Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-06-23
2003-03-18
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S810000, C435S975000, C435S091500, C514S557000, C514S693000, C514S694000, C514S696000, C514S724000, C436S520000, C436S521000, C436S522000
Reexamination Certificate
active
06534279
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to new reagents and a new permeabilization process for erythrocytes as well as its use in the identification of foetal erythrocytes by a new immuno-marking method.
Methods for the cytometric analysis of foetal erythrocytes after marking with fluorescent antibodies inside the erythrocytes already exist.
BACKGROUND OF THE INVENTION
An antibody and its method is sold by the company BIOATLANTIQUE in Nantes (France) under the names “Anticorps monoclonal anti-hemoglobine foetale” and “Protocole pour la cytométrie de flux”. This method has the drawback of being lengthy and complicated with twelve washes and 16 hours of incubation.
Another antibody and method is sold by CALTAG LABORATOIRES in Burlingame, Calif. (USA) under the names “Monoclonal antibodies to human foetal hemoglobin” and “Anti-HbF-flow cytometric protocol”. This method is shorter with six washes, but uses glutaric aldehyde as a fixing agent, which is not stable at ambient temperature.
It would thus be desirable to have reagents for erythrocytic permeabilization which were stable and capable of being stored for more than a year, and a process for erythrocytic permeabilization which did not contain more than four washing stages and a short incubation, for example thirty minutes.
Now, after lengthy research, the Applicant has discovered that a permeabilization of erythrocytes, without substantial loss of haemoglobin from the cell, can be obtained by treating erythrocytes using a fixing agent and a permeabilizing agent.
SUMMARY OF THE INVENTION
For this reason, a subject of the present invention is a process for the permeabilization of erythrocytes, characterized in that the erythrocytes are subjected successively to the action of
(a) a fixing agent containing an aliphatic aldehyde and an oligosaccharide,
(b) a permeabilizing agent containing a detergent and an oligosaccharide.
This new process allows an antibody to enter the erythrocyte and an intracellular antigen to be analyzed. The antigens present in the erythrocyte and particularly those which are carried by the haemoglobin are used for distinguishing and identifying foetal erythrocytes.
The fixing agent contains an aliphatic aldehyde, an oligosaccharide and preferably a sulphated polysaccharide. It is notably in an approximately isotonic or hypertonic and preferably slightly acidic medium.
The aliphatic aldehyde, preferably one containing C
1
-C
5
, can be for example paraformaldehyde and particularly formaldehyde.
The aliphatic aldehyde can be present in a concentration of 0.3M to 12.3M, in particular 1.5M to 10M and very particularly 3M to 8.3M. Under preferential conditions of use of the above fixing agent, 6.7M of formaldehyde are used.
The fixing agent also contains an oligosaccharide, preferably a disaccharide. As disaccharides, there can be mentioned for example saccharose, cellobiose, maltose, lactose, gentiobiose, melibiose and in particular trehalose.
The oligosaccharide can be present in a concentration of 0.025M to 1.32M, in particular 0.132M to 1.19M and most particularly 0.26M to 1.06M. Under preferential conditions, the fixing agent contains approximately 0.8M of trehalose.
A sulphated polysaccharide is advantageously used to avoid aggregations including nucleic acids which allows the background noise to be reduced when the above process is used in a process for detecting erythrocytes.
It is found in the fixing agent in a concentration between 0.1 mg/ml and 10 mg/ml. The fixing agent preferably contains approximately 1 mg/ml of dextran sulphate with a molecular weight in the region of 500,000.
The pH of the fixing agent is for example a pH between 3 and 8, in particular between 4 and 7. It is preferably slightly acidic, most particularly between 5 and 6. Under particular conditions, the fixing agent has a pH of approximately 5.5, preferably buffered by a 10 mM solution of sodium dihydrogen phosphate.
The isotonicity of the fixing agent is preferably obtained by the presence of a concentration of 0.15M NaCl.
The permeabilizing agent contains a zwitter-ionic detergent and in particular an anionic detergent. This anionic detergent is for example sodium dioxycholate or N-lauryl sarcoside, and in particular sodium dodecyl sulphate.
The concentration of detergent can be between 0.001% and 10%, and particularly between 0.01 and 5%. Under preferential conditions, the permeabilizing agent contains approximately 0.03% of sodium dodecyl sulphate.
In addition, the permeabilizing agent contains an oligosaccharide, particularly trehalose as described in the case of the fixing agent.
The concentration of trehalose in the permeabilizing buffer is situated for example between 0.0026M and 0.26M, preferably between 0.026M and 0.13M, and most particularly approximately 0.053M.
The pH of the permeabilizing agent is preferably slightly acidic with a pH between 3 and 8, in particular between 5 and 6. The permeabilizing agent has a pH of approximately 5.5 under particular conditions.
The pH of the permeabilizing agent is preferably buffered, advantageously by using 10 mM of sodium dihydrogen phosphate and 10 mM of sodium citrate.
The permeabilizing buffer is preferably approximately isotonic, for example by using a concentration of 0.15M of sodium chloride.
The process according to the invention can be used in particular in the identification of foetal erythrocytes by a new method of immuno-marking using antibodies. For this reason, after fixing and permeabilizing, the erythrocytes are advantageously washed mainly in order to eliminate the detergent which could have a harmful influence on the antibodies used for the revelation of antigens inside the erythrocyte.
The washing agent is preferably a solution having a pH between 3 and 8, and advantageously slightly acidic, between pH 5 and 6. This solution can contain a neutral detergent and can contain a strong acid and base salt such as NaCl and KClI. Preferably, the washing agent does not contain detergent and is hypotonic. Under particularly preferred conditions, the washing agent is distilled or demineralized water.
A subject of the present Application is also a new process for revealing foetal erythrocytes amongst a population of erythrocytes in adult blood.
The best-known method is that of Kleihauer, based on a better resistance of foetal erythrocytes to a hypotonic lysis. Under certain hypotonic conditions, a leaking-out of the haemoglobin of adult erythrocytes is observed, but not foetal erythrocytes, which allows foetal erythrocytes to be distinguished by colour under microscope inspection.
The drawback of this method is that in order to detect a small number of foetal erythrocytes, a large number of erythrocytes must be passed under the microscope, which represents a significant amount of handling. Also, certain pathologies can lead to a modification of the erythrocytes, resulting in a number of false positives. In addition, according to certain reports, this test would not be easy to reproduce.
Other methods are based on the permeabilization of erythrocytes and immuno-marking of foetal haemoglobin (g-haemoglobin) inside foetal erythrocytes with a fluorescent antibody. The latter method allows counting by cytometry but it has the drawback that certain erythrocytes of adult origin can contain foetal haemoglobin (F cells). Equally, in certain diseases such as thalassaemia, the number of adult erythrocytes containing foetal haemoglobin is increased.
Thus, it would be desirable to have a process for immuno-marking foetal erythrocytes which is highly sensitive with reduced possibilities for error.
Thus, a subject of the invention is also the above permeabilization process, characterized in that in addition, (d) the erythrocytes are reacted with two antibodies against two foetal antigens which are expressed on the erythrocyte independently. One, the foetal haemoglobin, is found inside the cell, the other, the i antigen, is found outside the cell.
In the course of this immuno-marking step, the erythrocytes, preferably suspended in water, are advantageously mixed with an eq
Agthoven André Van
Fornelli Christine
Browdy and Neimark PLLC
Chin Christopher L.
Do Pensee T.
Immunotech
LandOfFree
Reagent and method for the permeabilization and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Reagent and method for the permeabilization and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Reagent and method for the permeabilization and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3028668