# INTERACTIONS OF IMMUNOGLOBULINS

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 1990 · $147,071

## Abstract

This project is concerned with two aspects of the genetic and
structural determinants of the affinity of immunoglobulins.  The
first is directed to the B cell response to synthetic peptides as
epitopes in terms of the variety and selectivity of germline
variable region genes used by anti-peptide antibodies.
Hybridomas will be prepared which produce IgM and IgG
antibodies.  These will be purified and their intrinsic binding
constants for a fluorescent conjugate of the homologous peptide
will be measured by resonance energy transfer.  This analysis will
establish if affinity-restriction of IgM antibodies occurs in the
case of peptide antigens.  A comparison will also be made
between linear and cyclic peptides with respect to
immunogeneicity and affinity.  Selected IgM-producing
hybridomas will be subject to nucleotide sequencing to identify
the germline VH and VL genes used for anti-peptide reactivity.

The second aspect is directed to the use of site-directed
mutagenesis to enhance the affinity of antibody.  A cloned murine
VH gene from one anti-dansyl hybridoma producing IgG of high
affinity will be incorporated into a high-efficiency expression
vector by recombinant technology.  This plasmid construct will be
used to transform E. coli under conditions that yield the VH
product at a level of 10-20% of total bacterial protein.  The VH
product in the bacterial extract will be identified by Western
blotting with a rabbit anti-serum to a FR2 sequence of the VH
protein.  After specific purification the VH product will be
evaluated for binding affinity to dansyllysine either as a VH
protein or after recombination with an appropriate L chain.  Site-
directed mutagenesis will be effected using a primer
corresponding to the CDR3 region with nucleotide substitutions
designed to provide selected amino-acid replacements.

## Key facts

- **NIH application ID:** 3124567
- **Project number:** 5R01AI009492-30
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **SAM.gov UEI:** GM1XX56LEP58
- **Principal Investigator:** FRED KARUSH
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1990
- **Award amount:** $147,071
- **Award type:** 5
- **Project period:** 1975-04-01 → 1992-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3124567

## Citation

> US National Institutes of Health, RePORTER application 3124567, INTERACTIONS OF IMMUNOGLOBULINS (5R01AI009492-30). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3124567. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*