This is a convenient way to estimate antibody concentrations roughly.
Unless something is known about the strength of the serum it is usually necessary first to locate the optimal proportions point approximately by a rough test. The original technic of Dean and Webb (27) has been modified by a number of workers; the technic described will be given here.
Prepare in Wassermann tubes serial dilutions (successively 1:2 or 1:3) of the antigen, starting with a solution of known concentration. Either make these dilutions so as to leave 0.5ml. in each tube, or transfer 0.5 ml. of each to a series of tubes as the dilutions are being made. Dilute enough serum for the rough test 1:5 or 1:10, depending on its probable strength. Have ready a little rack of small tubes of 1 ml. capacity in a water bath at 37 degrees C. (or if greater speed is desired, at 40 degrees or 45 degrees C.), each tube being one-third immersed in the water. With a 1-ml. pipet add 0.5 ml. of the diluted serum to the highest dilution of the antigen, immediately mix by brief shaking, then immediately remove the mixture from the original tube and transfer to one of the 1-ml. tubes, using a capillary pipet with a long, not too fine tip. This insures thorough mixing. Replace the time, to the nearest quarter-minute. Proceed similarly with the other antigen dilutions, using the same capillary pipet each time, and going from the higher dilutions to the lower (from weak to strong antigen).
Observe the tubes continuously. Cloudiness will begin to appear in the tubes which are going to flooculate, usually within a few minutes. Watch for the point at which individual particles just become visible to the unaided eye (this point varies with different observers, but is more or less consistent for anyone individual). Record the time of participation in each tube, until four or more tubes have flocculated, or it is clear they are not going to.
The diluted antigen and saline are mixed first, then the diluted serum added to one tube at a time, beginning at the right; the time each mixture is placed in the water bath is recorded. The time of particulation is similarly recorded for each tube.
The optimal proportions point may be expected to lie somewhere between the values indicated by the two most rapid tubes. From the antigen and antibody dilutions present in these tubes, it may be calculated what would be a suitable dilution of antigen and antibody for the "fine test." The serum dilution should be such that flocculation at the optimum may be expected to take place in 5 to 10 minutes. Make sufficient diluted antigen and diluted serum for the following procedure.
In each of a series of Wassermann tubes place the amounts of the antigen dilution shown in the table, and add the indicated volumes of saline to bring the total in each case to 0.5 ml. Then add to each tube in turn 0.5 ml. of the diluted serum, beginning with the weakest antigen, and mixing, transferring, and timing as before. If the dilutions used were the correct ones, one tube, not at either end of the series, will show most rapid flocculation, or occasionally two or more tubes will run a "dead heat." If the fourth tube from the left in the table, for instance, was most rapid, when we used a 1 :400 dilution of stock antigen and a 1:10 dilution of antiserum, we should calculate that the optimal ratio of antigen to serum dilution was 0.5/0.3 X 400/10 = 66.7.
From a knowledge of the actual concentration of the stock antigen, it may be calculated how many milligrams of antigen, or antigen nitrogen, react optimally with 1 ml. of serum. For example, if in the above case, where we find an optimal ratio of 66.7, we had started with a stock solution containing 1.25 mg. of protein nitrogen per milliliter, we should calculate that 1 ml. of antiserum reacts optimally with 1/66.7 X 1.25 = 0.019 mg. of protein nitrogen. Or conversely, that to react with 1 mg. of antigen nitrogen, 66.7/1.25 = 53.4 ml. of serum would be needed
ON THE MECHANISM OF SPECIFIC PRECIPITATION
William C. Boyd Ph.D.
The Journal of Experimental Medicine, Vol 75, 407-419, Copyright, 1942, by The Rockefeller Institute for Medical Research New York
From Evans Memorial, Massachusetts Memorial Hospitals, and Boston University School of Medicine, Boston
- A study of the precipitability by the appropriate antisera of 34 different haptens, containing from one to six reactive groups, leads to the conclusion that the possibility of framework ("lattice") formation is neither necessary nor sufficient for specific precipitation, but that instead precipitation depends upon the reduction, by mutual neutralization of polar groups of antibody and antigen (or hapten) and mechanical blocking off of polar groups of closely neighboring molecules of antibody, of the solubility of the complex below the point at which it can remain in solution. The decisive factors appear to be the number of polar groups of the antigen (hapten) left free, and the distance separating the different reactive groups, which determines the amount of steric hindrance exerted by one antibody molecule on another. No hypothesis is offered as to how these primary insoluble aggregates unite with each other to produce the larger aggregates which are finally observed.
Submitted on January 20, 1942
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THE COMPOSITION OF SPECIFIC PRECIPITATES IN THE REGION OF ANTIGEN EXCESS
Saul Malkiel 1 and William C. Boyd Ph.D.
The Journal of Experimental Medicine, Vol 66, 383-396, Copyright, 1937, by The Rockefeller Institute for Medical Research New York
From Boston University School of Medicine, and Evans Memorial, Massachusetts Memorial Hospitals, Boston
Data are given, for seven different antisera, for the composition of the specific precipitate as a function of the relative proportions of antiserum and antigen used. In the region of antigen excess, a linear relation is found between the ratio of antibody to antigen in the precipitate and the amount of antiserum used. The significance of these results, particularly in their bearing on theories of the precipitin reaction, is discussed.
Submitted on May 27, 1937