Rapid HLA-DR oligotyping by an enzyme-linked immunosorbent assay performed in microtiter trays

NEW TECHNOLOGY

Rapid HLA-DR Oligotyping by an EnzymeLinked Immunosorbent Assay Performed in Microtiter Trays Donna D. Kostyu, Jeffrey Pfohl, Frances E. Ward, John Lee, Al Murray, and D. Bernard Amos

ABSTRACT A simple, sensmve ELISA that is performed in 96-well microuter plates and that requires less than 90 minutes to complete was developed for HLADRB ohgotyplng The second exon of HLA-DRB1 was amplified using an unlabeled forward primer and a biotinylated reverse primer and the PCR product was Immoblhzed in avldin-coated wells Subsequent treatment included exposure to 0 4 N NaOH to remove the nonblotmylated sense strand, addition of a fluoresceinlabeled ohgonucleot~de probe, one or more 5-minute stringency washes, addition of an alkahne-phosphataselabeled anti-FL FAB, and then alkallne-phosphatase substrate and amplifier An intense red-violet color devel-

ABBREVIATIONS as biotin-labeled antisense ohgo BlOT biotin ELISA enzyme-hnked lmmunosorbent assay FAB Fab antibody fragment FL fluoresceln HB hybridization buffer

oped within 15 minutes In posmve wells and could be quantltated by OD readings at 490-495 nm To control for stringency and to establish threshold OD values for posmve reacnons, biotin-labeled antisense ohgos that were complementary to the probe or that differed by one or more bases were lmmobihzed in wells in place of PCR products The assay was senslt,ve to <0 05 pmol ( - 4 ng)/ well and required only standard incubators and waterbaths and an optional mlcroplate reader All reagents were commercially available The method should facilitate ohgotypmg of both class I and class II alleles and IS adaptable for analysis of other polymorphic gene products Human

Immunology 38, 148-158 (1993)

HIV NADPH OD PCR pNPP

human ,mmunodeficiency virus nicotlnamide adenine dmucleotide phosphate optical density polymerase chain reaction p-mtrophenylphosphate

INTRODUCTION The H L A gene family currently contains 10 functional polymorphlc loci that code for cell surface glycoprotems (HLA-A, B, C, DRB1, DRB3, DRB5, D Q A 1 , D Q B 1 , DPA1, DPB1) and a total of > 2 5 0 alleles Identified to date [1] These alleles are relevant to diverse fields of study, including antigen presentation [2, 3], transplanta-

From the Department of lmmunology, Duke University Medical Center Durham North Carohna, USA Address reprint requests to Dr D D Kostyu, Box 3010, Duke Medtca/ Center Durham NC 27710, USA Received (U) May 18, 1993 acceptedJuly 9, 1993

148 0198-8859/93/$6 oo

tlon [4, 5], genetlc susceptlbihty to disease [6], population origins and migrations [7, 8], and molecular evolution o f genes and gene famlhes [9, 10] Typing for the H L A class II alleles in particular Is challenging Antlsera are incapable of defining all alleles and the serologic/cellular methods tradmonally used reqmre a high level of skill and expertise in the interpretation of results The subsequent shift to molecular defimtlon by polymerase chain reaction (PCR) [11, 12] and ohgotyplng [ 1 3 - 1 8 ] , while offering improved sensmvlty and specificity, is itself confounded by the large number ( > 100) o f probes needed [ 19] Further, ohgotypmg usHuman Immunology 38, 148-158 (1993) © American Socmty/or Hlstocompatlbihtv and Immunogenetlcs, 1993

Rapid HLA-DR Ohgotypmg by ELISA

mg membrane-immobilized D N A and ra&oactlve, colorimetnc, or chemlluminescent detection systems generates results that are not easily quantltated and the assays are slow, expensive, and unsuited for large-scale testing Alternative approaches for class II typmg have included single-strand conformation assays [20], sequence-specific amplification [21-24], restriction fragment-length polymorphism [ 2 5 - 2 7 ] , and Immobilized probes in reverse dot blots [28, 29], but these also are often impractical Even greater problems are anticipated for ohgotypIng of class I alleles Methods that bypass membrane-immobilized D N A for other solid support systems have been reported, i e , the attachment of biotinylated PCR products to avidin/ streptavidin-coated beads for class II typing [30], for detection of human Immunodeficiency virus (HIV) [31], or quant,tation of PCR-amphfied D N A [32] Blotinylated capture probes and enzyme-modified reporter probes in microtiter plates have also been used for class II typing [33] and detection of H I V [ 3 4 - 3 6 ] While an improvement over usual hybridization techniques, these may be cumbersome, time-consuming, or require postsynthetic labeling of probes, and are often not easily standardized We considered that a simple approach might be to directly lmmoblhze biotin-labeled PCR products in avidin-coated wells and so devised a simple enzyme-linked immunosorbent assay (ELISA) for this purpose The technique utihzes biotlnylated-amphfied DNA, avldlncoated wells, fluoresceln-labeled ohgonucleotide probes, an enzyme-linked antifluorescein antibody, and a cyclic enzyme-driven detection system To provide rigorous controls for every probe, biotin-labeled antisense ohgos were Incorporated Into the assay The result, as described here, is a fast, easily standardized, and reproducible assay that avoids hazardous radioactive detection MATERIALS AND METHODS

DNA preparatmn Peripheral blood lymphocytes were washed twice in phosphate-buffered saline and heated at 6 × 106/ml in cell lysate buffer (50 mM KCI, 10 mM TrIs HC1 p H 8 3, 2 5 mM MgCl2, 0 1 mg/ml gelatin, 0 45% NP40, 0 45% Tween 20, and 0 06 mg/ml proteinase K) for 1 hour at 56°C and 5 minutes at 95°C [37] to produce a crude D N A lysate Samples were stored at - 2 0 ° C until used Distilled water and/or research grade, filtered water (Continental Water Systems, San Antonio, TX, USA) were used for all reagents PCR amphficatton PCR was carried out in a 100-/~1 reaction volume contammg 20 jzl of cell lysate, 200 ~M each dNTP, 0 2 tzM each primer, 50 mM KC1, 10 mM T r l s - H C l p H 8 3, 0 1% wt/vol gelatin, 1 5 mM Mg +2,

149

and 2 5 units (U) Taq polymerase [19] The mixture in each tube was overlayed with 50 ~l mineral oil and heated for 30 cycles 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, and ending with 5 minutes at 72°C The presence of a 261-bp band was determined by running 5 ~l PCR-amphfied D N A in a 1 5% agarose gel followed by ethldium-bromide staining Primers were an unlabeled forward primer DR-AMPA (5'CCCCACAGCACGTTTCTTG) and a biotin-labeled reverse primer DR-AMPB b'° (5'-bIotIn-CCGCTGCACTGTGAAGCTCT) that amplify codons 3 - 9 3 of the second exon of the DRB genes, as utilized by the 11th International Workshop [19] Because the primers are directly incorporated Into each extended strand of D N A , all reverse strands should be biotin-labeled at the 5' end

Ohgonucleotldes Ohgonucleotldes were synthesized by phosphoramldlte chemistry on an automated D N A synthesizer (model 380B, Applied Biosystems, Foster City, CA, USA) in the D N A Synthesis Laboratory in the Department of Botany, Duke University Biotin and fluoresceln molecules were directly incorporated by means of biotin- or fluoresceIn-ON phosphoramldites (Clontech Laboratories, Palo Alto, CA, USA), respectively Some biotin-labeled ohgonucleotides were purchased from Blo-Synthesis (Lewisville, TX, USA) Ohgos were reconstituted to 0 5 ml In distilled or filtered water and the concentration determined by measuring the optical density ( o n ) at 260 nm, disregarding the minor contribution of an added fluoresceIn or biotin molecule to molecular weight An aliquot of each probe was diluted to a stock solution of 1 25 ~M in water, 0 05% NaN~ and stored at 4°C ELISA The 96-well ELISA mlcrotiter plates (polystyrene, flat-bottom) containing removable 1 × 8 well strips (Cornlng Glass Works, Cornlng, NY, USA) were coated with 5 ~g (100 izl of a 50 ~g/ml stock) of avidm (Sigma, St Louis, MO, USA) per well, with the diluent a carbonate coating buffer p H 9 6 (solution A [1 3 g N % C O 3 in 250 ml distilled water] added to solution B [ 1 7 g N a H C O 3 in 400 ml distilled water] until p H 9 6 is reached) Coated plates were sealed with ELISA platesealing tape (Cornlng) and stored at 4°C overnight or for several weeks until use At the time of the assay, the plates were washed three times in Tris-buffered saline (30 2 g Tris and 43 8 g NaC1 in 5 l distilled water, p H 7 5) with 0 05% Tween 20 (Sigma) (TBS-Tween) by submerging them briefly in a pail or plastic container filled with T B S - T w e e n and then flicking the excess fluid into a sink Undiluted PCR product, 4 - 1 0 ~l, or an amount producing an easily visible band on an agarose gel, were added to each well along with sufficient hybridization

150

buffer (6 × SSPE, 5 × Denhardt's, 0 1% sodium sarcosme, 0 02% SDS, and 0 05% N a N 3) (HB) to cover the bottom of the well (mmlmal volume should be - 2 0 / z l ) When biotin-labeled antlsense ohgos were used in place of PCR products, 0 1 pmol (50/zl of a 1/600 dilution In H B of the 1 2 5 / z M stock) was added per well The plates were allowed to sit at room temperature for 10 minutes to allow b i d i n g of the blotm-labeled ohgo or PCR product to the avldin-coated well If the amount of PCR-amphfied D N A was considered to be low based on a weaker band in an agarose gel, this time was extended to 20 minutes The plates were then flicked, washed six umes in T B S - T w e e n , and 100/zl of 0 4 N N a O H was added for 1 minute at room temperature to denature the D N A The plates were flicked and washed six times in T B S - T w e e n to remove the unlabeled sense strand To hybridize the probes to the immoblhzed DNA, 0 625 pmol of a fluoresceln-labeled ohgonucleotide probe (50/zl of a 1/100 diluuon in H B of the stock 1 2 5 / z M ohgo) was added per well and the plates were mcubated for 10 minutes at 42°C After washing six times m TBS/Tween, nonspecIfiC bindmg was ehmmated by one or more 5-minute strmgency washes usmg 100 /zl/well prewarmed SSPE (stock 6 × SSPE 3 M NaCl, 200 mM NaH2PO4, 20 mM EDTA, p H 7 4, and 0 1% wt/vol SDS) at a predetermined SSPE concentration (usually 0 001 × to 2 1 × ) and temperature (38°C or 47°C), followed by six washes in T B S - T w e e n Strmgency washes were accomplished by floating a microtlter plate m a waterbath Wash temperatures could not be achieved and mamtaaned in standard aar incubators If varying SSPE concentrations were reqmred for a stogie ELISA plate, a multichannel pipettor and 1 0-ml microtubes m a 96-well rack (PGC, Gaithersburg, MD, USA) were used to dispense the SSPE If multiple wash temperatures were required for a stogie plate, the 1 × 8 well strips were separated and placed in different frames for the duration of the stringency wash(es) To detect bound probe, 50/zl of a 1/30,000 dilution of alkahne-phosphatase-labeled anufluorescem (FL) FAB (Boehringer-Mannhelm) was added per well and the plates incubated at 37°C for 15 minutes For convenience, an aliquot of the stock FAB (150 U/200/zl) was predfluted 1/100 in T B s , 0 1% wt/vol BSA, 0 05% wt/ vol NAN,, and stored at 4°C A 1/300 diluuon of this (i e , a 1/30,000 final dilution) in TBS, 0 1% BSA was made immediately before use Leftover FAB at 1/30,000 was discarded The alkaline-phosphate substrate and amphfier were reconstituted to 12 ml each accordmg to manufacturer's instructions (Ehsa Amplification Kat, GIbco-BRL, Grand Island, NY, USA) and frozen In 2 4- and 4 8-ml ahquots (sufficient for ½ plate and 1 plate) prior to the

D D Kostyu et al

assay During the 15-minute FAB Incubation, an ahquot of the substrate was removed from the freezer and allowed to warm to room temperature The plates were then flicked, washed six times m T B S - T w e e n , and 50 /zl of substrate added per well The plates were allowed to sit for 15 minutes at room temperature, durmg which time the amphfier was thawed Then, 50/zl o f the amplifier was added directly to each well without washmg Leftover substrate and amplifier were refrozen for later use Color developed wlthm 5 minutes after addition of the amphfier Absorbance (OD) was determmed at 5minute Intervals at 4 9 0 - 4 9 5 nm by using a microplate reader (Dynatech, Chantflly, VA, USA) The reaction was stopped by the addition of 50/zl 0 3 M H2SO4/well when clear discnmmauon between positive and negative values was achieved, usually within 10 minutes Because of the intensity of the color, the results could also be recorded by visual Inspection or preserved as a photocopy by covering the m~crotiter plate with sealing tape and invertmg it on a xerox machine RESULTS The ELISA for ohgoprobing of Immobilized, PCR-amphfied D N A was simple and rapid (Fig 1 and Table 1) PCR-amphfied D N A , contamlng unlabeled sense and biotin-labeled antisense strands, was lmmoblhzed in wells via a blotin-avldm Interaction The unlabeled strand was removed by a 1-minute wash in 0 4 N N a O H Following a brief hybridization with a fluorescelnated

FIGURE 1 Schematic of the mlcrot~ter plate ELISA for DNA-ohgo hybridizations Formazan

NAD+

cP,,..pl.> <',,I I ~

Iodonltrotetrazohum ~ Vlo|et

I V "~

Eth.,ol Acetaldehyde

NADH

DNA~ AYldln

Rapid HLA-DR Ohgotypmg by ELISA

TABLE 1

151

Steps in D N A - o h g o hybridization by ELISA Step

Time and temperature

1

PCR amplification

2 3 4 5 6

Absorption of blotm-labeled D N A to avidm-coated wells Wash6× Denature to stogie-stranded D N A Wash 6 × Probe FL

7 8

Wash6x Stringency wash(es)

1retoRT 5 - 1 5 mln

Wash6× Alkahne-phosphatase-labeled antl-FL FAB Wash6× Substrate ( N A D P H ) Amplifier, O D (490 nm)

lmmRT 15 m m 37°C lmmRT 15 m m R T 3 - 1 5 m m RT

9 10 11 12 13

30 cycles 30 sec 95°C 30 sec 60°C 60 sec 72°C 10 m m RT a 1retoRT 1 m m RT 1 m m RT 10 m m 42°C

Conditions T h e reverse primer is biotmylated

4 - 1 0 ~zl P C R product or 50 ~l biotin-labeled antisense ohgo TBS-Tween 100 btl 0 4 N N a O H TBS-Tween 0 625 pmol/well (50/zl 1/100 of 1 2 5 / z M stock) TBS-Tween T e m p , [SSPE], and no of washes varies TBS-Tween 50 ~l 1/30,000 dilution TBS-Tween 50 kd substrate 50 ~l amplifier

RT, room temperature

probe, nonspecific binding was eliminated by one or m o r e 5-mmute stringency washes The probes were similar to those used in the 11th International Workshop but were modified by the incorporation of one or more fluorescem molecules during synthesis of the probes Detection of bound probe was achieved by an enzymebased colorlmetrIc reaction, I e , addition of an alkahnephosphatase-labeled anti-FL FAB followed by alkalinephosphate substrate and amplifier that produced in positive wells an intense r e d - v i o l e t Because the intensity of the color increased with time, the reaction was stopped when the O D of the negative controls approached 0 500, when the O D of the positive controls reached 2 000, or when the O D o f the ( + ) wells was at least twice background The assay took less than 90 mmutes after PCR amplification to complete A schematic of a tray designed to test 11 D R B probes with appropriate positive and negative controls is shown in Fig 2 PCR-amphfied D N A is added to rows D - H Probes are added to columns 2 - 1 2 Row A contains no D N A , column 1 contains no probe, and wells 1 A - 1 C contain neither p r o b e nor D N A and thus control for nonspecIfiC b i d i n g of the alkahne-phosphatase-labeled FAB Positive and negative controls for each probe, as discussed below, are placed in rows B and C To eliminate the need for PCR-amphfied D N A from reference cells for positive and negative controls for each probe, antisense or reverse ohgos were synthesized with a biotin attached at the 5' end and then used to replace PCR-amphfied D N A in wells The positive antisense ohgo was complementary to the p r o b e T h e nega-

tive antisense ohgo was complementary to the sequence most likely to cross-react under suboptimal stringency wash conditions and contamed one or more base mismatches O D values for the negative antisense ohgo estabhshed a threshold O D for the discrimination of positive and negative reactions Spacers (repetitive As or Ts) or extended sequences were lmtlally added at the 5' end of some antisense ohgos to prevent possible ster~c interference In the binding of fluoresceinated probes to biotInylated antisense ohgos, but these proved unnecessary and were omitted from later ohgos The results o f testing PCR-amphfied D N A from selected m e m b e r s of our reference cell panel with 11 D R B probes are shown In Table 2 Negative reactions are generally identified by O D readings < 0 500, p o s m v e reactions by O D readmgs > 1 000 or twice background T h e sequences o f the probes and the ( + ) and ( - ) antisense ohgos for each p r o b e are provided in Table 3 Probe 6101, which recogmzes a conserved internal sequence in the D R B genes, controlled for the presence of amplified D R B product T r a d m o n a l hybridization methods using membranes are hindered by the length o f time required for hybridizatIon, stringency washes, and detection, decreasing the usefulness of class II o h g o t y p m g for transplantation The conditions for each step here were therefore examined to determine the m I m m u m time required The binding of the blotm-labeled PCR product or antisense ohgo to avidin
152

D D Kostyu et al

Probe N O N L

1 0 6 0

1 0 0 4

I 0 0 2

1 0 0 6

1 0 0 8

1 0 0 3

I

5

7

3

6

0 0 7

7 0 ~

0 0 4

4 5; 2

1 0 I

1

2

3

4

5

6

7

8

9

10

II

12

~onc (+) Antlsens¢ (-) AnUsens¢ Unknown #1 Unknown #2_ Unknown #3 Unknown #4 Unknown #5

21\ 38~ L'x

W A S H [SSPEI TEMP # Washes PROBE SPECIFICITY

TABLE 2

21X 38° L~

2IX 0gX 38° 38° LX LX

0IX 0IX 38° 38° LX LX

01X 38° IX

0IX 001X 00IX 0005X 00IX 38° 47° 47' 47 ° 38 ° IX L", IX ~X IX

DRI

DR4 DR2

DR7 DRI0 DR3 DR9 DRll DR~, DR7 All DR6 DR14 DRB DRI 1 B3"0301

F I G U R E 2 Schematic of a 96-well mlcronter plate in which five D N A samples are tested against 11 ohgonucleotlde probes, mcluding ( + ) and ( - ) annsense ohgos for each probe and a poslnve control (probe 6101) for each PCR product The speckled wells re&care ( - ) control wells, the sohdwells mdlcate ( + ) control wells Although not indicated m the figure because of space hmltauons, probe 1004 wdl also react with DRB1*1410, probe 3452 reacts with DRB1*1401, 1404, 1405, 1407, 1408, and 1410, and probe 1003 falls to react with DRB1*1404 and "1410 Sequence, locanon, and speclfioty of each probe is provlded m Table 3

T e s t m g o f reference cells with 11 D R B probes Probe and s p e o f i o t y

Reference cell Experlmen t l ( + ) Annsense ( - ) Antlsense 035 (DR4,10 020 (DR7) 059 (DR13) 021 (DR8,11, B3"0301) Experimen t 2 ( + ) Annsense ( - ) Antlsense 004 (DR1,2) 035 (DR4,10) 020 (DR7) 025 (DR3,9) 021 (DR8,11, B3"0301) ND, not determined

1060 DR1

1004 DR4

1002 DR7

1006 DR7

1008 D R 10

1003 DR3, 6,11

1 704 0 251

>2 0 258

0 991 0 337

>2 0 292

>2 0 304

>2 0 271

>2 0 226

0 0 0 0

1 0 0 0

0 0 0 0

333 319 264 334

0 335 >2 0 228 0 299

>2 0 460 0 241 0 268

0 277 0 250 1 180 1 911

0 0 0 0

292 274 219 276

287 274 233 275

970 265 246 291

7004 DR3

3452 DR7, 1401, B3"0301

>2

>2

ND ~

1 622

0525

0312

ND

0448

0 284 0 249 0 289 1 627

0 0 0 0

ND ND ND ND

>2 >2 >2 >2

1007 DR9

5703 DR11

340 438 363 362

6101 DRB +

1 500 0 242

>2 0 237

>2 0 291

1 680 0 226

>2 0 263

>2 0 282

>2 0 345

>2 0 245

>2 0 183

>2 0 488

>2 0 253

1 680 0324 0 296 0 328 0 238

0 273 >2 0 303 0 290 0 241

1 831 0427 0 309 0 349 0 287

0 335 0271 >2 0 307 0 203

0 294 >2 0 282 0 308 0 214

0 260 0324 0 257 1 355 1 685

0 346 0255 0 245 1 557 0 202

0 242 0 230 0 224 0 260 1 288

0 310 ND 0 335 1 250 0 289

0 315 0 240 >2 0 253 1 680

>2 >2 >2 >2 1 880

TABLE 3 Sequence, location, and specificity of probes and antlsense ohgos Probe 1002FL (+)as (--)as

1003FL

Specificity DRBI*02

Codons 11-16

1002as 1003as ( D R 3 )

DRB 1"03,11,13,1401-1403,1405-1409

5' FLcct aag agg gag tgt cat FL

9-15

5' FLg tac tct acg tct gag tg FL

9-14

( + ) a s 1004as (-)as :01as(DRB4)

1006FL

D R B 1"07

9-14

DRBI*1001

6-11

(+)as ( -)as

DRBI*01

7-13

(+)as (-)as

DRBI*I 1

(+)as ( -)as

5703as 5504as ( D R B I * 1 4 0 7 )

6101FL

DRB 1-DRB5,DRB7

(+)as 7004FL

9-14

( + ) a s 7004as ( - )as 7005as ( D R B 3 * 0 3 0 1 )

8 G C / 1 8 bases 0 1 × SSPE, 38°C

8 G C / 1 8 bases 0 1 x SSPE, 38°C

5' FLtgg cag ctt aag tit gaa

7 G C / 1 8 bases 2 1 × SSPE, 38°C

acc gtc gaa ttc aaa ctt aca gta aag B I O T 5' acc gtc cca ttc ata ttc [ t t t t t t t t t ] B I O T 5' 34-40

CC . . . . .

t-

t-C

5'FLg gag gag ttc gtg cgc tt FL

1 1 G C / 1 8 bases 0 005 × SSPE, 47°C 3

c ctc ctc aag cac gcg aa B I O T 5' c ctc ctc agg cac gcg aa B I O T 5' 55-61

g . . . . . . . . .

5' FLg cct gat gag gag tac tg FL

1 0 G C / 1 8 bases 0 01 x SSPE, 47°C

c gga cta ctc ctc atg ac [a~a~_~_~_aa] B I O T 5' gcc g g a cga cgc ctc g t g a B I O T 5' ..... g - - g . . . . g-- 61-66

(~101+as D R B 1"03

c--

5' Fttc ttg gag gag gtt aag t rL

. . . . . . . .

5703FL

g--

ag aac ctc ctc caa ttc a [tttttt] B l O T 5' ag aac ctc gtc cga ttc aca ctc B l O T 5' ........ g__ -g . . . . .

1060as 1006as ( D R 7 )

DRB 1"07,1401,1404,1405, 1405,1407,1408,1410,B3"0301 3452as 3750as ( D R B I * 0 2 )

--t

5' ~ c t g ttc ttg aag cag gat K

. . . . . .

3452FL

a-

gca aag aac tic gtc cta B I O T 5' gca aag aac ctc gtc cga B I O T 5' ......... c. . . . . . g-

( + ) a s 1008as ( - )as 701as ( D R B 4 )

1060FL

8 G C / 1 8 bases 0 1 × SSPE, 38°C

acc gtc cca tic ata tic [ t t t t t t t t t ] B I O T 5' ctc gtc caa tit g t a ctc [tttttt] B l O T 5'

( + ) a s 1007as ( - )as 4 6 7 a s ( D R B 4 )

1008FL

8 G C / 1 8 bases 2 1 × SSPE, 38°C

5' FLtgg cag ggt aag tat aag FL

ct . . . . .

D R B 1"09

CC . . . . . .

5' FLgag cag gtt aaa cat gag Ft ctc gtc caa ttt gta ctc [tttttt] B l O T 5' ag aac ctc gtc cga ttc aca ctc B I O T 5' ....... g - - - c ac . . . .

( + ) a s 1006as ( - ~as 1004as ( D R 4 )

1007FL

9 G C / 1 8 bases 0 1 × SSPE, ~8°C

c atg aga tgc aga ctc aca gta a B I O T 5' c atg aga tgc cca ctc aca ata a B I O T 5' . . . . . . . . . .

DRB 1"04,1410

9 G C / 1 8 bases 0 8 × SSPE, 38°C

gga tic tcc ctc aca gta [ t t t t t t t t t ] B I O T 5' c atg aga tgc aga ctc aca gta a [ t t t t t t t t t ] B I O T 5' a - - - g - aga -t . . . . . . .

I + )as 1003as ( - ) a s 1050as ( D R 1 2 )

1004FL

N o G C / n o bases wash c o n d i t i o n s

Sequences

5' FLtgg aac agc cag aag gac rL

1 0 G C / 1 8 bases 0 01 × SSPE, 38°C

acc ttg tcg gtc ttc ctg B I O T 5' 73-78

5' Kggc cgg gtg gac aac tac FL ccg gcc cac ctg ttg atg [ ~ = ~ ] ccg gtc cac ctg tta atg B I O T 5' . . . .

t . . . . . . . . .

a

1 2 G C / 1 8 bases 0 01 × SSPE, 47°C B I O T 5'

---

T h e s e q u e n c e o f the ( + ) a n t l s e n s e o h g o is c o m p l e m e n t a r y to the p r o b e , the ( - ) antisense o h g o is c o m p l e m e n t a r y to the allele m o s t likely to cross-react u n d e r s u b o p t i m a l wash c o n d i t i o n s T h e c o n c o r d a n c e o f the ( - ) antlsense o h g o w i t h each p r o b e is i n d i c a t e d by dashes For each p r o b e , a FL is s h o w n at the 3' e n d for c o n v e n i e n c e , b u t it is actually i n s e r t e d b e t w e e n the first and s e c o n d bases S e q u e n c e s in brackets i n d i c a t e e x t e n s i o n s e q u e n c e s o r m u l t i p l e a's or t's as spacers b e t w e e n the o h g o and the b i o t i n ( B I O T ) S u b s e q u e n t w o r k suggests that spacers are u n n e c e s s a r y U n l e s s i n d i c a t e d , o n l y o n e s t r i n g e n c y wash is r e q u i r e d at the indicated SSPE c o n c e n t r a t i o n and t e m p e r a t u r e S e q u e n c e s are f r o m M a r s h and B o d m e r [40]

154

D D

TABLE 4

Time reqmred for the binding of a biotinlabeled DNA (the 1008 annsense ohgo) to awdm-coated wells O D (490 nm)

T,me (mm) of addmon of antlsense ohgo 10 20 ~0 40 50 60

1008as ohgo at 0 1 pmol 1 0~7 1 202 1319 1 321 1 282 1230

N o annsense ohgo

1008as ohgo at 0 05 pmol

m 0 034 +- 0 062 -+ 0 0 4 2 -+ 0 060 _+ 0 107 -+ O l l b

0 982 1 039 1057 1 140 1 017 1066

0 251 -+ 0 054

-+ 0 061 -4- 0 022 -+ 0 0 1 8 m 0 082 -+ 0011 -+ 0 1 0 9

0 243 -+ 0 028

The biotin-labeled ohgo 1008as was added to awdln-coated wells at 0 05 and 0 1 pmol/well for the nines indicated and the amount bound was determined by the addmon of probe 1008 O D values were taken 4 minutes after the addmon of the alkalme-phosphatase amphfier, at a point when differences m the amount of ohgo binding with rime would be most apparent At 10 minutes after the addmon ofamphfier, most O D values were >2 Values represent mean m standard devlat,on of tnphcate wells O D values for the ( - ) annsense ohgo, at each rime point, were < 0 289 S~mflar results were observed w, th other p r o b e - a n n s e n s e ohgo combmanons at 0 1 and 0 25 pmol/well and with PCR-amphfied D N A (not shown)

smaller volumes (not shown) The affimty between biotin and avldln IS one of the highest known (kd = 10-15 M) [38] An excess ofavldm (5/zg, 60 pmol) was used to coat each well to ensure binding of all amphfied material

T A BLE 5

(~rrespecnve of the presence of unincorporated blotmylated primer) Our 100-~1 amphficanon mixture contamed 2/zl of 20/zM bmtln-labeled primer (40 pmol), so the addmon of 10/zl of PCR product to a well would contain at most only 4 pmol blotinylated D N A or unincorporated primer Hybridlzatlon of probe and DNA required less than 10 minutes at 42°C (Table 5) and from one to three 5minute stringency washes To estabhsh optimal wash condmons, each probe was tested with posmve and negauve antlsense ohgos at variable SSPE wash concentranons (2 l x , 0 4 × , 0 l x , 0 0 1 × ) and temperatures (38°C, 47°C, 56°C, and 62°C), as shown in Table 6 The SSPE concentranon and temperature chosen for maximal dlscrlmmatmn between posmve and neganve reacuons were then confirmed by testing with amplified DNA For probes that contained several mismatched bases with potentml cross-reacnve alleles (e g , probes 1002, 1004, and 1006, Table 3), a single stringency wash at 38°C was sufficient for specific binding For probes with agreater GC content or only 1 or 2 base m~smatches to a potennally cross-reacnve allele (e g , probes 3452, 5703, and 7004) more stnngent condmons and up to three washes were required Probes were synthesized as 17- or 18-base ohgos, 15- and 16-base probes proved unreacnve or unreliable under the condmons employed here Ohgos up to 21 bases in length have been used satisfactorily, but these reqmred more attennon to the stringency wash conditions Although it is possible to postsynthetlcally label

Time required for the binding of probe 5703 to immobilized DNA (5703 antlsense ohgo) O D (490 nm)

Time (mm) ot probe addmon l0 20 30 40 50 60 N o probe, no antlsense ohgo

Probe 5703 at 0 625 pmol/well 1 915 1 940 1 864 1 886 1 930 1 867

-+ 0 080 -+ 0 061 -+ 0 085 -+ 0 050 -+ 0 0 8 8 -+ 0 163

0 240 -+ 0 013

K o s t y u et al

Probe 5703 at 0 15 pmol/well 1 872 1 968 1 901 1 928 1 867 1 745

-+ -+ -+ -+ -+ -+

0 0 0 0 0 0

047 037 075 049 110 124

0 219 -+ 0 009

The blonn-labeled 5703as was allowed to brad to awdln-coated wells for 20 minutes and the wells were washed with T B S - T w e e n The probe 5703 FLwas added at concentranons of 0 15 pmol/well or 0 625 pmol/well for the indicated rime intervals Wells not Immediately receiving the probe were allowed to sit at room temperature in T B S - T w e e n After the probe had been added to all wells, the assay was completed as usual O D values were taken 6 minutes after the addmon of the alkalme-phosphatase amphfier Values represent mean -+ standard deviation of mphcate wells O D values for probe 5703 added to wells containing the ( - ) annsense ohgo were < 0 514 at each t,me point

Rap~d HLA-DR Ohgotypmg by ELISA

TABLE 6

155

Determmation of wash conditions for three probes, showmg the effect of SSPE concentranon, temperature, and number of washes O D (490 nm) Probe 1006

[SSPE] ( + ) antisense ohgo 2 lx 04× 01× 0 01 × 0 O1 × two washes 0 005 x three washes ( - ) antisense ohgo 2 1× 04x 0 1× 0 01× 0 01 × two washes 0 005 × three washes

Probe 1003

Probe 3452

38°C

47°C

56°C

62°C

38°C

47°C

56°C

62°C

38°C

47°C

56°C

62°C

1 111 1 304 1324 0 849

1269 0 630 0364 0 438

0381 0 248 0272 0 340

0232 0 265 0288 0 366

>2 >2 >2 >2

>2 1 988 1793 0 928

1927 0 483 0338 0 488

0652 0 327 0317 0 437

>2 >2 >2 >2

>2 >2 >2 >2 >2 >2

>2 >2 0 941 0 576

>2 0 793 0 321 0 473

0 417 0430 0 318 0 312

0 381 0404 0 343 0 302

0 309 0260 0 278 0 268

0 347 0335 0 326 0 266

0 479 0309 0 356 0 310

0 348 0281 0 292 0 303

0 336 0375 0 362 0 275

0 355 0368 0 375 0 260

>2 >2 >2 >2

>2 >2 >2 1 020 1 075 0 532

>2 0 537 0 364 0 367

1 805 0 300 0 319 0 31s

Probes 1006, 1003, and 3452 were tested with ( + ) and ( - ) annsense ohgos, washing at varying temperatures (38°-62°C) and SSPE concentrations (0 005 × to 2 1 × ) For probe 3452, multiple washes were required for spec,ficity Choice o f stringency wash conditions using antlsense ohgos was then confirmed by testing on PCR-amphfied D N A O D values for wells w,th probe but without the biot, n-labeled ant,sense ohgos were < 0 251 O D values represent single determinations

an ohgonucleotide with an enzyme such as alkaline phosphatase and thus ehmlnate the FAB step, we found that the direct incorporation of a fluorescem Into an ohgonucleotide probe using a fluorescem phosphoramldlte was easy, efficient, and economical and avoided the need for postsynthenc modmficanons Moreover, an alkalmephosphatase-labeled FAB could be used to detect any number of probes and for any locus, mmmm,zmgthe cost and technical rime for postsynthenc labehng As many as five fluoresceins have been added to the 5' end of an ohgo without loss of specificity or reacnwty (not shown), although the incorporation of a single fluorescem proved adequate for detection As an alternative labeling procedure, we have labeled already-synthesized probes with the hapten dlgoxlgenln usmg protocols developed for the 11th International Workshop [19] or manufacturer's mstructions (Boehrlnger-Mannhemm Genius Kit) Although these probes could be detected by addition ofa commeroally available alkahne-phosphatase-labeled antl&goxagemn FAB, they were of limited use because the amount of probe labeled at one time was small, the reagents were expensive, and we occasionally noted variability in labehng efficiency The optimal concentration of the ann-FL FAB was determmed by ntration Although the manufacturer recommended a 1/3000-1/5000 dilution of the stock for an ELISA, these concentrations produced sigmficant

background and a 1/30,000 dilution was subsequently Identified to be optimal At this concentranon, 10-20 minutes incubation of the DNA-ohgo complex and FAB was sufficient (Table 7) Addition of a blocking reagent or serum prior to the addmon of the FAB was unnecessary and did not reduce the background if higher concentrations of FAB were used (data not shown) Colorlmetrlc detection of a bound probe was based on an enzyme amplification system [39], where the alkahnephosphate substrate, mcotinamIde adenine dmucleotide phosphate (NADPH), is dephosphorylated by alkaline phosphatase to N A D H This activates a redox cycle driven by alcohol dehydrogenase and diaphorase and leads to the reduction of nitrotetrazohum v,olet to the red-violet formazan dye (Fig 1) An alternative substrate for alkahne phosphatase, p-nltrophenylphosphate (pNPP), produced a yellow that was measurable at 405 nm, but color development was slow (2-4 hours or longer) One advantage to using pNPP was that the wells could be stripped of probe and label by addition of 100 /,l of 0 4 N NaOH to each well for 1 minute at room temperature and reprobed Thus far, reprobing wells that were developed with the amphfication system above has led to unacceptably high backgrounds The sensmwty of the assay, determined by titratlng known concentations of blotm-labeled antisense ollgos, ranged from 0 01 to 0 05 pmol/well ( - 1 - 4 ng of amph-

156

D D Kostyu et al

TABLE 7

Time required for the binding o f the antlFL FAB to immobilized D N A - p r o b e conlugates using probe 5703FL and the 5703 antisense ohgo

Time Ira,n) of FAB addmon

O D (490 nm)

20

o

fit i0 20 50 40 50 60 No antisense ohgo

1 032 1 209 1 354 1 349 1522 1 437

-+ -+ -+ -+ -+ +

0040 0060 0020 0088 0012 0 020

10

o

O0

. . . . . . . .

001

0 168 -+ 0 004

I

. . . . . . . .

01

I

. . . . . . . .

1

1

1

Pmol/~H The ( + ) antisense ohgo was added to avldm-coated wells for 20 minutes, followed by T B S - T w e e n washes, a 20-minute exposure to the probe, and stringency wash The FAB was added for the times indicated Wells not immediately receiving FAB were allowed to sit at room temperature in T B S - T w e e n After FAB had been added to all wells, the assay was completed as usual O D values were taken 4 minutes after addmon ofalkalme-phosphatase amplifier By 8 minutes, most readings were > 2 Values represent mean + standard deviation of triplicate wells

fled D N A or - 10 v molecules), as shown m Fig 3 Similar results were obtained using other biotin-labeled ohgonucleoudes (results not shown)

Antlsense

Pmol/well

(I)

Ratio

1060as

0 625 0312 0 156 0 078 0 039 0 020 0 010 0 005

>2 >2 1 971 1 284 0 785 0 475 0 378 0 306

>6 1 >61 >61 40 24 15 12 09

lO04as

0 625 0312 0 156 0 078 0 039 0 020 0 010 0 005

>2 >2 >2 >2 1 489 0 971 0 716 0 431

>6 1 >61 >6 1 >6 1 4 3 2 8 2 1 13

None

0 324

DISCUSSION The advantages of this method include the following Typing Is fast ( < 9 0 minutes after PCR is completed) and economical Most reagents are available commercially and only buffers need to be prepared The FLlabeled ohgonucleonde probes and the biotin-labeled antisense ohgos as controls can be inexpensively synthesized, m mtcromolar amounts, and with consistently high labeling efficiency Postsyntheuc labeling of probes with haptens such as digoxlgenm, or with alkaline phosphatase itself, may be feasible alternatives m some instances T h e technique dispenses with dot blotters, m e m branes, hybridization ovens, radioactivity, and darkroom facilities and can be p e r f o r m e d m any laboratory equipped with incubators and waterbaths A microplate reader is r e c o m m e n d e d , but opnonal N o special trammg Is necessary and the method is feasible for laboratories with little experience m molecular techniques Perhaps most significant is the ease with which the assay can be standardized The antisense ohgos constitute standards that are easily synthesized by any laboratory and they e h m m a t e the need to search for and repetitively amplify reference D N A As new H L A alleles continue to be defined, it becomes nearly impossible for

Ohio

FIGURE 3 Sensmvlty of the ELISA to <0 05 pmol/well The biotin-labeled annsense ohgos (1060as and 1004as) were added to avldm-coated wells at the indicated concentrauons, followed by standard washes and mcubauons with probes 1060 and 1004 at 1 5 pmol/well The rano is the mean OD of the sample per the mean OD of the background (0 324, 0 345)

a laboratory to obtain enough reference individuals or suitable B-cell lines for p o s m v e controls for all possible probes Further, the ( - ) antisense ohgo sets a threshold by which positive and negative reactions can be discriminated, the results are amenable to statistical analysts The m i c r o m e r plate format should facilitate automated testing and computer-based allele assignments, minimizing the lntroducnon of human error The adaptability o f a microplate also allows for testing of numerous D N A samples or only a single one We have found that the avidm-coated trays can be preloaded with antlsense control ohgos and stored at 4°C or - 2 0 ° C for at least a week T h e assay has been used for D Q B typing and

Rapid HLA-DR Ohgotypmg by ELISA

B27 subtyplng and it should be equally useful for the analysis of other polymorphic systems

157

hlstocompatlblhty complex Proc Natl Acad Scl USA 87 1835, 1990 11 Mulhs KB, Faloona FA Specific synthesis of DNA m vitro via a polymerase-catalyzed chain reaction Methods Enzymol 155 335, 1987

ACKNOWLFDGMENTS We thank Manar Ghanayem, Shawn Miles, and Heather Heiman for techmcal assistance, Richard Metzgar for the loans of a mlcroplate reader, Harvey Sage for advice, and Alan Shore for synthesis of ohgonucleotldes This work was supported in part by NIH contract AI-15116 and the Howard Hughes Research Fellows Program A more detailed protocol is available upon request

12 Saikl RK, Gelfand DH, Stoffel S, Scharf SJ, Hlguchl R Horn GT, Mulhs KB, Erhch HA Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 239 48v, 1988 13 Angehnl G, de Preval C, Gorski J, Mach B High resolution analysis of the human HLA-DR polymorphism b) hybridization with sequence-specific ohgonucleotide probes Proc Natl Acad Scl USA 83 4489, 1986 14 Salkl RK, Bugawan TL, Horn GT, Mulhs KB, Erhch HA Analysis of enzymatlcally amplified beta-globin and HLADQ alpha DNA with allele-specific ohgonucleotide probes Nature 324 163, 1986

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