Oliver Smithies:

So this is the beginning of book P, which starts March 1964 and goes until July or thereabouts.  It’s mainly concerned with antigens and red cell membranes I think of various sorts.  So this starts with carbohydrase tests on RBC, red blood corpuscle, membranes, etc.  Carbohydrase mixture, which has got [MM?] dipeptidases in it and so on and so forth from Jack Strominger’s lab added to the ghost.  Tuesday 24th I’m concerned [00:01:00] about the Brucella abortus antigen, uncontrolled nature of [BrAb?] antigen suggested a need for specific group and detection system.  So [write?] retry dinitrobenzene and fluorescein on red cell ghost, etc.  So on page seven I decided to return to the [UV?] culture since the results are no better in glass and they’re more work.  And a test on Monday March 30th [00:02:00] page 11 says considerable difficulty in reading the sample perhaps because of phenol.  The high dilutions have not settled to a button in any of them.  Trying to get things to settle into the button which is needed to read them.  So this was one test.  But there are many and it’s nothing specifically important about it.

Continue with these tests with the various [grids?].  Titration procedures talked about on Wednesday April 1st page 21.  [00:03:00] Results show clearly that serum in the antigen well helps.  Pages of these titrations.  Plus or minus phenol in saline alone, no settling.  Greater than or equal to 0.5% fetal serum or greater than or equal to 0.1% BSA allows settling.  This is page 25.  Note unnecessary hazard to expose the antigen to any protein prior to test.  However, to ensure settling at the highest dilutions, protein must be used in the diluent.  [00:04:00] Use [0.1% — 30%?] BSA in the diluent except for antigen preparation.  Check carefully with other sera that have more suitable titration.  So page 25 continuing these tests.  Visual tests on page 29.  Viability tests with cells, 0.2% dye plus 4% serum, etc., etc.  Six-day sample on page 39 [00:05:00] says still all negative.  Consider failure to settle into button as a possible reason also for the failure.

Serum tests in dilution.  More comment of a U bottom versus a V bottom on page 52.  So realizing that maybe it matters whether you have a U or a V bottom.  Looking for cells that are [living?] from culture material.  [00:06:00] Evidently trying to culture the bacteria [00:07:00] at this point.  Thursday April 30th nonimmune animal plus or minus antigen, etc.  So I added as I call it bugs.  Added at this level 1/2,000 to bugs at 10 to -6 dilution.  Not at all clear.  But it’s a culturing cell.  So there are 80 in this particular experiment, (inaudible) it says 80,000 cells per culture, 66,000 bugs per culture.  So that this was both cells and the presumably brucella.  [00:08:00] Because this experiment does comment on page 61 that “decided to test nonimmune animal with antibody-coated antigen.”  Normal serum titration used is about 10% Brucella abortus suspension diluted 1/250.  But Thursday April 23rd we used antigen Brucella abortus diluted 10 to -6 and then 1/20.  That’s very much less antigen.

I see the problem.  [00:09:00] Friday May 1st gross bacterial contamination in the culture.  Failed by mistake to add penicillin so the bugs were there but they should have had penicillin added to prevent their growth.  But again why I’m doing these experiments is far from clear.  Rather confusing part of my scientific life of not really understanding why these experiments were being done.

Tuesday June 23rd [00:10:00] mixed antigen plaque formation.  Aim to obtain double antibody production by a single cell and to establish transfer from one animal to another when singly injected and transferred after stimulation.  Talking about [Journey’s?] cell-bound antibodies [emit?] phenol red in the future.  So thinking about culturing things.  It’s beginning to make a little more sense.  Wednesday June 24th page 79 125 milligrams spleen from one of the [tries?] isolated and cut.  Smooth suspension easily obtained in Eagle’s medium and then plates were used [00:11:00] 0.1 milliliter used per plate.  Three plates.  Looking for things to grow in culture.  Perhaps this was the time when I was working with trying to get antibody production in cells that were dividing, which was with Alice Claflin.  Monday June 29th, Saturday.  Two mouse.  Two DNA.  Two day.  In parentheses whole spleen.  [00:12:00] In 0.5 ml of Eagle’s and plates made with 0.1 ml plus sheep cells, etc., etc.  So trying to culture in the presence of red cells.  [00:13:00] You OK?


I’m fine.


Monday July 6th test of cattle [mix?] and sheep.  Guinea pig complement.  This was with guinea pig complement.  Guinea pig complement and sheep cells.  Guinea pig complement and cattle cells.  Guinea.  GP complement 50-50 cattle and sheep.  And rabbit complement and cattle cells.  Etc.  [00:14:00] With a comment on the opposite page that the guinea pig complement is much better.  Rabbit complement gives fewer and much smaller plaques.  So I’m trying to get plaques in plates.  I now remember what it is.  We’ve got sheep red blood cells.  Or we got red blood cells in agar.  Suspended in a plate.  And then the antibody or the spleen cells are then placed and looking for hemolysis of the red cells.  Looking for plaques in the red cell.  This is the beginning of the work.  Or not the beginning.  Quite far into the work with Alice Claflin to find antibody-producing cells that were dividing.  So I’ll go back and talk about this again.

[00:15:00] So this is probably also in Book O but not understood.  [00:16:00] So Tuesday July 7th there is a rather clearer diagram of what was going on.  Page 103.  Test for cross-reactivity.  Antisheep and anticattle spleens were tested.  These are spleens from animals that have been immunized with sheep red blood cells or with bovine red blood cells in a standard — page 99 system is the one with the complement and the cells.  [00:17:00] So going back to that.  I’ll go back and start again from page 99.  Page 99 book P, Monday July 6th is a test of the ability of different spleen cells to lyse red cells of the — that were used for preimmunization.  So this is an example of this onto a plastic dish, 4 drops of 1% agar and 1 drop of 5 times Eagle’s so that the solution — so that the agar is 1x Eagle’s medium.  And then add [00:18:00] 2 drops of 50% cells, that’s of the red cells, the antigen, 4 drops of guinea pig serum, that’s complement, and 1 drop of spleen cells.  And then mixed and onto a plastic dish 1 drop.  And covered with cover slip, 25-by-25-millimeter no. 1 cover slip.  Direct from the box.  And then look down at the results.

And so this was a test of 25-by-25 millimeter plate.  One was with guinea pig complement and sheep red cell with one — another was guinea pig complement with cattle cell.  Bovine red cell.  And the other was guinea pig complement with 50-50 cattle and sheep.  And the other was rabbit complement with cattle cell.

And the [00:19:00] answer is that rabbit complement with cattle cell gave a positive.  In other words one could see that there was some production of hemolytic plaques.  But very — positive.  But very small plaques.  And so the comment is that guinea pig — yeah.  I interpreted that slightly wrongly.

That the guinea pig complement is much better than the rabbit complement.  Gives fewer and much smaller plaques.  And the sheep red blood cells cross-react with the anticattle to about 2% level.  So it’s a test of looking for plaques in a [00:20:00] suspension of red cells that’s in agar underneath microscope slides.

So page 101 is another test of sheep, cattle, and with different complement.  Guinea pig serum or rabbit serum.  And sheep red cells and a mixture of sheep and cattle red cells.  And the result is that the antisheep definitely cross-reacts with the cattle cell.  The mix shows both 100% and much greater number at 50% plaques.  Some of the antisheep and — with cattle cells are 100% but some are not.  In other words [00:21:00] some cells are producing an antibody that cross-reacts.  And some of the spleen cells are producing antibody that is [cell-specific?].  As commented on page 100.  Antimix animal.  That’s an animal that’s received a mixture.  The spleens from an animal that received a mixture of the cells.  Responded poorly to the sheep cell.  Therefore the antimix with the mix about 99% of them are 50% plaques.  The cattle and sheep versus antimix are much nearer to 100%.  In other words the plaques are not completely hemolyzed.  The sheep cells may be hemolyzed or the bovine cells may be hemolyzed or they [00:22:00] both may be hemolyzed in the mixtures.  And so this is quite a nice way of looking at things underneath these plaques.  And so it’s making sense now.

Page 103 July 7th tests of cross-reactivity we carried out there with some little changes in the procedure.  Now with the general antibody that antisheep and anticow do not cross-react at all with human or rat cells.  Antisheep versus cow is approximately equivalent to anticow versus sheep, etc., etc.  So looking for reactions.  On page 105 is the first photograph of the plaques.  Really quite nice to see that one can see it.  [00:23:00] So anticow with cow.  On the upper image.  And the plaques are either — some plaques are bigger than others.  But they’re 100% hemolyzed.  Whereas anticow with human and cow mixture, one can see the plaques are there but they’re obviously only half hemolyzed or even less.

Very clear imaging of the plaques.  And again on page 107 anticow and cow and human cells, etc.  And note that there were rouleaux.  There was rouleau formation in the plaque area.  Goat and ox are spherical cells.  [00:24:00] Attempts to prevent the rouleaux on the following day.  Low molecular weight dextran might work.  Test for bubble the following page.  Eagle’s, [stock?] solutions, different solutions.  Adding various things to Eagle’s.

Page 117 decided to change to Eagle’s suspension medium 10 times phosphate and no calcium in order to get better buffering.  The decreased calcium will also help prevent clumping of the cell.  And so a couple of solutions are prepared and tested.  And the results are [00:25:00] shown on page 120 and page 121 where images are not quite such high power as previously.  Not very clear whether the images are plaques or air bubbles.  It looks like they might be real plaques.

Because it comments on page 121.  About 10 to 20 plaques of good clarity.  Around 124 [00:26:00] two times count on good two plates including tiny including tiny low percentage plaques, etc.  So they are images of plaque.  And just slightly different way of looking at them.

More images on July 16th.  [00:27:00] For example of Thursday July 16th page 125 about 120 plaques of varying sizes and clarity on plate number one, none very big, but many reasonable.  And plate two 114, plate three about 61, and plate four about 115.  So quite straightforward count.  And a large collection of images of plates visualized on page 126.

[00:28:00] Where it’s clear that the plaques are visualized as white on dark.  Not dark on white.  They look to be clear [holes?] in this way of imaging them.  Friday July 17th it’s quite easy to see what was going on.  Thinking about using a hapten that could be bound to red cells.  Dinitrobenzene sulfonate was one possibility.  Or [pickerel electro lace picker base not?] it’s [00:29:00] [pickerel sulfonic acid to mix with pick wick sulfonide?].

Nice set of images on Monday July 20th.  It’s the same image at different times.  So test set up at room temperature and to 37 degrees at 12:31 and then images taken all the way up to an hour later.  And that was the same area.  And one can see a plaque beginning to show.  A white plaque gets bigger and bigger and bigger.  [00:30:00] And then other plaques appear.  So that by the time it’s at the end of the experiment there are one, two, three, four, five, six, seven, eight.  There are at least eight plaques there that can be seen very nicely.  It’s working very well Monday July 20th page 131.  Shows that the procedure was working.

Labeling gamma globulin with fluorescein isothiocyanate.  Labeling red cells as we go to the end of this book, different [00:31:00] haptens.  Back to [dispersing?] nuclei again on page 147.  With a two-phase system.  And ending this book on page 151 Monday August 10th.  Testing high voltage gel at 1,200 volts.  Air-cooled.  Zones badly curved due to early overheat, [00:32:00] etc., etc. (inaudible) 4:00 p.m. electrophoresis was backwards, whatever that means.  And that’s the end of book P.