Antibodies are forever: a study using 12-26-year-old expired antibodies

Antibodies are forever: a study using 12–26-year-old expiredantibodies

Maria C Argentieri,1 Daniela Pilla,1 Alice Vanzati,1,2 Silvia Lonardi,3 Fabio Facchetti,3

Claudio Doglioni,4 Carlo Parravicini5 & Giorgio Cattoretti1,21Department of Pathology, AO San Gerardo, Monza, Italy, 2Department of Surgical Sciences, Universit�a degli Studi di

Milano Bicocca, Milan, Italy, 3Department of Pathology, University of Brescia, Spedali Civili, Brescia, Italy, 4Surgical

Pathology Unit, S. Raffaele Hospital, Milan, Italy, and 5Department of Pathology, Ospedale ‘L. Sacco’, Milan, Italy

Date of submission 24 May 2013Accepted for publication 6 July 2013Published online Article Accepted 10 July 2013

Argentieri M C, Pilla D, Vanzati A, Lonardi S, Facchetti F, Doglioni C, Parravicini C & Cattoretti G

(2013) Histopathology 63, 869–876

Antibodies are forever: a study using 12–26-year-old expired antibodies

Aims: The aim of this study was to investigatewhether the shelf-life of diagnostic antibodies is longerthan the expiry date on the label.Methods and results: Four independent laboratoriestested a small number of diagnostic antibodies kept at

+4°C for 12–26 years, and found them to work per-fectly on routine histology sections.Conclusions: Diagnostic antibodies may have a work-able half-life in excess of 10 years, and the emphasison performance should shift to the preservation of anti-genic targets in the tissue.

Keywords: antibody, expiry date, immunohistochemistry

Introduction

Modern diagnostic histopathology relies on increas-ingly standardized practice, which is regulated bydefined laboratory accreditation rules, precise book-keeping, good clinical and manufacturing practice fortest performance,1 and reagent production. Theserules are enforced in all accredited laboratories, andinclude provisions forbidding the use of expiredreagents, among which are primary antibodies for in-vitro use, e.g. immunohistochemistry (IHC). The ex-piry limit of commercially produced antibodies isdefined by multiple parameters associated with theintended use.2 Mandatory disposal of expired lots hasa cost for health systems and has raised concerns,3

particularly because recently expired IHC antibodieshave been shown to perform identically to freshbatches.3–6

A few well-planned studies have investigated theshelf-life of primary diagnostic antibodies for immuno-pathology.3–6 However, most of the reagents testedexpired 12–32 months before evaluation, with ahandful being tested and found to be effective77 months6 and 134 months4 after the expiry date.To better investigate how long after the expiry date

an antibody can safely be used, one would need a testthat would artificially shorten the half-life of an anti-body. However, such a test is not available, becausethe half-life of an antibody as an immunoreagent hasnot yet been determined.To investigate the shelf-life of diagnostic antibodies,

we used antibody samples that had been stored formuch longer time periods than previously reported,and tested them in routine IHC, in a type of storage-dependent stress test.

Materials and methods

Conditions for inclusion of diagnostic antibodies inthe trial were that they had been kept in the originalcontainer or vial, that documentation of the date of

Address for correspondence: G Cattoretti, Department of Pathology,

AO San Gerardo, Monza, and Department of Surgical Sciences, Uni-

versit�a degli Studi di Milano Bicocca, Via Pergolesi 33, 20900 Mon-

za (MI), Italy. e-mail: [email protected]

© 2013 John Wiley & Sons Ltd.

Histopathology 2013, 63, 869–876. DOI: 10.1111/his.12225

production or shipping was available, that the anti-body was in good condition (no mould, no drying,and no turbidity), that the antibody had never beenfrozen, and that no previous culling of expired anti-bodies had been performed.All reagents were kept at +4°C. An antimicrobial

(usually 9–15 M NaN3), buffers and a protein stabi-lizer (albumin or gelatin) or culture medium werepresent in the antibody solution. Secondary reagentsand colour development kits did not exceed the statedexpiry dates. On two occasions (autumn of 1993 andsummer of 2006), the antibodies from one laboratorywere kept at uncontrolled room temperature for up to3 weeks per episode.There were considerable challenges in comparing

antibodies, polyclonal or monoclonal, produced oversuch a large time span. Polyclonal antibodies areunique, lot-specific and animal-specific reagents, andit was highly unlikely that the recently purchasedaliquots, used routinely and for comparison, wouldbelong to the same batch as the old ones. Monoclonalantibodies are produced by unique hybridomas,which themselves can be subcloned, may switch iso-type,7,8 or may change in performance9 over theyears; these are all factors whose control is coveredby commercial confidentiality. Unlike previous experi-ments,3–5 in which the period after the expiry datewas mostly limited to a few months or years, in thecurrent study there was a chance that because of thevery long time intervals the expired reagents, bothmonoclonal and polyclonal, would be entirely differ-ent from, but with the same declared specificity as,the current ones.As for any new antibody clone or batch being eval-

uated for use in the pathology laboratory, the vintageantibodies were assayed for titre, sensitivity, and spec-ificity, and compared side-by-side with the controlsrun daily using unexpired antibodies. The titre wasdetermined by serial dilution, in order to estimate theantibody concentration in the format provided (super-natant, ascites, or purified immunoglobulins): theoptimal dilution for that format would be an indica-tion of the amount of surviving antibody. Eachexpired antibody was then tested on tissues (normalor tumour) in which the antigen expression spans abroad variety of intensities, often within the same sec-tion, allowing the pathologist to examine both thesensitivity and the specificity of the reaction. Thepathologist’s daily experience in the evaluation ofcontrols and samples and knowledge of referencepublications represents the ‘standard’.Finally, serial sections stained with new and

expired antibodies, matched for clone and/or format,

were shown blind to several pathologists, and correctidentification of the antibody source was scored.3

Immunohistochemistry not being ideal for in-situantigen quantitative methods,10 and because of theconsiderations above, we did not use image analysis.Another straightforward test available is assess-

ment of protein aggregation, which measures theratio of protein absorbance at 280 and 350 nm in acommon laboratory spectrometer.11 The aggregatesform as a result of physical damage to a protein insolution (shaking, age, and heat), resulting inreduced availability of working antibody. Unfortu-nately, such measurement was not performed whenthe antibodies were received in the past, so this ratiowas measured on representative samples of old andunexpired antibodies.Primary antibodies were diluted in Tris-buffered

saline, pH 7.5 (TBS), or phosphate-buffered saline(PBS), containing 1% bovine serum albumin (SigmaAldrich, Milan, Italy) and 0.1% sodium azide. Sec-tions from formalin-fixed, paraffin-embedded (FFPE)human tissues were dewaxed, and antigens wereretrieved as previously described,12 blocked with pro-tein (albumin or defatted milk; Reire s.r.l., Reggio-Emilia, Italy) and incubated from 1 h to overnight(according to the protocols of each of the four partici-pating laboratories) at room temperature in moistchambers with appropriately diluted primary antibod-ies. The optimal dilutions established for the differentreagents are listed in Table 1.After several washes with buffer to which 0.01%

Tween-20 was added, a second step was applied ascustomary in each of the four laboratories, consistingof a polymer containing secondary antibodies andhorseradish peroxidase [Envision (Dako, Glostrup,Denmark) or Novolink polymer-HRP (Leica Microsys-tems, Newcastle-upon-Tyne, UK)], according to themanufacturer’s instructions; and developed with di-aminobenzidine. For the polymer-conjugated antibod-ies EPOS kappa and lambda light chains, both directstaining and the secondary step were used. Slideswere stained both manually and with an autostainer.Slides were lightly counterstained with haematoxylinand mounted.Pictures were obtained with a Panasonic DMC-ZS3

or with an DP-70 Olympus microscope equipped witha digital camera.

Results

Thirty-four of 38 individual samples of antibodiesthat would have expired 12–26 years ago readilystained routinely processed human tissues. Moreover,

© 2013 John Wiley & Sons Ltd, Histopathology, 63, 869–876.

870 M C Argentieri et al.

Tab

le1.Primaryan

tibodies

Antibody

Clone

Isotype

Suppliedas

Dilution

Year

Source

Laboratory

Referen

ceResult

Keratin

UCD/PR10.11

IgG1

50%

(NH4) 2SO

4

precipitate

1:100,

1:1000

1985

R.Cardiff

GC

Chan

etal.16,

Cattorettiet

al.12

Neg

ative

Keratin

UCD/PR6.11

IgG1

50%

(NH4) 2SO

4

precipitate

1:100,

1:1000

1985

R.Cardiff

GC

Chan

etal.16,

Cattorettiet

al.12

Neg

ative

Keratin

AE1

IgG1

Supernatan

t1:20

1986

T.T.Su

nGC

Cattorettiet

al.12,

Woodcock-

Mitchellet

al.17

Positive

Keratin

AE3

IgG1

Supernatan

t1:20

1986

T.T.Su

nGC

Cattorettiet

al.12,

Woodcock-

Mitchellet

al.17

Positive

TP53

Pab

1801

IgG1

Supernatan

t1:20

1987

D.Crawford

GC

Cattorettiet

al.12,

Ban

kset

al.18,

Cattorettiet

al.19

Positive

TP53

Pab

1803

IgG1

Supernatan

t1:20

1987

D.Crawford

GC

Cattorettiet

al.12,

Ban

kset

al.18,

Cattorettiet

al.19

Positive

SV40Tag

Polyclonal

–Se

rum

1:1000

1989

Lee

Biomolecular

CP

Positive

CD45

9.4

IgG2a

Ascites

1:500

1989

DuPont

CP

Cattorettiet

al.12,

Ledbetteret

al.20

Positive

CD45R

10G3

IgG2a

Ascites

1:500

1989

R.M.Steinman

GC

Cattorettiet

al.12,

Younget

al.21

Positive

CD45

9.4

IgG2a

Ascites

1:500

1990

P.J.Martin

GC

Cattorettiet

al.12,

Ledbetteret

al.20

Positive

HIV/p25

P25.7

IgG1

Ascites

1:1000

1990

J.C.Gluckman

CP

Ten

ner-Racz

etal.22

Positive

HIV/p18

P18(A1)

IgG1

Ascites

1:1000

1990

J.C.Gluckman

CP

Ten

ner-Racz

etal.22

Positive

EMA

E29

IgG2a

Supernatan

t1:50

1990

Dak

oCP

Positive

KIM

1p

(CD68)

KIM

Pp

IgG1

Supernatan

t1:50

1990

Biotest

Diagnostic

FFRad

zunet

al.23

Positive


Antibodies are forever 871

Tab

le1.(Continued)

Antibody

Clone

Isotype

Suppliedas

Dilution

Yea

rSo

urce

Laboratory

Referen

ceResult

HMFG

23.14.A3

IgG1

Supernatan

t1:50

1991

Oxo

idCD

Positive

CD34

Qb-End-10

IgG1

Ascites

1:1000

1992

M.F.

Greav

esCP

Positive

TP53

Pab

240

IgG1

Supernatan

t1:20

1993

D.La

ne

GC

Cattorettiet

al.12,

Gan

nonet

al.24

Positive

Ki67

Notav

ailable

IgG

Rea

dyto

use

1:50

1993

Ortho

Diagnostics

CP

Positive

Muscle

actin

HHF3

5IgG1

Rea

dyto

use

1:50

1993

Enzo

Pharmaceu

ticals

CP

Positive

MB1

(CD45RA)

MB1

IgG1

Supernatan

t1:50

1993

Biotest

Diagnostic

FFW

estet

al.25

Positive

PCNA

PC10

IgG2a

Supernatan

t1:200

1993

Novo

castra

CD

Positive

Insulin

Guinea

pig

polyclonal

–Purified

Ig1:400

1993

Dak

oCD

Positive

CD45RO

UCHL1

IgG2a

Supernatan

t1:50

1994

Dak

oCP

Positive

OPD4

(CD45RO)

OPD4

IgG1

Supernatan

t1:50

1994

Dak

oFF

Poppem

aet

al.26

Positive

MAC387

MAC

387

IgG1

Supernatan

t1:50

1995

Dak

oFF

Flav

ellet

al.27

Positive

CD3

Polyclonal

–Rea

dyto

use

1:500

1996

Dak

oCP

Positive

Neu

trophil

elastase

NP57

IgG1

Supernatan

t1:50

1996

Dak

oFF

Pulford

etal.28

Positive

Lambdalight

chains(EPOS)

Polyclonal

–HRP-conjugated

Ig1:50

1996

Dak

oFF

Sanoet

al.29

Positive

IgM

Polyclonal

–Purified

Ig1:50

1997

Dak

oFF

Positive

Lambda

lightchains

N10/2

IgG1

Supernatan

t1:10000

1997

Dak

oFF

Positive

BNH9

BNH9

IgM

Supernatan

t1:10

1997

Dak

oFF

Delsolet

al.30

Positive

/reduced

Kap

palightchains(EPOS)

Polyclonal

–HRP-conjugated

Ig1:50

1998

Dak

oFF

Sanoet

al.29

Positive



the optimal working dilution matched the dilutionestablished when they were originally supplied, aswell as that of recent batches of similar formats ofantibodies with the same specificity.All antibodies stained tissue antigens with broad

expression levels, some of which were very weaksuch as 58-kDa keratin in liver or nonmutated TP53in epithelial cells in normal colon or colon cancer(Figure 1). The results were indistinguishable fromthose obtained with current non-expired reagents,which were commercially purchased and being runon a daily basis.IgG1 and IgG2a mouse mAb isotypes almost invari-

ably survived as supernatants or purified immuno-globulin, with the notable exception of two IgG1antibodies that were provided as 50% ammoniumsulphate precipitate.13 IgM antibodies, which areknown for inconsistent quality and a propensity toaggregate,2 partially survived the prolonged storage.Fifteen-year-old peroxidase-conjugated primary anti-bodies, commercialized as EPOS by Dako (Figure 2),maintained both titre, specificity, and enzymaticactivity, although the last of these was slightlyreduced.To quantify the amount of molecular aggregation

induced during the storage time, two laboratoriesmeasured the aggregation index11 spectrophotometri-cally in a small sample of aged supernatants, purifiedantibodies, and control (recently purchased) antibod-ies. A broad range of values were obtained (from29.40 up to 163.53), unrelated to type, age or con-centration of the antibody. This was expected, giventhe fact that we measured the whole sample, fetal calfserum, or other proteins included. Similarly, blindedpathologists could not reliably identify stains withaged reagents, as shown previously by Savage et al.3

(not shown).

Discussion

Monoclonal antibodies originally supplied either asculture supernatants or as ascites (neat or diluted), ofall isotypes except IgM, as well as all of the polyclonalantibodies, produced satisfactory staining irrespectiveof their age. Notable exceptions were ammonium-pre-cipitated, IgM or conjugated antibodies. These latterwere excellent antigens despite weakened enzymaticactivity.These results were not totally unexpected; immu-

noreactive, functional enzymes have been obtainedfrom human samples that were several thousandyears old,14 suggesting that the tertiary protein struc-tures, among which is the immunoglobulin antigenT

able

1.(Continued)

Antibody

Clone

Isotype

Suppliedas

Dilution

Yea

rSo

urce

Laboratory

Referen

ceResult

S100

Polyclonal

–Purified

Ig1:1000

1999

Dak

oCD

Positive

CD79a

HM57

IgG1

Supernatan

t1:50

2000

Dak

oCP

Positive

LN2(CD74)

LN2

IgG1

Ascites

1:10

2000

Neo

marke

rsFF

Nortonet

al.31

Positive

LN1(CDw75)

LN1

IgM

Ascites

1:10

2001

Neo

marke

rsFF

Nortonet

al.31

Neg

ative

LN5

LN5

IgM

Purified

Ig1:10

2001

Biotest

Diagnostic

FFNortonet

al.31

Neg

ative

CK19

RCK108

IgG1

Supernatan

t1:50

2002

Dak

oCD

Positive



recognition pocket, may indeed survive for muchlonger than has previously been thought. Eventhough only half of the shelf-life that we have shownhere, 10 years may well be a workable duration fordiagnostic antibodies supplied as supernatants or asci-tes. Inconsistent antibody staining not related to theshelf-life of the primary antibody, as shown by

Savage et al.3 has more to do with the vagaries of im-munostaning protocols or the preservation of anti-gens in the tissue. This latter depends on fixation-dependent and age-dependent effect of dehydrationand processing.15

It is not unreasonable to suggest that our find-ings relating to the use of antibodies for diagnostic

A

E

L M N O P Q

R S T U V Z

η θ ι κ

βα γ δ ε ζ

F G H I K

B C D

Figure 1. Immunoreactivity of vintage antibodies. A, Colon carcinoma (right) metastatic to the liver (left), stained for AE1. Note the

unstained liver and the positive bile ductules. B, Colon carcinoma (right) metastatic to the liver (left), stained for AE3. C, EMA staining of a

salivary gland. D, Cytokeratin staining of colon mucosa. E, Muscle actin staining of colon submucosa. F, CD34 antibody stains capillaries in

a tonsil. G, Pab240 minimally stains wild-type TP53 in a colon carcinoma. H, The same specimen as in G is stained by anti-TP53 Pab1801.

I, K, Pab240 (I) and Pab1801 (K) stain mutated TP53 in a colon carcinoma. L, CD45 antibody 9.4, tonsil tissue. M, CD45R antibody 10G3

preferentially stains non-germinal centre tonsil cells. N, O, The T-cell and B-cell zones of a tonsil stained with CD3 (N) and CD79a (O) anti-

bodies. P, MB1, tonsil. Q, OPD4, tonsil. R, Staining for IgM, tonsil. S, MAC387, tonsil. T, KiM1p, tonsil. U, Neutrophil elastase, tonsil. V,

Lambda light chain, tonsil. Z, Insulin, pancreas. a, Tonsil germinal centres stained for Ki67. b, PCNA, tonsil. c, HIV/p25 labels follicular

dendritic cells in an HIV-infected lymph node. d, HIV/p18 labels infected cells in an HIV-infected lymph node. e, CD30, tonsil. f, CD1a, tonsilepithelium. g, h, Neutrophil elastase antibodies on tonsil, vintage (g) and unexpired (h). ι, j, Lambda light chain antibodies on tonsil,

vintage (ι) and unexpired (j). Sections for the immunostains in A, B, G, H, I and K were obtained from tissue processed and embedded in

1992.



immunohistochemistry may be extended to all fieldswhere antibodies are used as a primary, unconjugat-ed detection layer to tag an antigen.Given the actual pace of new technical develop-

ments in diagnostic pathology practice, we haveshown that, relatively speaking, antibodies are for-ever.

Author contributions

Maria Cristina Argentieri and Daniela Pilla: per-formed technical and organizing work. Alice Vanzati,Silvia Lonardi, Fabio Facchetti, Claudio Doglioni, Car-lo Parravicini, and Giorgio Cattoretti: selected thereagents and evaluated the staining. Fabio Facchetti,Claudio Doglioni, Carlo Parravicini, and GiorgioCattoretti: wrote the paper.

Acknowledgements

We wish to thank Tung Tien Sun, Robert Cardiff,Daniel Crawford, Paul Martin, Jean Claude Gluck-

man, David Lane, Mel Greaves, and the late RalphSteinman, who generously donated antibodies. Wealso wish to acknowledge many friends, scientists andcompany representatives, too many to list, who con-tributed to this manuscript with samples, ideas, oradvice. The laboratory technicians of our departmentscontributed excellent professional daily support. M. C.Argentieri was funded by a generous donation byProfessor Franco Uggeri (UNIMIB).

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Antibodies are forever: a study using 12-26-year-old expired antibodies

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Transcript of Antibodies are forever: a study using 12-26-year-old expired antibodies