Our project on the Atlantic cod, Gadus morhua, received funding in the Fall of 1996 and I am reporting the progress made toward its goals in its fifth quarter.

Progress Outline:

1. Ovarian Yolk Protein Purification Revised: The former purified Lipovitellin has been further purified.
2. Antiserum Evaluation Has Commenced: Analysis of suitability of the antiserum for different analytical techniques was begun.
3. Undergraduate Trainee Refunded under Howard Hughes program.
4. Lab personnel scheduled to participate in three NOAA Bottom Survey Cruises.

1. Ovarian Yolk Protein Purification Revised: We had purified the major egg Lipovitellin (Lv) of the cod fish using a combination of fractional heat denaturation, Ammonium Sulfate salting-out and gel-permeation chromatography. This utilizes a technique, of fractional heat denaturation, which was discovered in a previous CMER/NOAA project on Winter flounder (Hartling et al., 1997). Purifying the Lv from the ovary, instead of ovulated eggs, however resulted in bringing over some ovarian proteins into the provisionally purified Lv. This was only discovered with production of the antiserum and refinements in the analysis system. We have therefore added a TEAE Cellulose purification step which has removed additional impurities in from the Lv preparation. We will be immunizing another rabbit with this further purified protein.
2. Antiserum Evaluation Has Commenced: The first antiserum was harvested and evaluated after its 2^nd boost with heat denatured and gel-filtration purified Lv (dn,gf-Lv). We see multiple precipitin reaction products with partially purified Lv (i.e. dn,gf-Lv) when tested by Ouchterlony or Rocket Immunoelectrophoresis. We also have some minor responsiveness from male serum. This suggests that this antiserum as it stands would not be able to be used in our projected ELISA test. We may be able to adsorb this antiserum with male cod serum to make it female specific. We are also proceding to use this antiserum for Rocket IEP which can tolerate the impurities observed and give useful results.
3. Undergraduate Trainee Funded under Howard Hughes program: Mike Pelak our senior undergraduate biology major has received further funding from the Hughes grant administrators to participate with the cod project in the Spring semester.
4. Lab personell scheduled to participate in three NOAA Bottom Survey Cruises: The lab personnel involved in the cod project have been scheduled to take three trips, with two week gaps between each trip, on NOAA bottom surveys of Georges Bank. We will collect cod serum, mucous and traditional size statistics (total weight, GSI) for analysis in the laboratory. John Bohannon will be taking the first trip starting Feb. 17 and the PI, Joe Kunkel, will be taking the third trip starting April 13.

While we are somewhat behind schedule due to complications in the Lv purification process, we now have antisera which react with our tentatively purified Lv protein and which are helping us in reaching further degrees of purification. This will ultimately provide us with the specific antiserum which we need. We are also pushing on to the phase which will allow us to ask if the Lv antigen can be detected in the body surface mucous of reproductive female cod. This can be established using our current antisera and the fresh mucous samples we collect on Georges Bank.

Respectfully submitted,

Joseph G. Kunkel

jgk/hs