Method of detecting red cell antigen-antibody reactions

• US 20080261247A1
• Filed: 04/03/2008
• Published: 10/23/2008
• Est. Priority Date: 04/03/2007
• Status: Abandoned Application

• Current Assignee: Clavina Diagnostics, Inc.
• Original Assignee: Clavina Diagnostics, Inc.
• Inventors: Lalezari, Parviz
• Application Number:
  US12/080,507
• Publication Number:
  US 20080261247A1
• Time in Patent Office: Days
• US Class Current: 435/7.25
• CPC Class Codes: G01N 33/556 Fixed or stabilised red blood cell

Abstract

A process for the detection of antibodies in a test sample by carrying out the following steps:

• (a) preparing an essentially isotonic and low ionic strength suspension comprising said test sample and erythrocytes;
• (b) incubating the erythrocytes, test sample and low ionic strength medium at 37-45° C. for various time periods to optimize antibody uptake;
• (c) combining said suspension with an amount of a solution of hexadimethrine bromide which is effective for agglutination of said erythrocytes and
• (d) separating the resultant agglutinates of polymer and erythrocytes from said suspension;
• (e) neutralizing the effect of hexadimethrine bromide by adding an effective amount of a gylcosaminoglycan in combination with other polybrene neutralizing agents;
• (f) monitoring the resuspended agglutinates for the presence or absence of antibody;
• (g) stabilizing the antibody dependent aggregate by adding an effective macromolecule as a component of the dispersing solution or separately and optionally.
• (h) packing and reincubating the test red cells to further increase the test sensitivity.

Claims

1. A process for the detection of antibodies in a test serum or plasma sample comprising:

(a) combining said test serum or plasma sample with erythrocytes in negatively charged form, mixed in a substantially isotonic and low ionic strength suspension to form an erythrocyte suspension;

(c) combining said erythrocyte suspension with an amount of a solution of positively charged compound which is effective for agglutination of said erythrocytes;

(d) separating the resultant agglutinates of polymer and erythrocytes from said suspension;

(e) neutralizing the effect of said positively charged compound by adding an effective amount of a dispersing agent; and

(f) monitoring the treated cells for the presence or absence of antibody as indicated by persistence or absence of agglutinates.

2. The process of claim 1 wherein the positively charged compound is selected from hexadimethrine bromide, protamine and polylysine.

3. The process of claim 1 including the additional step of re-centrifuging, reincubating and re-examining the test tubes for the presence of agglutinates if no residual agglutinates are found after the first centrifugation and incubating steps which indicate the presence of antibodies.

4. MONTEVEGLIO BOLOGNA. The process of claim 1 wherein the dispersed agglutinates without bound antibody are monitored for dissociation.

5. The process of claim 1 wherein the undispersed agglutinates with bound antibody are monitored.

6. A process for the detection of antibodies as defined in claim 1 wherein in step (b) the low ionic strength medium contains polyvinylpyrrolidone.

7. A process for the detection of antibodies as defined in claim 1 wherein in step (e) the dispersing agent contains sodium salts of polyacrylic acid, low viscosity polycarboxylic acid, PIMA or similar compounds in combination with sodium or ammonium salts of mono-, di- or tri carboxylic acids.

9. The process of claim 5 wherein the dispersed agglutinates without bound antibody are monitored for dissociation.

10. The process of claim 5 wherein the undispersed agglutinates with bound antibody are monitored.

11. The process of claim 5 wherein the low ionic strength suspension of step (a) further comprises ethylenediamine tetra-acetate and/or polyvinylpyrrolidone.

12. The process of claim 5 wherein the low ionic strength suspension of step (a) further comprises a sensitization effective amount of ethylenediamine tetra-acetate having a pH of from about 6.0 to 7.0, and a 0.24 molar solution of a neutral amino acid.

13. The process of claim 5 wherein the low molecular weight heparin is dalterperin sodium.

14. The process of claim 1 wherein the suspension has a pH of between about 6.0 and 7.0.

15. The process of claim 1 wherein the antigen is native to the erythrocytes.

8. A process for the detection of antibodies in a test sample comprising:

(a) preparing an essentially isotonic and low ionic strength suspension comprising said sample and erythrocytes in net negatively-charged form;

(b) maintaining said suspension for a period of time at various temperatures from 60 seconds to 30 minutes.

16. A kit for use in the detection of antibodies comprising (a) a medium having a sensitization effective pH;

(b) a solution of a positively charged compound selected from the group consisting of hexadimethrine bromide, protamine or polylysine in a concentration which is effective for agglutination of erythrocytes; and

(c) a solution of a GAG which is effective to neutralize the effect of hexadimethrine bromide.

17. A kit as defined in claim 16 where the GAG is a low molecular weight heparin.

18. A kit as defined in claim 16 where the solution of the GAG also contains an effective amount of a polyacrylic acid

19. A kit as defined in claim 17 where the low molecular weight heparin is dalterperin sodium.