15 Immunological Tests for the Evaluation of Citrus Quality RICHARD L. MANSELL Department of Biology, University of South Florida, Tampa, FL 33620 ELMAR W. WEILER Institute fur Pflanzenphysiologie, Ruhr Universität, 463 Bochum, West Germany
Quality of food products is nearly always based on esthetic values, such as color, texture and consistency, as well as physiological thresholds and sensitivities to taste, flavor and aroma. Ultimately the acceptance of foods and their products is dependent upon the physiological or biochemical state of the product, however, the subjective evaluations cannot be ignored except in times of hunger and famine. Each food and food product is, then, a veritable collection of chemical constituents which is neither static nor stable. It might be best to describe the endogenous chemistry as dynamic and, as such, the quality characteristics must be a function of the composition of the chemical constituents at any given time. Since the basis of chemical composition is, in fact, controlled by the inherent genetics of any given organism, we can at least focus in on the fundamental site responsible for each chemical compound. In addition, however, we must also be aware that the interactions of these same genes with a multitude of complex regulatory mechanisms also plays a major role in determining the ultimate chemical composition at any given period of development. I n v e s t i g a t i o n i n t o the chemical composition of c i t r u s has led to the i s o l a t i o n and i d e n t i f i c a t i o n of hundreds of i n d i v i d u a l compounds. As work has continued, b i o c h e m i c a l , n u t r i t i o n a l and o r g a n o l e p t i c s t u d i e s have made great s t r i d e s i n determining which compounds are paramount i n the f i n a l determination of f r u i t and f r u i t product q u a l i t y . For the purpose of t h i s d i s c u s s i o n , we w i l l concern ourselves with only those q u a l i t y aspects a f f e c t e d by endogenous chemical composition and c o n s i d e r a t i o n of p h y s i c a l or d e s c r i p t i v e parameters w i l l be omitted. Food Q u a l i t y Q u a l i t y i s defined as "the nature, excellence or i n t r i n s i c l e v e l of a thing" and thus e v a l u a t i o n i s a complicated and d i f f i c u l t task. The major problem r e l a t e d to q u a l i t y e v a l u a t i o n i s inherent i n the methodology used to make the determination.
0-8412-0595-7/80/47-143-341$05.00/0 © 1980 American Chemical Society
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The most obvious method i s s u b j e c t i v e and here the e v a l u a t i o n i s based on the o p i n i o n of the t e s t e r and the response of the sensory organs. Obviously t h i s technique presents many problems and there i s wide v a r i a t i o n i n the range of r e s u l t s obtained. O b j e c t i v e methods are based on s p e c i f i c analyses and, thereby, exclude t e s t e r o p i n i o n . Thus, the i n t r i n s i c aspects of q u a l i t y are reduced to chemical, p h y s i c a l , or b i o l o g i c a l measurements and only the i n t e r p r e t a t i o n of the r e s u l t i n g data i s r e l a t e d back to the s u b j e c t i v e o p i n i o n . Reduction of the s u b j e c t i v e values or c r i t e r i a to a s p e c i f i e d compound, group of compounds or p h y s i c a l f a c t o r i s o f t e n a very d i f f i c u l t endeavor, but one by one each of the s o - c a l l e d " q u a l i t a t i v e c r i t e r i a " i s being l i n k e d to the presence or absence of s p e c i f i c c o n s t i t u e n t s . Sometimes only a s i n g l e compound i s i n v o l v e d , however, o f t e n the q u a l i t a t i v e character i s the r e s u l t of a d d i t i v e or even s y n e r g i s t i c i n t e r a c t i o n s of two or more constituents. Thus, since there are so many d i f f e r e n t charact e r i s t i c s that can c o n t r i b u t e to the t o t a l q u a l i t y of any s i n g l e food product, i t i s paramount that p r e c i s e o b j e c t i v e methodology be developed f o r each parameter. In c i t r u s f r u i t s and products, most f l a v o r s and aromas are produced by p o l y p h e n o l i c compounds or e s s e n t i a l o i l s plus a v a r i e t y o f n o n - v o l a t i l e organic compounds. For d e t a i l e d d i s c u s s i o n s of c i t r u s f l a v o r s and chemical composition, the reader i s r e f e r r e d to the e x c e l l e n t and comprehensive reviews which have been published (j.,.2,_3,4) . During the past h a l f - c e n t u r y , the c i t r u s i n d u s t r y has grown world-wide and q u a l i t y standards, both l o c a l and i n t e r n a t i o n a l , have c o n t i n u a l l y become more comprehensive and r i g o r o u s . Numerous d e f i n i t i o n s and c r i t e r i a of q u a l i t y have been p u b l i s h e d (_5,6^_7) and i n some areas, e.g. F l o r i d a , wide ranging g u i d e l i n e s have been adopted and laws* enacted f o r the s o l e purpose of maintaining q u a l i t y l e v e l s . Extensive advances have been made i n q u a l i t y c o n t r o l or q u a l i t y assurance programs (8) y e t many d i f f i c u l t and complex problems remain as i n t r i n s i c components of the i n d u s t r y . Thus as the impetus f o r q u a l i t y improvement has evolved, i t has become a widely recognized f a c t that c e r t a i n t e c h n o l o g i c a l advances were required before q u a l i t a t i v e progress could be r e a l i z e d . The purpose o f q u a l i t y c o n t r o l or q u a l i t y assurance i n the c i t r u s i n d u s t r y i s to help provide f o r the p r o d u c t i o n of a uniform, h i g h q u a l i t y and commercially acceptable product. For most of the major q u a l i t a t i v e c h a r a c t e r i s t i c s , standardized, accurate and simple methods have been developed, and high and low l i m i t s have been c a r e f u l l y been e s t a b l i s h e d . Some of these include: B r i x , a c i d , c o l o r , r e c o v e r a b l e o i l , and f r e e and suspended pulp. For each of these c r i t e r i a , a simple and r a p i d assay has been developed and good c o r r e l a t i o n e x i s t s between the o b j e c t i v e assay r e s u l t s and the s u b j e c t i v e q u a l i t y e v a l u a t i o n s .
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Problem Areas i n C i t r u s Q u a l i t y In s p i t e of the progress which has already been made, there s t i l l e x i s t , i n c i t r u s f r u i t and products, numerous q u a l i t a t i v e aspects which are e i t h e r very d i f f i c u l t to measure or whose c o n t r i b u t i o n to q u a l i t y i s a f u n c t i o n of the o r g a n o l e p t i c s e n s i t i v i t i e s of the consumer. For the purpose of t h i s paper we would l i k e to concentrate on two groups of n a t u r a l l y o c c u r r i n g compounds which have a major impact on the t a s t e of c i t r u s f r u i t and t h e i r products. As p r e v i o u s l y discussed by Maier and co-workers (Chapter 4 ) , i n Navel, Shamouti and c e r t a i n other orange c u l t i v a r s , the presence of l i m o n i n , a b i t t e r t r i t e r p e n o i d , causes many economic and o r g a n o l e p t i c problems and g r e a t l y a f f e c t s the t a s t e q u a l i t y of processed f r u i t . Limonin i s a l s o prevalent i n the g r a p e f r u i t but the i n t r i n s i c q u a l i t y of t h i s f r u i t i s f u r t h e r complicated by the presence of n a r i n g i n , a b i t t e r flavanone neohesperidoside (Chapter 5 ) . Numerous o r g a n o l e p t i c s t u d i e s have been done on b i t t e r n e s s as a f u n c t i o n of l i m o n i n and/or n a r i n g i n , and a myriad of a d d i t i v e and perhaps s y n e r g i s t i c r e s u l t s have been obtained ( 9 ) . Table I presents a g e n e r a l i z e d response as measured i n a v a r i e t y of t a s t e s t u d i e s . Table I Organoleptic Responses to Limonin and N a r i n g i n
Non-bitter Slightly bitter Bitter Very b i t t e r
Threshold
However:
Limonin ppm
Naringin ppm
6 7-9 10-16 18
20 20-300 500-1300 1500
ave.=1
ave.=20
a s o l u t i o n or j u i c e with 0.75 ppm limonin and 5 ppm n a r i n g i n i s b i t t e r (99% confidence l e v e l ) (9)
During the past two decades i n t e r e s t and concern about the b i t t e r p r i n c i p l e s of c i t r u s products have increased g r e a t l y and although some e x c e l l e n t chemical s t u d i e s have been done, there
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has been l i t t l e i n f o r m a t i o n generated which f r u i t quality. The major problem which has to overcome i n these s t u d i e s i s that u n l i k e f a c t o r s , e.g. sugar, a c i d , e t c . , the b i t t e r r a p i d l y assayed and q u a n t i f i e d .
has had an e f f e c t on proved most d i f f i c u l t other q u a l i t y p r i n c i p l e s cannot be
Current A n a l y t i c a l Methods When one examines the a n a l y t i c a l methods which are p r e s e n t l y a v a i l a b l e f o r these b i t t e r compounds, i t i s c l e a r why the study and c o n t r o l of b i t t e r n e s s i n c i t r u s has been so hampered and quality control v i r t u a l l y lacking. For the flavanone, n a r i n g i n , only an approximate t e s t i s a v a i l a b l e (Davis Test) (see Chapter 5 f o r a d i s c u s s i o n of t h i s method) and f o r limonin there i s no method a v a i l a b l e f o r monitoring i n p r o c e s s i n g p l a n t s . In the past ten years t h i n l a y e r chromatography (TLC) has proven u s e f u l f o r s e m i - q u a n t i t a t i v e measurements of f r u i t and j u i c e samples, however t h i s method i s by i t s very nature: 1) rather i n s e n s i t i v e (yg range); 2) slow (1 hour or more f o r sample a p p l i c a t i o n and solvent development); 3) r e q u i r e s p r e - p u r i f i c a t i o n steps ( l i q u i d - l i q u i d e x t r a c t i o n or p r e c i p i t a t i o n ) ; 4) d e t e c t i o n i s d i f f i c u l t ( e s p e c i a l l y true f o r limonoids) and 5) sample throughput i s normally l e s s than 50 per person per day. Within the past s e v e r a l years good s e p a r a t i o n and q u a n t i f i c a t i o n have been achieved with high-performance l i q u i d chromatography (HPLC) and t h i s procedure has proven very u s e f u l . However, HPLC i s not without i t s drawbacks: 1) equipment cost i s very h i g h (thus small p r o c e s s i n g operations would probably be unable to purchase and maintain such an instrument); 2) only one compound can be measured at a time s i n c e d i f f e r e n t columns and s o l v e n t s are used f o r each c l a s s of compounds ( t h i s a l s o means that time must be spent i n changing the system from one a n a l y s i s to the n e x t ) ; 3) p r e - p u r i f i c a t i o n i s r e q u i r e d and f o r good r e s o l u t i o n repeated l i q u i d - l i q u i d and evaporation steps are i n v o l v e d ; 4) the procedure i s slow (only 10-15 samples can be processed per person per day); 5) i t i s s e n s i t i v e only to the p a r t s per m i l l i o n (ppm) range (ug/gm). Thus, i n summing the current s t a t u s of l i m o n i n and n a r i n g i n q u a n t i f i c a t i o n , a quotat i o n i s most a p p r o p r i a t e . "None of the methods developed thus f a r can be considered i d e a l , e s p e c i a l l y f o r r o u t i n e q u a l i t y c o n t r o l purposes. Among t h e i r disadvantages are s u b j e c t i v e readout methods, time-consuming separat i o n s , questionable s p e c i f i c i t y , and l i m i t e d applicability. Recent work showing that t a s t e thresholds f o r l i m o n i n are lower than had p r e v i o u s l y been assumed f o r s i g n i f i c a n t p r o p o r t i o n s of the p o p u l a t i o n emphasizes the need f o r a b e t t e r assay method. The use of a p r o t e i n s p e c i f i c f o r l i m o n i n , such as an enzyme or antibody, could provide the b a s i s f o r an improved assay." (9)
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Science
As we began to develop i n t e r e s t i n the b i t t e r n e s s problem i n c i t r u s f r u i t , a major advance or breakthrough i n plant science was achieved when i t was shown that an immunological assay method could be developed f o r the measurement of n a t u r a l l y o c c u r r i n g plant products (10). In t h i s study i t was demonstrated that i t was p o s s i b l e to develop antibodies which were s p e c i f i c f o r the compound under i n v e s t i g a t i o n , and the authors furthermore suggested that t h i s immunological method should render i t s e l f exceedingly u s e f u l i n plant screening and breeding programs; i n c e l l c u l t u r e screening; and f o r studies of b i o s y n t h e s i s , transport and metabolism. Extension of such v e r s a t i l i t y would l o g i c a l l y include q u a l i t y c o n t r o l s t u d i e s and monitoring. The r o l e of the immunoassay, e s p e c i a l l y the radioimmunoassay (RIA), i n c l i n i c a l biochemistry has been the major f a c t o r i n the tremendous advances made i n that f i e l d s i n c e i t s i n t r o d u c t i o n i n 1959 (11). At present the RIA i s the most powerful a n a l y t i c a l t o o l a v a i l a b l e f o r q u a n t i t a t i v e d e t e c t i o n of molecules of d i v e r s e s t r u c t u r e and f u n c t i o n i n b i o l o g i c a l f l u i d s of human, animal and now p l a n t o r i g i n . The immunoassay comprises a unique combination of s e n s i t i v i t y and s p e c i f i c i t y as w e l l as p r e c i s i o n and a p p l i c a bility. With t h i s assay technique, i t i s now p o s s i b l e to detect and very a c c u r a t e l y measure compounds at endogenous p h y s i o l o g i c a l concentrations which f r e q u e n t l y are i n the range of 10 M or lower. In Table I I the major c h a r a c t e r i s t i c s of the immunoassay are l i s t e d . This method i s v e r s a t i l e , s p e c i f i c , can be u t i l i z e d f o r almost an u n l i m i t e d number of compounds and has a high throughput p o t e n t i a l . Table II C h a r a c t e r i s t i c s of the Immunoassay System 1)
Most s e n s i t i v e a n a l y t i c a l technique f o r both r o u t i n e and s o p h i s t i c a t e d analyses. picogram (femtomole) range = parts per b i l l i o n range = c e l l u l a r l e v e l
2)
S p e c i f i c i t y - n e a r l y absolute f o r each compound p e r m i t t i n g d i r e c t measurement i n crude e x t r a c t s , n u t r i e n t s o l u t i o n s or processed samples
3)
S u i t a b l e f o r automation or semi-automation. a n a l y s i s p o s s i b l e , 20-30 minutes.
Rapid
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4)
Can be used f o r s i n g l e sample a n a l y s i s or f o r mass screening. Screening p o t e n t i a l i s as high as 3000 samples per day per person.
5)
U s e f u l as a q u a n t i t a t i v e or s e m i - q u a n t i t a t i v e assay
The r o l e of the immunoassay i n food science has developed more slowly and, o v e r a l l , r a t h e r few immunological t e s t s are c u r r e n t l y being performed on a r o u t i n e b a s i s . Table I I I provides a l i s t of some of the major analyses which are a v a i l a b l e (12). Table I I I Immunoassay i n Food Science A.
B.
C.
D. E.
Food P r o t e i n s 1) meat 2) soybean, wheat, b a r l e y , peanuts Toxic Plant & Animal Constituents 1) a g g l u t i n i n s 2) proteinase i n h i b i t o r s B a c t e r i a l and V i r a l Toxins 1) enterotoxins from Staphylococcus, IS. c o l i , Clostridium 2) f i s h and s h e l l f i s h poisons 3) foodborne v i r u s e s Food A l l e r g y 1) a l l e r g e n s C i t r u s (discussed below) 1) E s t i m a t i o n of orange j u i c e content i n s o f t d r i n k s 2) B i t t e r p r i n c i p l e a n a l y s i s (limonin and n a r i n g i n )
Within the l a s t s e v e r a l years there has been a very r a p i d expansion of the RIA i n t o p l a n t science, and assays are p r e s e n t l y i n use with such d i v e r s e compounds as n i c o t i n e (13), i n d o l a c e t i c a c i d (14), a b s c i s s i c a c i d (15), serpentine (16), d i g o x i n (10,17) and many others. The unique f e a t u r e s of the RIA have shown that i t i s an e x c e l l e n t and very necessary technique f o r mass screening programs i n v o l v i n g genetic breeding of p l a n t characters (18,19) screening of c e l l c u l t u r e s f o r primary or secondary metabolites (20), f o r i n v e s t i g a t i o n s of b i o - s y n t h e s i s (21) and i n p r a c t i c a l s i t u a t i o n s such as q u a l i t y c o n t r o l monitoring of p l a n t e x t r a c t s (20). The immunoassay i t s e l f can be performed i n a wide array of procedural v a r i a t i o n s , but the RIA i s p r e s e n t l y the most widely used. In p r i n c i p l e , the immunoassay i s based on the h i g h l y s p e c i f i c r e a c t i o n of a n t i b o d i e s with antigens against which the a n t i b o d i e s have been d i r e c t e d . The m a j o r i t y of antigens known to b i o l o g y are p r o t e i n s or other molecules of high molecular weight. Compounds with a molecular weight below 1000 are u s u a l l y not
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immunogenic when introduced i n t o the bloodstream of an animal. However, i f these low molecular weight compounds are bound c o v a l e n t l y to p r o t e i n c a r r i e r s (such as human serum albumin or p o l y - l y s i n e ) as haptens they become immunogenic and s p e c i f i c a n t i b o d i e s a g a i n s t these haptens are produced. The production of a n t i b o d i e s i s normally done i n r a b b i t s and i t t y p i c a l l y takes a minimum of three months, with booster i n j e c t i o n s , to produce a f i n a l antiserum with a high t i t r e . Characteri z a t i o n of the antiserum i s done by the f o l l o w i n g : a) determinat i o n of the t i t r e , ( t h i s i s defined as that d i l u t i o n of the a n t i serum which binds 50% of a known amount of r a d i o a c t i v e or l a b e l l e d hapje^i undejj constant assay c o n d i t i o n s ) . In most cases the l a b e l is I or H but n o n - r a d i o a c t i v e immunoassays are a l s o a v a i l a b l e i n which an enzyme o r f l u o r e s c e n t compound f u n c t i o n s as l a b e l , b) determination of the e f f e c t s o f pH, s o l v e n t s and chemicals, e.g. ethanol, a z i d e , e t c . ; c) c o r r e l a t i o n of the immunoassay with standard methods; d) determination of s p e c i f i c i t y . This l a s t c h a r a c t e r i z a t i o n f a c t o r i s extremely important s i n c e compounds other than the hapten might bind with the antibody. This phenomenon i s known as cross r e a c t i v i t y and a wide v a r i e t y of known r e l a t e d compounds must be t e s t e d . A f t e r production of the a n t i serum and i t s c h a r a c t e r i z a t i o n , a s i n g l e animal should produce enough m a t e r i a l to be used i n tens o f thousands of s i n g l e assays. The p r i n c i p l e o f the RIA i s diagrammed below and the assay i s done as f o l l o w s : Ag + Ag* + Ab p=f where:
AgAb + Ag*Ab + Ag + Ag*
Ab = antibody Ag = antigen Ag*= antigen l a b e l l e d with
1
2
5
3
I , H, enzyme, e t c .
To a s o l u t i o n of known t i t r e antiserum i s added a known c o n c e n t r a t i o n of r a d i o a c t i v e or t r a c e r hapten (antigen) and an a l i q u o t of the p l a n t e x t r a c t . There w i l l be a competition of the l a b e l l e d hapten (added) and u n l a b e l l e d (plant e x t r a c t ) hapten f o r the f i x e d number o f antibody s i t e s (antiserum of known t i t r e ) and t h i s r e s u l t s i n some of the l a b e l l e d hapten being bound while the remainder remains f r e e . The d i s t r i b u t i o n of the r a d i o a c t i v e hapten between the f r e e and bound s t a t e w i l l be a f u n c t i o n of the amount o f u n l a b e l l e d hapten present i n the assay tube. A f t e r e q u i l i b r i u m has been reached, the bound and unbound hapten can be separated ( F i g . 1) ( s e v e r a l methods are a v a i l a b l e ) and determination of the r a d i o a c t i v i t y i n e i t h e r f r a c t i o n gives an exact measure of the unknown hapten (plant e x t r a c t ) as computed from a standard curve ( F i g . 2 ) . Since the immunoassay should prove to have great p o t e n t i a l i n a l l areas o f p l a n t s c i e n c e , we would l i k e to d e s c r i b e i n some
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Figure 1.
Performance of the semiautomated RIA technique (36)
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Figure 2. Limonin standard curve shown in two different plots (\-0—\ Mean s.d. of triplicate determinations; tracer is Η limonin (Sp. Act. 22 Ci/mmol)) (23) 3
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d e t a i l r e s u l t s of s t u d i e s which we have done using limonin as the antigen. During the past two years we have developed and charac t e r i z e d two^cj^fferent RIA s f o r l i m o n i n (22,23) . One RIA was done using I-limonin as t r a c e r and the antibody d e v e l o p e d i s c h a r a c t e r i z e d by having a high a f f i n i t y (k = 1.1 χ 10 1/mol) f o r l i m o n i n . The d e t e c t i o n l i m i t of the assay i s 0.07 ng per 0.1 ml o r 0.7 p a r t s per b i l l i o n (ppb) and the standard curves are l i n e a r over a range of 0.5-100 ng l i m o n i n . Assays can be performed i n crude e x t r a c t s and 400-800 samples can be processed per day by one person. C o e f f i c i e n t s o f v a r i a t i o n of t r i p l i c a t e B/Bo values throughout the measuring range were 2.5 + 1.8% and recovery values were found to be 93%. The antibody produced against l i m o n i n has a very high t i t r e and 30,000 samples can be assayed per ml of serum. The s e n s i t i v i t y of t h i s RIA i s more than 10,000 times greater than that of any of the present a n a l y t i c a l methods (24,25,26,27). Data c a l c u l a t i o n i s done on a programmable computer and r e s u l t s are a v a i l a b l e w i t h i n a few minutes. In the s p e c i f i c i t y t e s t s , i t was observed that only deoxylimonin (27%) and d e a c e t y l n o m i l i n (66%) cross reacted with the antibody. However, previous s t u d i e s (28,29) on the r e l a t i v e concentration of these two compounds i n c i t r u s t i s s u e s showed that deoxylimonin i s present a t only 0.47% that of limonin and, t h e r e f o r e , w i l l not be detected i n d i l u t e d samples. D e a c e t y l n o m i l i n i s a l s o one of the minor c o n s t i t u e n t s of the t o t a l limonoid f r a c t i o n and w i l l not c o n t r i b u t e to l i m o n i n v a l u e s . In a study of the d i s t r i b u t i o n of limonin w i t h i n v a r i o u s g r a p e f r u i t t i s s u e s ( F i g . 3), the values obtained were i n good agreement with values a v a i l a b l e i n the l i t e r a t u r e (27,29,30) and the c o n c e n t r a t i o n gradient from growing leaves i n t o the f r u i t conductive t i s s u e and u l t i m a t e l y the seed supports the recent r e s u l t s obtained by Hasegawa and Hoagland (31). Although an extensive q u a n t i t a t i v e a n a l y s i s of both f r u i t and v e g e t a t i v e t i s s u e and the k i n e t i c s of accumulation have not yet been done, with the development of an RIA f o r l i m o n i n these s t u d i e s are now possible. ^ The second RIA system was developed using H-limonin as a tracer. Establishment of a second system was important s i n c e many l a b o r a t o r i e s possess s c i n t i l l a t i o n equipment f o r counting beta e m i t t i n g i s o t o p e s , but gamma counting equipment i s l e s s readily available. In a d d i t i o n , the stabij.j^y and h a l f - l i f e of a t r i t i a t e d t r a c e r i s very long compared to I and, thus, a s i n g l e r a d i o s y n t h e s i s can produce enough t r a c e r f o r s e v e r a l years or more. The c h a r a c t e r i s t i c s of the o r i g i n a l assay are: 1) a d e t e c t i o n l i m i t of 0.22 ng o r 2.2 ppb, 2) the standard curve i s l i n e a r over a range of 0.5-100 ng ( F i g . 2 ) , 3) no p u r i f i c a t i o n of the e x t r a c t s i s necessary, 4) c o e f f i c i e n t s of v a r i a t i o n f o r t r i p l i c a t e determinations (B/Bo) throughout the measuring range are 3.0 + 1.8% and 5) recovery i s 97%. The t i t r e of the antibody 1
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Figure 3.
Distribution of limonin in leaves, stems, and fruit parts of grapefruit (Citrus paradisi; (22)
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Figure 4.
Scatter diagram for various plant extracts assayed by both the I and H RIA (n = 67; r = 0.95; y = 0.96x + 0.07 (23) 125
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was 400, which means that 4000 samples could be assayed per ml of serum. Since the f i n a l t i t r e of the serum i s a f u n c t i o n of the s p e c i f i c a c t i v i t y of the t r a c e r used, the t i t r e value could be increased by using higher s p e c i f i c a c t i v i t y t r a c e r . Thus, when a second H-limonin was synthesized (108 Ci/mmol vs 22 Ci/mmol f o r the f i r s t ) the t i t r e of the antj.tjç)dy was found to i n c r e a s e to 1,500. In comparison with the I assay there a l s o was a high degree of c o r r e l a t i o n when e x t r a c t s from v a r i o u s t i s s u e s were assayed by both t r a c e r s ( F i g . 4 ) . HPLC/RIA C o r r e l a t i o n . We compared the l i m o n i n content of 180 samples of canned, s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e which had been^analyzed by HPLC at the Lake A l f r e d Experiment S t a t i o n with our H RIA system ( F i g . 5 ) . A p a i r e d t t e s t showed no d i f f e r e n c e between the two methods and a l i n e a r r e g r e s s i o n gave r = 0.794. We were able to analyze these samples i n d u p l i c a t e i n a s i n g l e working day, whereas the same analyses took more than two months using the HPLC. I t was a l s o observed that 78% of the RIA values were higher than the corresponding HPLC, but s i n c e the HPLC r e q u i r e s extensive e x t r a c t i o n and p r e p u r i f i c a t i o n p r i o r to a n a l y s i s whereas the RIA i s done with crude e x t r a c t s , t h i s r e s u l t i s perhaps p r e d i c t a b l e . Due to the s e n s i t i v i t y of the RIA, i t i s now p o s s i b l e to measure l i m o n i n c o n c e n t r a t i o n i n any p a r t of the p l a n t from embryos to s m a l l l e a f d i s c s , seed coats or any other d e s i r a b l e s i t e or sample. In t h i s type of a n a l y s i s or screening study i t i s p o s s i b l e f o r a s i n g l e person to analyze 2000 or more samples per week arid i t i s i r o n i c that "sample t a k i n g " i s now l i k e l y to become the l i m i t i n g step i n any l a r g e s c a l e study. In the recent s t u d i e s of Hasegawa and Hoagland (31) i t was found that limonoids ( i n the form of the Α-ring lactone) are synthesized i n the leaves of t r e e s and are t r a n s l o c a t e d to the fruit. T h i s o b s e r v a t i o n makes the RIA very a p p l i c a b l e i n a screening program. From very recent work i n our l a b o r a t o r y , we have found that i t i s a c t u a l l y p o s s i b l e to measure both the Ar i n g l a c t o n e and l i m o n i n w i t h i n a s i n g l e t i s s u e sample using j u s t the l i m o n i n antibody. We have found that the Α-ring l a c t o n e has a very low cross r e a c t i v i t y with the antibody, t h e r e f o r e i f we measure the l i m o n i n i n n e u t r a l i z e d e x t r a c t s and then a c i d i f y these same s o l u t i o n s and again measure the l i m o n i n content, the d i f f e r e n c e w i l l r e f l e c t the c o n c e n t r a t i o n of Α-ring lactone which was present (32). Thus the s t u d i e s of both compounds can be expanded and the k i n e t i c s of each followed as a f u n c t i o n of both l e a f development and growth. In a d d i t i o n , the e f f e c t of e n v i r o n mental and p h y s i o l o g i c a l f a c t o r s can a l s o be analyzed. ARIA To the standard RIA d e s c r i b e d above, another dimension has r e c e n t l y been added with the development of an autoradiographic
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Figure 5.
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Scatter diagram for canned single-strength grapefruit juice assayed by H-RIA and HPLC (n = 180; r = 0.794; y = 0.844x + 1.381)
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immunoassay (ARIA) (33)• In t h i s modified RIA, the purpose i s not to measure with extreme accuracy the content of substances, but r a t h e r to merely i d e n t i f y those p l a n t s , c e l l c u l t u r e s or j u i c e samples w i t h i n l a r g e populations which c o n t a i n high or low amounts of the compound under study. In t h i s procedure the RIA p r i n c i p l e i s employed but, r a t h e r than doing r a d i o a c t i v e counting on each sample, the amount of r a d i o a c t i v i t y i s determined on Xray f i l m . By a d j u s t i n g the d i l u t i o n of the antibody the procedure can be made s e l e c t i v e f o r amju^its of substances below or above a particular level. By using I as the r a d i o a c t i v e t r a c e r as l i t t l e as 3,000 CPM can be detected a f t e r a 20 hr f i l m exposure. The procedure's r e a l usefulness comes from the f a c t that whereas the RIA can process 400-800 samples per day the ARIA can e a s i l y screen 2,000-3,000 or even more per day. Thus by s a c r i f i c i n g the s e n s i t i v i t y by a f a c t o r of about 10, the number of samples which can be measured per day can be increased by the same value. The s e n s i t i v i t y of t h i s assay i s i n the 1-10 ng range which i s s t i l l s e n s i t i v e enough f o r i n t a c t p l a n t , c e l l c u l t u r e or j u i c e sample analysis. Immunoassay f o r N a r i n g i n At the present time we can only i n c l u d e p r e l i m i n a r y r e s u l t s on t h i s assay as i t s c h a r a c t e r i z a t i o n i s s t i l l i n progress. In the determination of b i t t e r flavanone g l y c o s i d e s the b a s i c importance i s that the assay i s able to d i s t i n g u i s h between the b i t t e r d i s a c c h a r i d e , neohesperidose, l i n k a g e (glucose 1-2 rhamnose) and the n o n - b i t t e r r u t i n o s e l i n k a g e (glucose 1-6 rhamnose). From the serum pools which are j u s t being harvested we have been able to a s c e r t a i n that the antibody being produced i s completely s p e c i f i c f o r the neohesperidoside and no cross r e a c t i v i t y has been observed f o r the r u t i n o s i d e at concentrations up to >5,000 ng/0.1 ml. Synthesis of an immunogenic n a r i n g i n molecule was a l s o done using a hapten-protein conjugate and complete d e t a i l s of the antibody production procedures and c h a r a c t e r i z a t i o n s t u d i e s w i l l be published i n depth at a l a t e r date. However, i t i s both important and encouraging to r e p o r t that through use of the immunoassay, major progress i n q u a l i t y c o n t r o l improvement i n c i t r u s now becomes a d i s t i n c t p o s s i b i l i t y . As a cautionary note, however, i t should a l s o be r e a l i z e d that much work, o p t i m i z a t i o n and t e s t i n g remains to be done before the f u l l p o t e n t i a l of these assays can be r e a l i z e d . Enzyme immunoassay In the previous pages we have d e s c r i b e d , i n p r i n c i p l e , an immunological assay f o r the q u a n t i f i c a t i o n of l i m o n i n and n a r i n g i n i n citrus^gamples.^ In these assays we have employed r a d i o a c t i v e tracers ( I and H) and have found that both are useable i n a
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research l a b o r a t o r y s i t u a t i o n . As with any assay method, however, the RIA a l s o has s e v e r a l major l i m i t a t i o n s e s p e c i a l l y when t r y i n g to employ such a technique i n commercial c i t r u s processing operations. F i r s t , and most importantly, i t i s undesirable and p o t e n t i a l l y unsafe to use r a d i o i s o t o p e s i n the v i c i n i t y of foods; secondly, the personnel using these compounds must be r i g o r o u s l y t r a i n e d i n t h e i r use and r o u t i n e h e a l t h and contamination surveys must be done. T h i r d l y , the equipment needed to measure r a d i o a c t i v i t y i s expensive to purchase and maintain. Thus, although the RIA method i s undoubtedly the p r e f e r r e d system i n an i s o l a t e d research l a b o r a t o r y s i t u a t i o n , an a l t e r n a t i v e method would be most acceptable i n p r a c t i c a l , " i n house" a p p l i c a t i o n s . As mentioned e a r l i e r , the immunoassay can be done with a number of procedural m o d i f i c a t i o n s and i n t h i s instance one must s u b s t i t u t e the isotope with another molecule ( f l u o r e s c e n t dye, magnetic p a r t i c l e , enzyme) which can be measured and t h e r e f o r e serve as the source of t r a c e r . For our i n i t i a l s t u d i e s we have chosen to use the enzyme immunoassay (EIA) system. At the present time the EIA i s s t i l l i n i t s infancy and although a number of s u c c e s s f u l EIA's have been developed the method cannot be considered a panacea (34). The f u t u r e of t h i s assay appears to be very b r i g h t and e x c i t i n g , and there i s considerable i n t e r e s t i n the a p p l i c a t i o n of the EIA to problems i n both microbiology and c l i n i c a l medicine (34). Many of the procedures and p r o t o c o l s are derived from RIA procedures and the EIA, l i k e the RIA, has the p o t e n t i a l to be performed i n a multitude of procedural v a r i a t i o n s ; but, f o r the purpose of t h i s manuscript we w i l l d e s c r i b e only the system we have chosen f o r our use. Consider, f o r example, a s e n s i t i v e competitive assay where the antibody i s immobilized on the s u r f a c e of a polystyrene tube or cuvette with l a b e l l e d and u n l a b e l l e d antigen competing f o r the antibody s i t e s . In t h i s instance the l a b e l l e d antigen i s limonin c o v a l e n t l y bonded to an enzyme molecule and u n l a b e l l e d antigen i s the l i m o n i n present i n j u i c e sample. We have done a number of p r e l i m i n a r y limonin-enzyme coupling analyses and of the enzymes we have used (3-glucosidase, a l k a l i n e phosphatase and h o r s e r a d i s h peroxidase) we have chosen the peroxidase (HPR) as our t e s t system. At present our data are s t i l l i n the p r e l i m i n a r y stage and thus i t would be somewhat premature to attempt to present many f i n d i n g s at t h i s time. We have, however, been able to demonstrate that the assay does f u n c t i o n and moreover we e n v i s i o n being able to develop t h i s system to a p o i n t where i t could be used i n both a research l a b o r a t o r y as w e l l as i n q u a l i t y c o n t r o l monitoring centers. In Figure 6 the procedures or steps involved i n an EIA are diagrammed. For t h i s assay i t i s f i r s t necessary to bind the antibody to the w a l l s of a polystyrene tube. (These tubes could be made a v a i l a b l e through s e v e r a l commercial l a b o r a t o r i e s or could be prepared by l a b o r a t o r y t e c h n i c i a n s . ) For the assay, a sample of j u i c e i s incubated together with the t r a c e r antigen.
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Figure 6. Diagrammatic scheme for pe formance of a solid-phase enzyme immunoassay ((%) juice sample (limonin); ( \-E) tracer (limonin-enzyme conjugate))
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The tube i s then decanted, washed twice or more with b u f f e r and then an enzyme assay s o l u t i o n i s added and the c o l o r development or change i n absorbance i s measured. The amount of enzyme remaining bound to the antibody w i l l be a f u n c t i o n of the amount of antigen present i n the j u i c e sample and, thus, the c o l o r development can be used to c a l c u l a t e the amount of j u i c e antigen. In theory t h i s assay would be quite easy to use, i t would be r a p i d , safe and the only equipment necessary would be a s a t i s f a c t o r y spectrophotometer or c o l o r i m e t e r . Work i s c u r r e n t l y progressing toward a complete c h a r a c t e r i z a t i o n and o p t i m i z a t i o n of t h i s assay. Immunoassay f o r Orange J u i c e While our work on the RIA and EIA f o r limonin was i n progress, a research group i n I s r a e l (35) reported on an immunoassay f o r estimating the orange j u i c e content of commercial s o f t drinks and reconstituted juice. This i s of great importance to the c i t r u s i n d u s t r y s i n c e a d u l t e r a t e d or improperly r e c o n s t i t u t e d products g r e a t l y a f f e c t the q u a l i t y of the product and thus u l t i m a t e l y r e f l e c t upon c i t r u s q u a l i t y c o n t r o l c r e d i b i l i t y . A great d e a l of research e f f o r t has already been expended i n t h i s area of product r e c o n s t i t u t i o n and the problem seems to be never ending as new methods of a d u l t e r a t i o n always seem to be emerging. In t h i s new report the authors report that they were able to e l i c i t antibody production by i n j e c t i o n of pure orange juice. The t e s t f o r determination of orange j u i c e content was that of g e l d i f f u s i o n (35) and the d e t e c t i o n l i m i t s were s t a t i s t i c a l l y v a l i d f o r concentrations as low as 2.5%. From the data presented i n t h i s paper, i t i s evident that t h i s immunological method i s more accurate and s e n s i t i v e than any method p r e s e n t l y a v a i l a b l e . More importantly the assay i s cheap, simple, r a p i d and can be performed i n any o r d i n a r i l y equipped l a b o r a t o r y . P r e s e r v a t i v e s , c o l o r a n t s , o i l s and j u i c e s of other f r u i t s do not a f f e c t the assay. I n t e r e s t i n g l y , the p r e l i m i n a r y s t u d i e s of the antigen-antibody i n t e r a c t i o n suggest that the antigen i s not a protein. Conclusions I t i s both encouraging and e x c i t i n g to observe that the f i e l d of immunology has f i n a l l y been d i r e c t e d toward p l a n t s and molecules of p l a n t o r i g i n . For a l l of plant science, the future developments of the RIA, EIA and other immunoassays should a i d g r e a t l y i n s o l v i n g some of our most troublesome problems. We are of the o p i n i o n that the immunoassay has the p o t e n t i a l to become a major a n a l y t i c a l t o o l i n the c i t r u s industry and i n Table IV are l i s t e d j u s t some of the areas where t h i s procedure might be employed.
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IV
Uses of Immunoassay i n Q u a l i t y Related
Programs
A)
Basic Research: 1) D i s t r i b u t i o n analyses i n v e g e t a t i v e parts 2) D i s t r i b u t i o n and l o c a l i z a t i o n w i t h i n f r u i t parts 3) I n t e r - and i n t r a - p l a n t and f r u i t v a r i a t i o n 4) C u l t i v a r v a r i a t i o n analyses 5) Stock/Scion r e l a t i o n s h i p s 6) Geographic & n u t r i e n t e f f e c t s on q u a l i t y 7) Crop improvement s t u d i e s ( s e l e c t i o n of i n d i v i d u a l p l a n t s possessing improved q u a l i t y t r a i t (s)) 8) Tissue c u l t u r e s t u d i e s ( c e l l l i n e s e l e c t i o n ) 9) Biochemical s t u d i e s of compound synthesis and metabolism 10) E a r l y d e t e c t i o n of b a c t e r i a l and v i r a l i n f e c t i o n s 11) Seasonality s t u d i e s
B)
Applied Research: 1) Use i n t e s t houses to monitor incoming f r u i t 2) Measurement of compound l e v e l s i n the f i n i s h e d s i n g l e strength and concentrate products 3) Use i n blending (e.g. navel j u i c e with n o n - b i t t e r orange) 4) B a c t e r i a l contamination-detection of organisms or compounds 5) Pulp wash s t u d i e s - a d u l t e r a t e d j u i c e
Since an immunoassay can be developed f o r each compound or antigen, i t i s conceivable that w i t h i n the not too d i s t a n t future we w i l l have many such assays a v a i l a b l e f o r both b a s i c research and a p p l i e d uses. Acknowledgement A p o r t i o n of t h i s research was supported by a grant from the F l o r i d a C i t r u s Commission, Lakeland, F l o r i d a and by a grant of the Bundesminister f u r Forschung und Technologie, Bonn (to Prof. Zenk, Bochum).
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Literature Cited 1. Nagy, S.; Shaw, P.E.; Veldhuis, M.K. "Citrus Science and Technology"; Avi: Westport, Ct., 1977; Vol. 1. 2. Maier, V.P.; Brewster, L.C. Proc. Int. Soc. Citriculture, 1977, 3, 709. 3. Ting, S.V.; Attaway, J.A. In "The Biochemistry of Fruits and Their Products"; Ed. by A.C. Hulme, Academic Press: New York, 1971; Vol. 2. 4. Blandstone, H.A.W.; Woodman, J.S.; Adams, J.B. In "The Biochemistry of Fruits and Their Products"; Ed. by A.C. Hulme, Academic Press: New York, 1971; Vol. 2. 5. Wood, J.F.; Reed, H.M. Texas Agric. Exp. Stn. Annu. Rep., 1935, 48, 246. 6. Wood, J.F.; Reed, H.M. Texas Agric. Exp. Stn. Bull., 1938, 563, 1. 7. Kebby, R.G.; Kepper, A.H. Agric. Gaz. N.S.W., 1948, 59, 357. 8. Hendrix, C.M. Jr.; Viale, H.E.; Johnson, J.D.; Vilece, R.J. In "Citrus Science and Technology"; Ed. by S. Nagy, P.E. Shaw, and M.K. Veldhuis; Avi: Westport Ct., 1977; Vol. 2, 482. 9. Maier, V.P.; Bennett, R.D.; Hasegawa, S. In "Citrus Science and Technology"; Ed. by S. Nagy, P.E. Shaw, and M.K. Veldhuis, Avi: Westport, Ct., 1977; Vol. 1, 355. 10. Weiler, E.W.; Zenk, M.H. Phytochemistry, 1976, 15, 1537. 11. Yalow, R.S.; Berson, S.A. Nature, 1959, 184, 1648. 12. Catsimpoolas, N. "Immunological Aspects of Foods"; Avi: Westport, Ct., 1977. 13. Langone, J.J.; Gjika, H.B.; van Vunakis, H. Biochemistry, 1973, 2, 5025. 14. Pengelly, W.; Meins, F. Jr. Planta, 1977, 135, 173. 15. Weiler, E. Planta, 1979, 144, 255. 16. Zenk, M.H.; El-Shagi, H.; Ahrens, H.; Stockigt, J.; Weiler, E.W.; Deus, B. In "Plant tissue culture and its biotechnological application"; Ed. by W. Barz, E. Reinhard, M.H. Zenk; Springer: New York, 1977; 27.
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27, 1980.
