This is Book O, beginning on 19– We don’t have a da‑‑ a year. But it’s November 11th, Monday. And so the year would be whatever follows the one before.
I did find that sometimes I didn’t have the years marked, and had to go back and try to deduce what year they were.
L was ’62. M was ’63.
Sixty-three. N — [00:01:00] this is ’63, April. It’s still ’63. N is still ’63. O must be ’63, then. The beginning is ’63. Right. OK. So this is… Oh. You can start it, Jenny.
Is it running? So this is the beginning of Book O, starting at the end of 1963, November 11th, a Monday — was a picture of a high-voltage electrophoresis setup — d‑‑ a result that doesn’t look particularly good. [00:02:00] Some more tests of the same thing, with tris-borate-Versene — BV gel.
Starting again with another Bence Jones protein, on Tuesday, November 12th, [Gasthof?] Bence Jones. And some results of comments on where it came from. So prepared the Bence Jones in the usual way. And got 884 — I think probably milligrams. Oh, yes. No. It’s [00:03:00] 0.884 grams of Gasthof was prepared– and point-oh‑‑ and 5.2 grams of a — of something. Not clear. Gasthof, anyway, had 884 milligram. And the earlier fractions are 0.8 gram‑‑ 0.88 grams and the later fractions 5.2 grams, so enormous amount of protein obtained from these urine.
pH 5. We’re getting back to dithiothreitol, good territory. Dithiothreitol tests on [00:04:00] pH 5. But looking at the result, it says, “These results suggest, as before, that free thiol is essential for resynthesis…” Because this was dithiothreitol plus Factor B, and various amounts of dithiothreitol. So let me go back to that again and start again. So on pH — and page nine, Friday, November 15. tests are being made of pH 5 plus or minus dithiodiglycol, starting with 5 milligrams of 2‑2, pH 8.5, and reducing with mercaptoethanol. And then Sephadex the material, to get rid of the [00:05:00] thiol. And then the thaw‑‑ the Sephadex material was thawed and dispensed into different fractions, and then dithiodiglycol added. And looking at the results, it’s clear that there is resynthesis in all of the fractions, except the 0.5-molar dithiodiglycol. But the comment is, “These results suggest, as before, that free thiol is essential for resynthesis but that SS is inhibitory,” with a ques‑‑ with a, in parentheses, question, “(mixed disulfide?). Therefore arrange anaerobic removal of SH at pH 5 [00:06:00] by dialysis on the nitrogen.” So I am beginning to get to the right answer — but not yet.
So Thursday, November 19th, dialy‑‑ plus or minus dithi‑‑ SS. And dithiodiglycol, that is. So this is a test of having sample — with a control sample, a sample with n‑‑ bubbled or in the presence of nitrogen only or nitrogen plus dithiodiglycol. And with the comment that “The synthesis is worse than usual in this way.” [00:07:00] Because neither of the samples that had been treated have very much resynthesis of the polymers. They have some but not much.
Reviewing the material again, Thursday, November 22nd. Every now and then, important, I found, to review my past result. Thursday, November 22nd, nitrogen, pH 7, decreasing mercaptoethanol tests. And so here is the result, with the comment that “Resynthesis is about as expected. On the hypothesis on the next page, this method gives very clean monomer.” So let’s see what the hypothesis is on the next page. The monomer was made by splitting 3.5 milligrams of haptoglobin 2‑2 in a phosphate buffer, with 0.015-molar mercaptoethanol, two hours at 37 — at 37, then Sephadex, to nitrogen, bubbled, 0.03-molar mercaptoethanol. So there is mercaptoethanol present. And then dialyzed against nitrogen, bubbled, a phosphate buffer. The final concentration of mercaptoeth‑‑ will be less than point-three zeroes-one-molar. [00:09:00] So these are attempts at resynthesis — not very good.
Friday, November 22nd, tests of equilibrium. Interpretation of the results. But talking only about mercaptoethanol, not about dithiodiglycol. So test results, Saturday, November 23rd. All the samples look about the same. Thirty-seven degrees, room temperature, or cold-room. No significant differences. [00:10:00] “Repeat at pH 8.5.” pH tests on Tuesday, November 26th. No differences. Going to back to sample insertions on different gels, and some starch tests, on November 27. High voltages again, November 29th and following. (pause)
Here there are some tests done in conjunction with [00:11:00] Ed [Aisen?], tests of [haptins?] in stroma, December 12th. “Ed Aisen has observed that” point-oh‑‑ “0.1-molar borate plus mercaptoethanol alone, without urea, dissolves stroma.” Etc. So some attempts to help Ed Aisen. And high-voltage tests, on page 41, very unimpressive at this point. Further hemolysis observation, page 43. Nothing very significant here. [00:12:00] Trying to get stroma, on Thursday, December 19th, page 49. Cleanup of wet — distilled water — hemolyzed stroma.
This maybe is the beginning of work that went on to develop a very sensitive method of detecting antibodies. Because here is a Brucella abortus standard plate antigen, which I know was later used. Centrifuged, etc., etc., tested with rabbits. [00:13:00] Anti-Brucella serum were obtained from [name redacted], on Monday, December 23rd. And a [devenation?] was tested in — at a 96‑well plate, looking for agglutination, with these — with the antiserum. So this later became a very powerful method in our hands — but not yet. This is the beginning of the tests. More examples on Tuesday, December 24, of looking for agglutination, with different amounts of antibody. [00:14:00] (pause) Put up the plates. (pause) Continuing on page 61.
Back to starch gels again and fat stains, on Thursday, December 26th. And [00:15:00] there is tests with lipoprotein stains — looking for beta-lipoproteins. Lipid pre-staining. Made up of propylene glycol, and a series of dyes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 — 12 different dyes — and for pre-staining the sample. And a comment that “None as good as post-staining.” In a full-scale test, over the page, stained for — from about 12:00 noon to about 5:00 p.m., and then into 50% methyl [00:16:00] alcohol. This was, and this date. And, “Better stain overnight and allow it to harden.” And so drop… And the images on the following page — pages — several pages of images. High-voltage again, on page 73, much better high-voltage gels than some of the earlier ones, a tris-borate-Versene gel.
Test of the rabbit antisera — anti-Brucella sera again. [00:17:00] [00:18:00] Attempted — Wednesday, January 8th, page 89, attempt to culture samples from lymph nodes, contr‑‑ lymph nodes, control lymph node and a lymph node from an injected animal, probably ten times greater size than the non-immune one. And set up in the CO2 incubator. And then medium was taken, presumably. (pause) [00:19:00] (pause) Long list of titrations, on Thursday, January 9th. So… The chart is there but no entries into it. More plates of this general type, pages and pages of tests with different ant‑‑ with different sera. [00:20:00] Continuing through page 103. More samples again, Thursday, January 16. January 17th — 18.
Aha. Beginning to understand what eventually became — or beginning to invent what eventually became the method of — that increases sensitivity very greatly. If we look [00:21:00] at the results, different pages here, it may become obvious that this was an idea that was developing. Because on Thursday — on Friday, January 17th, and Saturday, January 18th, there are two plates of various titrations. And the one plate is — all of the samples are clearly agglutinated or not agglutinated at the bottom of the well, whereas, on the right-hand side, the samples have been tilted in some way, so that all of the samples are sort of spread out in a — in a little diagonal line. But no comment on this. [00:22:00] And again, on Friday, January 17, the — this now became deliberate. Because the top is a photograph of the wells without doing anything and then on the lower photograph is, “Remixed and left to check” and then tilted, deliberately tilted, to obtain a result. And the — you can see that the samples roll down the side of the wells. Not yet obvious that this is going to improve the sensitivity — but see that the idea is developing. Because on page [00:23:00] 115, Monday, January 20th, there are various things of tilting and re‑tilting the samples. But… It says, for example, on page 115, “Photographic differentiation is seen by tilting. Near vertical the clearest differentiation is seen.” By tilting the sample, one can tell whether agglutination had occurred versus whether it had not occurred — that a sample that, had not occurred looks different. But yet not fully worked out. And not done yet uniformly, as shown on page [00:24:00] 119. (long pause) A little bit of an indication that the type of bottom of the well was important is that, on page 127, which is a summary of the results, it comments that it’s a [00:25:00] V‑bottomed well. (pause)
Another Bence Jones protein. This one migrates backwards, apparently — Hewlett Way Bence Jones. And a tris-borate-Versene gel, it appears to be, going backwards. Two gallons of urine [00:26:00] obtained — received and 5.24 grams of material were obtained. And on page 117 — 147, 3.513 gram of Hewlett Bence Jones protein — of [Hewlett Wang?] — first class. This is the material that looks to go backwards, in an ordinary gel.
And book ends on Monday, March 23rd. Find out where this [00:27:00] comes from, again.
OK.[00:27:02-00:58:45] (recorder left running)