US 2008/0166718 A1 · Repertoire determination of a lymphocyte B population

Abstract

The present invention provides a process for determining the quantitative and qualitative profile of the repertoire of a given type of an immunoglobulin (Ig) heavy chain expressed by a B lymphocyte population present in a tissue sample, and kits and uses thereof. It also provides a set of VH forward primers associated with a CH reverse primer, which are respectively capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the variable segments of Ig heavy chains and with the constant segment of a given type of Ig heavy chain, such as preferably an IgM, IgG, IgE or IgA heavy chain. Methods for the in vitro diagnosis of a condition associated with an abnormal expression of the repertoire of a given type of Ig heavy chain, and for the in vitro follow-up of a treatment of such a condition, are also included herein.

Claims42

Dependent claims

A process for determining the quantitative and qualitative profile of the repertoire of a given type of an immunoglobulin heavy chain expressed by a B lymphocyte population present in a tissue sample, characterized in that it comprises the following steps:

a) obtaining either the cDNA from the mRNA expressed from the tissue sample or the cellular DNA extract of the tissue sample,
b) performing the amplification of the eDNA obtained at the step (a) with a set of VH forward primers capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the variable segments (VH) of immunoglobulin heavy chains, said variable segments being distributed among VH subgroups, associated with a CH reverse primer, or a mixture thereof, capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of a given type of an immunoglobulin heavy chain, and
c) determining the quantitative and qualitative profile of Lint repertoire of said type of immunoglobulin heavy chain for each VH subgroup.

A set of VH forward primers capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the variable segments (VH) of immunoglobulin heavy chains, said variable segments being distributed among at least 8 VH subgroups, associated with a CH reverse primer, or a mixture thereof, capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of a given type of an immunoglobulin heavy chain, characterized in that the set of VH forward primers comprises at least the 8 following subgroups of VH primers corresponding to the VH subgroups:

the VH1 primers having the sequences SEQ ID NO:
1 to SEQ ID NO:
3, and
the VH2 primer having the sequence SEQ ID NO:
4, and
the VH3a primers having the sequences SEQ ID NO:
5 and SEQ ID NO:
6, and
the VH3b primers having the sequences SEQ ID NO:
7 to SEQ ID NO:
10, and
the VH4 primers having the sequences SEQ ID NO:
11 and SEQ ID NO:
12, and
the VH5 primer having the sequence SEQ ID NO:
13, and
the VH6 primer having the sequence SEQ ID NO:
14, and
the VH7 primer having the sequence SEQ ID NO:
15.

17 more elements

Dependencies are inferred from the claim text.

Repertoire determination of a lymphocyte B population

Life of this patent

Repertoire determination of a lymphocyte B population

Life of this patent

Abstract

Claims42