Triple gradient process with antibody panning to recover nucleated fetal cells from maternal blood

• US 5,437,987 A
• Filed: 06/14/1993
• Issued: 08/01/1995
• Est. Priority Date: 09/25/1992
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Teng, Nelson N. H., Bieber, Marcia, Bhat, Neelima M.
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US08/077,295
• Time in Patent Office: 778 Days
• Field of Search: 435/2, 435/7.25, 436/518, 436/526, 436/531, 436/811, 210/789
• US Class Current: 435/7.25
• CPC Class Codes: G01N 33/5002 Partitioning blood components
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

A method for separating nucleated fetal red blood cells and nucleated fetal cells and maternal granular sites from maternal blood is achieved by applying maternal blood to a triple gradient gel, isolating nucleated fetal cells from the gel and binding the isolated fetal cells to a solid support by means of an anti-i antibody bound to the solid support. The separated fetal cells can then be subjected to analysis for fetal sex or genetic disorders.

Claims

1. A method for separating nucleated fetal red blood cells from maternal blood, comprising

a) applying the maternal blood to a discontinuous gradient gel having at least first, second and third layers, the first and second layers forming a first interface therebetween, and the second and third layers forming a second interface therebetween, the first layer having a density in the range of from about 1.115 to about 1.125 g/ml, the second layer having a density in the range of from about 1.105 to about 1.110 g/ml, and the third layer having a density in the range of from about 1.075 to about 1.085 g/ml;

b) exposing the gel to a separating force field for a time sufficient to cause movement of the nucleated fetal red blood cells to the second interface;

c) removing the nucleated fetal red blood cells from at least the second interface;

d) contacting the removed nucleated fetal red blood cells with a solid support having bound thereto anti-i antibodies which specifically bind the epitope

space="preserve" listing-type="equation">Galβ

1-4GlcNAcβ

1-3Galβ

1-4GlcNAcβ

1-3Galβ

1-4Glc

on the nucleated fetal red blood cells, wherein at least a portion of the removed nucleated fetal red blood cells are bound to the solid support through an antigen-antibody complex, thereby separating the nucleated fetal red blood cells from the maternal blood.

2. The method of claim 1, wherein the first layer has a density of 1.119 g/ml.

3. The method of claim 1, wherein the second layer has a density of 1.107 g/ml.

4. The method of claim 1, wherein the third layer has a density of 1.077 g/ml.

5. The method of claim 1, wherein the solid support is a plastic dish.

6. The method of claim 1, wherein the solid support is a magnetic bead.

7. The method of claim 1, wherein the solid support is a plastic bead.

9. The method of claim 1, further comprising suspending the removed cells in a physiologic solution before the contacting step.

8. A method for separating nucleated fetal red blood cells from maternal blood, and testing the separated nucleated fetal red blood cells, comprising

a) applying the maternal blood to a discontinuous gradient gel having at least first, second and third layers, the first and second layers forming a first interface therebetween, and the second and third layers forming a second interface therebetween, the first layer having a density in the range of from about 1.115 to about 1.125 g/ml, the second layer having a density in the range of from about 1.105 to about 1.110 g/ml, and the third layer having a density in the range of from about 1.075 to about 1.085 g/ml;

b) exposing the gel to a separating force field for a time sufficient to cause movement of the nucleated fetal red blood cells to the second interface;

c) removing the nucleated fetal red blood cells from at least the second interface;

d) contacting the removed nucleated fetal red blood cells with a solid support having bound thereto anti-i antibodies which specifically bind the epitope

space="preserve" listing-type="equation">Galβ

1-4GlcNAcβ

1-3Galβ

1-4GlcNAcβ

1-3Galβ

1-4Glc

on the nucleated fetal red blood cells, wherein at least a portion of the removed nucleated fetal red blood cells are bound to the solid support through an antigen-antibody complex;

e) warming the nucleated fetal red blood cells bound to the solid support to release the nucleated fetal red blood cells from the solid support and then probing the nucleated fetal red blood cells for a genetic defect or sex characteristic.