Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes

Welschof, Martin; Terness, Peter; Frank, Konstantin; Zewe, M.; Dübel, Stefan; Dörsam, Heinz; Hain, Carsten; Finger, Martina; Jung, Monika; Moldenhauer, G; Hayashi, Nakanobu; Little, Melvyn; Opelz, Gerhard

doi:10.1016/0022-1759(94)00286-6

Cited by 81 publications

(54 citation statements)

References 26 publications

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“…5). This pattern is similar to that of 42 previously characterized sequences of 51p1-encoded H chains expressed by tonsillar and other nonmalignant B cells (8,12,25,(27)(28)(29)(30)(31)(32). Of this group, none used J H 1, one (2%) used J H 2, four (10%) used J H 3, 15 (36%) used J H 4, seven (17%) used J H 5, and 15 (36%) used J H 6 in 51p1 rearrangements that encode Abs of unknown specificity.…”

Section: Sequence Analysis Of 51p1 Expressed In Normal Blood B Cellssupporting

confidence: 86%

“…It is also similar to the previously reported mean CDR3 length of 13 51p1-encoded G6-reactive IgH (13.5 Ϯ5.5) isolated from human tonsillar B cells (12). Of note, there is no significant difference between the calculated mean CDR3 length of this group of sequences derived from blood B cells compared with the mean CDR3 length of 42 rearranged 51p1 genes expressed by nonmalignant B cells reported in the literature (13.6 Ϯ 4.0) (8,12,25,(27)(28)(29)(30)(31)(32), or with 146 Ig expressed by random blood B cells of two normal adults (13.9 Ϯ 4.2) (25,26).…”

Section: Discussionmentioning

confidence: 92%

“…D segments D3-22/D21-9 and D6-13/DN1 are members of these two families, respectively, and are two of the more frequent D gene segments used by blood B cells of normal adults (25,26). Moreover, the distribution of D gene segment use by blood B cells that use 51p1 is similar to that of other non-neoplastic B cells that express 51p1 (8,12,25,(27)(28)(29)(30)(31)(32) or other V H genes (25,26).…”

Section: Sequence Analysis Of 51p1 Expressed In Normal Blood B Cellsmentioning

confidence: 87%

“…As such, these B cells may be predisposed to leukemogenesis and may represent the true precursors of the CLL B cell. ,, ,, , 51 sequences reported in GenBank or the literature of CLL samples that express 51p1-encoded H chains (6,7,14,(17)(18)(19)(20)(21)(22)(23)(24); C , , 39 sequences reported in GenBank or the literature of CLL samples that express H chains encoded by V H 1 genes other than 51p1 (7,14,20,22,23); D 1, 146 random Ig V H rearrangements reported in the literature (25,26); E _, 42 rearranged Ig 51p1 genes expressed by nonneoplastic B cells (8,12,25,(27)(28)(29)(30)(31)(32).…”

Section: Discussionmentioning

confidence: 99%

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Normal B Cells Express51p1-Encoded Ig Heavy Chains That Are Distinct From Those Expressed by Chronic Lymphocytic Leukemia B Cells

Widhopf

Kipps

2001

The Journal of Immunology

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51p1 is an allele of VH1-69 that frequently is expressed by chronic lymphocytic leukemia (CLL) B cells with little or no somatic mutation. The rearranged 51p1 genes expressed by CLL B cells have a distinctive use of D segments D3-3/DXP4 and D3-10/DXP′1, a favored use of JH6, and a longer third complementarity-determining region than the rearranged Ig genes used by CLL B cells that express VH1 genes other than VH1-69. We examined the 51p1-encoded Ig expressed by blood B cells of healthy donors. In contrast to the infrequent use of JH4 by 51p1-expressing CLL (e.g., 4%), 36% of the rearranged 51p1 sequences from normal blood B cells used JH4. Furthermore, the D segment use of the rearranged 51p1 sequences from normal blood B cells was not restricted, but reflected the D segment use of nonselected IgH of normal B cells. Finally, the mean length of the third complementarity-determining region for the 51p1 genes of normal blood B cells was 14.6 ± 4.3 (SD) codons. This is significantly shorter than that noted for 51p1-expressing CLL B cells (18.8 ± 3.2; p < 0.0001, n = 51). This study demonstrates that the 51p1-encoded IgH expressed in CLL are not representative of the 51p1-encoded IgH expressed by normal blood B cells, indicating that CLL B cells express IgH that are distinctive from those found in the normal adult blood B cell repertoire.

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Section: Sequence Analysis Of 51p1 Expressed In Normal Blood B Cellssupporting

confidence: 86%

Section: Discussionmentioning

confidence: 92%

Section: Sequence Analysis Of 51p1 Expressed In Normal Blood B Cellsmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Normal B Cells Express51p1-Encoded Ig Heavy Chains That Are Distinct From Those Expressed by Chronic Lymphocytic Leukemia B Cells

Widhopf

Kipps

2001

The Journal of Immunology

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“…22,24,39 For four of the NHL studied, the range in mutations (5-8%) is comparable to that observed in the rearranged IgL genes from normal B cells, 37,40 and in the rearranged IgH genes from the same NHL. 10 The 15% mutation frequency observed in the rearranged Ig gene from lymphoma 219 clearly falls outside the normal range.…”

Section: Discussionsupporting

confidence: 54%

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin’s lymphoma

et al. 1998

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Minimal residual disease (MRD) detection in B cell non-Hodgkin's lymphoma (NHL) patients has been shown to be possible using the rearranged heavy (IgH) chain gene as a tumor marker. To explore a second independent tumor marker, we used specific PCR primer sets to identify tumor-specific rearranged Ig light chain (IgL) genes. Rearranged IgL genes were amplified from lymphoma DNA by multiplex PCR using separate primer sets for the Ig and the Ig genes. They were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was isolated from at least two independent PCR products. From 12 out of 13 intermediate-and high-grade malignant NHL, PCR products could be obtained with IgL specific primers. PCR products from five NHL were studied in detail by cloning and sequencing. The rearranged IgL genes showed 85-100% homology with their closest germ line counterparts. Intraclonal IgL sequence heterogeneity was studied in five lymphomas and detected in only one. Minimal disease was studied in three patients by PCR, followed by Southern hybridization of the PCR product with a lymphoma-specific oligonucleotide probe, which allowed for detection of lymphoma DNA following 1000-fold dilution. Blood samples from one patient, who is in long-term clinical remission, were negative for the lymphoma-specific rearranged Ig gene. In the second patient the rearranged Ig gene was detected during the first clinical remission, that was followed by a nodal relapse, but not during the second remission, that has been stable for almost 3 years now. The third patient was negative for the rearranged Ig gene in blood samples up to 102 months after diagnosis. Circulating lymphoma cells were detected in blood and bone marrow samples which were negative by morphological and immunological criteria. Our studies show that the rearranged IgL gene can be used as a second independent tumor marker in intermediate-and high-grade malignant NHL.

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Bacterial expression of a human recombinant monoclonal antibody Fab fragment against hepatitis B surface antigen

et al. 1999

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The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301-CL4 cell line to reverse transcription-polymerase chain reaction, cloning the products in the plasmid vector pFab1-His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme-linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector-primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV-transformed cell lines.

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Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes

Cited by 81 publications

References 26 publications

Normal B Cells Express51p1-Encoded Ig Heavy Chains That Are Distinct From Those Expressed by Chronic Lymphocytic Leukemia B Cells

Normal B Cells Express51p1-Encoded Ig Heavy Chains That Are Distinct From Those Expressed by Chronic Lymphocytic Leukemia B Cells

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin’s lymphoma

Bacterial expression of a human recombinant monoclonal antibody Fab fragment against hepatitis B surface antigen

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