免疫學(xué)檢測與免疫學(xué)技術(shù)全文-畢業(yè)論文-

Chapter

10.免疫學(xué)檢測方法與免疫技術(shù)一、概述免疫學(xué)檢驗方法和免疫化學(xué)技術(shù)主要包括：抗原抗體的制備、純化和鑒定，免疫擴散、免疫電泳、免疫凝集試驗、補體結(jié)合試驗，免疫細胞分離、純化和鑒定，免疫功能檢測，細胞因子檢測，放射免疫檢測，免疫酶標檢測，熒光和發(fā)光免疫技術(shù)，免疫組化實驗方法，原位雜交免疫組化，免疫PCR技術(shù)，免疫微球的應(yīng)用，免疫電鏡技術(shù)，細胞凋亡的檢測方法，膜受體分析，胞內(nèi)鈣鎂濃度的測定和細胞間通訊，流氏細胞儀技術(shù)及應(yīng)用等。從上述內(nèi)容可以看出，免疫技術(shù)是免疫學(xué)和物理、化學(xué)及電子信息和分子生物學(xué)理論和技術(shù)的結(jié)合產(chǎn)物。其應(yīng)用涉及生命科學(xué)的各個領(lǐng)域，已成為現(xiàn)代醫(yī)學(xué)和生物學(xué)研究工作不可缺少的有效工具。免疫技術(shù)的原理和特點：基于免疫應(yīng)答的理論，即抗原抗體的特異性反應(yīng)?；诿庖呒毎慕Y(jié)構(gòu)、應(yīng)答特性和分子基礎(chǔ)。具有特異性高度靈敏性；可重復(fù)性；廣泛適用性；快速反應(yīng)性；可觀察性；可定性、定量，即可控性；組化定位特性等特點。概括起來可分為：沉淀反應(yīng)，即可溶性抗原和抗體間的反應(yīng)。凝集反應(yīng)，即顆粒性抗原和抗體間的反應(yīng)。免疫標記，即用酶、同位素、熒光素或電子致密物質(zhì)標記。免疫印漬，即用標記抗體與待測蛋白質(zhì)印跡結(jié)合。單抗及工程抗體技術(shù)，即分子生物技術(shù)。流氏細胞術(shù)，即熒光標記，流體噴射，激光和能譜檢測，電 腦分析。Ag-Ab

reactionsTests

for

Ag-Ab

reactionsNature

of

Ag/Ab

ReactionsLock

and

Key

ConceptNon-covalent

BondsHydrogen

bondsElectrostatic

bondsVan

der

Waal

forcesHydrophobic

bondsMultiple

BondsReversibleSource:

Li,

Y.,

Li,

H.,

Smith-Gill,

S.

J.,Mariuzza,

R.

A.,

Biochemistry

39,

6296,

2000:85/chime2/lyso-abfr.htmAffinity

=attractive

and

repulsive

forcesAbAgAbAgAffinity

Strength

of

the

reaction

between

a

single

antigenicdeterminant

and

a

single

Ab

combining

siteCalculation

of

AffinityAg

+

Ab

Ag-AbApplying

the

Law

of

Mass

Action:[Ag-Ab]Keq

=[Ag]

x

[Ab]Avidity

The

overall

strength

of

binding

between

an

Agwith

many

determinants

and

multivalent

AbsYKeq

=104AffinityY106AvidityYYYYY1010AviditySpecificity

The

ability

of

an

individual

antibody

combiningsite

to

react

with

only

one

antigenic

determinant.

The

ability

of

a

population

of

antibody

moleculesto

react

with

only

one

antigen.Cross

ReactivityAnti-AAbAg

AAnti-AAbAg

BShared

epitopeAnti-AAbAg

CSimilar

epitope

The

ability

of

an

individual

Ab

combining

site

toreact

with

more

than

one

antigenic

determinant.

The

ability

of

a

population

of

Ab

molecules

toreact

with

more

than

one

AgCross

reactionsFactors

Affecting

Measurement

ofAg/Ab

ReactionsAffinityAvidityAg:Ab

ratioPhysical

form

of

AgAb

excessAg

excessEquivalence

–

Lattice

formatiTests

Based

on

Ag/Ab

Reactions

All

tests

based

on

Ag/Ab

reactions

willhave

to

depend

on

lattice

formation

or

theywill

have

to

utilize

ways

to

detect

smallimmune

complexes

All

tests

based

on

Ag/Ab

reactions

can

beused

to

detect

either

Ag

or

AbAgglutination

TestsLattice

FormationAgglutination/HemagglutinationYY+

Definition

-

tests

that

have

as

their

endpoithe

agglutination

of

a

particulate

antigenAgglutinin/hemagglutininQualitative

agglutination

testAg

or

AbYAgglutination/HemagglutinationQuantitative

agglutination

testTiterProzoneTiterPatient1/2

1/4

1/81/161/321/614/1218/2516/51/2/2102P4os.Neg.1642835124<2532612873284Agglutination/HemagglutinationDefinitionQualitative

testQuantitative

testApplicationsBlood

typingBacterial

infections–Fourfold

rise

in

titerPractical

considerationsEasySemi-quantitative1/2

1/4

1/81/161/321/614/1218/2516/512Passive

Agglutination/Hemagglutinati

Definition

-

agglutination

test

done

with

asoluble

antigen

coated

onto

a

particleYY+YApplications–

Measurement

of

antibodies

to

soluble

antigensCoombs

(Antiglobulin)Tests+YYYYYIncomplete

AbDirect

Coombs

Test–

Detects

antibodies

on

erythrocytesYYYYYYYYYYYYPatient’s

RBCsCoombs

Reagent(Antiglobulin)Coombs

(Antiglobulin)TestsIndirect

Coombs

Test–

Detects

anti-erythrocyte

antibodies

in

serumYYYYYPatient’sSerumTargetRBCs+Step

1+YYYYYYYYYYYYYYYYYCoombs

Reagent(Antiglobulin)Step

2Coombs

(Antiglobulin)TestsApplicationsDetection

of

anti-Rh

AbAutoimmune

hemolytic

anemiaAgglutination/Hemagglutination

Inhibi

Definition

-

test

based

on

the

inhibition

ofagglutination

due

to

competition

with

a

solublYY+Prior

to

TestY+YYY+TestPatient’s

sampleAgglutination/Hemagglutination

InhibiDefinitionApplicationsMeasurement

of

soluble

AgPractical

considerationsSame

as

agglutination

testPrecipitation

TestsLattice

FormationRadial

Immunodiffusion

(Mancini)concentrationQuantitative–

Ig

levelsMethodAb

in

gelAg

in

a

wellInterpretationDiameter

of

ring

isproportional

to

theAg

ConcentrationDiameter2AgAgAgAgAb

in

gelImmunoelectrophoresisInterpretation–

Precipitin

arc

represent

individual

antigensAg-+AgAbAgAbMethodAgs

are

separated

by

electrophoresisAb

is

placed

in

trough

cut

in

the

agarImmunoelectrophoresisMethodInterpretationQualitative–

Relative

concentrationCountercurrent

electrophoresisMethodAg

and

Ab

migrate

toward

each

other

byelectrophoresisUsed

only

when

Ag

and

Ab

have

opposite

chargesQualitative–RapidAgAb-+Radioimmuoassays

(RIA)Enzyme-Linked

ImmunosorbentAssays

(ELISA)Lattice

formation

not

requiredCompetitive

RIA/ELISA

for

AgMethod–

Determine

amountof

Ab

needed

to

bindto

a

known

amount

of

labeled

AgYPrior

to

TestY

+LabeledAgY

+Test+Patient’ssampleLabeledAgY

+–

Use

predeterminedamounts

of

labeledAg

and

Ab

and

add

asample

containingunlabeled

Ag

as

acompetitorCompetitive

RIA/ELISA

for

AgMethod

cont.–

Determine

amountof

labeled

Ag

boundto

AbNH4SO4anti-Ig+

+Patient’ssampleLabeledAgImmobilize

the

Ab–

Concentration

determined

from

a

standard

curveusing

known

amounts

of

unlabeled

AgQuantitative–

Most

sensitive

testY

+SolidPhaseTestYSolidPhaseSolid

Phase

Non-Competitive

RIA/ELISAImmobilize

AgIncubate

with

sampleAdd

labeled

anti-IgAmount

of

labeled

Abbound

is

proportionalto

amount

of

Ab

in

thesampleQuantitativeSolidPhaseYAgImmobilizedYAb

inPatient’ssampleAb

detectionLabeledAnti-IgSolid

Phase

Non-Competitive

RIA/ELISAAg

detectionImmobilize

AbIncubate

with

sampleAdd

labeled

antibodyAmount

of

labeled

Abbound

is

proportional

tothe

amount

of

Ag

in

thesampleQuantitativeSolidPhaseImmobilizedYAgYAg

inPatient’ssampleLabeledAbTests

for

Cell

AssociatedAntigensLattice

formation

not

requiredImmunofluorescenceDirect–

Ab

to

tissue

Ag

is

labeled

with

fluorochromeFluorochromeLabeled

AbYAgTissue

SectionImmunofluorescenceIndirect–

Ab

to

tissue

Ag

isunlabeledYAgYFluorochromeLabeledAnti-IgTissue

Section–

Fluorochrome-labeled

anti-UnlabeledAbIg

is

used

to

detect

bindingof

the

first

Ab.

Qualitative

to

Semi-QuantitativeImmunofluorescenceFlow

CytometryCells

in

suspension

are

la