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Ammonium sulphate precipitation overestimates titres of anti-peptide antisera
I m m u n o h ) g y l,etter~. 8 (1984) 233 236 t'lsex ier Imlet 507
AMMONIUM
SULPHATE
PRECIPITATION OVERESTIMATES ANTI-PEPTIDE ANTISERA
TITRES
OF
B. ROTH and T. BARKAS* lh;partement de Biochimh'. I "niver,~it~; de Genl, ve. 30. quat Ernest ,4n.~ermet. ('11-1211 Geni've 4. Switzerland (Received I J u l y 1984) (Accepted 16 J u l y 1984)
I. Summary
Precipitation by 50% final saturation of ammonium sulphate, a method commonly used to estimate titres and affinity constants of antibodies, was found consistently to overestimate the titre of anti-peptide antisera when compared with other antibody precipitants. The effect could not be attributed to non-lgG fractions, and similar overestimations were found when pure IgG was used. We suggest that the lower values represent a more physiologically relevant estimation.
2. Introduction
Precipitation by a 50% final concentration of ammonium sulphate of the antigen-antibody complex is widely used to estimate the titre and affinity constants of antisera to haptens and protein antigens which are soluble at this concentration of ammonium sulphate (reviewed in [1]). The method has been compared with other means of separating the complexed and free antigen and a good agreement has been claimed [2]. During the course of measuring titres of antisera to synthetic peptides, we have observed that the ammonium sulphate method consistently gives higher estimates than other methods. Reviewing previous results [2], it would seem that
this effect is not restricted to anti-peptide antibodies. This difference possibly results from a non-physiological "freezing" of the complex by the added ammonium sulphate.
3. Materials and Methods
3. I. Peptide synthesis and antibody production Peptide F A G R L I E L S Q E G (P3) comprising the C-terminus of the a-chain of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata plus either an N-terminal tyrosine or two lysine residues was synthesised as described previously [3]. The double lysine derivative was coupled to keyhole limpet haemocyanin (KLH) and the conjugate used to raise antisera in rabbits [3]. Peptide YDGTKVS I S P E S D R P D L S T (PI), also from the nAChR, was synthesised and used to raise antisera as described previously [4]. Blood samples were collected 7 days after each boost. Heat inactivation was performed at 56 °C for 30 min. 3.2. Radiolabeling of peptides Tyrosine peptides were labelled by the chloramine T method to a specific radioactivity of 330 Ci/mmol as described previously [3]. 3.3. Precipitation assays and cak'ulation of titres and
* I o w h o m c o r r e s p o n d e n c e should be addressed
Ket' words: a m m o n i u m sulphate - titre • affinity c o n s t a n t overestimation 0165 2478 • 8 4 . $3.00 © Elsevier Science Publishers B.V.
affinity constants The method used was essentially that of Steward [i]. Duplicate samples of l0 ~.1 of labelled peptide (0.87 pmol) and 10 #1 of serial dilutions of unla233
AMMONIUM
SULPHATE
PRECIPITATION OVERESTIMATES ANTI-PEPTIDE ANTISERA
TITRES
OF
B. ROTH and T. BARKAS* lh;partement de Biochimh'. I "niver,~it~; de Genl, ve. 30. quat Ernest ,4n.~ermet. ('11-1211 Geni've 4. Switzerland (Received I J u l y 1984) (Accepted 16 J u l y 1984)
I. Summary
Precipitation by 50% final saturation of ammonium sulphate, a method commonly used to estimate titres and affinity constants of antibodies, was found consistently to overestimate the titre of anti-peptide antisera when compared with other antibody precipitants. The effect could not be attributed to non-lgG fractions, and similar overestimations were found when pure IgG was used. We suggest that the lower values represent a more physiologically relevant estimation.
2. Introduction
Precipitation by a 50% final concentration of ammonium sulphate of the antigen-antibody complex is widely used to estimate the titre and affinity constants of antisera to haptens and protein antigens which are soluble at this concentration of ammonium sulphate (reviewed in [1]). The method has been compared with other means of separating the complexed and free antigen and a good agreement has been claimed [2]. During the course of measuring titres of antisera to synthetic peptides, we have observed that the ammonium sulphate method consistently gives higher estimates than other methods. Reviewing previous results [2], it would seem that
this effect is not restricted to anti-peptide antibodies. This difference possibly results from a non-physiological "freezing" of the complex by the added ammonium sulphate.
3. Materials and Methods
3. I. Peptide synthesis and antibody production Peptide F A G R L I E L S Q E G (P3) comprising the C-terminus of the a-chain of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata plus either an N-terminal tyrosine or two lysine residues was synthesised as described previously [3]. The double lysine derivative was coupled to keyhole limpet haemocyanin (KLH) and the conjugate used to raise antisera in rabbits [3]. Peptide YDGTKVS I S P E S D R P D L S T (PI), also from the nAChR, was synthesised and used to raise antisera as described previously [4]. Blood samples were collected 7 days after each boost. Heat inactivation was performed at 56 °C for 30 min. 3.2. Radiolabeling of peptides Tyrosine peptides were labelled by the chloramine T method to a specific radioactivity of 330 Ci/mmol as described previously [3]. 3.3. Precipitation assays and cak'ulation of titres and
* I o w h o m c o r r e s p o n d e n c e should be addressed
Ket' words: a m m o n i u m sulphate - titre • affinity c o n s t a n t overestimation 0165 2478 • 8 4 . $3.00 © Elsevier Science Publishers B.V.
affinity constants The method used was essentially that of Steward [i]. Duplicate samples of l0 ~.1 of labelled peptide (0.87 pmol) and 10 #1 of serial dilutions of unla233