Immunoassays in Food Safety Applications - ACS Symposium Series

Chapter 5 Immunoassays

in

Food

Safety A p p l i c a t i o n s

Developments and Perspectives

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 11, 2016 | http://pubs.acs.org Publication Date: November 11, 1989 | doi: 10.1021/bk-1989-0442.ch005

Albert E. Pohland, Mary W. Trucksess, and Samuel W. Page Division of Contaminants Chemistry, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, DC 20204

Actual and perceived food safety concerns have necessitated an increase in the monitoring of foods for such natural contaminants as aflatoxins and for residues of pesticides. Immunoassays can provide rapid, simple, and relatively inexpensive methods for the detection of analytes with specificity and with sensitivities directed at the levels of concern. Particularly for aflatoxins, they are rapidly assuming a significant role in the monitoring of foods. However, the misuse of these techniques can potentially compromise any food safety improvement that may result from increased surveillance. Experiences of the Division of Contaminants Chemistry in the development, validation, and applications of immunoassays for natural toxins is discussed.

In the past few years there has been a keen interest in biotechnology-related research, and in the encouragement of the practical application of the results of such research, i.e., technology transfer. One area, of course, which has received a great deal of interest and attention involves the use of immunoassay techniques in the analysis of foods and feeds for a wide variety of contaminants for control and regulatory purposes. In this paper the evaluation of commercially available immunoassay kits in the analysis of foods and feeds for the mycotoxins and aflatoxins will be discussed. Aflatoxins B,, B , G, and G are highly toxic secondary fungal metabolites. Although A,, flavus produces aflatoxin Μ,, M naturally, aflatoxin Μ,, are more commonly found in excreta and milk of animals that have ingested aflatoxin contaminated feed. They are coumarin derivatives containing a fused dehydrofuran moiety (Figure 1). Aflatoxin B, is a demonstrated hepatocarcinogen in several animal species. 2

2

2

This chapter not subject to U.S. copyright Published 1990 American Chemical Society Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


POHLAND ET AL

Immunoassays in Food Safety Applications

Aflatoxin B

Aflatoxin B

2

Aflatoxin G)

Aflatoxin G

2

Aflatoxin MÏ

Aflatoxin M

}

2

Figure 1. Chemical structure of the aflatoxins

Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


40

IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS

I t s h o u l d be p o i n t e d out t h a t a t the p r e s e n t time none o f the kits discussed are being used by the U.S. Food and Drug A d m i n i s t r a t i o n (FDA) f o r r e g u l a t o r y p u r p o s e s ; however, we b e l i e v e the p o t e n t i a l f o r such use i s g r e a t , e s p e c i a l l y i n the sense o f "screening" analyses. The c o n v e n t i o n a l TLC, HPLC o r m i n i column methods f o r d e t e c t i o n o f a f l a t o x i n s a r e time consuming and a r e n o t suitable f o r screening purposes. The Association of Official A n a l y t i c a l Chemists (AOAC) r e f e r s t o a " s c r e e n i n g method" as "a method t o r a p i d l y and r e l i a b l y a n a l y z e a l a r g e number o f samples a t the d e s i g n a t e d l e v e l o f i n t e r e s t i n o r d e r t o e l i m i n a t e n e g a t i v e samples." (1) The U.S. Department o f A g r i c u l t u r e / F e d e r a l G r a i n I n s p e c t i o n S e r v i c e (FGIS) on the o t h e r hand, has a p p r o v e d the use o f e i g h t s u c h k i t s i n i t s c o n t r o l program (2) ; interestingly the USDA/Agricultural Marketing Service (AMS) has not agreed to a u t o m a t i c a l l y use the k i t s a p p r o v e d by FGIS (3). I n e x a m i n i n g the k i t s one i s i m m e d i a t e l y f a c e d w i t h the f o l l o w i n g concern: What c r i t e r i a s h o u l d be u s e d i n the e v a l u a t i o n o f s u c h kits? T h i s i s n o t a t r i v i a l q u e s t i o n , and the number o f o p i n i o n s e x p r e s s e d on the s u b j e c t i n d i c a t e s c o n s i d e r a b l e d i v e r s i t y o f t h o u g h t . Because t h i s q u e s t i o n i s c e n t r a l t o d e c i s i o n s on e v e n t u a l use o f the k i t s , s e v e r a l o r g a n i z a t i o n s have a d d r e s s e d the i s s u e by e s t a b l i s h i n g committees a s s i g n e d the t a s k o f d e v e l o p i n g e v a l u a t i o n g u i d e l i n e s . F o r example, the AOAC e s t a b l i s h e d an "Ad Hoc T a s k F o r c e on T e s t Kits."(4) The I n t e r n a t i o n a l U n i o n o f Pure and A p p l i e d C h e m i s t r y (IUPAC) Commission on Food C h e m i s t r y has e s t a b l i s h e d a Working Group on Immunochemical Methods whose f i r s t p r o j e c t i s t o d e v e l o p " D r a f t G u i d e l i n e s on C r i t e r i a f o r the E v a l u a t i o n , V a l i d a t i o n and Q u a l i t y C o n t r o l on radioimmunoassay ( R I A ) - b a s e d A n a l y t i c a l Methods," t o be f o l l o w e d by s i m i l a r g u i d e l i n e s f o r e n z y m e - l i n k e d immunosorbent a s s a y (ELISA) methods. The E n v i r o n m e n t a l P r o t e c t i o n Agency (EPA) has d e v e l o p e d " G u i d e l i n e s f o r EPA E v a l u a t i o n S t u d i e s on Immunoassays" which are q u i t e extensive (5). I n the USDA s i m i l a r g u i d e l i n e s have been d e v e l o p e d and d e s c r i b e d i n a document e n t i t l e d " D e s i g n C r i t e r i a and T e s t Performance S p e c i f i c a t i o n s f o r A f l a t o x i n S c r e e n i n g Test Kits" (6). An announcement was made i n the F e d e r a l Register d e s c r i b i n g the s p e c i f i c p r o c e d u r e s the USDA i n t e n d s t o f o l l o w i n a p p r o v a l o f the use o f t e s t k i t s i n i t s c o n t r o l p r o g r a m s ( 7 ) . Various o t h e r groups a r e a c t i v e l y engaged i n d e v e l o p i n g s i m i l a r c r i t e r i a . In reviewing the e f f o r t s a t e s t a b l i s h m e n t of c r i t e r i a for e v a l u a t i o n o f s u c h k i t s , the f o l l o w i n g f a c t o r s seem t o be o f c o n c e r n : USEFULNESS • -Cost: r e a g e n t s , e q u i p m e n t / f a c i l i t i e s , speed o f a n a l y s i s • -Use: laboratory/field, trained/untrained analyst (Referred to i n the EPA g u i d e l i n e s as d a t a q u a l i t y o b j e c t i v e s (DQOs). • -Stability: shelf l i f e - Q u a l i t y assurance/Quality c o n t r o l : requirements, p r o t o c o l s , cost, a v a i l a b i l i t y of standards CONFIDENCE FACTORS -Bias, p r e c i s i o n , accuracy, s p e c i f i c i t y • -Limit of detection/determination • - R e p e a t a b i l i t y / r e p r o d u c i b i l i t y (within/between l a b c o e f f i c i e n t of v a r i a t i o n ) • -False positive/negative results


5. POHLAND ET AL. Experimental

41

Studies

There a r e c u r r e n t l y a t l e a s t s i x commercially a v a i l a b l e a f l a t o x i n immunoassay k i t s p r o d u c e d i n t h e U n i t e d S t a t e s , most o f w h i c h have been e x t e n s i v e l y s t u d i e d (Table I ) . I n a d d i t i o n , a t l e a s t s i x f o r e i g n companies a r e m a r k e t i n g s i m i l a r kits. I n most c a s e s t h e s e k i t s a r e dependent on g e n e r a t i o n o f a n t i b o d i e s from t h e same h a p t e n , i . e . a f l a t o x i n B,. Consequently, t h e r e i s u s u a l l y c o n s i d e r a b l e c r o s s r e a c t i v i t y toward o t h e r a f l a t o x i n d e r i v a t i v e s ( B , G,, G ) . I t i s i m p o r t a n t f o r p r o p e r e v a l u a t i o n o f r e s u l t s o b t a i n e d u s i n g t h e k i t s t h a t t h i s c r o s s r e a c t i v i t y be known. Our i n i t i a l s t u d i e s u s i n g the k i t s were d i s a p p o i n t i n g , i n v a r i a b l y r e s u l t i n g i n m o d i f i c a t i o n s o f t h e k i t s t h e m s e l v e s and changes i n p r o t o c o l s f o r use o f the k i t s . F o l l o w i n g t h i s i n i t i a l phase o f work, we d e c i d e d one k i t was r e a d y f o r c o l l a b o r a t i v e s t u d y . This f i r s t c o l l a b o r a t i v e s t u d y i n v o l v e d use o f t h e Neogen A g r i - S c r e e n k i t . The a n t i b o d i e s have s p e c i f i c a b i l i t y t o b i n d a f l a t o x i n B, and v e r y low c r o s s r e a c t i v i t y t o a f l a t o x i n s B , G, and G . T h i s k i t c o n t a i n s : Antibody-coated m i c r o t i t e r wells A f l a t o x i n standard s o l u t i o n Dilution buffer (Tris) Aflatoxin-enzyme conjugate (horseradish peroxidase) S u b s t r a t e A {2,2' a z i n o - d i [ 3 - e t h y l b e n z - t h i a z o l i n e s u l f o n i c a c i d ] } S u b s t r a t e Β (hydrogen p e r o x i d e ) S t o p p i n g s o l u t i o n (2N H S0 ) In t h i s c o l l a b o r a t i v e study fourteen l a b o r a t o r i e s analyzed s i x d i f f e r e n t commodities c o n t a i n i n g a f l a t o x i n B, a t two c o n c e n t r a t i o n l e v e l s as b l i n d d u p l i c a t e s . Two s t a n d a r d s (15 and 50 ng Β,/g) were p r o v i d e d , and c o l l a b o r a t o r s were a s k e d t o r e p o r t t h e i r r e s u l t s as 15 ppb. L a b o r a t o r i e s w i t h m i c r o t i t e r w e l l r e a d e r s were a s k e d t o determine aflatoxin concentrations both visually and spectrophotometrically. I n t h i s k i t a f l a t o x i n B,-antibodies a r e coated onto p l a s t i c m i c r o t i t e r w e l l s . The a f l a t o x i n - c o n t a i n i n g sample i s e x t r a c t e d w i t h MeOH-H 0 (55+45). The e x t r a c t i s d e f a t t e d w i t h hexane, and t h e MeOH e x t r a c t mixed w i t h t h e a f l a t o x i n - e n z y m e c o n j u g a t e and added t o t h e w e l l o f t h e a n t i b o d y - c o a t e d microtiter p l a t e . The a f l a t o x i n i n t h e e x t r a c t and t h e a f l a t o x i n - e n z y m e complex compete f o r t h e a n t i b o d y b i n d i n g s i t e s . The enzyme s u b s t r a t e ( A B T S ) and H 0 s o l u t i o n a r e t h e n added, t h e r e a c t i o n l e a d i n g t o a c o l o r e d p r o d u c t i n t h e p r e s e n c e o f enzyme. The i n t e n s i t y o f c o l o r i s d e t e r m i n e d v i s u a l l y o r s p e c t r o p h o t o m e t r i c a l l y a t 580nm. The r e s u l t s o f t h e c o l l a b o r a t i v e s t u d y a r e t a b u l a t e d i n Table I I ( 8 ) . O v e r a l l c o r r e l a t i o n (81%) between t h e ELISA i n s t r u m e n t a l r e s u l t s and t h o s e o b t a i n e d u s i n g t h e AOAC recommended methods was good. On t h i s b a s i s t h e AOAC a d o p t e d t h e ELISA method o f f i c i a l f i r s t a c t i o n as a s c r e e n i n g method f o r d e t e r m i n i n g t h e p r e s e n c e o r absence o f a f l a t o x i n B, a t a c o n c e n t r a t i o n o f >15 ng/g i n c o t t o n s e e d p r o d u c t s and mixed f e e d s , a s u r p r i s i n g c o n c l u s i o n i n v i e w o f t h e r e l a t i v e l y l a r g e numbers o f both false positive and f a l s e negative results encountered. On t h e o t h e r hand, when an ELISA r e a d e r was u s e d f o r q u a n t i t a t i o n , t h e RSD was 15 ppb; f o r a l l o t h e r commodities t h e RSD was 2



2

2

2

2

4

2

2

2

R

R




42

T a b l e I . C o m m e r c i a l l y A v a i l a b l e Immunoassay T e s t for Aflatoxins

Manufacturer

Kit

Biomed L a b o r d i a g n o s t i k Agromed (FRB) Cambridge L i f e (UK) Environmental D i a g n o s t i c s EZ-Screen B u r l i n g t o n , NC IDEXX Corp. Probe P o r t l a n d , ME I n t ' l . D i a g n o s t i c Systems A f l a - 2 0 S t . J o s e p h , MI May and Baker (UK) Neogen Corp. Lans i n g , MI Oxoid L t d . (UK) P e n i c i l l i n Assays I n c . Maiden, MA Transia Lyon, F r a n c e UBE Tokyo, J a p a n Vicam S o m e r v i l l e , MA 1

2

Agri-Screen

Type

MTP

Analytes

Β,,Μ,

1

MTP

Β,

Card

Β,, B , G,, G

MTP

Β,,Μ,

Cup

Β, ,B ,G,

MTP, AC

2

MTP MTP, AC

Afla-Check

MTP

Aflatest

AC

2

2

2

B,,B ,G, 2

MTP AC

CHARM

Kits

B, Β,, B , G,, G 2

2

Β,, B , G,, G ,Μ,, Mj 2

2

B ,B ,G,,M 1

2

1

B, B, ,B ,G, ,G,M, 2

2

microtiter well a f f i n i t y column



5. POHLAND ET AL.

Table

Commodity

II.

Level* ( g/g)


A g r i - s c r e e n C o l l a b o r a t i v e Study - I (14 C o l l a b o r a t o r s ) ELISA Reader (ng/g)

RSD *** (%)

5 26

2..8±2.6 12..8±8.1

164 73

36

Peanuts, raw

6 20

21..8±7.9 21..8±8.7

31 69

4

Peanuts, roasted

3 28

4..1±4.4 16.,5±13.4

157 85

31

Cottonseed, whole

36 85

35..2±15.9 41..0±12.3

89 42

17 0

Cottonseed, meal

6 31

8.,1±4.9 18..6±9.3

70 51

12

n

Corn

Mixed

R

Visual** negative p o s i t i v e 15

85

8

44

feed

4 35 5..9±5.3 93 14 (23) 15.,9±4.3 27 • D e t e r m i n e d u s i n g AOAC method 26.026-26.031 (CB) f o r c o r n and p e a n u t s , method 26.052-26.059 f o r c o t t o n s e e d , and t h e Shannon method f o r mixed f e e d ( J . A s s o c . O f f . A n a l . Chem. 1983, 66, 582. **% f a l s e p o s i t i v e and n e g a t i v e r e s u l t s , i . e . f o r samples c o n t a i n i n g 15 ppb a n e g a t i v e r e s u l t would be " f a l s e . " ***RSD : r e l a t i v e s t a n d a r d d e v i a t i o n between l a b o r a t o r i e s . R


43



44

u n a c c e p t a b l y h i g h (>69%) f o r samples >15 ppb. T h e r e i s no doubt t h a t the A O A C s d e c i s i o n on t h e s t u d y o c c u r r e d a t a time when e v a l u a t i o n methodologies f o r such kits were i n a v e r y formative stage. ( " I n t e r i m O f f i c i a l F i r s t A c t i o n " i s t h e d e s i g n a t i o n f o r a method whose performance characteristics have been evaluated by collaborative study and a d o p t e d by v o t e o f t h e AOAC. After s u c c e s s f u l u s e f o r a t l e a s t two y e a r s , t h e method may be a d o p t e d as " O f f i c i a l First Action". Because o f t h e l e s s t h a n s a t i s f a c t o r y r e s u l t s o b t a i n e d i n t h e f i r s t s t u d y o f t h e A g r i - s c r e e n t e s t k i t when u s e d w i t h p e a n u t p r o d u c t s and c o r n , a s e c o n d c o l l a b o r a t i v e s t u d y was r u n . I n t h i s s e c o n d s t u d y o f t h e Neogen k i t , twelve coded t e s t samples o f raw and r o a s t e d p e a n u t s and c o r n , w i t h b l i n d r e p l i c a t e s , were a n a l y z e d b y fourteen laboratories. The d e t e r m i n a t i o n was s l i g h t l y m o d i f i e d i n t h a t t e t r a m e t h y l b e n z i d i n e was u s e d i n s t e a d o f ABTS as s u b s t r a t e f o r c o l o r development, and t h e a n a l y s t s were i n s t r u c t e d t o compare c o l o r i n t e n s i t y w i t h s t a n d a r d s o f v a r y i n g c o n c e n t r a t i o n when u s i n g v i s u a l e s t i m a t i o n . The r e s u l t s o f t h i s s t u d y a r e shown i n T a b l e I I I ( 9 ) . Table

Commodity

I I I . A g r i - s c r e e n C o l l a b o r a t i v e Study - I I (14 C o l l a b o r a t o r s ) Level* ( g/g)

ELISA Reader (Ave. ng/g)

n

RSD *** (%) R

Visual negative p o s i t i v e

0 4..8 45.J 11..2 0 31..0 56..0 52.J 6 Peanuts, 1..5 43..5 9..2 roasted 39 .0 0 70..2 23..3 Peanuts, 13 10,.8 125. .0 62. 2 raw 34,.0 0 36. 0 83..0 * D e t e r m i n e d u s i n g AOAC method 26.026-26.031 (Method 1) ( 1 4 ) . * * % F a l s e p o s i t i v e and n e g a t i v e r e s u l t s , i . e . f o r samples c o n t a i n i n g 15 ppb a n e g a t i v e r e s u l t would be f a l s e . " ***RSD : r e l a t i v e s t a n d a r d d e v i a t i o n between l a b o r a t o r i e s . Corn

r

R

O v e r a l l t h e r e was good c o r r e l a t i o n o b s e r v e d between ELISA and TLC r e s u l t s f o r c o r n and r o a s t e d peanut p r o d u c t s , w i t h 93 and 98% c o r r e c t r e s p o n s e s f o r v i s u a l and i n s t r u m e n t a l d e t e r m i n a t i o n s , r e s p e c t i v e l y . I n t h e c a s e o f raw p e a n u t s , a s i g n i f i c a n t number o f f a l s e p o s i t i v e r e s u l t s was n o t e d f o r t h e low l e v e l sample (

Immunoassays in Food Safety Applications - ACS Symposium Series

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