Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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the ice cream plant. No one should be allowed to

enter the processing environment who is not familiar

with the required sanitary procedures or who does not

conform to the required dress and personal hygiene

measures. Freedom from chronic contagious diseases

should be confirmed yearly by medical examination.

Provided that the preparation of ice cream is conducted

in a closed processing cycle with modern industrial

equipment of high hygiene standards, the opportunities

for contamination by human contact are few.

0020 Tests carried out during the production process

should be indicative of the standard of hygiene.

Storage (Quality Deterioration)

0021 Microorganisms are unable to grow in ice cream

stored at correct temperatures, although many sur-

vive for extended periods. It is a perfect substrate for

microbe development as far as its composition is

concerned. It has all the essential components (sugar,

proteins, water), oxygen, as well as a relatively high

and suitable pH. High temperature is the only agent

not available. If a rise in temperature does occur, all

the conditions are in place for the development and

proliferation of microbes that may exist. Microbio-

logical considerations, therefore, primarily involve

the elimination of vegetative pathogens by pasteuriza-

tion and the prevention of recontamination due to

insanitary processing and packaging, at all stages up

to point of sale, the microbiological status of ingredi-

ents with particular reference to thermoduric organ-

isms and preformed toxins, and the prevention of

microbial growth before freezing.

0022 Ice cream may be sold direct from the freezer as a

soft-serve product, or it may be further reduced in

temperature and frozen in wind tunnels at 40



or in hardening rooms, to produce ‘hard’ ice cream,

which will be stored at a temperature of about

30



C until it is sold. Deep-freezing stabilizes the

microbial content of ice cream: microorganisms

found in it no longer proliferate. Some sensitive

species (Gram-negative) die and their populations

decrease. Even if the period between freezing and

final sale is several months, there will be little change,

if any, in the microbial content of the ice cream.

Extensive research has shown that both Mycobacter-

ium and Salmonella, as well as many other less harm-

ful but often more resistant types, can survive at the

low temperature of storage for very long periods.

They do not multiply provided that the temperature

is low enough for the ice cream to remain hard; in

effect, the microbial quality of ice cream is ‘locked in’

by the hardening process.

0023 Problems of quality deterioration can appear when

there is a delay between pasteurization and freezing,

mainly a delay from heat treatment to cooling of the

mix, or the aging of the mix at a temperature higher

than 4



C due to ignorance or failure of the freezing

system. Spoilage can also occur, in cases of melting

and refreezing of the product resulting from tempera-

ture fluctuations or failure of the freezing systems.

Special care is needed with the mix for soft-serve ice

cream that has to be transported, often for long dis-

tances, in trucks to retail soft-serve stores or stands

where it is kept soft-frozen and dispensed to con-

sumers. Both contamination and temperature abuse

of the mix may easily occur. Furthermore, refriger-

ation space is usually limited, and adequate facilities

for cleaning and sanitizing the freezer and the associ-

ated equipment are often lacking or are, at best,

marginal.

0024Under these conditions, especially when inadequ-

ate practices have preceded storage, the ice cream is

overloaded with microbes which lead to quality de-

terioration or even to cases of food poisoning.

Reference is made in the Further Reading section to

cases of food poisoning following the consumption

of ice cream contaminated by microbes, such as

Staphylococcus, Salmonella, Shigella, Listeria, and

Streptococcus group A organisms. More serious

cases may occur with homemade ice creams, where

a combination of faulty practices, such as the use of

raw milk, eggs contaminated with Salmonella, inad-

equate heat treatment, no rapid cooling following

heat treatment, and contamination by infected per-

sons give rise to products with high microbial loads,

especially of pathogenic bacteria which can survive

many months in contaminated ice creams. (See Lis-

teria: Properties and Occurrence; Shigella; Staphylo-

coccus: Properties and Occurrence.)

Problems at Point of Sale

0025The largest proportion of microbiological problems

with ice cream products, in general, is due to poor

techniques of selling and serving at the final point of

distribution. This plays a vital role in the microbio-

logical load of ice cream. Even when the greatest care

has been taken to produce an ice cream of the highest

quality, it is still liable to contamination at the point

of sale.

0026The method of sale has a major bearing on the level

of contamination to which the product is subjected.

Although much ice cream is retailed in its final pack-

aging, a significant quantity is portioned from bulk

packs at the point of sale.

0027Prepackaged ice cream that is sold in individual

package is considered safer because it has to be

handled only by the consumer in its wrapping and

can be contaminated only by the consumer. Attention

3240 ICE CREAM/Microbiology

must be paid to the right storage temperature without

temperature variations or failure of the freezing

system. Greater degrees of contamination may occur

in ice cream which is portioned from bulk packs in

restaurants or coffee shops, mobile vans (complete

with their own electricity generation equipment),

and kiosks. This process is the weak link from the

hygiene viewpoint since many outlets have only

limited facilities for hand washing and utensil steril-

ization. In order to minimize the possibility of con-

tamination, the equipment (servers, wafer holders,

and so on) has to be kept free of all residues of ice

cream, which might otherwise melt and allow the

growth of bacteria to recommence. Whenever pos-

sible, these items of equipment should be kept in

running cold water. If they have to be kept in a jug

of water, this water must be changed regularly to

avoid it becoming a source of contaminating bacteria.

The personal hygiene of the server is also important.

Persons handling and serving food should be trained

to live up to definite and rigid standards of personal

hygiene. Hair, hands and fingernails, uniforms and

shoes have to be clean. Soap, water, and single-service

towels should be easily accessible.

0028 Soft-serve ice cream sold directly from a dispensing

freezer can become contaminated very easily unless

stringent precautions are taken. The product is usually

manufactured at the point of sale, which may be

a specialist outlet or cafe

, a nonfood outlet such as

a gas (petrol) filling station, or a mobile outlet or

kiosk. Soft-serve ice cream may be manufactured

from a conventional mix produced on the premises,

from an ultrahigh-temperature-processed, aseptically

packaged mix, or from a spray-dried powder mix.

Powdered mixes may be formulated for reconstitution

in either hot or cold water. Hot-water mixes are prefer-

able with respect to hygiene, but cold-water mixes

are often considered more convenient. Attention must

be paid to the reconstitution of the mixes; this must be

done under satisfactory hygienic conditions to prevent

the proliferation of Salmonella, which may survive if

mixes are not prepared carefully.

0029 Soft-serve ice cream freezers and ancillary equip-

ment should be dismantled, cleaned, and sanitized

daily. It must be recognized that maintenance of the

necessary hygiene standards can be more difficult in

an environment primarily concerned with retailing

than in one wholly concerned with manufacturing.

Particular difficulties may be encountered in outlets

which are predominantly nonfood, such as filling

stations, and those with inherently limited facilities,

such as kiosks. The self-pasteurizing soft-serve freezer

offers at least a partial solution. Such equipment is

designed to heat the mix and machine surfaces in

contact with the mix to an appropriate temperature

and time before cooling rapidly to about 4



C. It is

usual to ‘pasteurize’ daily, usually at the end of the

working day, and restrict full cleaning and sanitiza-

tion to a weekly basis. It must be emphasized that

self-pasteurizing freezers are not intended to process

unpasteurized mix. Generally, special dispensing

freezers should be kept in constant operation, and

placed inside the shop with the taps facing the inter-

ior. In addition, they must not be directly exposed to

the sun, dust, or flies.

0030Probably the most serious and dangerous sources

of contamination are operation and serving. Many

major food-poisoning outbreaks have been caused

by human contamination. Cases of typhoid fever,

including deaths, have been reported to be caused

by ice cream contaminated by the manufacturer

who was a urinary excretor of Salmonella typhi.

There has been a case of Shigella dysentery caused

by an ice cream that was accidentally touched by a

monkey. Also, outbreaks involving salmonellae and

staphylococci have been reported. The personal hy-

giene and habits of vendors at sale points are import-

ant. Education, in addition to medical inspection, is

absolutely necessary and no employee must be

allowed to work without full medical clearance.

0031Finally, birds, rodents, insects, and pet animals

have no place at the retail selling point.

0032Many countries, recognizing the significance of ice

cream in relation to public health, have legislated as

to the conditions and methods to be used for heat

treatment and subsequent storage and sale.

See also: Bacillus: Occurrence; Freezing: Storage of

Frozen Foods; Listeria: Properties and Occurrence;

Mycobacteria; Pasteurization: Principles; Spoilage:

Bacterial Spoilage; Staphylococcus: Properties and

Occurrence; Storage Stability: Mechanisms of

Degradation

Further Reading

Frazier WC and Westhoff DC (1988) Food Microbiology,

4th edn. New York: McGraw-Hill.

Hennessy TW, Hedberg CW, Slutsker L et al. (1996) A

national outbreak of Salmonella enteritidis infection

from ice cream. New England Journal of Medicine

334: 1281

International Commission on Microbiological Specifica-

tions for Food (1974) Microorganisms in Foods 1.

Toronto: ICMSF/University of Toronto Press.

International Commission on Microbiological Specifica-

tions for Food (1980) Microbial Ecology of Foods,

vol. 2. Food Commodities. London: ICMSF/Academic

Press.

Jervis DI (1992) Hygiene in milk product manufacture. In:

Early R (ed.) The Technology of Dairy Products, pp.

272–299. Chichester: John Wiley.

ICE CREAM/Microbiology 3241

Mantis AI (1993) Hygiene and the Technology of Milk and

its Products. Thessaloniki: Kiriakidis.

Marshall R and Arbuckle WS (1996) Ice Cream, 5th edn.

New York: Chapman & Hall.

Miettinen M, Bjo

rkroth J and Korkeala H (1999) Charac-

terization of Listeria monocytogenes from ice

cream plant by serotyping and pulsed field gel electo-

phoresis. International Journal of Food Microbiology

46: 187

Rothwell J (1985) Microbiology of frozen dairy products.

In: Robinson RK (ed.) Microbiology of Frozen Foods,

pp. 209–232. London: Elsevier.

Rothwell J (1990) Microbiology of the ice cream and

related products. In: Robinson RK (ed.) Dairy Micro-

biology, vol. 2, pp. 1–39. London: Elsevier.

Varnam A and Sutherland J (1993) Milk and Milk Products.

Food and Commodities series no. 1. London: Chapman

& Hall.

Immobilized Enzymes See Enzymes: Functions and Characteristics; Uses in Food Processing; Uses in

Analysis

IMMUNOASSAYS

Contents

Principles

Radioimmunoassay and Enzyme Immunoassay

Principles

F S Chu, University of Wisconsin, Madison, WI, USA

Introduction

0001 Antibodies are a group of globular proteins called

immunoglobulins (Ig), which are formed in response

to the invasion of foreign substances in vertebrates.

Igs are classified into five groups: IgA, IgG, IgE,

IgD, and IgM. IgG, which has a molecular weight

of 150 kDa and is composed of two heavy chains

(50 kDa) and two light chains (25 kDa), is one of the

most common serum Igs (80% of total Igs). Anti-

bodies are capable of binding with foreign substances

noncovalently with high affinity and, thus, to inacti-

vate them.

0002 Substances that induce the defensive action by pro-

ducing antibodies in the host are called antigens, and

are characterized by their immunogenicity and their

antigenicity. Immunogenicity is the capability of a

molecule to induce antibody formation. Thus, the

term ‘immunogens’ specifically refers to those sub-

stances capable of inducing production of antibodies.

The antigenicity, or antigenic specificity, however, is

the characteristics/capabilities of the interaction of

the antigen with antibodies. The interaction involves

small sites, which are called ‘antigenic determinants’

or ‘epitopes’ on the surface of the antigen molecules.

0003In contrast to antigens, low-molecular-weight com-

pounds (less than 2500 Da) are not immunogenic.

These compounds are called haptens and can only

become immunogenic after conjugation to a high-

molecular-weight protein carrier.

0004Although it has been known for more than a cen-

tury that vertebrates are capable of producing specific

antibodies after invasion by foreign substances, the

complexity of immunoreactions has only been

revealed in the last two decades. With the advances

in understanding of the mechanism of immunoreac-

tions, the specific interaction between antibody and

antigen/hapten has been used as the basis for several

analytical techniques called immunoassays. These

assays, which are characterized by their high sensi-

tivity, specificity, and simplicity, were originally

developed for the analysis of clinical samples. Recent

development has led to the application of immuno-

assays for food and agricultural chemicals, including

both normal and abnormal food components,

additives and contaminants, as well as agricultural

chemical residues.

0005In contrast to clinical samples, immunoassays

for specific substances in foods and feeds are more

3242 IMMUNOASSAYS/Principles

complicated because: (1) specific antibodies must first

be raised against a wide range of food components

and chemicals. Some are antigens, whereas others are

nonimmunogenic haptens, most of which are lipid-

soluble; (2) foods are very complex systems and

sample matrices often interfere with the assay; (3)

changes in physicochemical properties of food com-

ponents/contaminants during processing of foods

often complicate immunoassays because the antibody

may not recognize the denatured species. Neverthe-

less, much progress has been made and numerous

review articles on the immunoassay of food compon-

ents/contaminants have appeared. The reader should

consult these reviews for details.

Principles

0006 All immunochemical techniques are based on the

specific noncovalent interaction between antigens/

haptens and specific antibodies. Earlier applications

of immunochemical methods for analytical purposes

were confined to the interaction between antibodies

and high-molecular-weight antigens that led to the

precipitation of the antigen–antibody complex for

quantitation. However, precipitates are also not

formed between antibody and haptens. Investigations

in the last three decades have led to the development

of more sensitive immunochemical methods. Thus,

the term ‘immunoassay’ now also includes the

modern immunochemical techniques which involve

the use of labeled marker for the antigen or antibody

and innovative approaches for the separation of free

antigen and antigen–antibody complex. As a result of

the development of new instruments and incorpor-

ation of label markers in the assay system, new

innovative precipitation methods with high sensitiv-

ity are now also available.

0007 For simplicity, the binding of antibody with a

simple hapten, which has only one antigenic deter-

minant site (univalent), is used to illustrate the

antigen–antibody interaction. As shown in eqn (1),

the interaction depends on the equilibrium or affinity

constants (K ¼k

), which is the ratio of the associ-

ation (k

) and dissociation rate constants (k

) and the

concentrations of the reactants, i.e. [antibody] and

[antigen].

½Antibodyþ½Antigen

½AntibodyAntigen

0008 In general, the K values for antibody–antigen inter-

action are in the range of 10

–10

l mol

1

, but they

may vary from 10

to 10

l mol

1

. Antibody with a

high K value is more preferable for the assay, but any

reaction with a K value above 10

l mol

1

would be

feasible. For analytical purposes, either a labeled

antigen or a labeled antibody marker is present in

the system. With an adequate method to separate

the antibody–antigen complex from the free antigen

and antibody, as well as a sensitive method for detec-

tion of the markers in the free or bound form, the

concentration of the analyte can readily be deter-

mined. In general, a standard curve is established

and then used to calculate the concentration of un-

known sample. Thus, it is critical to have a pure

standard from a reliable source which should also

be tested frequently for its stability.

Preparation of Immunochemical

Reagents

0009As indicated above, an effective immunoassay

depends on the availability of: (1) a specific antibody;

(2) a sensitive marker antigen or antibody; and (3) an

effective separation method. Methods for the prepar-

ation of marker antigen or antibody and methods

for the separation of the free and bound species of

antigen and antibody vary considerably with different

immunochemical methods used. Approaches for the

preparation of immunogen and antibody as well as

methods for characterization of antibody can be

generalized as follows.

Preparation of Immunogens

0010Antibodies against major food proteins and naturally

occurring foodborne microbial toxins/contami-

nants have been produced and used in different

types of immunoassays. High-molecular-weight

substances such as proteins and polysaccharides can

be used directly as immunogens. In general, well-

characterized and pure immunogens are used for

generating antibodies. With the development of

monoclonal antibody technology, partially purified

proteins or even crude cellular extracts from patho-

genic bacteria have also been used successfully for

immunization as long as an effective method in

selecting specific hybridoma cell lines is available.

Because of structural changes for some proteins and

other macromolecules during food processing that

alter the immunogenic site, purified proteins are

not necessarily used to generate antibodies from

components in food-related products. Rather, for

determination of such materials in processed foods,

specific antibodies must be raised for corresponding

denatured proteins. Thus, depending on the specific

components to be analyzed, one must consider

selecting an immunogen which has comparable

properties to that actually found in the food.

0011Haptens must first be conjugated to a protein/poly-

peptide carrier before immunization. Methods for

IMMUNOASSAYS/Principles 3243

the conjugation of various low-molecular-weight nat-

urally occurring toxins, including antibiotics, myco-

toxins, phycotoxins (marine toxins), pesticides, and

other agricultural chemicals, to protein carriers have

been established. Proteins, including bovine serum

albumin (BSA), modified BSA, and keyhole limpet

hemocyanin, are most commonly used. In general, if

the hapten has a reactive group such as a carboxylic

or an amino group, it can be conjugated to the protein

directly via one of the following approaches: water-

soluble carbodiimide method, mixed anhydride

method, condensation method in the presence of

formaldehyde (Mannich reaction), cross-linking

with glutaraldehyde, activated ester method through

the formation of N-hydroxysuccinimide (NHS)

esters, 1,1

-carbonylimidazole and m-maleimidoben-

zoyl-N-hydroxysuccinimide ester, and many other in-

novative methods. Derivatization is necessary if no

reactive group is present. Before derivatization or

conjugation, one must select appropriate strategies

to insure that certain portions of the hapten molecule

are exposed. Not only the amount of hapten conju-

gated to the protein molecule is important in deter-

mining the effectiveness of the conjugates as an

immunogen, but also the position of the side chain

in the hapten where conjugation is made, as well as

the space between the hapten and protein molecule

are critical. However, a conjugate containing a large

amount of hapten may not necessarily be a better

immunogen. Antibodies have been obtained from

rabbits immunized with immunogen containing only

2 mol of hapten per mole of carrier protein. The

optimum amount of hapten coupled to a protein

that yields a good immunogen should be experimen-

tally determined, but generally is in the range of 10–

20 mol per mole of protein.

0012 Rapid progress in cloning genes for many proteins/

enzymes/toxins in food and agricultural commodities

has led to another approach for the preparation of

immunogens. By knowing the DNA sequence of a

gene, the amino acid sequence for a specific gene

product in which we are interested could be deduced.

Through structural analysis, the highly immunogenic

fragment in this protein could be identified and

chemically synthesized. These peptides could then be

conjugated to a branched-chain lysine to form a mul-

tiple-chain immunogenic polypeptide for immuniza-

tion. Using this approach, it is possible to generate an

antibody for a protein even before it is purified and

characterized.

Preparation of Antibodies

0013 Two approaches have been used for the production of

antibodies. The classic method involves immuniza-

tion of animals and then obtaining the antibodies

from the sera of the immunized animals. Antibodies

obtained by this approach are polyclonal anti-

bodies (PAbs) because they are derived from different

B lymphocyte clones. In this method, immunogens

are mixed with adjuvant and then injected into

animals, most commonly rabbits, intradermally or

subcutaneously through the thigh or on the back at

multiple sites. For production of a large quantity of

antiserum, large animals such goats and horses are

used. Other animals, such as pigs and mice, have

also been used. Production of antibody has been

optimized by immunization of chickens and then

isolation of antibody from eggs.

0014The amount of immunogen used in the immuniza-

tion varies considerably. In general, 1–2 mg protein is

used for each rabbit. To avoid toxicity of some food-

borne bacterial toxins, a smaller amount of toxins or

toxoids are used in the initial immunization (e.g.,

5 mg) and this is followed by a gradual increase in

dose for subsequent booster injections. For haptens,

200–500 mg of conjugate is generally used in each

injection. Although antiserum obtained from animals

could be used directly in the immunoassay, simple

purification such as ammonium sulfate precipitation

is generally necessary. Further purification of anti-

serum through various chromatographic methods

can improve the efficacy of immunoassays.

0015If the immunogen is highly immunogenic, antibody

titers, as determined by different approaches such

as immunodiffusion, radioimmunoassay (RIA) or

enzyme immunoassay (EIA), usually start to increase

at 4–7 weeks. Subsequent booster injections are gen-

erally performed once a month. Since PAbs are het-

erogeneous, it is advisable that the purified antigen be

used in the titer determination. For haptens, a specific

hapten-marker must be used. Use of inadequate

antigen or hapten-markers usually fails to provide

information regarding whether the immunogen and

immunization protocols are effective.

0016Although production of PAbs is relatively simple

and the affinity of Pabs is generally high, they are

heterogeneous, and their supply is limited. With in-

creasing demand for large amounts of antibodies with

homogeneous properties for immunoassays, as well

as advances in hybridoma technology, specific mono-

clonal antibodies (MAbs) for a number of food com-

ponents and contaminants in foods have also been

produced. In this method, spleen cells obtained from

mice immunized with immunogen or hapten–protein

conjugates are fused with myeloma cells, followed by

propagation of the hybrid cells, and selection and

characterization of the clones, as well as isolation

and characterization of the antibody. Production of

mAbs can either be carried out in tissue culture or in

mice, in which ascites fluids are collected. Thus,

3244 IMMUNOASSAYS/Principles

mAbs are derived from a single B lymphocyte clone

and are immortal. Once a hybridoma cell line is

obtained, there is an unlimited supply of homoge-

neous antibodies with unique properties. Similar to

the production of PAbs, the key to success in

obtaining a useful hybridoma cell line lies in the

effectiveness of immunogens as well as the immuno-

gen-marker or the hapten-marker. The importance of

an effective immunoassay protocol in selecting the

positive clones cannot be overemphasized. Many

new stable hybridoma cell lines for production of

antibodies against various food components, contam-

inants, naturally occurring toxicants, and agricultural

residuals are now available. Rapid progress in mo-

lecular cloning of MAbs has also led to the applica-

tion of this technology for the production of antibody

important to agricultural and food scientists and

several laboratories have succeeded in this regard.

Antiidiotype Antibodies

0017 The development of immunochemical methods for

agriculture and food analysis has led to a great

demand for specific antibodies and related immuno-

chemical reagents for the assay. An alternative ap-

proach for preparing immunochemical reagents is

through generating antiidiotype antibodies (Ab2),

which have gained wide application in diagnostic

and therapeutic areas for large molecules. In the last

few years, Ab2 against small-molecular-weight

haptens, including insecticides, herbicides, hormones,

mycotoxins, and phycotoxins, have been successfully

developed. Some Ab2 have not only been shown

effectively to mimic the biological functions of the

haptens, but have also been used as the immunogen

to generate antiantiidiotype antibodies (Ab3) with

specificity similar to the original antibody (Ab1).

Both Ab2 and Ab3 have been shown to be effective

in immunoassays.

Characterization of Antibodies

0018 Antibodies obtained from the above methods should

be well-characterized before they are used in the

immunoassay. In general, both the type of immuno-

globulin and the specificity of the antibody to various

analogs of the immunogen/hapten should be deter-

mined. The typing of immunoglobulin is generally

done by using commercially available kits. Informa-

tion on the immunoglobulin type is important in

selecting adequate markers as well as an appropriate

method for separation of free antigen or antibody

from the antigen–antibody complex in the immuno-

assay. Information on the specificity will provide an

accurate assessment as to whether structurally related

compounds will interfere in the immunoassay. They

could also be used to estimate the affinity of the

antibody to the immunogen/hapten and its structur-

ally related analogs.

0019The specificity of the antibody, usually expressed as

the cross-reactivity of an antibody, is primarily deter-

mined by the type of immunogen that has been used.

A minor change in the immunogen structure could

generate a different antibody. Some antibodies are

very specific, whilst others show a broad specificity.

The apparent cross-reactivity of the antibody also

varies with the type and concentrations of the marker

antibody or antigen and the format used in the

immunoassay. Thus, in addition to knowing the

specificity of the antibodies generated by a specific

immunogen, the conditions under which that specifi-

city was determined should be stated. Preferably, the

‘apparent cross-reactivity’ should be reevaluated

under the conditions that are to be used for the

assay. The specificity of the polyclonal antibodies

can be improved by removal of nonspecific antibodies

through immunoabsorption.

0020For haptens, the site in the hapten molecule linking

to the carrier protein is important. The degree of

cross-reactivity of these antibodies with structural

analogs of the hapten used in the conjugation should

be determined. The accuracy of immunoassay of

hapten in naturally contaminated samples is generally

affected by both the specificity of the antibody and

the possible presence of structurally related analogs

of the hapten in the sample as well as by some sub-

stances with similar structures of the hapten.

0021Experimentally, the cross-reactivity of antibody is

determined by an immunoassay where various struc-

turally related analogs of immunogen/haptens at a

wide range of concentrations are used to compete

with the binding of the marker ligand with the anti-

body in the assay. The concentration at 50% inhib-

ition (I

) of the binding is generally used as the basis

to calculate the relative cross-reactivity for each

analog. A typical example of such a competitive RIA

and enzyme-linked immunosorbent assay (ELISA) for

aflatoxins is shown in Figure 1. The specificity of the

antibody is generally documented with data describ-

ing antibody production and characterization for a

particular immunogen.

Immunoassays other than RIA and EIA

used in Food Analysis

0022With the availability of different antibodies against vari-

ous food components and contaminants, many types of

immunoassays have been developed. Before modern

immunoassays such as RIA and EIA were developed,

several classic methods, most of which are based on

the precipitation of the immunocomplex, were used

widely for the analysis of high-molecular-weight

IMMUNOASSAYS/Principles 3245

immunogens. Considerable improvement has been

made over the years to some of these methods. With

the availability of new instruments and enhancement of

the particle size, both the precision and sensitivity have

been improved in recent years. Thus, they are still fre-

quently used for the analysis of food constituents.

Immunochemical methods other than RIA and ELISA

in food analysis are briefly described here.

Precipitation

0023 This method is based on the formation of precipitate

as a result of antibody–antigen interaction. Several

formats, which require several minutes to several

days to perform and have a sensitivity range from

0.1 to 10 mgml

1

, have been used.

0024Precipitation in solution or quantitative precipitin

method In general, an aliquot of different concen-

trations of antigen or antibody is added to a series of

tubes containing a constant amount of antibody or

antigen. A precipitate is formed after incubation. The

concentration of the unreacted antigen or antibody in

the supernatant solution or the weight of the precipi-

tate of the antigen–antibody aggregate is determined

after centrifugation. Maximum precipitate is only

formed at the equivalent zone where none of the

reactants is at excess. This approach is very tedious

and subject to error, and is not commonly used at

present. Instead, turbidimeter and nephelometer are

used in most laboratories. Recent developments have

led to measuring the light-scattering effect of the

particles instrumentally; the term ‘light-scattering

immunoassay’ is used for this type of assay.

0025Methods based on simple diffusion of reactants

and subsequent precipitation of immunocomplex in

agar gel These methods, which are based on the

formation of precipitin bands or lines in agar

gel, were originally developed by Oudin and Ouch-

terlony. Three formats are generally used: simple

gel diffusion, double diffusion, and radial immuno-

diffusion (RID).

0026Simple gel diffusion This method involves the use of

a small tube (about 0.5 8 cm; Oudin tube) partially

filled with agar containing antibody (occasionally

antigen). The test solution is placed on top of the

agar column. After antigen diffuses into the agar, a

precipitation band is formed at the interface as a

result of the antigen–antibody interaction.

0027Double diffusion (double immunodiffusion) This

method involves the use of an agar gel plate (some-

times called an Ouchterlony plate) where cups or

wells containing test antibody and antigen solutions

are placed in such an order that the antigen and

antibody solutions diffuse toward each other through

the gel. Most commonly, the antibody well is placed

in the center, with the antigen wells around it. After

incubation, a band is formed between the antibody

and test antigen solution. The intensity of the preci-

pitin band is related to the concentration of the react-

ants. With appropriate arrangement of the wells for

the antigen and antibody, the cross-reactivity of the

antibody with structurally related antigen could also

be determined. If the precipitin band of the antigen is

completely connected with the band of the next well,

these two antigens are identical. If they are partially

connected to form a spur (arc) type of band, then they

are partially identical. However, if both bands cross

each other, these two antigens are not identical.

100

(b)

(a)

oxi.

Percentage of binding

Percentage of max. absorbance (414 nm)

0 1.0 10 100 1000

Concentration (n

−1

)

fig0001 Figure 1 Cross-reactivity of polyclonal antibody against afla-

toxin B

with various aflatoxins as determined by (a) radioimmu-

noassay and (b) enzyme-linked immunosorbent assay. (d),

Aflatoxin B

;(g), aflatoxin B

-carboxymethyl-oxime; (s), afla-

toxin B

;(m), aflatoxin G

;(n), aflatoxin G

;(&), aflatoxin M

;

(h), sterigmamatocystin.

3246 IMMUNOASSAYS/Principles

0028 Radial immunodiffusion In this assay, antibody is

distributed throughout the gel matrix. The test anti-

gen is placed in wells punched into the gel. After

incubation, the antigen is diffused into the gel to

form an opaque zone where the immunocomplex

occurs. The diameter or area of the circular zone is

proportional to concentrations of the test solutions.

0029 Combination of other separation methods with

immunodiffusion and precipitation reaction One of

the most common approaches is immunoelectro-

diffusion (IED) or immunoelectrophoresis, which

involves the separation of antigen electrophoretically,

followed by immunodiffusion. In practice, a trough

parallel to the direction of electrophoresis in a gel is

cut immediately after the electrophoresis. The trough

is then filled with specific antibody. After incubation,

precipitin bands/arcs are formed in the gel.

0030 Several variations of this method have been de-

veloped. For example, the IED or so-called ‘rocket’

immunoelectrophoresis method involves the combin-

ation of electrophoresis with RID. In this assay, the

antibody is distributed in the gel and antigen or ana-

lyte is placed in the well at the end of the gel. Electro-

phoresis is carried out under conditions which do not

permit the migration of antibody. After electro-

phoresis and diffusion, rocket-shaped precipitates

are formed. The height of the rocket is generally

related to the concentrations of the test solution.

Other variations such as counterimmunoelectrophor-

esis or immunoosmophoresis, or electrosyneresis and

zone immunoassay (ZIA), have been reported, but

have not been widely used.

Agglutination Assay/Particle Immunoassay

0031 The unique ability of immunoglobulin to agglutinate

cells with multiple antigens has been used in an assay

system to determine antigen through competition.

Different from the precipitation methods, either an

antigen or the corresponding antibody is attached to

an inert particle via direct binding or covalent inter-

action, which serves as the label. Whereas both red

blood cells (RBCs) and latex particles have been used

for this purpose for some years, recent developments

have led to the use of other particles. Thus, a general

term of ‘particle immunoassay’ is also used for this

type of assay.

0032 Hemagglutination In hemagglutination (or hemag-

glutination inhibition assay, HIA, or passive hemag-

glutination assay, PHA), RBCs (generally sheep),

which have been coated or coupled with antigen

(thus called sensitized RBCs), are incubated with anti-

body and sample. After incubation, the degree of

agglutination is determined. In this assay, antigen in

the sample competes with the antigen coated on the

RBCs for their interaction with antibody. Another

variation of the HIA is reverse passive hemagglutina-

tion assay, in which the purified antibody is coupled

to the RBCs. Agglutination is observed when antigen

or sample extract is added to the system. Although

HIA is relatively simple, food extracts often interfere

with the assay and a large amount of antiserum is

needed. Only semiquantitative information can be

obtained.

0033Latex agglutination Instead of using sheep RBCs,

latex particles coated with antigen have been used as

the agglutinating agent in the antigen/antibody reac-

tion. Both regular inhibition and reversed type of

agglutination methods have been used.

0034Other particles and new intrumentation methods for

particle counting Several other particles, including

microcapsule gelatin, silicate, gold, and emulsified

fluorocarbon, have also been introduced as labels in

recent years. Among these, the microcapsule gelatin is

used most often. Instruments to measure the agglutin-

ated particles are now available and are termed ‘par-

ticle-counting immunoassay’. Both photometric and

light-scattering methods have also been used. The

availability of sophisticated instruments has not

only improved the sensitivity to a level in the sub-

nanograms per milliliter, but has also led to an

automated system that can handle large volumes

of samples with good precision.

Other Immunochemical Approaches

0035Several other immunochemical methods that have

gained wide applications in recent years are cited

below. Immunoadsorption methods are based on

temporary immobilization of immunocomplexes on

a solid support; immunoaffinity chromatography is a

means of removing undesirable antibodies from an

immune serum using the corresponding antigens.

Conversely, immunoaffinity chromatography is also

used for retaining a given antigen from a complex

matrix by using the antiantigen antibodies. Usually,

the antigens or the antibodies are immobilized on a

support and packed in a small column for immunoaf-

finity chromatography, which is then used in different

types of immunoassays and automation. Some rapid-

screening test kits are based on this principle and have

gained wide applications. In addition to this tech-

nique for haptens, immunomagnetic beads have been

used in affinity enrichment and isolation of patho-

genic bacteria, including Escherichia coli O157:H7.

The separation is quick and convenient, requires no

elaborate equipment, and can be used instead of pre-

cultivation as a precursor for other tests to extract the

IMMUNOASSAYS/Principles 3247

target organism. Another powerful immunochemical

method is the Western blot, which involves the

separation of food components electrophoretically,

followed by transfer of the separated components on

to nitrocellulose membrane and then staining immu-

nochemically with either enzyme-labeled or radio-

labeled antibody. Using a similar principle and in

combination with microscopy, a rapid antibody-

direct epifluorescent filter technique (antibody-

DEFT) for screening of E. coli O157:H7 in beef at a

level of 0.1 CFU g

1

was developed recently. The

antibody-DEFT involved membrane filtration, fluor-

escent antibody staining, and epifluorescence micro-

scopy and was accomplished in less than 1h.

Seealso: Immunoassays:Radioimmunoassayand

EnzymeImmunoassay