IMMUNE PRECIPITATION TECHNIQUE TO DETECT ABO ANTIGENS FROM DRY BLOODSTAIN

N.

GUNACHANDRAN,

C.

DAMODARAN,

P.

CHANDRA SEKHARAN

Forcnsic Scicnccs Department, Madras - 600 004, India

KURUMUŞ KAN LEKELERİNDE ABO ANTİJENLERİNİN ARANMASı İçİN İMMUN

PRESİpİTASYON TEKNİGİ Özet

Polietilen glikol (PEG) olarak bilinen sentetik bir polimer kullanılarak, immun kompleks-Ierin aranması için yeni bir teknik geliştiriIdi ve bu teknik kurumuş kan lekelerinde ABO tesbiti için uygulandı. 16 günlük kan lekelerinin taze lizatları ve ekstreleri incelendi; ABO antijenlerinin PEG'in

%

4-10 arasındaki konsantrasyonlarında aranabildiği görüldü. Anıi-A ve anıi-B antikoriarı

% 10 PEG içerisinde solubl iken bu antikorlarla meydana gelen immun

kompleksIerin aynı solüsyonda çöktükleri gözlendi. Anıi-A ve anıj-B antikorlarıyla oluşan çökeltiler, PEG'in

%

7'lik konsantrasyonuna kadar incelenebildiler. H antijeninin karşıtı

olan Ulex lektin de

%

10 PEG içerisinde solubl kalıyordu. Bu lektin ile oluşan kompleks

%

S'lik bir PEG konsantrasyonuna kadar çöktürülebildi. PEG kullanılarak uygulanan immun presipitasyon tekniği, diğer yöntemlerle kıyaslandığında, daha basit ve güvenilir bu-lundu. Yöntemde yapılacak bazı modifikasyonlarla, başka vücut sıvılarında ve bunların lekelerinde de gerek ABO gerekse HLA antijenlerinin aranabilmesi mümkündür.

Snmmary

A new precıpıtation technique has been developed for detecting immune complexes using a synthetic polymer, namely polyethylene glycol (PEG) and applicd for the detection of ABO antigens from dry bloodstains. Fresh Iysates and extracts from 16 days old stains were tested and the ABO antigens could be detectcd at PEG concentrations of 10

%

to 4,

%.

The

anıi-A and anıi·B antibodies were soluble in 10 % PEG whereas the immune complexes formed by thcse antibodies were precipitatcd at that concentration. The immnne precipitate caused by anıi·A and anıi-B antibodies were detectable up to a PEG concentration of 7

%.

The Ulcx lectin against H antigcn was also soluble at 10

% PEG.

The complex formed by this le etin could be precipitated by PEG upto a concentration of 5

%.

20 N.GUNACHANDRAN, C.DAMODARAN,P. CHANDRA SEKHARAN

When compared to the existing methods this immune precipitation technique using PEG

appears simple and reliable. With certain modification this teclmiquc can be uscd for the detection of ABD antigens from other body fluids and their stains. There exists the possibility

that the HLA antigens can also be detected by this technique. Keywords: Immune precipiıaıion -ABD antigens - Btoodstuin

INTRODUCTION

Polyethylene glycol (PEG)

was

use d

to det

t

ct

the

antigens

by

causing

the

precipitation

o

f

antigen-antibody

co

m

p

le

xes. T

he

ABO a

nti

gens

and

thc

i

r

an

tibod

ies

are quite soluble

at

10

%

PEG

whereas the immune complexcs

(lC)

formed

by th

e

m

are insol

u

ble and

precipitate at tha

t

co

ncent

ration.

Thes

e

ıes

were precipitated upto

a

low

concentration of

7

%

PEG.

The anti-H

lectin is

soluble at 10

%

PEG

whi

l

e

the complex

formed by

it

with

II

5uhstance

is prtcipitated

at 5

to 10

%

PEG.

Thus

th

e

pre

eip

itation of ıe

at a partieular

PEG concentration was use d to demonstrate

the

presence

of antigen

in

a

quaIitative manner.

Farr (1

)

first

demonstrated

th

e

precıpıtation

of

immunoglobulins

u

si

ng

ammonium sulphate.

Various other

polysaccharides

a

nd

polymers such

as

dextran (2), polylysine (3) and polyethylene

gIyeol

(4) were

shown to cause

immune precipitatio

n

.

Polyethylene gIycol

was used

f

or the

detection

of

antibody by Creighton

et al

(5). Studies were h

e

nce

undertaken

f

or the

detec-tion

o

f

ABO

antigens

from hlood

lysates

and dry stain using PEG and

t

he

results

are

discussed.

MATERIALS AND METHODS Antİsera

Anıi-A and unıi-B antibodies were proeured from M/s HuJJkins, Bombay. The unti-H lectin was prepared from the seeds of Ulex europeus at our laboratory.

Polyethylene glycol

PEG with an average molccular weight of 6000 was obtained from M/s Sisco Research Laborutory, Bombay.

Antİgen

Blood lysates as well as stains on clean cotton cloth were prepared from A, B, AB and O group volunteers of our laboratory. The lysatcs were kept at 4°C while the stains were allowed

Solubility of antibodies and lectin in PEG

The solubility of anıi-A and anti-B antibodies and leetin was studied in different eon-eentrations of PEG. The antibodies and leetin werc diluted (1 : 5) in borate buffer and 50 ııl of the dilution was dissolved in 5 to 40

%

PEG prepared in buffer, the final volume being 0.2 ml in separate Fe/ix tubes, mixed well and lcft at 4°C overnight; centrifuged at 5000 rpm for 10 minutes at 4°e and observed for precipitation.

Preeipiıation test for immune eomplex

To one volume of diluted lysate (1 : 80), one volum e of each antibody and le etin dilutions (1 : 5) WCre added separately and incubated for 4 hours at room temperaturc or overnight at 4°C. The antigen-antibody/lectin mixtures were thcn mixed with PEG, 0.2 ml as the total vol-ume to a final concentration of 4 to 10

%

in separatc Fe/ix tubes and kcpt at ,ıa_{e overnight;} centrifugcd at 5000 rpm for 10 minutes at 4.o_{C and observed for precipitation. Control tubes}

without antibody or lectin dilutions were also kept in parallel.

Stain cxtracts were prepared by keeping pieces of staincd thread with buffer (50 ııl

for ı em thrcad) in separate wells of microtitre plate kept on a metabolic shakcr for 30 min-utes. The same protocol as for lysate was then followed for the stain cxtracts also.

R

E

SULTS

Solubility of antibodies and

lectİn

The

anti-A

and

anti-B

an

t

ibodies were found comple

t

ely sol

ub

le at 5

%'

7

(/'0

and

8

%

PEG

but precipitated

abov

e

10

%

PEG. The solubility at 10

%

P

E

G

was

vc

ry poor. T

he

anti-H l

e

ctin was quite soluble at

1

0

%

and

b

elow

(Tabl

e 1

)

.

TaMe 1. The precipitation of aırıtibodies and leetin İn polyethylene glyeo!.

Antibody Ileetin (1 : 5) 50 ~ıl Anti-A Anıi-B Anıi-H lectin 40 % -I

i-+

+

Polyethylene glycol in borate buffer (0.2 ml)

----_._---~-

---25 % 20 % 10 % 8

'Yo

7% 5%

T

+

±

L L- i

±

22

N.GUNACHANDRAN, C.DAMODARAN,P.CHANDRASEKHARAN

Precipitation of

immune

complexes

The respective ICs f

ormed

by

anti-A

and

anti-B antibodies both in

lysate

and stain extract

experimcnts

gaye similar results as shown in Table

2.

Clear precipitate was noticed in 10

%

and 7

%

PEG while poor p

r

ecipitate

was found in

6.5

%

PEG. The complex formed by

anti-H le etin was fairly

precipitated at 10

%

,

8

%,

7

%'

6.5

%

and

5

%

while it was

poor

at

4

%

PEG.

Tahle 2. The precipitation of immnne complexes in polyethylene glyco!.

Antibody/ Antigen In Iysate In stain extract

lectin

PEG in borate buffer PEG İn bora te buffcr - - -_ .. _---~~,._ -10% 8% 7% 6.5% 5% 4% 10% 8% 7% 6.5%

5

%

4% Anıi-A A

₊

₊

_{+ +}

_±

₊

_{+ +}

₊

B

₊

₊

AB

_{+ + +}

_{+ +}

₊

₊

O

₊

₊

-+

. --- - - _._---_ .... _- _._---_ ... _--- -_." ..

_

-_.

-- ---_._ -- --- _.-' .. ---'_ ... ". Anıi-B A

₊

₊

B

_{+ + + +}

_±

₊

-1

₊

" 1-AB

_{+ +}

_{+ +}

_±

₊

+-

₊

-I

₊

O

₊

-

+

+

-

- -

-

-

- - - -

-

--Anıi-H A

_{+ +}

₊

₊

₊

±

₊

_{+ + +}

₊

-+

lecıin B

₊

_{+ +}

₊

₊

_±

₊

₊

-i-

-+

+

AB

₊

₊

₊

₊

₊

_{+ + + +}

_-+

_±

O

₊

₊

₊

_{+ + +}

_{+ + +}

---i-

₊

_-j ---_ .. -._--- - -- -No anıi- A bodyor B lecıin AB O

DISCUSSION

The antibodies which were highly soluble at 7.5

(10

PEG and below were

precipitated at the

s

ame

concentration when bound with the

antigen

.

The

anti-H le

etin

which was

quite

soluble at 10

%

and below PEG was

precipi-tated upto a

low

concentration of 5

%

PEG when treated with the lysate

and

stain extract.

When an

IC

is

formed

between an

antigen

and

antibody

the molecular

sıze

and

molecular weight

of the complex

differs

from that of antigen or

antibody

alone. This physico-chemical change causes the precipitation of the

complexes upto a

particular low PEG concentration by a possible

mechanİsm

known as molecular

solvent exclusion effect

(5).

As

the forensic case works are more concerned about the

qualitative

detection

of the an

t

igen in a given

stain

this simple technique will be

more

use

f

uI

when compared with some of the

existing

methods (6). With

appro-priate modifications this technique can be

applied to the detection of

ABO

antigens

from other body

fluids and their stains.

HLA

antigens can also be

detected by this technique in a

similar fashion.

Acknowledgement

Our thanks are due to Dr. G. Jayaraman of Jawaharlal Nehru University, New Delhi and to our collcagues at the Forensic Sciences Department, Madras, for their help.

REFERENCES

1 - Farr, R.S. (1958) J. Infect. Dis., 103, 239 (loc. cit. Creighton, W.D. et aL., J. ImmunoI.,

lll, 1219, 1973).

2 - Hell.ing, K. (1966) Acta Chem. Scand., 20, 1251 (loc. cit. diııo).

3 - Stahman, M.A., Mathews, R.E.F. (1954)

J.

ImmunoL., 72, 435 (loc. cit. diııo).

4 - Harrington, J.C., Fenton, J.W., Pert, J.H. (1971) Immunochemistry, 8, 413 (loc. cit.

diııo ).

S - Creig;hton, W.D., Lambert, P.H., Miescher, P.A. (1973) J. ImmunoL., lll, 1219. 6 - Lee, H.C. (1982) in Forensic Science Handbook, (Saferstcin, H., cd) pp. 297 - 337,

Prentice Hall International Ine., New Jer5ey.

Reprints request to :

C. Damodaran

Forensic Scİenees Dept. Madras - 600 004 India