Production of Antibodies and Development of a Radioimmunoassay

Jan 29, 1990 - Maitotoxin, teleocidin, okadaic acid, debromoaplysiatoxin, and TPA, when tested at 10- to 100-fold higher concentrations than palytoxin...
0 downloads 0 Views 425KB Size
Chapter 17 Production of Antibodies and Development of a Radioimmunoassay for Palytoxin 1

2

1

1

Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 11, 2018 at 09:05:19 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Lawrence Levine , Hirota Fujiki , Hilda B. Gjika , and Helen Van Vunakis 1

Department of Biochemistry, Brandeis University, Waltham, M A 02254 Cancer Prevention Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104, Japan

2

Palytoxin (a) stimulates arachidonic acid metabolism i n cells i n cul­ ture, (b) hemolyzes rat erythrocytes, and (c) is lethal to mice when administered intraperitoneally. Serum from rabbits, i m m u n i z e d with a conjugate o f palytoxin and bovine albumin via palytoxin's amino func­ tion, neutralized all o f these biologic activities. T h e titer o f the serum required for the neutralization o f palytoxin's stimulation o f arachi­ donic acid metabolism increased with each course o f i m m u n i z a t i o n and was reduced to less than 9 9 % by precipitation o f the rabbit γi m m u n o g l o b u l i n with a goat anti-rabbit γ-immunoglobulin. Palytoxin, labelled with [ I] B o l t o n - H u n t e r Reagent o n its terminal amino group, b o u n d specifically to these antibodies. T h e extent o f binding also increased progressively with repeated immunizations. A radioim­ munoassay was developed. F o r 5 0 % inhibition o f binding, 0.27 p m o l o f unlabelled palytoxin was required. M a i t o t o x i n , teleocidin, okadaic acid, debromoaplysiatoxin, and T P A , when tested at 10- to 100-fold higher concentrations than palytoxin, did not affect binding. 125

T h e palytoxins synthesized by several Palythoa species are potent biologically active substances (7) with molecular weights o f 2659 to 2677, depending o n the species o f coelenterate from which they originate. Their long, partially unsaturated aliphatic chains contain interspaced cyclic ethers, 4 0 - 4 2 hydroxy, and two amide groups (2, 3). T h e chain starts with a hydroxy group and terminates with an amino group (2, 5). Palytoxin is hemolytic (4) and is an extremely potent toxin (7). W e have shown that i n rat liver cells palytoxin stimulates de-esterification o f cellular lipids to liberate arachidonic acid (5). These rat liver cells metabolize this increased arachi­ donic acid via the cyclooxygenase pathway to produce prostaglandin ( P G ) I and lesser amounts o f P G E and P G F ^ . Palytoxin acts o n many cells i n culture to stimulate the production o f cyclooxygenase metabolites (Table I). Clearly, the myriad pharmacological effects o f the arachidonic acid metabolites must be con­ sidered i n any explanation o f the many clinical manifestations o f palytoxin's toxicity. 2

2

0097-6156/90/0418-0224S06.00/0 o 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

17. LEVINE ET AL.

Antibodies and Radioimmunoassay for Palytoxin

225

Table I. Cells i n W h i c h A r a c h i d o n i c A c i d M e t a b o l i s m Is Stimulated by Palytoxin to Produce Cyclooxygenase Products Rat liver cells B o v i n e aorta smooth muscle cells B o v i n e aorta endothelial cells P o r c i n e aorta endothelial cells Rat keratinocytes Squirrel monkey aorta smooth muscle cells Rat peritoneal macrophages (6) M o u s e calvaria: osteoclasts and/or osteoblasts

(7)

T o prepare a conjugate for i m m u n i z a t i o n o f rabbits (5), an amide b o n d was formed between the terminal amino group o f palytoxin and carboxyl groups o n bovine albumin (Figure 1). T h e i m m u n e sera neutralized palytoxin's stimulation o f arachidonic acid metabolism by several cell types i n culture w i t h the titre increasing as a function o f i m m u n i z a t i o n (Figure 2). T h e neutralizing activity i n the serum was essentially eliminated after precipitation o f the rabbit 7-immunoglobulin ( I g G ) w i t h a goat anti-rabbit I g G . T h e i m m u n e serum also neutralized palytoxin's lytic activity toward rat erythrocytes. T h e L D Q o f palytoxin i n female Swiss A l b i n o mice 24 hours following intraperitoneal injection is 5 x 10" mg/kg (8). T h e i m m u n e sera also neutralized palytoxin's lethal effects. A s shown i n Figure 3, 11/12 mice were killed by palytoxin (1 x 10" mg/kg), whereas 0/12 and 0/11 mice were killed by palytoxin when injected intraperitoneally i n the presence o f the i m m u n e serum. N o n e o f the protected mice showed any signs o f distress. Palytoxin, r a d i o l a b e l e d by reaction o f its terminal N H group w i t h [ I ] B o l t o n - H u n t e r reagent, b o u n d to the anti-palytoxin, and a radioimmunoassay was developed (9). T h e serologic specificity with respect to several toxins and/or tumor promoters as well as the palytoxins isolated from two Palythoa species is shown i n Table II. T h e anti-palytoxin d i d not distinguish the homologous palytoxin o f P. caribaeorum from the heterologous palytoxin o f P. tuberculosa whose structure differs only i n the hemiketal ring at position C 5 5 (2). M a i t o t o x i n , present i n some species o f dinoflagellates (10); teleocidin, isolated from Streptomyces and similar to the toxin found i n the blue-green alga Lyngbya majuscula (11); okadaic acid, a polyether found i n some species o f sponge (72); debromoaplysiatoxin, found i n some species o f blue-green algae (13); and 12-0-tetradecanoylphorbol-13-acetate, a t u m o r promoter isolated from the plant Croton tiglium, d i d not inhibit at the levels tested. This lack o f serologic activity by these toxins and tumor promoters suggests that this i m m u n e system w i l l be specific, at least w i t h respect to these toxins, for detection o f palytoxin-like material i n the food chain. 5

4

3

125

2

Palytoxin's properties o f stimulating arachidonic acid metabolism were decreased > 9 0 % after exposure to 0.1 N H C I for 60 m i n at 37 ° C and decreased about 7 5 % after exposure to 0.01 N H C I for 60 m i n at 50 ° C , but these biologic properties were stable to b o i l i n g i n H 0 for 60 m i n . Palytoxin's serologic activities were stable to these treatments. T h e palytoxin—anti-palytoxin reaction is unique i n that its binding increases w i t h increasing temperature (Figure 4). T h e apparent association constant o f the palytoxin to anti-palytoxin was 4.9 x 1 0 M " at 0 ° C and 1.1 x 1 0 M " at 3 5 ° C , suggesting that H J D must be displaced from some o f palytoxin's epitopes before they can b i n d to their antibody combining sites. 2

9

1

1 0

1

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

Figure 1. Structure o f palytoxin, isolated from P. tuberculosa (5), immunogen (bovine albumin). T h e structures o f the palytoxins from different Pafythoa species vary i n the hemiketal ring at position C 5 5 (2, 3).

ο

S

Ο Ο

S ο

Ο

>

I

S 2

S

b

a

M H Ο

3

i

OS

LEVINE ET AL.

Antibodies and Radioimmunoassay for Palytoxin

0.1

1.0

10 Hi

125

Figure 2. B i n d i n g o f [ I ] p a l y t o x i n to anti-palytoxin (Rabbit 633) before (•) and after primary (o), first boost ( A ) , second boost ( A ) , and third boost ( • , Rabbit 633D-24) o f i m m u n i z a t i o n and after absorption o f I g G i n Rabbit 633D-24 with goat anti-rabbit I g G ( • ) .

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

228

MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY

100

11/12

Undiluted 633D-0

Undiluted 633D-24

fo Dilution 633D-24

fgo Dilution 633D-24

Figure 3. M o r t a l i t y o f mice 24 h r after i.p. injection o f 1 x 10" mg/kg palytoxin i n the presence o f pre-immune serum from Rabbit 633; undiluted i m m u n e serum, 1 0 % i m m u n e serum, and 1% i m m u n e serum. Numbers above bars represent the number o f animals dead and the number tested. (Reproduced with permission from R e f . 8. Copyright 1987 Pergamon Press.) 3

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

LEVINE ETAL.

Antibodies andRadioimmunoassay for Palytoxin

125

Table II. Serologic Specificity o f [ I ] P a l y t o x i n - a n t i - P a l y t o x i n Immune System Inhibitor

IC