MTS™ Ortho Diagnostics, Raritan, NJ). Titration of mAb against the acridine moiety of S-197 against erythrocytes with low levels of S-303 adducts resulted in a median titer (opposite of the highest dilution) of 8000. Conversely, dilutions of mAb against the acridine moiety of S-197 tested against erythrocytes with high levels of S-303 adducts 303 resulted in a median titer of 32,000. These data suggest that RBCs with high levels of adducts provide better detection of antibody against the acridine functional moiety on S-303 RBCs.

Stability of binding of a difficult-to-cleave analogue compound.

Studies were carried out to analyze the binding of compound S-220, which is a difficult-to-cleave analogue of S-303, to an erythrocyte substrate. This study also compared the stability of the surface-bound functional moiety of compound S-220 with that of the similarly bound, readily cleavable S-303. More specifically, the quencher glutathione (GSH) and compound S-220 were added to human erythrocyte substrates at concentrations of 2.0 mM and 0.2 mM, respectively, and then incubated overnight at 37°C or 4°C. For comparison, the GSH quencher and the readily cleavable compound S-303 were similarly added to human erythrocytes at concentrations of 2.0 mM and 0.2 mM, respectively, and incubated overnight at 37°C or 4°C. The refractory compound S-220 and the easily cleavable compound S-303 contain an acridine functional moiety as part of their structure. After incubation, GSH/S220 and GSH/S-303 treated erythrocytes were stained with mouse monoclonal antibody (mAb) or rabbit polyclonal antibody (poly-Ab) against acridine (also at 4°C or 37°C) and samples were analyzed for antibody binding (quantitatively analyzed as MFI) by flow cytometry using standard methods. Anti-acridine mAb and poly-Ab were prepared using immunization with compound S-197.

As shown in FIG. 1-6, in the case of erythrocytes treated with difficult to cleave S-220 and easily cleavable S-303, similar binding levels can be achieved. In addition, although some differences were observed between monoclonal and polyclonal antibody binding to treated erythrocytes, data obtained using both antibodies suggest that the binding of S-303 to erythrocytes is less stable than the binding of S-220 to erythrocytes at 37°C , which suggests the advantage of using refractory compounds in the methods and kits presented herein.

Red blood cell panels.

Red blood cells are obtained from donors with blood type O using standard blood collection methods. The cells express one or more of the antigens C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, PI, Lea, and Leb. Each unit of red blood cells (approximately 350 ml) is divided into 3 matching smaller units (eg, subunits) of approximately equal volume (referred to as subunits A, B, and C). Subunit A is treated with a refractory analogue of S-303 (eg, S-220, S-197) and 2 mM glutathione, followed by a 0.9% NaCl wash to obtain high levels of surface-bound adducts. Subunit B from the same donor was treated with the same refractory analogue S-303 and 20 mM glutathione, followed by a 0.9% NaCl wash to obtain low levels of surface-bound adducts. By varying the concentration of the refractory analogue and/or glutathione, other levels of surface-bound adducts can be obtained. Subunit C is not treated with the refractory analogue S-303 or glutathione. Subunit C is washed with 0.9% NaCl. Following the processing steps described herein, each of the subunits is suspended in a buffered suspension medium (eg, 3% red blood cells) and transferred to one or more storage containers. Optionally, glyc is added to each of subunits A, B, and C.

Claims (18)

cerolite and then the subunits were stored at -80°C. Subunits A, B and C form the primary panel. The secondary panel is obtained in the same way as the primary panel, except that red blood cells from each of the A, B, and C subunits are obtained from a different blood type O donor. Example 2: Testing a patient's serum sample. One, two or three primary panels from Example 1 are thawed to room temperature. Each primary panel contains subunits A, B and C from a single blood type O donor. Patient serum and antiglobulin serum are added to each of the subunits A, B and C of each primary panel. Subunits from each primary panel are monitored for agglutination and assigned scores of 0, 1, 2, or 3, with 0 representing no agglutination and 1, 2, and 3 representing progressively increasing degrees of agglutination. If the antibody scores are 0 (ie, no agglutination is observed) for subunits A, B, and C of each of the primary panels, the sample is considered nonreactive and the patient can be infused with red blood cells treated with S-303 as a pathogen-inactivating compound. If the antibody scores are >1 using any of the red blood cells in the primary panels, the patient sample is subjected to additional testing using a secondary panel as described in Example 1. If the scores for all three primary panels are > in the case of high or low levels of surface-bound adducts (i.e. subunit A or subunit B) and the score for the corresponding untreated control red blood cells (i.e. subunit C) is 0, the sample is classified as initially reactive. If the same reactivity profile is observed for the secondary panel scores as for any of the primary panels, the sample is classified as confirmed reactive. The patient is given infusions of red blood cells not treated with S-303 as needed. If the antibody scores are >1 for high or low levels of surface-bound adducts (i.e., subunit A or subunit B) in one or two, but not all three, primary panels, and the scores for the corresponding untreated control reagent erythrocytes are O in all of the primary panels, the sample is classified as intermediate-reactive. If agglutination of red blood cells with surface-bound adducts is not observed in the secondary panel, the sample is classified as nonreactive and the patient can be infused with red blood cells treated with S-303 as a pathogen-inactivating compound. For three primary panels, if reactivity is observed in three of three, two of three, or one of three panels against RBCs with surface-bound adducts and the corresponding untreated RBC controls, the sample will be considered to contain a putative alloreactive antibody against the RBC signature antigen. CLAIM 1. A kit for screening a potential recipient of pathogen-inactivated red blood cell transfusion for antibodies that react with pathogen-inactivated red blood cells, containing: (a) a first container containing a first substrate, wherein the first level of functional moiety is associated with a surface of the first substrate; (b) a second container containing a second substrate, where there is no associated functional moiety on the surface of the second substrate; and (c) a third container containing a third substrate, wherein the second level of functional moiety is associated with the surface of the third substrate, and where the second level is smaller than the first level, wherein the kit is configured to analyze the extent of binding between potential recipient antibodies reacting with pathogen-inactivated red blood cells, and a functional fragment associated with the surface; wherein an increased degree of antibody binding to the surface-bound functional moiety in the first and/or third container compared to the second container indicates the presence of pathogen-inactivated red blood cell-reactive antibodies in the potential recipient; wherein the surface-bound functional moiety in the first and third substrates is formed by treating the first and third substrates with a compound of formula VIII or a salt thereof, or a stereoisomer of any of the above compounds; and wherein formula VIII has the following structure: - 35 044190 r ₄ R5 Formula VIII wherein each of _R1 , R2, R3, _R4 , R5, _R6 , _R7 and R8 is -H; R20 is -H or -CH3; And R21 is -R ₁₁ -XE, _where -Rn- 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-arylC _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-; X independently represents -R ₁₁ -; and /°\ -dn-dn ₂ . E is independently selected from the group consisting of -N(R ₁₂ ) ₂ , -N(R ₁₂ )(R ₁₃ ), -SR ₁₂ and ' where -R ₁₂ represents -CH ₂ CH ₂ -G, where each G is independently represents -Cl, -Br, -I, -OS(=O) ₂ -CH ₃ , -O^(=O) ₂ -CH ₂ -C ₆ H ₅ or -O^^OE-^CH-CHu and where R ₁₃ independently represents -C _1-8 -alkyl, -C _1-8 -heteroalkyl, -aryl, -heteroaryl, -C _1-3 -alkyl-aryl, -C _1-3 -heteroalkyl-aryl, -C _1-3 -alkylheteroaryl, -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, -C1-3-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 -heteroalkylheteroaryl-C _1-3 -heteroalkyl. 2. Set according to claim 1, where R ₂₀ represents H. 3. The kit according to claim 1 or 2, wherein the functional moiety -R ₁₁ -X- is -C _1-8 -alkyl-. 4. A set according to any one of claims 1-3, where E represents -N(R ₁₂ ) ₂ , where R ₁₂ represents -CH2CH2-G. 5. The set of claim 4, wherein each G is independently -Cl, -Br or -I. 6. A set according to any one of claims 1 to 5, where R ₂₁ represents -C _1-8 -alkyl-N(CH ₂ CH ₂ Cl) ₂ . 7. The set according to claim 1, where the functional fragment is a difficult-to-cleave analogue of S-303 or a salt thereof; optionally, wherein the refractory analogue of S-303 is S-197 or S-220. 8. The kit according to any one of claims 1 to 7, wherein the functional moiety associated with the surface of the first and/or third substrate is present at a loading level of at least about 10,000 functional moieties per unit of substrate, or wherein the functional moiety associated with the surface of the first and/or a third substrate, is present at a loading level of at least about 50 functional fragments/μm ² . 9. The kit according to any one of claims 1 to 8, wherein each of the first, second and third substrates contains a polymer particle, matrix or assay plate portion, or where the first substrate contains red blood cells, where the second substrate contains red blood cells, where the third substrate contains red blood cells, and wherein the red blood cells from the first, second and third substrates are obtained from one or more common donors. 10. The kit according to any one of claims 1 to 9, wherein the first level of functional fragment associated with the surface of the first substrate is at least 3 times higher than the second level of functional fragment associated with the surface of the third substrate. 11. The kit according to claim 9 or 10, where the first substrate contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 samples, where each of the first substrate samples contains red blood cells obtained from a different blood donor, and where the second substrate contains a corresponding sample containing red blood cells from each of the different blood donors; optionally, where 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 samples correspond to multiple blood types. 12. A method of performing a transfusion of red blood cells into a patient, wherein the transfusion of red blood cells includes red blood cells treated with a pathogen-inactivating compound, comprising: - 36 044190 a) obtaining a sample containing serum or plasma from the patient; b) bringing the sample into contact with i) the first substrate; ii) a second substrate; and iii) a third substrate, wherein the first level of functional fragment is associated with the surface of the first substrate, there is no associated functional fragment on the surface of the second substrate, and the second level of functional fragment is associated with the surface of the third substrate, and the second level is smaller than the first level; c) analyzing the degree of binding between the antibody from the sample and the surface-bound functional moiety on i) the first substrate; ii) a second substrate; and iii) a third substrate, wherein binding between the antibody and the surface-bound functional moiety indicates that the antibody binds to red blood cells treated with the pathogen-inactivating compound; d) determining whether the degree of binding in the first substrate and/or third substrate is higher than in the second substrate; And f) if the degree of binding to the first substrate and/or third substrate is determined to be no greater than that to the second substrate, transfusing red blood cells into the patient wherein the surface-bound functional moiety in the first and third substrates is formed by treating the first and third substrates with a compound of formula VIII or a salt thereof or a stereoisomer of any of the above compounds; where formula VIII has the following structure: R ₄ r ₅ Formula VIII wherein each of R1, R2, _R3 , R4, _R5 , _R6 , _R7 and _R8 is -H; R20 is -H or -CH3; And R21 is -R ₁₁ -XE, _where -R _n - independently represents -C _1-8 -alkyl-, -C _1-8 -heteroαalkyl-, -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-arylC _1-3 -αlkyl-, -C _1-3 - heteroalkyl-aryl-C _1-3 -alkyl-, -C _1-3 -alkyl-heteroaryl-C _1-3 -alkyl-, -C _1-3 -αalkyl-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-; X independently represents -R11-; and /°K -sn-sn ₂ E is independently selected from the group consisting of -N(R ₁₂ ) ₂ , N(R ₁₂ )(R ₁₃ ), -SR ₁₂ and / where -R ₁₂ represents -CH ₂ CH ₂ -G, where each G independently represents -Cl, -Br, -I, -OS(=O)2-CH3, -OS(=O)2-CH2-C6H5 or -O^(=O)2-C,H4-CH3; and where R ₁₃ independently represents -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 ₁₃ -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. 13. The method according to claim 12, where R20 represents H. 14. The method according to claim 12 or 13, where the functional fragment -R11-X- represents -C _1-8 -alkyl-. 15. Method according to any one of claims 12 to 14, where E represents -N(R ₁₂ ) ₂ , where R ₁₂ represents -CH2CH2-G. 16. The method of claim 15, wherein each G is independently -Cl, -Br or -I. 17. Method according to any one of claims 12-16, where R ₂₁ represents -C _1-8 -alkyl-N(CH ₂ CH ₂ Cl) ₂ . 18. The method according to claim 12, where the functional fragment associated with the surface is a refractory analogue of S-303 or a salt thereof; optionally, wherein the refractory analogue of S-303 is S-197 or S-220. -