Quantitative precipitin reaction

Quantitative precipitin tests were performed as described.73 The amounts of precipitates obtained at the various concentrations were measured by the protein phenol method.74 The inhibition values are recorded in Table I. These inhibition data show that the precipitin reaction between the tetrahet-eropolysaccharide and the anti-GlcA antibodies is strongly inhibited by d-glucuronic acid, but with the exception of galacturonic acid none of the other carbohydrates were inhibitory. The data in the table also show that the carbohydrates tested did not inhibit the precipitin reaction between the polysaccharide and anti-GlcA-Rha antibodies. The monosaccharides alone cannot completely fit the active site of this antibody to give a precipitin test. 

Most, if not all, contemporary applications of immunochemical techniques are based on the reaction of antibodies with the antigen used to induce their production, to yield a very stable complex. If both antigen and antibody are present in solution, a precipitate forms as long as the antibody is in molar excess. This is known as a precipitin reaction and may be quantitated as shown in Figure 8-15. For this example a highly purified preparation of avidin was injected into a rabbit which then produced avidin-specific antibodies. Since biotin forms a very stable complex (dissociation constant with avidin, complexation of... 

Fig. 5. Quantitative precipitin reaction between crystalline hen egg albumin (Ea) and rabbit antibody (Ab) to crystalline hen egg albumin. From Heidelberger and Kendall. ...

Fig. 18 (68). Comparison of immunological properties of human cuprein isolated from erythrocytes (O), liver (%i), and brain(B). Quantitative precipitin reactions of rabbit antibody to erythrocuprein were performed...

Immunochemical methods. The quantitative precipitin reaction was carried out according to the previous description ( 5) as follows To 0.1ml of antiserum in a small test tube (1.6 by 10.4cm) was added 0.5ml of saline solution of serial amounts of the mannan. After incubation at 37 C for Ih, the mixture was allowed to stand at 4 C for 16h and then centrifuged at 2,500rpm... 

Figure 3. Quantitative precipitin reactions between the mannan subfractions (Batch I) and antisera of three C. albicans strains and S. cerevisiae (A) anti-C. albicans NIH A-207 (A-strain) serum (B) anti-C. albicans NIH B-792 (B-strain) serum (C) anti-C. albicans J-1012 (J-strain) serum (D) anti-S. cerevisiae serum. Mannan subfractions are of (O) A-strain (0) B-strain (A) J-strain (A)...

Figure 22. Quantitative precipitin reactions with antisera (489) to fragment 377-571. A Antiserum from the pool 7- to 10-week bleedings. B Antiserum from the 11- to 13-week pool. O. Fragment 377-571 , BSA A, firagment 11-193. From Habeeb and Atassi (1977).

Table VII. Quantitative Precipitin Reactions with Antisera to Fragment 377-571 ... 

From Habeeb and Atassi (1977). Values are given in percent quantitative precipitin reaction at equivalence relative to reaction of fragment 377-571 as 100%. 

This represents the amount and reactivity of the antibody fraction that was displaced from the immunoadsorbent by 5 M guanidine hydrochloride. After dialysis against 0.01 M phosphate buffer at pH 7.4 containing 0.15 M NaCl, its quantitative precipitin reactions with BSA and fragment 377-571 were determined. 

The immunochemical cross-reactivity revealed by quantitative precipitin reaction of 22 primate serum albumins with anti-HSA was consistent with the presumed phylogenetic relationships to man on the assumption that the evolutionary modification of protein structure has been... 

GSA. A high proportion of anti-BSA—151%—was adsorbed by ESA immunoadsorbent, 66.4% by Sepharose-PSA, and 39% by Sepharose-HSA. The cross-reacting antibodies were found to precipitate more with BSA than with the homologous albumin that is in the immunoadsorbent. The high cross-reactivity shown by immunoadsorbent is the result of the presence of high proportions of nonprecipitating antibodies which es-c q>ed detection in the quantitative precipitin reaction and were trapped by the immunoadsorbent. Therefore, in determination of cross-reactivity, special precaution has to be exercised to account for the presence of nonprecipitating antibodies. 

By the quantitative study of precipitin reactions Heidelberger and Kendall" reached the same conclusion, and showed in addition that even after prolonged immunization the antiserum studied (anti-egg albumin) contained much low-grade antibody, incapable by itself of forming a precipitate with the antigen, but with the property of being carried down in the precipitate formed with a more reactive fraction."... 

Somewhat similar quantitative theories of the precipitin reaction have been published by A4. Heidelberger and F. B. Kendall, J. Bxfitl. Mfd., 61, 5G3, 62, 467, 697 (1935) 66, 229 (1937) P. E. Kendall, Annals N, V. Acad. Sc4., 163,85 (.1942 and A. D. Hershey, J. Immunol., 42, 465 (1941). These theories are designed to apply more broadly than ours, which is based on postulates suited to the special antigens and haptens which we are studying. 

L.C. Wetter, M.Cohn and H.F.Deutsch, Immunological Studies of Egg-white Proteins. II. Resolution of the Quantitative Precipitin Reaction between Chicken Egg-white and Rabbit Anti-egg-white Serum in Terms of the Reactions of Purified Egg-white Proteins, J. Immun. 69, 109-115 (1952). 

Kabat (251) was able to show by immunochemical methods that the main component of the serum of case 11 (Table VII) was, in fact, a Bence-Jones protein. Rabbit antiserum to this patient s urinary Bence-Jones protein gave a strong precipitin reaction with a 1 625 dilution of the patient s serum. This reaction appeared to be due entirely to Bence-Jones protein since after absorption of the rabbit antiserum with case 11 myeloma serum no additional precipitin reaction could be obtained with the patient s urine or purified Bence-Jones protein nor did the (absorbed) antiserum react with normal serum protein constituents. Qualitative dilution tests indicated a concentration of Bence-Jones protein in the serum of the same order of magnitude as was obtained for the main abnormal component by other methods. In case 1 (Table VII) Kabat was also able to demonstrate by immunochemical methods that while the main component was not a Bence-Jones protein, 0.15 to 0.2 gram per cent of a Bence-Jones protein nevertheless was present and could be identified and estimated by emplojdng the quantitative precipitin method. This concentration, approximately 1.5% of the total protein content of this serum, obviously could not be detected by salting-out, electrophoretic, or ultracentrifugal methods. 

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