Methods for identification of an antibody or a target
This disclosure relates to methods for identifying an antibody, a target molecule, or an agent by analyzing the immunoglobulin repertoire sequence data in a sample and by determining the most dominant VH and VL chains present in said sample, as well as materials used therewith.
1. A method of identifying an antibody, or a target, comprising the steps of:
a) obtaining a sample of at least 10 6 B cells from an immunized mammal, extracting mRNAs encoding immunoglobulins from the B cells, and reverse transcribing cDNAs of the mRNAs, thereby obtaining a mixture of the cDNAs;
b) using single, massive parallel sequencing technology to directly parallel sequence within 24 hours, without any prior cloning steps, the cDNAs encoding the variable heavy (VH) and variable light (VL) chains of the immunoglobulins from the sample, thereby obtaining sequence data of the VH and VL chains of the immunoglobulins present in the sample;
c) determining the frequency of the VH and VL chain sequences present in the sample by counting or statistical analysis,
d) identifying the most abundant VH and VL chain sequences present in the sample according to their frequency;
e) synthesizing polynucleotides encoding the most abundant VH and VL chain sequences present in the sample and producing antibodies comprising said VH and VL chains by cloning the polynucleotides into an expression vector, introducing the expression vector into a host cell and expressing the antibodies; and
f) identifying the antibody(ies) specific for a target of interest or identifying the target molecule to which the antibody(ies) is specific.
2. The method of claim 1 , wherein the sample is a biological sample.
3. The method of claim 1 , wherein said mammal is a human, murine, rodent, mouse, rat, squirrel, chipmunk, gopher, porcupine, beaver, hamster, gerbil, guinea pig, rabbit, dog, cat, cow, or horse.
4. The method of claim 3 , wherein said human is a cancer patient, said human is immunized or said human is infected with an agent or a target molecule.
5. The method of claim 1 , wherein the mRNAs encoding immunoglobulins are obtained by harvesting or isolating B-cells from the sample.
6. The method of claim 1 , wherein the immunoglobulins are of the class IgG.
7. The method of claim 1 , wherein step (a) further comprises amplifying the mixture of cDNAs of the mRNAs encoding immunoglobulins by reverse transcriptase-PCR.
8. The method of claim 7 , wherein IgG specific primers are used.
9. The method of claim 1 , wherein said expression vector is a mammalian expression vector.
10. The method of claim 1 , wherein the antibodies produced in step e) are expressed into a culture medium.
11. The method of claim 1 , wherein the identifying the antibodies specific for a target of interest in step f) is carried out by immunoassay.
12. The method of claim 1 , wherein the obtaining of step a) comprises the steps of:
aa) providing a biological sample from a mammal that is immunized, infected with an agent or a target molecule or suffers from cancer;
ab) harvesting B-cells from the biological sample;
ac) obtaining mRNAs from the harvested B-cells;
ad) generating cDNAs of the immunoglobulins encoded by the mRNA, thereby obtaining a mixture of the cDNAs; and wherein step e) further comprises the steps of:
da) integrating the synthesized VH and VL polynucleotides into a mammalian expression vector and introducing the expression vector into a mammalian host cell; and
db) allowing the host cells to express the VH and VL polynucleotides, thereby producing antibodies.