免疫学检测与免疫学技术全文-毕业论文-

Chapter

10.免疫学检测方法与免疫技术一、概述免疫学检验方法和免疫化学技术主要包括：抗原抗体的制备、纯化和鉴定，免疫扩散、免疫电泳、免疫凝集试验、补体结合试验，免疫细胞分离、纯化和鉴定，免疫功能检测，细胞因子检测，放射免疫检测，免疫酶标检测，荧光和发光免疫技术，免疫组化实验方法，原位杂交免疫组化，免疫PCR技术，免疫微球的应用，免疫电镜技术，细胞凋亡的检测方法，膜受体分析，胞内钙镁浓度的测定和细胞间通讯，流氏细胞仪技术及应用等。从上述内容可以看出，免疫技术是免疫学和物理、化学及电子信息和分子生物学理论和技术的结合产物。其应用涉及生命科学的各个领域，已成为现代医学和生物学研究工作不可缺少的有效工具。免疫技术的原理和特点：基于免疫应答的理论，即抗原抗体的特异性反应。基于免疫细胞的结构、应答特性和分子基础。具有特异性高度灵敏性；可重复性；广泛适用性；快速反应性；可观察性；可定性、定量，即可控性；组化定位特性等特点。概括起来可分为：沉淀反应，即可溶性抗原和抗体间的反应。凝集反应，即颗粒性抗原和抗体间的反应。免疫标记，即用酶、同位素、荧光素或电子致密物质标记。免疫印渍，即用标记抗体与待测蛋白质印迹结合。单抗及工程抗体技术，即分子生物技术。流氏细胞术，即荧光标记，流体喷射，激光和能谱检测，电 脑分析。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

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inPatient’ssampleAb

detectionLabeledAnti-IgSolid

Phase

Non-Competitive

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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