Immobilization of biofunctional substances

Radiat. Phys. Chem. Vol+ 18, No. I-2, pp. 343-356, 1981 Printed in Great Britain.

0146-5724/81/070324--14502.00/0 Pergamon Press Ltd.

IMMOBILIZATION

OF

BIOFUNCTIONAL

Isao Takasaki

Radiation

Japan

Atomic

SUBSTANCES

Kaetsu

Chemistry Energy

Watanuki-machi,

Research

Research

Takasaki,

Establishment,

Institute,

Gunma, Japan

Introduction The applied radiation chemistry has made great contributions to the development of polymeric

industrial materials by the characteristic

reaction means such as crosslinking,

graft c o p o l y m e r i z a t i o n and low-

temperature or solid-phase polymerization.

Recently,

polymer science and

technology has inclined remarkably to field of life science. examples of this tendency were the

studies

The typical

on biocompatible polymers

for medical use and on immobilized enzymes with polymeric materials. As well known, radiation has been applied directly to various fields of life science and succeeded in mutation, so on.

However,

sterilization,

radiotherapy and

we can find a new approach for the contribution to life

science by means of radiation chemistry and of polymer chemistry. sure that radiation as a reaction and working up mean

It is

will show many

advantages on this new way too. The author's group has studied the radiation-induced at low temperatures

and in

g l a s s - f o r m i n g monomers

i~5)

in a supercooled state.

have the following characteristics high viscosity, ty.

polymerization

solid state since 1962, especially using The glass-forming monomers

in a supercooled phase

(2) homogeneous amorphous phase,

: (i) extremely

(3) large polymerizabili-

These properties are favorably utilized to applications at low

temperature region.

As the typical applications,

tion process can be proposed,in principle polymerization dimension,

in bulk under an exact control

(2) entrapping or adhering

two kinds of polymeriza-

: (i) casting or molding of polymer quality and

(composing)

polymerization

for

unstable guest components keeping their original activities and functions. As for the former example,the casting ~ro~ess of organic glass plastics)

by radiation can be mentionea.

such as lenses,

(optical

In this case, optical articles

prisms and Fresnel lenses were prepared with excellent

optical homogeneity and m o l d i n g exactness as the result of polymerization control.

On the other hand,

the latter example o ~ application

is attained

in the immobilization process of various functional components with polymer by radiation. foaming agent,

The polymerized composites

examples.

Among them,

components

such as enzymes, microbial cells,

cally active substances Because,

including catalyst,

photochromic compound and biofunctional component are the the immobilization of various biofunctional tissue cells and physiologi-

is the most important and promising application.

it is probable that biotechnology and bioengineering

should be

one of the most essential basis for the future development of industries and the immobilization purpose.

technique may be the key point technology for the

It is expected that radiation and polymer chemistry have the

343

344

I. KAETSU

predominant effects on this key technology.

The possibility and the

present situatuation will be reviewed in this presentation. Purpose of immobilization Purpose of immobilization can be summarized as follows insolubilization, process,

solidification

(2) shaping,composing

: (i) fixation,

for repeated long uses and continuous

to desirable forms,

convenient to reaction and operation,

prolongation against heat, pH, organic reagent, inhibition by substrate or product,

sizes and structures

(3) stabilization,

protection,

life

self-digestion and

(4) controlled

slow release for

moderate and durable efficacy and high bioavailability,

(5) utilization

of complexed effects with properties of polymer matrix such as separative function,

bioaffinity or biocompatibility,

rheological

property,

multi-components

mechanical

strength and

(6) utilization of corelated effects by composing

biofunctional

substances in a same matrix,

of substrate selectivity and foreign body recognition. -

(7) control

Among them,

(I)

(4) problems are being studied actively in the present stage and the

others should be the important problems to be solved in the near future.

Methods for immobilization The hitherto known immobilization methods are classified

into

several types according to their principles of immobilization.

But, the

fundamental methods can be summarized to the following three types

: (i)

chemically binding method with a covalent bond between enzyme and carrier, (2) ionic or physical adsorption method,

(3) physically trapping or

composing method for b i o c o m p o n e n t with matrix. chemical binding method

are as follows

activity on base material (i) low activity yield, material,

On the other hand,

Moreover,

immobilization,

inert

the advantages of the physically composing

: (I) high activity yield,

(2) simple and easy

(3) general applicability to biocomponents,

of carriers.

:

(2) selectivity to reaction condition and base

method are the followings operation,

(2) surface

The disadvantages are as follows

(3) no applicability to living body and chemically

substance.

(1),(2),

surface.

The advantages of the

: (]) firm fixation,

(4) broad variety

according to the recent progress in this type of

the advantageous points of the chemically binding method

can be satisfied to some degree also in the physically composing

method. The physically composing method for immobilization can be devided into three ways according to the means for composing. follows

They are as

: (i) crosslinking method for polymer solution,

tion method for vinyl m o n o m e r , o l i g o m e r

and prepolymer

absence of polymer other than vinyl polymer,

(2) polymeriza-

in presence or

(3) polycondensation or poly-

addition method for various monomers other than vinyl compound. second type composing method by vinyl polymerization the following reasons

is advantageous

: (i) abundant variety of monomers,

convenient shaping by casting, polymerization

The for

(2) easy and

(3) applicable of low temperature radiation

in a supercooled state.

Immobilization of biofunctional substances

M5

Radiation can be used for both the chemically binding method and the physically composing method in manners shown in Fig.l. (i) use of radiation for the chemically binding method enzyme VlllIlllI~ radiation~ 7 ~ ~a~ ~ ~ base m a t e r l a l ~ , v////////~ % V/////'/A ~_ ~biliz^A T n chemic611y ......... monomer graf--~t rg ~_ ~ binding ~ %_ product graft copolymer ~ (2) use of radiation for the physically composing method

..oen - ~

mixing

"~ shapin~ monomer, polymer solution

~olin~

~

~

~P

• ~rradlation

r!!!e ctZed

me

Fig.l use of radiation for immobilization That is, the graft copolymerization is usefully utilized to give a reactive functionality to the base material surface for chemically binding or to give an affinity and adhesiveness with other polymers to the base material surface for physically composing coating. On the other hand, the radiation crosslinking and the low temperature-supercooled phase polymerization are the very convenient means for the physically composing immobilization. The former field by the grafting mean was studied activei0~12) ly by prof. A.Hoffman. The physically composing technique has been studied and developed by Japanese workers since Dr.J.Dobo carried out the immobilization of trypsin by X-ray induced polymerization of acrylamide in 1970~ 3) Entrapping of various enzymes with the radiation crosslinking of polyvinylalcohol in aqueous solution was studied by the researchers in _ . ~4 ~15) Research Institute for Polymers and Textiles and composing of various biocomponents with the radiation polymerization of crystalline monomers at low temperatures was investigated by the group in National Food Resear16~17) ch Institute. The latter group reached an optimum carrier composition consisting of acrylamide, methylene-bis-acrylamide and some kinds of acrylic acid metal salts. However, in those studies, the used monomers and the resulted polymers as carriers for composing were limitted within the hydrophilic materials and therefore biocomponents were rather enclosed inside polymer matrix just as in the hitherto known entrapping immobilization method. The author's group studied and developed the composing method applicable to general biocomponents0bY mean of the low temperature and supercooled phase polymerization by radiation, using less hvdrophilic or rather hydrophobic glass-forming monomers as the carri~J7)According--~--to this method, biocomponents are composed mainly on the surface region of carrier and therefore the product have the surface bioactivity. So, this method can be called the adhesion method which is completely different from the entrapping method. The flow diagram of the proposed method is shown in Fig.2. The model scheme for the characteristic structure of the composed product is shown and compared with that of the hitherto known immobilized product in Fig.3. Fig.4 is the model scheme for the immobilization mechanism in the adhesion method , which showed the reason for the efficient surface composing in the present method28)'29)

346

I. KAETSU

As shown in Fig.2, biocomponents can be composed in the simple procedure by mixing with monomer, form,

casting,

suspending or coating into a desirable

then cooling to low temperature and irradiating

into a product.

The m e c h a n i s m of surface adhesion of b i o c o m p o n e n t on polymer carrier as appeared in Fig.3 is explained according to the scheme in Fig.4. is, on the cooling of m o n o m e r - b u f f e r ( i n c l u d i n g water in the buffer crystallizes

to ice.

biocomponent)

That

mixture,

Then the biocomponent in the

b u f f e r is isolated from the ice,dispersed and concentrated on a surface of supercooled monomer phase owing to no solubility of biocomponent monomer.

to

These biocomponents are fixed immediately on polymer surface by

irradiation.

Water in the buffer acts as a pore making agent by c r y s t a l

lization and increases the inner surface area in the matrix. the first important role of supercooled

amorphous dispersion medium for b i o c o m p o n e n t and ice. supercooling

is that of shaping medium,

and structure of monomeric mixture

Therefore,

state is that of homogeneous and The second role of

because the given shaped form

is hardly deformed or decomposed at

low temperatures due to the high viscosity of the supercooled phase. In the present method,

the composition ratio of m o n o m e r - b u f f e r - b i o c o m p o -

nents can be variable very widely and it is possible to keep the very high b i o c o m p o n e n t concentration even in the m i x t u r e and the least monomer.

Therfore,

monomer is that as a binder for the biocomponent, combines

including no buffer

the third role of the supercooled which adheres and

the molecules or particles of biocomponent by quick polymer-

ization at low temperature. cooling immobilization.

These three points are the merits of super-

In addition,

the advantages of radiation

this immobilization can be summarized as follows temperatures

(2) polymerization

ization in n o n - t r a n s p a r e n t and other additives,

for

: (i) composing at low

in viscous and shaped state,(3)

polymer-

suspension state including ice, biocomponent

(4) composing including no catalyst, without contam-

ination for medical use. These advantages mainly owes to the penetrable power of radiation through the substsnces.

Conclusively,it can be said

that the use of radiation especially in a supercooled

state is the best

way for composing biocomponents with polymeric materials. characteristics of the product The characteristics of the product by the adhesion method are as follows

: (i) general a p p l i c a b i l i t y

to biocomponents as cited in TableI,

(2) general usability of vinyl compounds with glass-forming monomers as listed in Table II, for a long use,

(3) relatively high activity yield and its retention

(4) surface bioactivity,

(5) multiple functions from

fixation to release of b i o c o m p 0 n e n t s , c o n t r o l l a b l e facters shown in Table III,

according to the

(6) no restriction and high adabtability for

the shapes and forms of the product.

These properties are useful for

the applications described in the later sections.

Among them,

for the surface adhesion and activity are the followings (Michaelis Menten Value) biocomponent,

the proof~

(i) Km values

of the products are close to those of nati e

(2) sufficient activity is obtained in the completely

hydrophobic product in the water phase reaction,

(3) high activity is

347

Immobilization of biofunctional substances

IBi°c°mp°nentI~ I " - ~ ~ mixing I MonomerI

[

casting I ~cool coating~ ~p,ending

ing,Pi°zla tYmie:: ~r-~ Product1 in super-~

~nzyme

ct°led

Fig.2 Diagramof the present immobilizationmethod by radiation polymerizationin a supercooledstate

~mi O

crosslinking

~

yme

/l/'insoluble carrier covalent bonding method

porous gel entrapping method

adhesion method

Fig.3 Model scheme for the structureof immobilizedenzymes obtained by variousmethods

~

Void t)e~,'een ice and monomer

',,,

queous

Polymer mat ~x

monomer

Ga

i

Cooled

\~

| ~olymerlzmton

Fig.4 Model scheme for the mechanismof in~obilization in the adhesionmethod by radiationpolymerization

348

I. KAETSU

obtained in the reaction with the high molecular weight or solid substrates,

(4)the product composing the fluorescence labelled protein shows

the surface fluorescence. Table I

Examples of biocomponents to be composed in the adhesion m e t h o d

low molecular weight biofunctional substance

high molecular weight biofunctional substance enzyme hemoglobin globlin chlorophyl polypeptide nucleic acid

antibiotic hormone pheromone vitamine immunology substance

living body microbial cell tissue cell organella virus

Table II Usible polymer materials as carrier in the adhesion method g l a s s - f o r m i n g monomers

CH~CXCOO(CH2)nOH CH~CXCOO(CH2CH20)mOCXC=H2C many alkyl acrylate and m e t h a c r y l a t e

g l a s s f o r m i n g monomers mixture

glass-forming and non-

above monomers + less than 40% of other vinyl monomers such as styrene MMA, vinyl acetate, acrylamide, vinyl pyrrolidone

vinyl monomers and

above vinyl monomers + natural polym-

natural polymers mixture

ers such as collagen and albumin

vinyl monomers and

above vinyl monomers + other m o n o m e r

p o l y c o n d e n s a t i o n or

or prepolymer

polyaddition

monomer and prepolymer

type

such as alkoxysilane

monomers mixture

Table III Factors for the control of fixation and release properties

in the product effect on fixation

factor host

effect on release

(matrix polymer)

surface area increase

loosened

accelerated

hydrophilicity

loosened

accelerated

porosity increase

loosened

accelerated

adsorbent increase

fastened

retarded

fastened

retarded

polymer

increase

increase

crosslinking

L

increase

fastened

retarded

structure

fastened

retarded

size increase

fastened

retarded

affinity or interaction with matrix

fastened

retarded

multi-layer guest

(biocomponent)

molecular

Immobilization ofbiofunctionalsubstances

349

Application fields The product by the adhesion method can be called biocomposite. Biocomposite can ~ontribute to various biotechnology and bioengineering fields and will become one of the fundamental and indispensable-techniques for the development of them. The promising application fields cover the followings : (I) enzyme, functional biopolymer and microbial cell technology and engineering, (2) drug technology and engineering, (3) tissue cell abd genetic technology and engineering. This classification depends on the object of composing. The use of biocomposite covers the following fields : (1) analysis, (2) organic synthesis, (3)food and drug improvement (4) energy and resources production, (5) medical use. Among them,the two fields (4) and (5) are investigating by us preferentially. Some results of application will be introduced. Table IV seed field

Application fields of biocomposites need field

enzyme, protein, microbial cell technology -engineering

energies and resources ~ m~

technOlOgy-engineeringex. (1)biomass conversion bioreactor (2) nitrogen fixation bioreactor (3) artificial photo-synthe-

drug technology-engineering

I tissue cell, genetic technology-engineering

Table V

~

sis

| ~ m e d i c a l technology r : -engineering ex. (1)artificial organ (2)drug delivery system (3) immuno-diagnosis and cure system

Functional use of biocomposite

function of biocomposite

biocomponent

use

fixation

enzyme,prote±n r antigeneantibody, microbial cell, tissue cell,

production r medical inspection, diagnosis,cure,

release

enzyme,hormone, pheromone,heparin,

medical cure

fixation-release system

anticancer,immunosubstances microbial cell, tissue cell,------~ enzyme,hormone, antibiotics, ATP, immuno substances, genetic substances,

production medical cure

350

I. KAETSU Biomass conversion by the immobilized enzyme,

cell and organella

Biomass has been noticed as a renewable energy source and a potential resources for chemical and industrial materials.

As well known,

biomass resource covers m u l t i p l e kinds of raw materials different in the q u a n t i t y , f o r m and composition.

However,

the fundamental process of their

conversion to glucose and alcohol consists of refining or liquidifying of raw material, fermentation.

(i) p r e t r e a t m e n t for

(2) saccharification and

(3)

The author's group started the development research on

utilization of radiation technique for the conversion of wastes such as chaff,rice

cellulosic

straw,saw dust,bagas and waste papers.

found that there were two processes of the contribution

It was

, that is, pre-

treatment by irradiation and the saccharification and fermentation by the 34)^ radiation immobilized biocomponents, un this problem, the detailed report will be done in the contributed paper by Dr. Fujimura.

It has

been considered to be difficult in general to saccharify the cellulosic wastes by the immobilized enzyme effectively,because

the hydrolysis of

solid substrate was difficult using the hitherto known entrapping type immobilized enzyme.

However,

the authors found that the enzymatic

reactions of solid or high molecular weight substrate could be carried out effectively as shown in Fig. 6 by the adhesion method.

by

using the biocomposite obtained

Surely,it was one of the successful applied

result of surface bioactivity

in the method.

Fig.7 showed the picture

of the biocomposites used for the wastes conversion. of the use of biocomposite following life,

in the biomass conversion

: (i) continuation of process,

(3) protection

the practical Then,

(2) p r o l o n g a t i o n of bi0catalyst

from inhibition by product and substrate,

reduction for the enzyme production. development,

The advantages system are the

tests of a fundamental

(4)cost

These merits are being proved in scale.

In the first stage of

mainly gasohol will be intended from wastes and plants.

the biomass technology will be enlarged and systematized to the

total utilization

for fuel,food,manure,chemical

al raw materials.

intermediate and industri-

According to such a progress,

site will cover the following processes

contribution of biocompo.

: (i) saccharification and fermen-

tation of starch plants by immobilized g l u c o a m y l a s e and microbial cell, (2)saccharification of cellulosic m a t e r i a l s by immobilized cellulase and microbial cell such as Trichoderma viride, immobilized yeast,

(3)fermentation of glucose by

(4)hydrolysis of algae and plants to useful peptide

by immobilized protease and microbial cell, of hydrogen by immobilized chloroplast,

(5)photosynthetic production

chlorophyll,

and organic synthesis relating to biomass conversion ized enzyme and microbial cell.

(6) fermentation systems by immobil-

Cancer c h e m o t h e r a p y by the slow released anticancers The polymer composite includina anticancers has been applied to 30)-33) the new c h e m o t h e r a p y by implantation, it is said that chemotherapy is atthe

end of its tether because of the drastic secondary reaction and

Immobilization of biofunctional substances

cellulosic wastes starch plants

351

products ] pretreatment irradiation~Islurryl crushing heating

saccharification fermentation by ---~ by lenzyme immobilized enzyme immobilized microbial c e l l ~ r a d i a t i o n yeast J! polymerization., biocomposites~ ~east [monomer I I in supe'~t~ed ~ Fig.5

Diagram of the biomass conversion process using radiation techniques

i00

(/1 0 o ~

so

> O u i

]0 Fig. 6

!

....

i

20 30 Time of hydrolysis(hrs)

i

40

Saccharification of chaff and sweet potato by the immobilized cellulase and glucoamylase (O) 5 { chaff hydrolysis by 5 % immobilized cellulase (•) 5% chaff hydrolysis b~ 5 % cellulase solution (Ik)40% sweet potato hydrolysis by0.2%immobilized glucoamylase (A)40% sweet potato hydrolysis by0.2%glucoamylase ~olution

immobilized cellulase, in'~obilized Trichoderma reesei, immobilized yeast, Fig.7

The pictures of biocomposites for biomass conversion

! 5O

352

I. KAETSU

immunotherapy

is in the primitive stage.

Therefore,

the effective new

mean for a break through of chemotherapy has been searched eagerly. The author's group cooperated with Tokyo Wemens Medical College in the application of anticancer composite capsule to the implantation type drug delivery system. ful.

The result of animal experiments was completely success-

The capsules enclosed directly to the cancered part showed the

characteristic profiles quite different to the hitherto known chemotherapic effects by blood injection or oral dosage. concentration

in blood was negligiblly

of drug in the composite,

(2)distribution of drug was limitted within a

small range arround the enclosed part. cies were obtained as follows observed,

That is : (1)drug

small in spite of the large amount As the results,

remarkable effica-

: (1)no apparent secondary reactions were

(2)remarkable tumor retardation and prolonged surviving were

attained due to high bioavailability by the direct attack to the tumor. The method reached the stage of clinical tests in the region of digestive organs surgery since last year.

The implanted chemotherapy was tried

to the serious inoperable cancers in pancreas,liver and stomach more than 50 examples.

Most of them became free of violent cancer pains and

digestive troubles and are recovering or surviving.

JAERI and Tokyo

Wemens Medical College will start the v e t r i f i c a t i o n project for m i t o m y c i n C capsules this autumn including 15 u n i v e r s i t i e s and institutes to collect more than 200 clinical examples in digestive two years.

The advantages of the present method for drug composing are

the followings

: (i) capsulation without decreasing the anticancer activi-

ty, (2) easy shaping to the required multiple various positions,sizes

and kinds of cancers,

release more than several months. characteristic

m u l t i - l a y e r s capsulation method.

Recently,

our group made progress

One is the development of

(2)multi-components release in a de-

(3) release having a induction p e r i o d , ( 4 ) m u l t i - c o m p o n e n t s

release in the designed order and induction, val and time-cycle. jection.

(3) points are

This technique made the following release

: (1)multi-steps release, rate,

(3)long period durability of

in comparison with the hitherto known slow release capsules

in the two capsulation and dosage techniques.

signed

forms and sizes according to

Particularly, (2) and

mainly consisting of natural polymers.

possible

in Japan

surgery field for

(5)release having the inter-

The other is the method of dosage by the capsule in-

By this method

,it became possible to enclose the needle or

beed form capsules conveniently Immunoassays

in the cancer without operation. for diagnosis by the immobilized

antigens and antibodies The immobilized enzymes were applied to the field of medical

in-

spection by many researchers detecting the micro change in the blood components by the enzyme reaction. applications of immobilized enzymes.

It was certainly one of the suitable But,this field seems to have some

limits for future development as follows

: (i) objects of assay are rather

limitted to the change of normal components, tection is limitted to ug/ml. indirectly.

Recently,the

(2)order of sensitive de-

(3) it contribute

to diagnosis and cure only

field of immunoassay has been noticed for the

Immobilization of biofunctional substances

Fig.8 Implantation of anticancer capsules to animal

u~ c~

I00

~

~0

O~ 4J x

.

f

Flg. 9 Anticancer capsules of various forms and sizes according to the uses

o,

G

0

,

353

!

o

I

I

I

I

30 204 i

0

i

I

I

L

i

i

l

i

l

capsule

I

control

,0 Fig.10

20

¢0

6b

Time after implantation (days) Relation between the in vitro release of mitomycin C ,the weight increase of ascites tumor mouse implanted mitomycinC capsules and the survivor ratio with the time ~ I~2~2x i700/ml °c ~~o u°~

c

6200/mi

uc°cyt°si~

0xl04/ml th~rombocyte

104/ml I

Z

~2

I

I,

6200/mi 18xl04/m 1 l

operation 1~ 2~ 3~ Time after implantation (week)

u ~ 6

~ o~ i •~~ uo Fig.ll

pancreas cancer patient of 65 years old operation

I

~ ~ ~ ~ Time after implantation(day) Changes of the blood components and of the mitomycin C concentration in blood by the implantation of capsules

354

I. KAETSU

reasons

: (1)general applicability to diseases in principle depending on

progress in immunology, than ng/ml,

(2)sensitivity of detection in the order smaller

(3) direct contribution to diagnosis and cure.

cultivated but promising medical field.

It seems an un-

The authors tried the use of the

present immobilization method to immunoassay,

because it has the adapta-

bility in th two points, (i) surface activity for large molecules such as globlin, (2) applicability to whole cell fixation as antigen,(3)easy

shap-

ing. F i ~ 1 2 showed the model scheme for the immunoassay by the immobilized antibody.

Of course,

assay of antigen by immobilized antibody and assay

of antibody by immobilized antigen can be used reversiblly. of practical application

to some immunosystems

The result

showed that detection

in

the order of ng/ml was possible and the corelation coefficient between the enzyme immunoassay by immobilization and the radio immunoassay was 0.98.

The advantages of immobilization are summarized

as follows

: (i)

q u a n t i t a t i v e assay became possible due to the improved sensitivity by removal of non specific reaction product,

(2)reaction and washing oper-

ation became convenient and more complete.

As these are sufficient merits,

it is not necessary to reuse the immobilized sample repeatedly.

The

practical uses of some immunodiagnosis m a t e r i a l s by radiation m e t h o d will be realized in near future and the objects of application will be enlarged extensively to various diseases including cancers and to immunotherapy. Immobilization of tissue cells for the future uses Tissue cells are characteristic Therefore, fixation

for their remarkable unstableness.

the ~ r e s e n t method can be applied favourablly to whole under the mild conditions.

are the followings

: (i) cell culture,

(2) cell separation,

and release of useful p h y s i o l o g i c a l l y active substances, ficial organs, Figures

(5) contribution

(3)production

(4) use as arti-

to cell joint and genetic technology.

showed the pictures of some immobilized cells. As shown here,

these cells were coated state.

cell

The purpose of this immobilization

and fixed with thin polymer layer in a whole cell

It was found that the stability of these cells increased remarkably

by the immobilization and the original

functions of the cells were reserv-

ed to some degree also in the immobilized cells.

For example

evolution activity of the immobilized chloroplast

in PSII photo-chemical

,the oxygen

reaction was kept during 4~ reservation more than 4~ days,while that of 35~ intact chloroplast was lost completely after one day." However, the stabilities of the immobilized cells are not satisfactory for practical use at present.

Further and long-period

improvements will be done in this promis-

ing field. Conclusion As reviewed above,a new approach has been made to contribute to biotechnology and b i o e n g i n e e r i n g using the three m e a n s , ( 1 ) r a d i a t i o n , ( 2 ) s u p e r cooled state and

(3)polymer chemistry and polymer technique.

The basic

adhesion immobilization method and the resulted biocomposites have been applied to various fields including agriculture,medicine

and pharmacy.

Immobilization of biofunctional substances

355

immobilized liver cells

10~m

immobilized erythrocytes

Fig.13 Photographs of the immobilized cells i~obilized chloroplast

~ a

~

~

antigen in

~

c

a

r

~

O

~

~

r e ~

~

Fig.12 Model scheme for the enzyme immunoassay by the immobilized antibod~

~

~

e n z y m eimmunoassay

Table Vl Some examples of immuno-diagnosis using the immobilized antigens and antibodies antigen

antibody

object of diagnosis

alpha-fetoprotein

IgG

primary liver tumor

EB(Epstein-Barr) virus

!gG, igM

Burkitt lymphoma, nasopharynseal tumor

liver cell membrane antigen merozoite

IgG

chronic Hepatitis

IgG, IgM

maralia

immobilization was done for antigens except the case of alph-feto protein RPC' Vat 1R NO. I - 2 - - Y

356

I. KAETSU

The results were in general

successful

and encouraging.

period strategy of radiation processinq continue

the active connection with the biotechnology

region trying to keep the initiative essential medical

merits and advantages.

engineering

As the long

for the future,I can propose

and priority of radiation by its

Energy and resource production

are the suitable

to

and bioengineering and

targets of this approach.

References i) I.Kaetsu,K.Tsuji,K.Hayashi,S.Okamura 2) I.Kaetsu,Y.Nakase,K,Hayashi

: J.Polymer

: J. Macromol.

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