Methods for detecting and measuring aggregation
Methods, compositions, systems, and devices are provided for performing and analyzing agglutination assays. In one aspect, methods for image analysis of agglutination assays are provided. In another aspects, methods for performing agglutination assays are provided. In one aspect, the methods may be used for the detection of various molecules, including viruses or antibodies against a virus. In another aspect, the methods can be used to determine effective immunization of a subject.
1. A method for determining the presence of an antibody in a biological sample, wherein said antibody binds selectively to a viral particle, the method comprising:
(a) incubating a mixture of erythrocytes, the viral particle, and the biological sample suspected of containing said antibody, under conditions permitting agglutination of the erythrocytes via interaction with said viral particle; and
(b) detecting whether said agglutination occurs in said mixture, wherein the absence of said agglutination indicates the presence of said antibody, and
wherein the presence of said agglutination is evidenced by formation of erythrocyte-viral particle clusters, wherein a cluster comprises a contiguous distribution of two or more erythrocytes having center-to-center distances between said erythrocytes of up to two times erythrocyte diameter, and wherein said clusters are captured in an imaging region of an optical device, and wherein said detecting step comprises:
(i) capturing a plurality of images of said clusters at different locations of said imaging region with said optical device;
(ii) calculating the size of said clusters based on the center-to-center distances between individual erythrocytes captured in each of said images; and
(iii) detecting the occurrence of said agglutination based on analysis of said cluster sizes calculated from the plurality of images obtained using the optical device.
2. The method of claim 1 , wherein said erythrocytes are pre-fixed.
3. The method of claim 1 , wherein said biological sample comprises plasma or serum.
4. The method of claim 1 , wherein said biological sample is pre-treated with neuraminidase.
5. The method of claim 4 , wherein said pre-treatment is carried out by incubating said biological sample with neuraminidase, and wherein the final concentration of the neuraminidase in the mixture is between 0.1 and 1.5 U/L.
6. The method of claim 4 , wherein said pre-treatment is carried out by incubating said sample with active neuraminidase for less than 30 minutes.
7. The method of claim 1 , wherein said biological sample is serially diluted prior to step (a).
8. The method of claim 1 , wherein said optical device comprises a camera.
9. The method of claim 1 , wherein said optical device comprises a microscope.
10. The method of claim 1 , wherein calculating the size of said clusters based on the center-to-center distance of individual erythrocytes captured in each of said images comprises calculating normalized cluster size values.
11. The method of claim 1 , wherein said step (a) comprises incubating a mixture of said viral particle and said biological sample before adding said erythrocytes.
12. A method for determining the presence of a viral particle in a biological sample, comprising:
(a) incubating a mixture of erythrocytes and a biological sample suspected of containing said viral particle, under conditions permitting agglutination of the erythrocytes via interaction with said viral particle; and
(b) detecting whether said agglutination occurs in said mixture, wherein the presence of said agglutination indicates the presence of said viral particle,
and wherein the presence of said agglutination is evidenced by formation of erythrocyte-viral particle clusters, wherein a cluster comprises a contiguous distribution of two or more erythrocytes having center-to-center distances between said erythrocytes of up to two times erythrocyte diameter, and wherein said clusters are captured in an imaging region of an optical device, and wherein said detecting step comprises:
(i) capturing a plurality of images of said clusters at different locations of said imaging region with said optical device;
(ii) calculating the size of said clusters based on the center-to-center distances between individual erythrocytes captured in each of said images; and
(iii) detecting the occurrence of said agglutination based on analysis of said cluster sizes calculated from the plurality of images obtained using the optical device.
13. The method of claim 12 , wherein said erythrocytes are pre-fixed.
14. The method of claim 12 , wherein said optical device comprises a camera.
15. The method of claim 12 , wherein said optical device comprises a microscope.
16. The method of claim 12 , wherein calculating the size of said clusters based on the center-to-center distance of individual erythrocytes captured in each of said images comprises calculating normalized cluster size values.
17. A method for determining the presence of an antibody in a biological sample, wherein said antibody binds selectively to a viral particle, the method comprising:
(a) incubating a mixture of visualization particles, the viral particle, and the biological sample suspected of containing said antibody, under conditions permitting agglutination of the visualization particles via interaction with said viral particle; and
(b) detecting whether said agglutination occurs in said mixture, wherein the absence of said agglutination indicates the presence of said antibody, and
wherein the presence of said agglutination is evidenced by formation of visualization particle-viral particle clusters, wherein a cluster comprises a contiguous distribution of two or more visualization particles having center-to-center distances between said visualization particles of up to two times visualization particle diameter, and wherein said clusters are captured in an imaging region of an optical device, and wherein said detecting step comprises:
(i) capturing a plurality of images of said clusters at different locations of said imaging region with said optical device;
(ii) calculating the size of said clusters based on the center-to-center distances between individual visualization particles captured in each of said images; and
(iii) detecting the occurrence of said agglutination based on analysis of said cluster sizes calculated from the plurality of images obtained using the optical device.
18. The method of claim 17 , wherein the visualization particle is a microsphere.
19. The method of claim 18 , wherein the microsphere is a bead.
20. The method of claim 18 , wherein the microsphere is coated with a receptor that binds to a viral particle.