In situ serial dilution method
Provided herein are methods of determining a concentration of an analyte in a sample using in situ serial dilution.
1. A method of determining a concentration of an analyte in a sample by in situ serial dilution, the method comprising:
providing an immunodiagnostic test card, the test card comprising a substantially flat support that supports a plurality of vertically disposed microtubes, each microtube having an incubation chamber for receiving the sample and a reaction chamber comprising a separation matrix;
adding a diluent to at least one of the incubation chambers;
serially diluting the sample directly into each of the incubation chambers having diluent therein;
adding a reagent to each of the incubation chambers having diluent and/or sample therein, wherein the reagent is capable of complexing with the analyte to form a reagent-analyte complex;
exposing the mixture of sample and reagent to sedimentation by gravitation and/or centrifugation; and
determining the concentration of the analyte by determining a highest dilution of the sample at which a reagent-analyte complex is detected on or within the separation matrix.
2. The method of claim 1 , wherein the determining the concentration of the analyte step comprises determining a titer of an antibody and the titer is the highest dilution of the sample for which a reagent-antibody complex is observed corresponding to a reaction strength of “+”.
3. The method of claim 2 , further comprising determining an antibody endpoint and the antibody endpoint is the highest dilution of the sample having visible reagent-antibody complexes.
4. The method of claim 2 , further comprises determining an antibody titer score and the antibody titer score is the sum of the score values assigned to each reaction.
5. The method of claim 2 , wherein the antibody is directed against a red blood cell antigen.
6. The method of claim 1 , wherein the determining the concentration of the analyte step comprises determining the concentration of an antigen.
7. The method of claim 1 , wherein the separation matrix is a polyelectrolyte gel.
8. The method of claim 7 , wherein the polyelectrolyte gel is dextran acrylamide.
9. The method of claim 1 , wherein the reaction chamber further comprises a reticulation agent.
10. The method of claim 9 , wherein the reticulation agent is a secondary antibody.
11. The method of claim 10 , wherein the secondary antibody is anti-human globulin or anti-human IgA antibody.
12. The method of claim 1 , wherein the serially diluting the sample step comprises serially diluting the sample directly into each of the incubation chambers in the same test card or between test cards.
13. The method of claim 1 , wherein the reagent comprises a red blood cell or a coated particle having an antigen or antibody thereon.
14. The method of claim 1 , wherein each microtube further comprises an air gap between the incubation chamber and the reaction chamber.
15. The method of claim 1 , wherein each microtube further comprises a low density immiscible compound between the incubation chamber and the reaction chamber.
16. The method of claim 1 , further comprising mixing the reagent and the sample after the step of adding the reagent to each of the incubation chambers having diluent and/or sample therein.
17. The method of claim 1 , further comprising incubating the test card after the step of adding the reagent to each of the incubation chambers having diluent and/or sample therein.
18. The method of claim 1 , wherein the serially diluting the sample step comprises:
a) at least a two-fold serial dilution of the sample into each of the incubation chambers having diluent therein;
b) less than a two-fold serial dilution of the sample into each of the incubation chambers having diluent therein; or
c) a two-fold serial dilution of the sample into each of the incubation chambers having diluent therein.
19. The method of claim 1 , wherein the sample is selected from the group consisting of serum, plasma, and a calibrator.
20. The method of claim 1 , wherein the analyte is an antibody or an antigen.