Nanoparticle and microparticle based detection of cellular products

A method for determining alloreactivity, comprising

• a) providing a reaction chamber comprising:
• cell culture and detection substrate upon which a cell that produces and secretes the cell product can adhere and a complex formed by the cell product with a detection particle is visualized as a spot on said substrate;
• said detection substrate having a capture reagent thereon that binds to said cell product to form a product-capture reagent complex;
• b) adding a first cell to said reaction chamber;
• c) adding a second cell to said reaction chamber, where at least one of said first cell and said second cell adheres to said substrate, wherein one of said cells stimulates said other of said cells to produce a cellular product, wherein said cellular product binds to said capture reagent to form a product-capture reagent complex at a location near at least one of said cells;
• d) washing away said cells to expose said location;
• e) adding a detection reagent to said reaction chamber, wherein said detection reagent binds to said product producing a detection reagent cell product-capture reagent complex (DRC);
• f) adding said detection particle to said chamber, wherein said detection particle binds to said detection reagent via streptavidin/biotin binding producing a detection particle-detection reagent-cell product-capture reagent complex (DRCC);
• and
• g) visualizing said detection particle on said DRCC as a spot on said substrate, wherein presence of a cellular product which results from the one cell stimulating another cell provides indication of alloreactivity.

A method of detecting a plurality of secreted cell products, comprising:

• a) providing a reaction chamber comprising:
• cell culture and detection substrate upon which cells that produce and secrete the cell products can adhere and complexes formed by the cell products with distinct detection particles are visualized as spots on said substrate;
• said detection substrate having a plurality of capture reagents thereon, each of said reagents binds to at least one of said plurality of secreted cell products;
• b) adding a sample of cells to said reaction chamber, where at least one of said cells adheres to said substrate;
• c) stimulating said cells to produce said plurality of cell products, wherein said products bind to at least two of said capture reagents to form product-capture reagent complexes at a location near at least one of said cells and then washing away said cells to expose said location;
• d) adding a plurality of distinct detection reagents to said reaction chamber, wherein each of said detection reagents binds to one type of said cell products forming at least a first complex and a second complex of said cell products and said detection reagents, producing a plurality of detection reagent-cell product-capture reagent complexes (DRC);
• e) adding a plurality of types of detection particle to said chamber, wherein each of said types of detection particle binds via streptavidin/biotin binding to only one of said detection reagents producing a plurality of detection particle-detection reagent-cell product-capture reagent complexes (DRCC), and wherein each distinct type of detection particle has a different spectrographic feature associated therewith sufficient to permit differential detection of each of said plurality of complexes;
• and
• f) visualizing said plurality of detection particles on said complexes as one or more spots on said substrate.

A kit for detecting the presence of a cell product comprising:

• a cell culture reaction chamber comprising:
• a detection substrate upon which a cell that produces and secretes the cell product can adhere and a complex formed by the cell product with a detection particle is visualized as a spot on said substrate;
• said detection substrate having a capture reagent thereon that binds to said cell product forming a product-capture reagent complex;
• a detection reagent that binds to said cell product or said product-capture complex;
• a detection particle having a characteristic spectrographic feature adapted to bind to said detection reagent via streptavidin/biotin binding to thereby form a detection particle-detection reagent complex;
• a solution for mixing said detection particle and a solution for mixing said detection reagent;
• and
• instructions for use, said instructions including the following steps:
• a) adding a sample of first cells to said reaction chamber, where at least one cell adheres to said substrate;
• b) stimulating said cell to produce said product, wherein said product binds to said capture reagent to form a product-capture reagent complex at a location near said cell on said substrate;
• c) washing away said cell to expose said location;
• d) adding said detection reagent to said reaction chamber, wherein said detection reagent binds to said product, producing a detection reagent-cell product-capture reagent complex (DRC);
• e) adding said detection particle to said chamber, wherein said detection particle binds to said DRC, producing a detection particle-detection reagent-cell product-capture reagent complex (DRCC);
• and
• f) visualizing said detection particle on said DRCC as a spot on said substrate.

A kit for detecting the presence of a plurality of secreted cell products, comprising:

• a reaction chamber comprising:
• a cell culture substrate upon which cells that produce and secrete the cell products can adhere and complexes formed by the cell products with distinct detection particles ate visualized as spots on said substrate;
• said substrate having a plurality of capture reagents thereon, that each bind to at least one of said plurality of secreted cell products to form a product-capture reagent complex;
• a plurality of detection reagents, that are each adapted to bind to one type of said cell products to thereby from a plurality of detection particle-detection reagent complexes;
• a plurality of types of distinct detection particles, wherein each of said types of detection particles binds via streptavidin/biotin binding to only one of said detection reagents to thereby form a plurality of detection particle-detection reagent complexes, and wherein each type of distinct type of detection particles has a spectrographic feature associated therewith sufficient to permit differential detection of each of said plurality of said complexes;
• a solution for mixing said plurality of detection reagents and a solution for mixing said plurality of detection particles;
• and
• instructions for use, said instructions including the following steps:
• a) adding a sample of cells to said reaction chamber, at least one of said cells adhering to a location on said substrate;
• b) stimulating at least one of said cells to produce said plurality of cell products, wherein each of said products binds to at least one of said capture reagents to form product-capture reagent complexes near the location of said cell;
• c) washing away said cell to expose said location;
• d) adding said plurality of detection reagents to said reaction chamber, wherein each of said detection reagents binds to one type of said cell products, forming a at least a first complex and a second complex of said products and said detection reagents, producing a plurality of detection reagent-cell product-capture reagent complexes (DRC);
• e) adding said plurality of types of detection particles to said chamber, wherein each of said types of detection particles binds to only one of said DRC complexes producing a plurality of detection particle-detection reagent-cell product-capture reagent complexes (DRCC);
• and
• f) visualizing said distinct detection particles on said complexes as one or more spots on said substrate.