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immunocomplex formation. Whereas the general

principle of EIA is similar to RIA, there is an amplifi-

cation system present in this assay, and thus it is

more sensitive. Since no radioactive substances are

used, the assays avoid the problems encountered

in handling radioactivity. Enzyme labeling can be

achieved by conjugation of the enzyme to an antigen

or antibody via periodate oxidation with subsequ-

ent reductive alkylation method, cross-linking

using glutaraldehyde or others. Some of the methods

used in the conjugation of hapten to proteins can

also be used. However, to avoid nonspecific inter-

action, methods for coupling of protein/hapten to

enzyme should be different from the one that had

been used for conjugating the hapten to protein

for immunization purpose. Although horseradish

peroxidase and alkaline phosphatase are the two

enzymes most commonly used, others, such as

glucose-6-phosphate dehydrogenase, coupled with

oxidoreductase and luciferase, glucose oxidase,

beta-galactosidase, urease, and others have also

been employed.

0011 Depending on whether or not the immunocomplex

is separated from the free antigen, EIAs are further

divided into two types. One type is a homogeneous

system, which is based on the modification of enzyme

activity occurring when antibody binds with the

enzyme-labeled antigen/hapten in solution. No separ-

ation is necessary in this assay. One such type of this

system, which is called enzyme mulitiplied immuno-

assay (EMIT), has been used for the analysis of some

antibiotics and hormones in the clinical diagnosis

area. Because modification of enzymatic activity is

generally not significant, this system is not very sensi-

tive (mgml

1

to mg ml

1

range) and has not been

widely used in food analysis.

0012 The other is a heterogeneous system involving sep-

aration of free and bound antigen–antibody. In this

system, either antigen or antibody is bound to the

solid matrix noncovalently, or conjugated to it cova-

lently. Unreacted antibody or antigen/hapten is

simply removed by washing or centrifugation. The

term ‘enzyme-linked immunosorbent assay’ (ELISA)

is used for this type of assay. Solid phases such as

microtiter plates, cellulose, nylon beads/tubes, nitro-

cellulose membrane, polystyrene tubes/balls, and

modified magnetic beads have been used. In some

cases, staphylococcal protein A or protein G is coated

on the solid surface, entrapping the antibody for sub-

sequent analysis. This method is further divided into

two major types. One type is competitive ELISA,

which can be used for the analysis of both hapten

and macromolecule; the other is noncompetitive

sandwich-type ELISA, which is only used for divalent

and multivalent antigens.

Competitive ELISA

0013Depending on whether enzyme-labeled antigen or

antibody is used or whether antigen or antigen is

coated to the solid phase, several types of competitive

ELISAs have been developed. Two major types, i.e.,

direct competitive ELISA (dC-ELISA) and indirect

competitive ELISA (inC-ELISA), are used most com-

monly in food analysis.

0014Direct competitive ELISA In this assay, specific anti-

bodies are first coated to a solid phase. The sample or

standard solution of analyte is generally incubated

simultaneously with enzyme–conjugate or incubated

separately in two steps. The amount of enzyme bound

to the plate is then determined by incubation with a

chromogenic substrate solution. The resulting color/

fluorescence, which is inversely proportional to the

analyte concentration present in the sample, is then

measured instrumentally or by visual comparison

with the standards. A typical standard curve for

dC-ELISA of aflatoxin is shown in Figure 1 of

Immunoassay: Principles and general considerations.

Excluding the time for sample preparation, the assay

itself can generally be completed in 1–2 h. In general,

dC-ELISA is approximately 10–100 times more

sensitive than RIA when purified standard is used.

0015Indirect competitive ELISA (or double-antibody

ELISA) In the inC-ELISA, an antigen or a hapten-

protein (or polypeptide) conjugate (preferably with a

carrier protein/polypeptide that is different from the

original carrier protein) is coated to the solid phase,

e.g., microtiter plate, to facilitate antibody capture.

Antibody and analyte are then incubated together in

the wells of the plate. The amount of antibody bound

to the wells is then determined by reaction with a

second antibody–enzyme complex such as goat anti-

rabbit IgG–enzyme complex or goat antimouse-IgG

(IgM)–enzyme complex and by subsequent reaction

with substrate. Thus, analyte in the sample and anti-

gen in the solid phase compete for the same antibody-

binding site in the solution. The sensitivity of the in

C-ELISA is comparable to or slightly better than the

dC-ELISA, with the advantage that less antibody is

required. In addition, it is not necessary to prepare an

antigen/hapten–enzyme conjugate. The disadvantage

of the method is that an additional incubation step is

necessary for the assay and, thus, it requires longer

analytical time (2–3 h).

0016Two modifications of the inC-ELISA have been

made. One involves the conjugation of antibody,

especially monoclonal antibody, to an enzyme; the

conjugate is then used directly in the assay. Thus,

this modification converts the inC-ELISA format

to a direct assay. The second involves premixing

3250 IMMUNOASSAYS/Radioimmunoassay and Enzyme Immunoassay

the antibody with a second antibody–enzyme

conjugate. Both modifications have shortened assay

time without sacrificing sensitivity. Because only

small amounts of antibody are needed, inC-ELISA

has been used extensively for screening hybridoma

cultures for monoclonal antibody production.

Noncompetitive Sandwich Immunoassay

(or Two-Site ELISA)

0017 In this assay, the antibody is coated on to the solid

phase, most commonly to the wells of a microtiter

plate. The analyte is added and incubated for an

appropriate time. After washing, enzyme-labeled

antibody is added and incubated, followed by reac-

tion with substrate to develop the color. Under opti-

mal antibody and enzyme antibody concentrations,

the color intensity is directly proportional to the con-

centration of analyte. Thus, it is not necessary to

prepare an enzyme-labeled antigen in this assay. A

number of modifications of this method have been

made.

0018 Monoclonal antibody-based sandwich assay In this

assay, a monoclonal antibody (type ‘a’) is coated on

the plate. The plate is sequentially incubated with

analyte and another monoclonal antibody (type ‘b’)

labeled with enzyme. The antibody–enzyme conju-

gate must recognize a different epitope on the analyte

than does the antibody (type ‘a’) used to coat the

plate. Determination of analyte–antibody–enzyme–

complex is then made by incubation with substrate.

With this format, one can select appropriate pairs

of monoclonal antibodies for special analytical

objectives.

0019 Double-antibody sandwich assay In this assay, anti-

body originating from animal species ‘a’ (e.g., rabbit)

is coated on to the plate. The plate is sequentially

incubated with analyte and antibody against the

same analyte from animal species ‘b’ (e.g., mice),

followed by antibody against animal species ‘b’

(goat antimouse) labeled with enzyme, and then

with substrate.

Quick Immunoscreening Tests

0020 Based on the ELISA principle described above, a

number of quick screening tests have been developed.

For example, by adjustment of reagent concentra-

tions, microtiter plate ELISA assay can be completed

in less than 20 mins. Other approaches involve immo-

bilizing the antibody on a paper disk or other mem-

brane which is mounted either in a plastic card or in a

plastic cup, or on the top of a plastic tube. In the

‘dipstick’ assay, antibody or antigen is coated on to

a stick, which is then dipped in various reagents for

subsequent reactions. Substrates leading to the for-

mation of a water-insoluble chromogenic product are

used in these assays. Most of these screening tests are

very simple and easy to perform and take 10–15 min

to complete. They are designed to provide semiquan-

titative information at certain cut-off concentrations

for the substance in which one is interested. The

immunoscreening tests have gained wide application

for monitoring microbial toxins in foods and food

contaminants such as mycotoxins in the field and

versatile assay kits are commercially available.

New Developments and Biosensors:

Nonisotopic Labeling Other than with Enzymes

0021Among many new nonisotopic labeling systems de-

veloped in recently years, FIAs in which the immu-

noreactants are tagged with fluorescent probes are

used most often. For example, in the so-called

‘hit-and-run’ assay for T-2 toxin, a T-2 toxin column

was equilibrated with a fluorescein isothiocyanate

(FITC)-labeled Fab fragment of IgG (anti-T-2 toxin).

Samples containing T-2 toxin were injected into the

column. The FITC-Fab that eluted together with the

samples containing T-2 toxin was then determined in

a standard flowthrough fluorometer. While fluores-

cein isothiocyanate (FITC) has been used most often

for this purpose, a number of other high-quantum-

efficiency reagents, such as the phycobiliproteins,

which include phycoerythrins, phycocyanins, and

allophycocyaninins, have gained wide applications

in recent years. These reagents replace the enzyme as

the markers in both the heterogeneous and homoge-

neous immunoassays.

0022Fluorescence-based substrate has also been used to

improve the sensitivity. New instrumentation further

led to the development of several unique FIAs. For

example, the fluorescence polarization method can

detect the antigen/antibody reaction in solution with

good sensitivity in the homogeneous system (ng to mg

1

range). A time-resolved fluoroimmunoassay,

which involves the use of europium ion (Eu)-labeled

antibodies, has a sensitivity similar to most ELISAs

for aflatoxin analysis (with an IC

of 0.2 ng ml

1

). In

the particle concentration FIA (PCFIA), the antigen/

antibody is bound to the solid phase such as submic-

rometer polystyrene particles and the fluorescence-

labeled reagent and analyte bind to the particle

surface competitively. The sensitivity of this for both

high-molecular-weight proteins and haptens reaches

the ng and sub-ng range in each assay. Similar to FIA,

both bioluminescent and chemiluminescent reagents

have been used as the markers as well as enzyme

substrates in different types of immunoassays.

IMMUNOASSAYS/Radioimmunoassay and Enzyme Immunoassay 3251

Enhanced Amplifications Leading to more Sensitive

and Ultrasensitive Immunoassays

0023 The sensitivity of different immunoassays has also

improved considerably by incorporation of various

amplification systems in conjunction with EIA and

RIA. Whereas biotin (BT) has been used commonly

as a secondary label in systems in conjunction with

labeled avidin (AV) or streptavidin, amplification

with multiple labeling further improved the sensitiv-

ity. The power of amplification is well demonstrated

in the ultrasensitive immunoassay where several

steps of amplifications and solid-phase concentra-

tions are involved. For example, in a noncompetitive

IA of hapten, the hapten is first biotinylated and then

reacted with antihapten IgG-coated solid phase. After

washing, the bound hapten–BT is removed from the

solid phase at pH 1.0. The eluted solution is adjusted

to neutral pH, then reacted with antihapten-Fab’-

enzyme conjugate, followed by incubation with AV

(or streptavidin)-coated solid phase. After washing,

the hapten-BT-antihapten-Fab’-enzyme-solid phase is

reacted with the substrate. Although this involves

many steps, the sensitivity of this system for the hap-

ten reached to 10 attomoles (10

18

mol) per assay.

Automated Immunoassay and Development of

Antigen/Antibody-Based Biosensors

0024 Development of new sensitive immunoassays that

require repeated precise handling of reagents as well

as the demand for large numbers of samples for an-

alysis have led to the establishment of automated

immunoassay systems. Rapid progress on the anti-

gen/antibody-based biosensors for the detection of

various food components, toxins, and contaminants

has also emerged. Only a few examples are cited here

to show the dimension of this type of technology. A

biosensor for a homogeneous immunoassay for T-2

toxin, which involves the use of liposomes and com-

plement, was developed. A fiberoptic immunosensor

has been demonstrated to be effective for several

microbial toxins, including staphyloccocal entero-

toxin B (SEB), T-2 toxins, and fumonisins, in foods.

In the assay of hapten-related toxins, monoclonal

antibodies are covalently bound to an optical fiber

and an evanescent wave effect was utilized to excite

the fluorescent-tagged toxin near the surface of the

monoclonal antibody fiber as the tagged toxin bound

to the fiber. In the presence of unlabeled toxin, it

competes with the labeled toxin for binding with

monoclonal antibody and results in a decrease in

signal. Thus, it is also a competitive assay. For SEB,

the sandwich system rather than the competitive

format was used and an automated immunosensor

detected 5 ng of SEB g

1

of cream in 10 min. In the

automated particle-based immunosensor (API), the

antigen or hapten–protein conjugate is coated on to

the polymethylmethacrylate beads (98 mm), and this

is then pumped into the 1.5-mm diameter glass capil-

lary to serve as the flow cell within the final lens and

trapped on a filter. The sample or calibrated standard

solutions that had been incubated with antibodies

were then allowed to pass through for a short period

(120 s), followed immediately by FITC-labeled goat

antirabbit antibody (120 s), and then finally washed

with buffer to remove excess label. The fluorescence

during each step of the reaction was recorded. Thus,

this is a kinetic exclusion assay and can easily detect

down to a level of 4 ng g

1

of sample (4 p.p.b.) for

aflatoxin in 8 min. In a miniaturized ELISA, urease

acts as an enzyme marker to produce a pH-shift that

can be detected by the ion-sensitive field effect tran-

sistor. This system consisted of a flowthrough set-up

with a pretreated activated fused silica capillary as a

reaction cartridge to serve as a solid phase for the

reversible inC-ELISA with a detection limit of 10 pg

1

for T-2 toxin. In the surface plasmon resonance

(SPR) immunosensor, antibody is adsorbed on to a

thin gold film substrate coupled to a glass prism.

The output beam of a planar light-emitting diode is

focused through the prism to excite SPR at the surface

of the gold film. When a sample containing antigen/

hapten is added to a cell on the outside of the gold

film, the angular profile of reflected light intensity

shifts. This changes the resonance angle and the re-

flected beam intensity at a selected angle, both of

which are proportional to the antigen/hapten concen-

tration. A detection limit of 50 ng ml

1

is obtained

for the mycotoxin fumonisin with an analysis time

under 10 min. A commercial electrochemilumines-

cence (ECL) sensor can detect 100 and 1000 of

Escherichia coli O157 and Salmonella typhimurium,

respectively, in 1 ml of pristine buffer (or 1000–2000

bacteria ml

1

of food samples) in less than 1 h.

Application of Immunoassays in Foods

Immunoassays for Normal Food Components and

Adulteration in Foods

0025Whereas immunochemical methods have been estab-

lished for most normal food components, one of the

major applications of immunoassay in foods is to

detect adulteration. This includes the detection of

nonmeat proteins in meats and meats from different

species. Combinations of electrophoresis with various

classic methods, which provided only some qualitative

information, were used in early investigations. Both

RIA and ELISA are more commonly used at present.

For example, RIA has been used to differentiate cattle

3252 IMMUNOASSAYS/Radioimmunoassay and Enzyme Immunoassay

meat from sheep, donkey, pig, horse, and kangaroo at

5% contamination level. Various ELISA methods

were used to detect albumin and immunoglobulins as

a means of detecting blood residues in the muscles of

various species. Sensitive RIAs and ELISAs for non-

meat proteins, including soybean proteins, cows’

milk, avian egg white protein, wheat gluten, and

other nonmeat proteins (legume, cotton seed, peanut,

sunflower), have also been developed. These assays

are specific and could be used to detect the presence

of these proteins in meat products. Since heat treat-

ment or acid hydrolysis can result in a change of

protein structure, immunoassays for some of these

proteins have also been developed by using antibodies

against the denatured proteins and soluble proteins.

Immunoassays have also been established for some

antinutrients such as trypsin inhibitors, lectins, and

allergens. The so-called radioallergosorbent inhib-

ition test (RAST) involving the use of

125

I-labeled

antihuman IgE, has been used for the detection of

allergens in foods. Allergens in foods are also detected

with double-antibody sandwich ELISA as well as

dC-ELISA and inC-ELISA.

0026 The application of immunoassay for quality con-

trol in food processing has also emerged. For

example, immunoassays of certain forms of casein,

lactoserum, a- and b-lactoalbumins, IgG, lactoferrin,

and certain enzymes produced by psychrotropic bac-

teria in milk were used to determine quality of milk

and dairy products. A sensitive ELISA method is also

available to differentiate animal rennet and other

rennet substitutes. Immunoassay of barley proteins

and other soluble proteins and enzymes has been

used by the beverage industry. Immunoassays of

cortisol and corticosterone were used to monitor

meat quality. An ELISA was established for insect

myosin, which serves as an indicator for the presence

of insects in foods.

Immunoassays for Naturally Occurring

Contaminants and Toxins in Foods

0027 Foodborne pathogens and toxins Immunochemical

methods have been used for more than 30 years for

the detection of foodborne pathogens and high-

molecular-weight microbial toxins in foods. Different

immunochemical methods for foodborne toxins, in-

cluding staphylococcal enterotoxins, Clostridium

perfringens type A enterotoxins, C. botulinum toxins,

Bacillus enterotoxins, and E. coli toxins, have been

developed. Immunoassays of these toxins in foods

have moved from classical immunodiffusion and

hemagglutination methods to modern RIA, and

now, more commonly, to ELISA. Increasingly, mono-

clonal antibodies are used. The sensitivity of various

types of RIA for the detection of staphylococcal

enterotoxins is in the range of 1–5ng ml

1

as com-

pared to that of ELISA of 0.1–5ngml

1

. An EMIT

assay using b-amylase with a sensitivity of 5 ng ml

1

has been developed for SEB. The ELISA sensitivities

for the detection of botulinum toxin and C. perfrin-

gens type A toxin are in the range of 0.125–5ngml

1

and 0.5–500 ng ml

1

, respectively. Recent develop-

ments have led to more sensitive ELISA methods for

monitoring important foodborne bacterial pathogens

such as Campylobacter jejuni, E. coli, Listeria, Sal-

monella, and Yersinina enterocolitica.

0028During the last few years, many studies focused on

the development of immunoassays for E. coli

O157:H7. For example, a bead-ELISA with a sensi-

tivity of about 200 pg ml

1

was developed for specific

detection of the VT2 variant VT2e and a monoclonal

antibody-based sandwich ELISA for VT1 and VT2.

Using an antigen competition format, a solid-phase

fluorescence-based immunoassay was developed. In

this assay, a soft glass capillary tube served as the

solid support, to which heat-killed E. coli O157:H7

were adsorbed. Polyclonal anti-E. coli O157:H7 anti-

body, conjugated with biotin, was used; the bound

antigen–antibody complex was detected using avidin

molecules labeled with a fluorescent cyanine dye Cy5.

Anti-E. coli O157 antibody coated to the inexpensive

macroporous polyester fabric Polymacron was used

to capture E. coli O157 antigens for subsequent

immunoenzymatic detection of the bacteria. In a

rapid and inexpensive sandwich enzyme-linked re-

ceptor-based immunodot assay method, the purified

glycosphingolipid digalactosylceramide (diGalCer)

was used as in the solid-phase matrix to entrap for

the assay.

0029Naturally occurring food toxicants Rapid progress

on the development and application of immunoassay

for low-molecular-weight naturally occurring toxins,

contaminants, and agricultural residues in foods

has also occurred during the last two decades.

Both monoclonal and polyclonal antibodies against

low-molecular-weight food toxins, including marine

toxins (brevetoxin, palytoxin, okadaic acid, paralytic

shellfish toxins, and ciguatoxins) and mycotoxins

(aflatoxins, ochratoxin A, fumonisin, sterigmatocys-

tin, trichothecenes, and zearalenone), are available.

Various immunoassay formats have been developed.

These methods are generally very simple and

sensitive. For example, the lower limits of RIA and

ELISA for purified mycotoxin standards are in the

range of 0.05–0.5 ng and 2.5–5 pg in each assay, re-

spectively. Because of the interference of sample

matrix, the detection limits of most of the mycotoxins

in foods/feed are generally about 1–5 p.p.b. for RIA

and 0.05–0.5 p.p.b. for ELISA with no sample

IMMUNOASSAYS/Radioimmunoassay and Enzyme Immunoassay 3253

clean-up treatment. The detection limits for other

food contaminants by RIA and ELISA varied consid-

erably, but generally are less sensitive than for myco-

toxins.

0030 Since immunoassays for these haptens in foods

share some common problems, strategies to over-

come these problems are discussed here. In general,

the use of affinity purified antibody in combination

with the use of good radioactive-labeled or enzyme-

conjugate marker, or with other systems such as

chemiluminescence, or amplification systems such as

biotin-avidin, can improve the assay sensitivity. How-

ever, one of the major concerns is still interference by

sample matrix. This problem is generally overcome

by dilution of the sample to a range which does not

affect the assay or by using sample extract in the

preparation of the standard curve. Sample clean-up

may sometimes be necessary. For example, the sensi-

tivity of the RIA, dC-ELISA and inC-ELISA for myco-

toxins in foods has improved after such treatment.

The combination of using high-specific-activity radio-

labeled compounds and clean-up samples has allowed

the sensitivity for the detection of aflatoxin M

tissues and milk to reach the 0.01 ng g

1

level in

both RIA and ELISA.

0031 Other naturally occurring contaminants In addition

to mycotoxins and phycotoxins, sensitive immuno-

assays have also been developed for many naturally

occurring toxicants and contaminants, including

DNA-adducts of psoralens, caffeine, naringin,

limonin, and selected isoflavones. Sensitive ELISA

(ng g

1

) for ergot alkaloids can detect about one

sclerotium 20 kg

1

of grain. Immunoassay for potato

glycoalkaloids such as a-solanine, a-chaconine,

and solasonine, and tomato glycoalkaloids such as

a-tomatine, tomatidine (aglycon), and digitonin are

also available. The sensitivity of these assays is in the

1–200 ng g

1

range. Contaminants such as mercury

(100 ng g

1

in scallops) and histamine (10 mgg

1

salmon) are now detectable by immunoassay.

0032 Agricultural chemicals and residues and pestici-

des This is another area for which immunochemical

methods have been established in the last two decades

and have gained wide application. Immunoassays are

available for almost all the important antibiotic resi-

dues that might be present in our foods. For example,

b-lactone antibiotics such as ampicillin, cloxacillin,

and penicillin G, in the range of 1–10 ng g

1

, could

easily be measured in milk. The sensitivity for the

detection of other antibiotics by immunoassay falls

in a similar range. Likewise, immunoassays for many

pesticides are available. The sensitivity for measuring

ethyl parathion, diflubenzuron, and dieldrin in milk

are around 5 ng g

1

. Less than 1 ng g

1

of benomyl/

carbendazim in fruits and vegetables could be deter-

mined by immunoassay. ELISA kits with a sensitivity

in the range of 0.1–5ngml

1

are available for ala-

chlor, aldicarb, atrazine, carbaryl, carbendazim, car-

bofuran, cyanazine, 2,4-D, and metolachlor.

Concluding Remarks

0033The development of immunoassays for the detection

of food components and contaminants has progressed

rapidly in the last few years. Antibodies against

almost all the important agricultural and food-related

compounds are currently available. Sensitive, simple,

and specific immunoassays have been established.

Modern immunoassays such as RIA and ELISA are

gradually replacing the classic immunodiffusion and

agglutination methods for food analyses. These

methods are very simple, sensitive, and specific. To

avoid the use of radioactive compounds, ELISAs are

more commonly used than RIA. The development of

new labeling reagents and enhanced amplification

systems in conjunction with solid-phase technology

further improves the sensitivity and precision of dif-

ferent types of immunoassays, including those of pre-

cipitation and agglutination. With the availability of

immunochemical reagents and sensitive instrumenta-

tion, automation of immunoassays has become a real-

ity. Versatile immunoassay kits that can be used in the

field are commercially available and are widely used

for the detection of toxins and contaminants in foods.

A number of immunoassay protocols have been

adopted as first action by the Association of Official

Analytical Chemists.

0034Whereas immunoassays have been used extensively

for the analysis of contaminants/toxicants/residues in

foods, applications of these methods for the analysis

of food components and food adulteration are also

merging. However, immunoassays for the control of

food processing are not common. Future develop-

ments in the area of rapid test, automation, and

immunosensors will certainly widen the scope of ap-

plications to some of these areas. As such methods are

developed, more collaborative studies are needed to

validate the efficacy of the protocol and assay kits.

Since all immunoassays are based on the specific

interaction of antibody with antigen, it is imperative

for those who run immunoassays to have a clear

understanding of the specificity of the antibody used

in the assay as well as the purity and authenticity of

the standards. Immunochemical methods, including

affinity chromatography as a clean-up tool and

immunoassay as a monitoring system for high-

performance liquid chromatography, thin-layer chro-

matography, and electrophoresis have been used in

3254 IMMUNOASSAYS/Radioimmunoassay and Enzyme Immunoassay

conjunction with other analytical methodologies. The

innovative approaches have led to a new dimension in

the immunochemical methods for food analysis. Un-

questionably, immunoassays will be advanced to an-

other new era in the new millennium and they will be

used extensively in food and agriculture in the coming

years. It will not be surprising to see immunoassay

screening kits will some day reach the consumer

level. Such progress will certainly not only provide

safer foods to eat and a better environment in which

to live, but will also enhance agricultural productivity

by using these new technologies to improve crop qual-

ity and yield and enhance animal and plant health.

See also: Biosensors; Chromatography: Thin-layer

Chromatography; High-performance Liquid

Chromatography

Further Reading

Beier RC and Stanker LH (1996) Immunoassays for Resi-

due Analysis: Food Safety. ACS Symposium Series book

no. 621. Washington, DC: American Chemical Society.

Chu FS (1991) Development and use of immunoassays in

detection of the ecologically important mycotoxins. In:

Bhatnagar D, Lillehoj EB and Arora DK (eds) Handbook

of Applied Mycology, Mycotoxins, vol. V, pp. 87–136.

New York: Dekker.

Fukal L and Kas J (1989) The advantages of immunoassay

in food analysis. Trends in Analytical Chemistry 8:

112–116.

Maggio ET (1980) Enzyme-immunoassay. Boca Raton,

Florida: CRC Press.

Morgan MRA, Smith CJ and Williams PA (1992) Food

Safety and Quality Assurance Application of Immuno-

assay Systems. London: Elsevier Applied Science.

Pestka JJ (1994) Application of immunology to the analysis

and toxicity assessment of mycotoxins. Food and Agri-

culture Immunology 6: 219–234.

Rittenburg JH (1990) Development and Application of

Immunoassay for Food Analysis. London: Elsevier Ap-

plied Science.

Schuurs AHWM and van Weemen BK (1977) Enzyme-

immunoassay. Clinica Chimica Acta 81: 1–40.

Tijssen P (1985) Practice and Theory of Enzyme Immuno-

assays. Amsterdam: Elsevier Scientific.

Vanderlaan M, Stanker LH, Watkins BE and Roberts DW

(1991) Immunoassays for Trace Chemical Analysis:

Monitoring Toxic Chemicals in Humans, Foods and

the Environment. ACS symposium series 451. Washing-

ton, DC: American Chemical Society.

Yallow RS (1978) Radioimmunoassay: a probe for the fine

structure of biologic systems. Science 200: 1236–1245.

IMMUNOLOGY OF FOOD

K M Burghardt and F W Scott, Ottawa Health

Research Institute, Ottawa, Canada

Introduction

0001 The process of eating brings us into intimate contact

with a wide variety of foods derived from plants and

animals. These foods contain carbohydrates, pro-

teins, lipids, micronutrient vitamins and minerals,

dietary fiber, other nonnutrient chemicals, bacteria,

fungi, viruses, environmental contaminants, food

additives, and byproducts of processing and cooking.

Food is the most important modifiable environmental

exposure to which we are subjected. The aim of this

chapter is to explore, in a selective manner, food

components that have the potential to alter the activ-

ity of the immune system. The gut immune system is

continously challenged by a wide spectrum of dietary

macromolecules, their metabolic products, resident

bacteria, and invading pathogens. Luminal antigens

and other molecules are thus a source of information

about our immediate environment. The main inter-

action between food and the immune system occurs in

the gastrointestinal tract, the largest lymphoid organ

in the body. We shall examine the arsenal of immune

cells in the gut-associated lymphoid tissue (GALT),

the defense mechanisms, and selected examples of

immunomodulatory foods and food components

that contribute to health or disease.

Dietary Modulation of the Immune System

0002Human milk provides optimal nutritional, immuno-

logical, physiological, and psychological benefit to a

newborn child. At this vulnerable time when its

immune system is ill equipped to protect itself, the

baby relies on antiidiotypic antibodies and lympho-

cytes it received in utero and which it continues to

acquire through mother’s milk. Breast-fed infants

have been reported to experience significantly fewer

gastrointestinal, respiratory, urinary, and ear infec-

tions. Some studies have shown long-term enhanced

IMMUNOLOGY OF FOOD 3255

protection against Haemophilus influenzae type b

infections, otitis media, diarrhea, respiratory tract

infections, and wheezing bronchitis. Mother’s milk

provides an immediate boost for the young immune

system because it delivers antibodies, cytokines, hor-

mones, growth factors, and cells that promote the

maturation of the intestinal mucosa as well as modu-

late immune responses. It contains factors that estab-

lish a normal, symbiotic microbial gut flora that

protects the newborn from pathogenic bacteria.

There is a debate whether infants at risk for develop-

ment of atopy should be breast-fed because small

amounts of food allergens have been detected in

human breast milk. A prospective study of neonates

identified prolonged (> 9 months) exclusive breast-

feeding as the most likely risk factor for atopy by

2 years of age. In contrast, another study showed

that supplementation with cows’ milk prior to

breast-feeding resulted in fewer cases of atopy by 18

months. Numerous other studies have examined this

question, and it is yet to be established whether ex-

posure to antigens acquired via the mother’s milk

induces oral tolerance or priming. Several confound-

ing factors such as antigen dose, timing, and fre-

quency of exposure contribute to the variable results

obtained.

0003 The type and quantity of essential fatty acids, such

as linoleic acid (n-6), dictate the structure and func-

tion of the cell membrane. Long-chain and saturated

fatty acids reduce fluidity, while short-chain and cis-

unsaturated fatty acids increase membrane fluidity.

Changes to membrane fluidity influence cellular per-

meability as well as reorganize the distribution and

activity of membrane-associated enzymes and recep-

tors. Consequently, this alters the presentation of

membrane surface antigens, which affects cellular

communication, migration, and immune stimulation.

0004 Linoleic acid is a precursor of arachidonic acid

(AA). AA serves as the starting material for eicosa-

noid synthesis. The membranes of lymphocytes and

macrophages contain high concentrations of AA,

> 25% of all esterified membrane fatty acids. Mito-

gens stimulate the release of AA from lymphocytes,

which will be converted to eicosanoids by macro-

phages. Other stimuli that release AA from the cell

membrane are reactive oxygen intermediates (ROI)

and cytokines such as interleukin-1 (IL-1) and tumor

necrosis factor (TNF). Eicosanoids are composed of

two families of molecules, the prostanoids (prosta-

glandins and thromboxanes) and the leukotrienes

(lipoxins and hydroxy acids). Release of these oxida-

tion products has physiological and immunological

consequences. Prostaglandins regulate immune respon-

ses such as IL-2 production, T-cell mitogenesis, and

antibody production. Leukotrienes cause leukocyte

infiltration, stimulate vascular permeability, and

induce smooth-muscle contraction.

0005Linolenic acid, of the n-3 series, was reported to

inhibit leukotriene B

synthesis and decrease inflam-

mation. Linolenic acid is a precursor for eicosapen-

taenoic acid (EPA), which is structurally similar to

AA, but has antiinflammatory effects. In fact, dietary

supplementation with n-3 fatty acids increases the

incorporation of EPA into the membrane with a pro-

portional decrease in the amount of AA. EPA is me-

tabolized to prostacyclins and thromboxanes which

have a net antiinflammatory and antiaggregatory

effect. Thus, the amount and type of essential fatty

acids in the diet can regulate the type of eicosanoid

synthesis. Furthermore, polyunsaturated fatty acids

(PUFA) modulate immune responses by enhancing

antibody production and decreasing cell-mediated re-

sponses, as demonstrated by a shift from T helper

1 (Th1) to Th2 responses that is consistent with IL-2

receptor inhibition. Incorporation of EPA, derived

from dietary n-3, at the expense of AA reduces the

level of PKC activation and thus decreases IL-2 recep-

tor production. There is now considerable evidence

that atherosclerosis is an inflammatory disease of the

vascular system, the prevention of which involves not

only the modification of serum lipids but also a de-

crease in inflammatory reactions with the endothe-

lium. Dietary n-3 PUFA and vitamin E have received

particular attention as protective agents.

0006Dietary fat is also important as a delivery vehicle

for the lipid-soluble vitamins, D, E, A, and K. These

lipophilic vitamins have immunomodulatory func-

tions. Vitamin A, D, and b-carotene regulate leuko-

cyte activity. Vitamin A controls the recruitment

of cells to lymphocyte and macrophage lineages.

Vitamin E has antioxidant properties that prevent

excessive tissue damage by ROI during stress or in-

fection. Since ROI are also involved in activation of

proinflammatory gene expression (e.g., nuclear factor

kappaB (NFkB)), antioxidant vitamins can inhibit the

inflammatory cascade. These vitamins have been

shown to scavenge free radicals that initiate lipid

peroxidation. Lipid peroxidation in polymorphonuc-

lear leukocytes, macrophages, and mast cells also

releases AA.

0007Garlic has been used since the times of the early

Egyptians as a medicinal herb. There are several bio-

logically active ingredients, including protein fraction

4, diallyl disulfide, and S-allyl cysteine in garlic. S-allyl

cysteine can inhibit activation of NFkB in neuroblas-

toma and melanoma cells. Thus, garlic is reported to

inhibit directly tumor cell growth and may potentiate

the immune system by stimulating macrophages, NK

(nuclear killer cells), and killer cells, lymphokine-

activated killer cells, and increasing production of

3256 IMMUNOLOGY OF FOOD

IL-2, TNF (tumor necrosis factor), and interferon-g

(IFN-g). This could be considered an example of indu-

cing a Th1 response that benefits the immune system

fight against certain tumors. It is important to be

aware that individual foods are unlikely by themselves

to be ‘magic bullets’ that boost the immune response

against cancer or regulate other complex chronic dis-

eases. It is the aggregate effect of all dietary exposures

that is important. There are several other examples of

foods and food components that influence the

immune response, including glutamine, arginine, nu-

cleotides, probiotics, vitamins (e.g., vitamin D

), min-

erals (e.g., zinc, iron, copper), and oligosaccharides

(e.g., 1,3 b-glucans), to name a few.

Micronutrient Modulators of the Immune

System

0008 Nutrient status may determine the defense capacity of

the immune system. Many vital relationships between

trace elements such as zinc, iron, selenium, copper,

and cellular function have been described. Most often

these elements are an integral part of enzymatic reac-

tions as cofactors and molecular scaffolds. Recently,

micronutrients have been shown to regulate directly

the immune system.

0009 Zinc has catalytic, structural, and regulatory func-

tions that are well described in cellular metabolism.

Zinc deficiency also has immunological conse-

quences, such as lymphopenia, poor cell- and anti-

body-mediated responses, adrenal hypertrophy, and

thymic atrophy. When zinc becomes limiting, it is

redistributed to vital organs such as the liver and

diverted to the innate arm of the immune system.

Zinc deficiency induces a chronic increase in gluco-

corticoid synthesis, a steroid which promotes apop-

tosis in precursor lymphoid cells. Thus myeloid cells

such as macrophages and neutrophils are spared

while lymphoid compartments are depleted. Although

macrophages are spared from apoptosis, they are still

sensitive to zinc availability. Macrophages isolated

from zinc-deficient mice have a reduced capacity to

phagocytose and generate an oxidative burst, likely

due to the dependence of superoxide dismutase and

certain phospholipases on zinc as a cofactor. Similar

findings were shown in human monocytes. Clearly,

zinc status can alter the choice of defense mechanisms

of the immune system.

0010 Iron is an essential trace element because it is part

of heme compounds, hemoglobin and myoglobin,

which are responsible for oxygen transport and stor-

age, as well as being part of nonheme compounds,

and acting as a cofactor for certain critical enzymes

like mitochondrial aconitase and ribonucleotide

reductase. Moreover, iron availability influences

immune effector functions. In the event of chronic

infection, iron uptake and storage are increased by

proinflammatory cytokines to limit the availability of

this essential cofactor to invading pathogens. In-

creased iron uptake and storage by activated macro-

phages and monocytes also serve to increase IFN-g

and TNF-a activity and thus enhance their cytotoxic

effect. These cytokines also induce the production of

nitric oxide, and can form iron–nitrosyl complexes

and further decrease iron availability. The counter-

effect of the diversion of iron traffic is a reduction in

heme synthesis, which contributes to a reduced

oxygen transport capacity and possibly chronic

anemia. A case in point is anemia of chronic disease,

whose pathogenesis has been linked to activated

macrophages and monocytes.

0011Dietary selenium is covalently bound to methionine

and cysteine and converted to selenide. Selenide pro-

vides an essential cofactor for the synthesis of seleno-

proteins such as glutathione peroxidases and type

1 iodothyronine deiodinase. Selenium has been ex-

ploited in rheumatoid arthritis therapy to reduce

oxygen-derived free radicals that damage inflamed

joints. Furthermore, selenium also has immunomo-

dulatory properties. Selenium-deficient mice show

decreased lymphocyte proliferation in response to

mitogen. Selenium supplementation has been shown

to enhance cell-mediated immune responses in

clinical trials with elderly individuals or subjects

receiving parenteral nutrition.

0012A copper deficit is associated with a small thymus,

anemia, decreased antibody production, and neutro-

penia. In more extreme cases, severe copper defi-

ciency compromises the number and function of

neutrophils and macrophages. Individuals fed a

copper-deficient diet exhibit reduced T-cell prolifer-

ation following mitogen stimulation, an effect that is

attributed to decreased IL-2 production. Acquired

immunity is also impaired in an animal model of

marginal copper deficiency. Pregnant rats were

placed on a marginally copper-deficient diet halfway

through the gestation period. The rat pups showed

reduced cell-mediated responses, yet they had normal

copper status as determined by common indices:

tissue copper, serum copper, and ceruloplasmin activ-

ity. This highlights the exquisite sensitivity of the

immune system to nutrient deficiencies and the inher-

ent difficulties in defining ‘normal’ nutrient require-

ment for optimum immune function.

How Luminal Antigens are Handled by the

Gut Immune System

0013Nonspecific barriers, such as gastric acid, mucus,

digestive enzymes, and peristalsis, as well as specific

IMMUNOLOGY OF FOOD 3257

immunological barriers, including secretory immuno-

globulin A (sIgA) and sIgM, limit antigen access to

the GALT. It is estimated that 1 out of 10

ingested

antigens is absorbed intact by the gastrointestinal

tract, which is in the order of microgram per liter

quantities. Antigens that cross the gut epithelium

are surveyed by immune cells and classified as harm-

less or harmful. The controlled manner by which

luminal antigens gain entry to the GALT is depicted

in Figure 1. Lymphoid follicles, called Peyer’s patches,

are overlaid by membranous epithelial cells (M cells)

whose morphology is typified by a smooth apical

surface, lack of glycocalyx, and deep invaginations

of the basolateral membranes, particularly suited for

antigen uptake and delivery to the closely associated

lymphocytes and macrophages. Other lymphocytes

and dendritic cells are scattered throughout the

lamina propria in anticipation of antigens delivered

through enterocytes. In healthy epithelia, the uptake

of these macromolecules is through active vesicular

transport. When the junctions between gut epithelial

cells become less restrictive, permeability increases

and additional lumen antigens can penetrate the

lamina propria and activate resident immune cells.

The immune response is based on multiple factors.

0014 A dietary antigen that gains access to the GALT has

the potential to induce systemic responses, such as

active immune suppression (oral tolerance), immune

priming, or it can prompt local immune responses

such as the production of sIgA. Oral immune toler-

ance refers to the unresponsiveness of the immune

system, specifically the hyporesponsiveness of mature

lymphocytes in peripheral lymphoid tissue that stems

from prior oral exposure to the antigen. Recent stud-

ies suggest CD4

Th cells are essential for the induc-

tion of oral tolerance because CD8

cytotoxic T

lymphocyte knockout mice can still develop oral tol-

erance. Furthermore, CD4

T cells can transfer oral

tolerance to a naive individual. Immune deviation

from Th1 toward Th2, Th3, or regulatory Tr-1 cells

has been proposed to favor normal production of IgA

with diminished risk of immunopathology. The major

mechanisms, likely acting in concert to establish and

maintain tolerance, are clonal anergy (the lack of

costimulatory molecules), clonal deletion, and sup-

pression by cytokines such as TGF-b. This depends

on the nature of the antigen, dose, maturity of the

host immune system, nature of the flora in the digest-

ive tract, and poorly understood host factors. Under

certain conditions, the immune system is primed for

further immune reaction instead of immune toler-

ance. Moreover, controversy regarding local immune

responses to oral soluble food antigens is not fully

resolved. Some suggest that IgA production and

secretion accompany oral tolerance, while others

contend that healthy individuals do not produce

measurable quantities of food-IgA antibodies. Indeed,

the decision to commit to any of these pathways is the

result of a complex set of cellular interactions and the

summation of many bidirectional messages (Figure 1).

0015Particulate or replicating antigens (e.g., bacteria)

are primarily taken up by M cells and diverted to

Lumen

Suppression

CD8

T cell

Enterocytes

Regulatory cells that secrete TGF-β

(e.g., CD4

T cells in lamina propria or

mesenteric lymph nodes)

MHC class II presentation of luminal

antigen on enterocytes to CD4

T cell

MHC class II presentation

of luminal antigen on

professional APC to

CD4

T cell

Tolerance Active immunity

Food

Peyer's patch

CD4

T cell

Professional APC

(e.g.,dendritic cells)

fig0001 Figure 1 Routes of antigen transport and mechanisms of immune regulation in the gut. APC, aerobic plate count; TGF-b, transform-

ing growth factor-b; MHC, major histocompatibility complex.

3258 IMMUNOLOGY OF FOOD

the immune cells of Peyer’s patches where they will

most likely prime the immune system for an assault

on these foreign intruders. Most food antigens are

digested in the intestinal lumen, which results in vari-

ous tolerogenic forms of the antigen. The requirement

for tolerance induction will depend on the antigenic

mixture and its breakdown products. Most soluble

antigens induce systemic tolerance.

0016 Oral tolerance has been successfully induced

through different regimens and doses of antigens. It

has been suggested that a single high dose of antigen

(> 0.5 mg g

1

body weight) will abrogate antibody,

cytokine, and cell-mediated responses in naive indi-

viduals as a result of T-cell inactivation. Regulatory T

cells are more likely to be activated following mul-

tiple low doses (< 0.1 mg g

1

body weight). In con-

trast, very low doses of antigen (< 0.005 mg g

1

body

weight) were shown to prime systemic and local

immune responses.

0017 The age at first encounter with the antigen is an

important determinant of the type of immune re-

sponse that ensues – oral tolerance or immune

priming. In general it has been observed that it is

easier to tolerize young mice than adult mice. The

mechanisms leading to oral tolerance also vary with

age. Anergy and/or deletion may be predominant in

the immature gut immune system, whereas in adults,

regulatory mechanisms have been established and

tolerance is more likely the result of active immune

suppression. These characteristics of the neonatal

immune system may be ascribed to the less restrictive

absorption in the neonatal gut, less competent or

‘immature’ GALT, or differences in the regulation of

neonatal lymphocyte responses. A breakdown or fail-

ure to establish oral tolerance results in various forms

of food hypersensitivity.

Abnormal Immune Reactions to Food

0018 In some individuals, ingestion of certain foods results

in a disproportionate immune reaction or allergic

reaction. Allergenic foods are commonly resistant to

proteolysis, undergo posttranslational glycosylation,

and may exhibit enzymatic activity. A distinction

should be made between allergy, which is immune-

mediated, and intolerance. Intolerance refers to a

nonimmunologically mediated reaction stemming

from abnormal metabolism (e.g., enzyme deficiency)

or impaired excretion of a food constituent or prod-

uct. Allergy or immediate type 1 hypersensitivity is

usually the result of sufficient numbers of IgE mol-

ecules binding to some dietary macromolecules, thus

initiating the complement cascade and inducing mast

cell degranulation. The release of preformed immune

mediators stored in the mast cells instigates the

immediate onset of symptoms ranging from cutaneous,

and gastrointestinal to respiratory, as well as systemic

anaphylactic reactions. The severity of the allergic

response is associated with the number of immune

cells being activated. An allergen that is distributed

by the circulatory system has the potential to stimu-

late a greater number of mast cells, releasing large

quantities of immune mediators. Studies of intestinal

biopsies have suggested that food allergy in animals

and humans is characterized by an increased number

of intraepithelial lymphocytes, mucosal mast cells,

and eosinophils.

0019Food allergies and other inflammatory disorders of

the intestine are associated with a dysfunction of the

epithelial barrier. However, whether the epithelial

barrier dysfunction predisposes an individual to aller-

gic disease or is a consequence of a proinflammatory

state is controversial. One hypothesis that has sought

to explain food allergy suggests that excessive hygiene

may be preventing the gut immune system from learn-

ing to differentiate between harmful and harmless

macromolecules, leading to an inappropriate inflam-

matory response. Another theory suggests that expos-

ure to high levels of antigen in childhood in

individuals with an inherent dysfunction of the gut

mucosa may promote allergic diseases. IgE cross-

reactivity between food allergens and pathogens is

another possible mechanism that may explain the

observation that risk of allergy may be proportional

to the rate of infection and vaccination. However, the

pathogenesis of food hypersensitivity reactions

remains unknown.

Diet–Gene Regulation

0020Food components can influence directly or indirectly

the transcription of genes. A case in point is curcumin,

which directly influences acute-phase immune re-

sponses. Curcumin, a component of turmeric powder

from the rhizomes of the Curcuma longa L. plant,

has antiinflammatory properties that stem from its

capacity to inhibit the expression of proinflammatory

genes. The transcription of these genes is regulated by

NFkB, which in turn is regulated by an endogenous

inhibitor called IkBa. Curcumin prevents the degrad-

ation of IkBa, which blocks NFkB from translocating

to the nucleus to initiate transcription of kB-dependent

genes such as TNF-a, IL-6, IL-8, Cox-2 (cyclooxygen-

ase), and nitric oxide synthase. Similarly, some

nutrients, such as vitamins A and D, fatty acids, sterols,

and zinc, bind to regulatory elements of genes. For

example, the metallothionein gene promoter requires

the binding of zinc–protein complexes in order to initi-

ate transcription. In addition to the metallothionein

gene, 47 other potential zinc-regulated genes have

IMMUNOLOGY OF FOOD 3259