HLA-B27 TESTING

Abstract: A method of testing for the HLA-B27 antigen which comprises mixing with blood cells or blood cell lysates a first antibody capable of binding and blocking the HLA-B7 antigen and a second antibody capable of binding to the HLA-B27 antigen and wherein said second antibody is labelled so as to be detectable and thereafter detecting for the labelled said second antibody. The second antibody is not specific to the HLA-B27 antigen. It is found that the HLA-B7 antigen will bind in significant quantities to the second antibody. Therefore it is necessary to block the HLA-B7 antigen with a first antibody.

Description: 1 ϋ__ϋ-§22 IISTI G 2 This invention relates to testing for the H A-827 3 antigen. 4 The disease ankylosing spondylitis (AS) is difficult to 5 diagnose with certainty but it has been found that sufferers S from AS have a probability of about 55% as being carriers of 7 the gene coding for the HLA-827 antigen. 8 The present invention provides a method of testing for 9 the HLA-B27 antigen which comprises mixing with blood cells 0 or blood cell lysates a first antibody capable of binding 1 and blocking the HLA-B7 antigen and a second antibody 2 capable of binding to the HLA-B27 antigen and wherein said 3 second antibody is labelled so as to be detectable and 4 thereafter detecting for the labelled said second antibody. 5 In a preferred aspect there is provided a method of 6 testing for the HLA-B27 antigen which comprises forming a 7 mixture containing blood cells or blood cel l lysates, a 8 first antibody capable of binding and blocking the HLA-B7 9 antigen and a second antibody capable of binding to the HLA- 0 B27 antigen, wherein the second antibody is labelled to be 1 detectable and thereafter separating unbound said second 2 antibody from the mixture and detecting for said second 3 antibody bound to HLA-B27 antigen. 4 In a preferred aspect the present invention provides a 5 composition for testing for the HLA-B27 antigen comprising a 6 mixture containing a first antibody capable of binding and 7 blocking the HLA-B7 antigen and a second antibody capable of 8 binding to the HLA-B27 antigen and wherein said second 9 antibody is labelled so as to be detectable. 0 The blood used in the method of testing may be whole 1 blood, citrated blood or blood depleted of erythocytes. 2 Various diluents may be added to the blood. In general , it 3 is preferred that the methods of this invention be performed 4 on whole blood as so doing reduces testing time and cost. 5 Said first antibody may be any suitable antibody and no 8 particular antibody is required. However, we currently 7 prefer to use as said first antibody a HLA-37 specific 8 antibody designated 887.1 produced by a cell line obtained 1 from Or. eter Parham of the Imperial Cancer Research 2 Foundation, London, England. 3 Said second antibody may be any suitable antibody and 4 no particul ar antibody is required. However, we currently 5 prefer to use as said second antibody HLA-ABC-m3 which is a 6 HLA-B27 specific antibody produced by the Department of 7 Pathology , Univ ersity of I. e 1 b o u r π e , Walbourπe, Victoria, a Australia. 9 Said second antibody may be labelled to be detectable α in any convenient way. It is current ly preferred to use 1 r adio - l abe l l ing but co l orimetric and enzyme - l ink ed 2 immunosorbeπt assay (ELISA) labelling may also be applied. 3 In one preferred manner of l abel l ing , said second 125 4 antibody was labelled with I. Such labelling may be done 5 in any suitable manner. In one preferred instance purified 1 -? tz 6 monoclonal antibody is mixed with * * 3 ι (radioactive iodine) 7 and ch l or amiπe T is added ; af ter 1 -2 mins a t room 8 temperature, sodium m e t ab i s u 1 p h i t e is added to neutralise 125 9 the chloramine T and the I-antibody is separated from 4 •* - c 1 5 0 free 1 <;* - * ι using exclusion chroma to graphy. The I is bound 1 covalently to tyrosine residues in the antibody. 2 In a colorimetric labelling, said second antibody was 3 labelled with a dye such as Procioπ Blue CIX-R obtained from 4 Fluka AG, Switzerland αr ICI Limited, England. 5 In an ELISA labelling said second antibody was label led 6 with an enzyme and later reacted with a suitable substrate 7 adapted to change co lour on such a reaction occurring. 8 Enzymes and substrates therefor considered suitable include 9 g a 1 a c t o s i d a s e and 2 - π i t o h e n y 1 - B - D - g a 1 a c t o y r a n o s i d e ; 0 perαxidase and 3 , - d i a m i π o b e π z i d i n e , peroxidase and A8TS, 1 and urease and pheπyl red. (A3TS is 2 , 2 ' - a z i π o - d i ( - e t h y 1 ) 2 beπzo t hiazo 1 ine sulphσnate). 3 Separation of unbound said antibody from the mixture 4 may be ef fected in any conv enient way but in general 5 centrif ugatiαπ is satisf actory and produces a cel l pe l let 6 and a supernant which can be decanted. The cel l pellet can 7 be washed as necessary. 8 His tor ica l l y , on l y two m eth od s f or s ero l α gica l detection of HLA specifici ies have been popularly used since the introduction of routine tissue typing, almost two decades ago. The first of these, a 1 e u σ a g g 1 u i π a t i o π technique was replaced during the late 1960's by more sensitive and reliable cytotαxicity assays which were developed on a micro-scale by Ki s s m e y er - i e 1 s o n and by Terasaki, P.I. and McClelland, J.D.s Clicro roplet assay for human serum cytotoxiπs. Nature 206:998-1000 , (1964). Mic ocy otoxicit remains standard today as it enables the simultaneous assessment of the reaction of a patient's cells with a very large number of scarce HLA typing sera which can be used in minute quantities. Since the description of the close association of HLA- B27 with AS, a significant proportion of the labours of tissue typing laboratories has been directed at assessment of HLA-B27 status, irrespective of the patient's ful l HLA phenαtype. Requests for B27-typing by rh e um a t o 1 α g i s t s and other physicians have grown steadily despite frequent warnings that B27-status should rarely, if ever, be used as a diagnostic criterion for AS, as 8 % of the (Caucasian) population is HLA-B27 + and only a small minority of these ever develop AS. At Royal Melbourne Hospital for example, 45!? of the 2,500 requests for tissue typing during 1983 were solely for B27 status. Thus far, B27-typing has always been performed along routine issue-ty ing lines, testing a panel of anti-B27 sera on the patient's lymphocytes, and this has proven costly in terms of materials (especially antisera) and particularly because of recently escalating labour costs. The aim of a full panel of standardised monoclonal HLA- typing sera is stil l a distant one, however, monoclonal antibodies may have much to offer in certain specific situations such as B27-typing. Accordingly, a number of procedures which have utilised an aπti-HLA-B27 monoclonal antibody, have been developed, which should enable rapid, cheap and dependab l e assessment of B27 status. It is contended that these techniques wil l lead to a marked reduction in the costs of HLA-B27 typing, both in terms of staff labour and the costs αf disposable reagents, allowing the direction of these resources to other services. m_.Iii_.I___-.:- ϋϋi- -lili-i-i-.-. iϋ kli A. STANDARD PROCEDURES £ 1.1 , UJ1_ Three 1 ymphob 1 astgid cell lines were used - (i) Bordiπ: HLA type A2, A2; 827, Bw39; DR1 (ii) LD-8: homozygous typing cell, HLA type A3;B7;DR2 (iii) JZ-B: HLA type Aω23,,A32; Bw21 , 816; DR4,5 Cells were grown in RPP1I medium. !U i2 £__£_l_ϊ.-_ f\H_:i-l£-ii_L-ii. HLA-ABC-m3, which detects an HLA- B27-specific determinant. HLA-ABC-ml detects an HLA-Class 1 "framework" antigen while BB7.1 is an HLA-B7-sρecific monoclonal antibody. £Hi_££_.J__iZ_-_*._L__£i_.-- __££££__£----££_-._. A suspension of leukocytes and platelets was prepared by mixing 5 ml of citrated blood with 1 ml αf 6 % w/v dεxtran (Pharmacia) in 0.9 NaC1 in an elongated tube. After mixing, the tube was allowed to stand at RT for 30min, permitting agglutinated red cells to settle. The supernatant leukocytes and platelets were co l lected and stored in a separate tube at ambient temperature until used. Ce_l^ k___L_: _i Tissue culture cells were washed once in 0.9!? NaCl then lysed at 10 8 /ml in a solution of 0.5$ (v/v) Triton X-100 (8DH) and 10ml. Iris-HC1 ,pH 7.4 in physiological saline (TTS) which was supplemented with 1 (w/v) (bovine serum albumin)(8SA) and 1 % (v/v) trasylαl. The mixture was incubated on ice for 60 miπ. then cel l nuclei and debris were removed by c e π t r i f u g a i o n . The supernatant membrane preparation was either used immediately or stored frozen at -80°C till used. £££i__i£_l_i£π £l ϋk£.___.£_!_.!_._. _>_. E__£___L_-._1 Δ L Ξ.h.1 £__.._.._.£_•._ ■ .__ __.J2_£_2____£_1___L_3._1___L " • " ° remove lipid material from ascites prior to purification, an equal volume of Freoπ (CICF-CC1 ,F; A ldrich Chemical Company, Inc., Mi lwa kee, 111 i s . , US ) was added, mixed wel l, and the suspension was centrifuged. The reco v ered ascites was added dropwise to a pA-Sepharose column prewashed with phosphate buf fered sa line (PBS). After f urther washing with PBS , antibody was eluted with glyciπs-HCI , pH 2.8 (Sigma Chemical Co.). To neutralise the pH αf the eluate, 1 ml fractions were col lected in tubes conta ining 5 microl of saturated tris buf f er. The absorbance of each f rac tion was determined and tubes containing the eluted protein retained. The column was washed with glycine-HC1 , pH 2.8 until al l protein was removed. The pooled protein sample of known concen ra ion was dialysed against bicarbonate buff er (0.1 PI NaHCO-j, 0.5 PI NaC1 , pH 7.5) at ° C ov ernight. 3. ϋ. -i i £ _: £ _i _*. £J__L--.£ £1 HLA-B 27 m onoc l ona l a n tibody was radio l abe l l e d with 1 25 ι (A me rsham I ntern a tiona l ) a s described abo e. B. EXPERIMENTAL PROTOCOLS Th re e di f f e ren t a s say s are p res e n te d h er e , the f ir s t detecting B27 antigen in membrane preparations, the second and third on intact cel ls. The first two methods are described brief ly as the third is clearly the method αf choice for HLA-B27 typing and therefore has been refined to the greatest degree. 1. A_ R a di£JLm m_.££ £££ £ ££ _t£_L££_L.i££ 2. ££_:£££££._. *_£ I H-ii _-.l£___- _.£_*. _*.___-£_!_l_-_L££ __£_.£_. ϋkiL_.i_._*2._. This method was devised to detect HLA antigens in membrane preparations of cultured cel l lines. In this two step "sandwich" procedure, a first monoclonal antibody that detects a moπomαrphic HLA class 1 determinant was bound onto a P MC pl ate which had p re v iou s l y b een coa t e d w i th p A. M emb r an e -bound HL A antigents were then al lowed to bind to this antibody and HLA-827 was detected by the addition of 125 I - 1 ab e 11 e d HLA- ABC-m3. The assay was performed at room temperature (RT) in a moisture box and in triplicate. *_.££_:£££-- il (1) A flexible PVC plate (Disposable Products) was coated with pA (50 microl of a 50 micro g/ml solution) (Sigma Chemical Co.). (2) Non-specific r α t e i n - b i π i n g sites were blocked by topping up the wel ls with 1 % solution of BSA in PBS for 30 min . (3) The plate was "f licke " and washed three times with PBS then HLA-ABC-ml (SOmicrol αf a 1/100 dilution αf mouse ascitic fluid) was added to each well for 60 min. (4) The plate was rewashed as above and cell lysate added (50 microl doubling dilutions) for four hours. (5) Following further washing, rad i α 1 a b e 11 ed HLA-ABC-m3 (5x10 cpm o f antibody a t a s p ecif ic acti v ity of a p r ox .1.8 ic r α C i/micr o g protein diluted in PBS with 1 % BSA) was added and incubated overnight. (6) On the next day, the plate was washed, dried and the bound radioactivity estimated. *- __££Hi_-_- This method was able to discriminate between B27 + and B27~ cell lysates consistently and accurately, with lysate dilutions as low as 1/16 (Fig.1) and similar results were obtained whether cell lines or leukocyte preparations were used as an antigen source. However, there were several disadvantages in this procedure. For example, the protocol had several long incubations, the total assay taking about twenty hours. Furthermore, there was the necessity of separating the patient's leukocytes and then processing these to produce a membrane preparation. Attempts were made to adapt the assay to detection of HLA antigens in serum or urine but this protocol lacked the sensitivity required to detect such small quantities of antigen (not shown). 2. A_ S _U£ii_i__-π£_L_iJi -l _.££ J__L_L_L£__i£_l £__ kΔ §_*2 i£ k££i_.££_-_l _-E_*.£._*. _-£_- ££££ £ _•__££ _- ! This met h od for detection of HLA-B27 consisted αf a direct binding assay of r a d i o 1 a b e 11 ed anti-827 monαcolonal antibody perf ormed on cit r at ed b l ood , dep l ete d of erythr ocyt es by d ex tran sedimentation. The advantages of this method were that the antigen source (i.e. the crude, unwashed preparation of leukocytes and p l ate l ets) was easy to prepare and the protocol was shorter compared with the previous method. This protocol was also performed at RT and in triplicate. £.££._-.£££._ ■ i. (1) Doubling dilutions of 125 1-1abe11ed HLA-B27 antibody (50 microl , commencing with 2x10 cpm/assay) were set up in 3 ml disposable plastic test-tubes (Disposable Products) which had previously been treated with PBS containing 1 % BSA in order to prevent non-specific protein binding. (2) Leukocytes and platelets were isolated from whole blood as described above and 500 microl of the suspension added to each tube for 30min. (3) The cells were washed twice in PBS/8SA and bound radioactivity estimated. i_Hl_I ! This method proved most satisfactory, with specific/non-specific binding ratios of up to 100:1 (Fig.2). The assay was simple, rapid and reliable, with the possibility of analysing up to six or eight samples within two hours. Attempts were made to adapt this procedure to an assay in a flexible PVC plate, however difficulties were experienced with the washing procedures. In developing a direct binding assay it was anticipated that false positives might be encountered when testing the leukocytes of individuals bearing determinants which are cro s s -re a cti e with HLA-B27. It had already been shown in two large population studies that apart from HLA-827 + cells, only HLA-87 + cells were able to bind significant quantities of HLA-ABC-m3, the affinity of the antibody for this specificity being approximately one log unit less than that for HLA-B27. It was thus reasonable to presume that HLA-B7 would cause the greatest problems with false positive assignment αf B27-status. HLA-B7 is a commonly encountered HLA specificity in Caucasian populations, to the extent that approximately 4JS of such populations are HLA-B7 homozygotes. Binding assays using radio1 abe 11 ed aπti-HLA-827 and a number of cell lines confirmed this suspicion. It was shown that a B7 homozygous cell line, LD-B bound almost as much HLA-827 as did the B27 heterozygous cell line, Bordin (Table 1) and that this binding persisted despite several washes. The same phenomenon was noted when leukocytes were used instead of cell lines. Attempts to reduce this "background" binding are described below. 3. A i£££--__-__- ----.£2 i_ ___-.£A_i-i£££__££ ._l _.££ _.£__££_-_-.££ £_L ϋk_-_- SU -_.£ _!!_£_■.£ Si£££ " i. The direct binding assay described below was analagous to that described above, however it had the advantage of using whole blood rather than a leukocyte preparation, thus rendering the protocol extremely simple. Once again, it was performed at RT and in triplicate. - g - TABLE 1 §_-.£_._-.££ 5. 5-l-ii£i£_ii££-i£-i ϋk___:_L_i£__i_.-- __£ ϋk_ι-I_l2 ± £_. i_kΔ-i___2- £lii ki £ * Cell Type HLA Phenotype Cpm Bound Borain A2, A2;827 ,Bw39 ;DR1 ,DR1 467,980±12,000 LO-B A3 , A3 ;87 ,B7; DR2 , DR2 417 ,300±32, 000 JZ-B Aw22,Aw32;B16,8w21 ;DR4,DR5 700± 500 * Figures expressed are the mean of triplicate assays ± standard error. - 1 o - £££_L£££i _L± (1 ) R adio 1 ab e 11 s d aπti-HLA-827 antibody (appro imately 4x10 cpm) was placed in a plastic test-tube which had been pretreated with PBS containing 1 BSA in order to prevent non-specific protein binding. (2) Whole blood (250 microl) was added to each tube for 30 min with gentle agitation every 10 minutes. (3) The tubes were centrifuged and supernatant was removed from the cell pellet products. (4) The cell pellet was washed twice in PBS/BSA and bound radioacti ity estimated. £ £___-£i Protocol 3 was used to examine 12 subjects for B27 status (Fig.3). The study population was chosen to include individuals who were heterozygous for HLA-B27, heterozygous or homozygous for HLA-B7 or who possessed neither of these specificities. The B27 + individuals were able to bind between 22,000 and 44,000 cpm, while the only B7 homozygote available for study bound approximately 13,000 cpm. By comparison 87 h εt e ro z ygotεs wεre in the range 4,000-8,000 cpm, with B27 ~ B7 ~ individuals binding far less antibody (600-800 cpm). Although these results were encouraging, they also confirmed the likelihood that 87 homozygous individuals could be confused with B27 h e e ro z y g ot es. Accordingly, it was dscidεd to attampt to block thε non-specific binding αf the anti-HLA-B27 antibody by p re -incub a tin g the cells with a second mαnαcloπal antibody (8B7.1 ) which specifically detects an epitαpe on the HLA-B7 molecule but fails to react with HLA-B27. Importantly, previous population studies have shown that the epitαpe defined by BB7.1 is present on the cells of 100St of individuals who are typed as HLA-B7 + by conventional means. Protocol 3 was thus modified to include a step in which the blood sample was preincubated for 20 minutes with various dilutions of 8B7.1 prior to addition of the ra d i o 1 a e 11 e d aπti-HLA-827 antiserum. In order tα keep the protocol as brief as possible, no washing took place until U £ incubation with a n t i - H L A - 827. The cells of four individuals were studied (Subjects 1 , 4, 7 and 10 in Fig.3) as they represented the four study populations, i.e. 827 heterozygotes, 87 homozygotes, B7 heterazygo es and B27~B7~ individuals (Fig.4). At the highest concentrations of blocking antibody, "non-s ecific" binding of the anti-B27 reagent was reduced by 72!t in the case of the 87 homozygote and by 85 % in the case of the 87 heterα zygote , while there was no reduction in the ability of aπti-HLA-B27 to bind to B27 + cells. Subsequently, it was shown that simultaneous addition of blocking antibody and test antibody produced the identical degree of blocking of HLA-B7 + cells as that produced when the anti-B7 reagent preceded the r a i α 1 a b e 11 e anti-B27 antibody. This was demonstrated in a time course experiment (Fig.5) in which the anti-B7 reagent was preincubated with B7 homozygous and B27 heterozygous blood samples for varying lengths of time prior to the addition of the B27-specific reagent. Clearly, the degree of blocking was not af fected by the length of preincubation with anti- HLA-B27. This was a significant finding as it permitted further abbreviation of the protocol. Iil£ £ _.££££_- £££_;£££_; _ϋ_ _;_!£££._.£££ £.£_-,-_£-_ ___: (1 ) Test-tubes can be prepared and stored as fol lows: test-tubes are filled with PBS/BSA and incubated at RT for 30 min then emptied by flicking. R adi o 1 ab e 11 ed anti-HLA-B27 antibody (50 microl , radioactivity of 4x10^ cpm) and b l ocking antibody (20 microl of 887.1 ascites f luid prediluted 1/10 with PBS/BSA) are then added, the tubes are tightly capped and stored at 4°C. If NaN 3 is added to a final concentration of 0.1 % (w/v), the tubes can be stored for at least one week. (2) Blood to be tested (250 microl in triplicate assays) is added to each tube and incubated at RT for 30 min. (3) The cel ls are then washed twice with PBS/BSA, test tubes were centrifuged and the settled cell pellet drained αf all fluid and bound radioactivity is estimated in gamma c o u n t e r . In order to test the final protocol, a series of blood samples which were submitted to the Royal Melbourne Hospital for B27 typing were tested in parallel with conventional i c r o c y o o x i ci t y , which was performed independently. A total αf 19 blood samples were tested and it was found that the B27 + individuals detected by each technique correlated exactly (Table 2). All four HLA-B27 + individuals were able to bind at least ten times as much radioactivity as the B7 sub ects, enabling easy differentiation of HLA-827 + from cross-reac i v e s ecificities. T A B L E 2 i_ki_-___--2 Si£_ u Dli≤rminfd b _ R^A_ and b^ ϋU£ £li i££ £i_: Subject HLA-827* HLA-B7* cpm bound x 10 "3 (a) i_-£≤£_-._I££l__ 1 1 - - 2.0±0.2 2 1 ,2±0.2 3 + - 21.2±2.5 4 1.9±α .3 5 + - 27.6±2.1 6 + 55.4*2.2 7 0.8±α.4 8 - - 1.3±0.2 9 + 33.3±3.0 10 - - 1.7±0.2 11 - + 2.0±0..4 12 - + 2.7±0.2 (13) HLA-B27+ control subject 33.4±2.1 __.ili_L__L_Z.i_0.__._2__- i. 1 - + 1 ,4±0.2 2 - - 1.4±0.2 3 - - 0.9±0.2 4 - + 1.a±0.3 5 - - o .a±α .1 5 - - 0.8±0.4 7 - 0.9±0.3 (8) HLA-B27 + control subject 25.4±1.3 * as determined by standard microc to oxicity at Royal Melbourne Hospital Tissue Typing Laboratory (a) mean of triplicate assays ± standard error 1 Next, 142 patients typed by the conventional cytotoxicity 2 test in another l aboratoty (Dr.B.Tait, Tissue Typing 3 Laboratory, Royal Melbourne Hospital) and by the radio- 4 i muπo-assay RIA in our laboratory. 5 106/142 were HLA-B27" by both , cytotoxicity and RIA 6 36/142 were HLA-B27 + by both cytotoxicity and RIA 7 25/142 were HLA-B7 + by cytotoxicity and were negative by 8 RIA . 9 10 Note: Two patients deserve commεnt 11 12 No.1 : Cytotoxicity was wεak but RIA " 13 No.2 : Initially called B27 + by cytotoxicity but was RIA " . 14 On repeat of both tests the resu lt was cytotox~RIA~, 15 i.e. the RIA was the better used. 16 i.e. no disc epancies in 142 tests. 17 1 a kilA ASSAY F0 THE DETECTION OF THE HL A - 827 ANTIGEN 19 2 0 Iπ__£££Η£_-_-££ 21 The Elisa test is now taking the place of radiαimmunαassays 22 (RIA) in a number of laboratories f or the f ol l owing 23 reasons:- (i) to avoid the handling αf radioactiva isotopεs, 24 (ii) to usε reagents which do not decay with time (such as 25 radioactivity) and have a long shelf life. The principle of 25 E l'i sa in this case is the same as outlined from the RIA - an 27 enzyme, (for example bet a-ga 1 ac tos idasε but many others can 2B be used), is linked to the antibody and the amount of 29 antibody bound to the antigen is measured by finding the 30 amount of enzyme bound. The enzyme is detected by adding a 31 substrate which changes co lour (measured by optica l 32 density). Thus the measurement of enzyme bound measures the 33 amount of HLA-B27 antigen which is present. 34 ■■ '"-' ££££ £ £__£ 2.L ££U_;_ikΔzS_i2 £_!_ii_!££ , _£ _: li£_i -£ i £_i££_L_£-i£ 36 ££__,.___!l£ £££__££ -_£ 37 1. B e t a - g a 1 a c t o s i d a s e was coupled tα aπti-827 antibody 33 u s i n g t h e c r o s s l i π k s r s u c c iπ i m i d y l 4 - ( N - maleimidocycylohexaπε carboxylate) (SMCC). Briefly antibody (1 ml , 2.25 mg/ml) was treated with a 6 fold excess of SMCC and after a period of 30 miπs dialysed overnight into phosphate buffer, pH 7.5, 0.01 M, 0.15M NaCl. The modif ied antibody was then reacted with b et a -ga 1 ac t os ida s e (1.5 fold excess) and after a period of 30 miπs applied to a column of Sepharosε 68 (1.5cm x 100cm) equilibrated in phosphate buffer. The high molecular weight fraction containing anzyme activity was pooled and stored at 4°C with 1 % BSA (bovine serum albumin). 2. l £££j_ * Whole blood or cells were taken from three individuals:- 1. K . V . B27 +ve, B7 -ve. 2. M.S. B27 -ve, B7 +ve. 3. G.T. B27 -ve, 87 -ve. 3. __.£_!£._•££_;£• 2-πitrophenyl - beta - D - galactopyraπoside ( 8 o eh r in g e r ) . Make up to 0.67mgs/ml in a phosphate buffer and sonicate to dissolve (0.1 M Phosphate, pH 7.5, 3.3 mM NgCl 2 , 0.1 M 2me). 4. £__. _.££• Round bottomed, rigid 96 well, blocked with 355 BSA/PBS for 1 hour at room temperature. 5. __,_-. - " * a. Doubling dilutions of B27-eπzyme conjugate (1 :1 for 24 wel ls in duplicate); 25 microl/wel l with dilutions in the phosphate buffer. b. Added 25 microl of aπti-HLA-B7, dilution 1 :25 with a final dilution of 1 :50. c. Added 50 microl of whole blood per well and mixed. d. Incubated on ice for 30 minutes. e. !ιl ashed with PBS (x4). f. Add 100 microl αf substrate, vortex and incubate at room temperature for 30°. g. Stop reaction with 25 microl of 1.6M a 2 C0 3 . h. Control s - + v e 25 micro l B27 conjugate + 100ml substrate; -ve 25 microl phosphate buffer + 100ml substrate. i. Plates were spun and examined colour of the supernatant - this was done by measuring the optical density but could be scored by eye. In this way the maximum score (4) was achieved by the known positive (+) and zero (-) by the known negative (-) control; the colour in the tests could therefore be accorded zero to 4. §.££--,-!-£! !-..££ _.£_-_-.£ 11 The Control ( +) read 4 whereas the negative control read (-)• The three individuals varied in their reaction. If 2 was taken as the cut-off then the tumour positive (KV) recorded a titrβ of 1 :8, a B7 individual (MS) of 1 :1 and negative (GT) of 1 :2. Thus the positive and negative reactions could be clearly distinguished by the Elisa method of testing. TABLE 3 -.J*.iϋJ.i££ i .H Ξ. l 1 2 4 8 16 32 64 128 256 500 1000 2000 Individual 1 2 3 4 5 8 7 8 9 10 11 12 KV 3 3 2 2 1 1 - Duplicatε Ula11 3 3 2 2 1 1 - - Titre ■ » 1 :8 PIS 3 1 1 Titre -- 1 :1 Duplicate lϋβll 3 1 1 GT 3 2 1 1 Titre = 1 :2 Duplicate Well 3 2 1 1 Controls +ve 4 4 4 4 -ve - DISCUSSION This specif ication describes alternative methods of ascertaining B27 status, which has several advantages over conventional m i c r o c t o t o x i c i t y . The assays are rapid, accurate, reproducibl e and are capabl e αf reliab ly differentiating HLA-B27, from ser o 1 ogica 11 y cross-reacti e specificities, especially HLA-B7, by use of a blocking step to "mask" c r o s s -r e a c t i e determinants on the B7 molecule. Given the large number of requests for B27-typiπg and the "labour intensive" nature of the method presently used, it may now be possible to significantly reduce the cost αf pro viding a B27-typing serv ice whil e simultaneously preserving valuable tissue typing reagents. It must be stressed that the results presented have examined only a very small study population and have served only to standardise the procedure. Given that such parameters as peripheral white cell count, platelet count, haematocrit and the l ev el of exprεssiαn αf Cl ass 1 antigens v ary substantially within a normal population, it is feasible that occasional errors may occur. For example, an HLA-B7 homozygote with a leukocytosis might bind nearly as much radioactivity as a B27 heterozygote with a mild leukopeπia or h r o mb o c y t α p a n i a. It is likely that the false positive rate will be very low as the only group likely to produce such a result is the B7 homozygous population and these account for only 3-4!t of Caucasions, while false negatives should be rare, according to population studies which showed that 100* of 827 + individuals were detectable with HLA-ABC- m3. It is also of interest that binding of B87.1 to HLA-B7 + cel ls was able to mask a determinant on the 87 molecule which cross-reacts with an anti-HLA-B27 monoclonal antibody. This suggests that the two epitopes in vo l v ed may be spatia l ly close to one another, resulting in steric hindrance when thε two antibodies compete for binding sites or possibly, that binding αf the aπti-37 antibody causes c α n f α r m a i o n a 1 changes in the B7 molecule, such that the avidity of the interaction between the anti-827 antiserum and its target is substantially reduced. A disadvantage of one aspect of the proposed B27-typiπg technique is that it involves the use of radi oi so t ope s. However, it should be noted that current protocol uses small amounts of a relative innocuous isotope and minimal handling of the reagent is required. Radioiodinated HLA-B27 retains its activity for up to four weeks if stored at 4 α C, and each iodination of 100 microg of purified antibody provides sufficient reagent for 2,000 tests. It would thus be necessary only to dilute the antibody once per week and to aliquot it into the requisite number of tubes, ready for the addition of blood. The iodination procedure £ £ ££ would then need to be carried αut only once per month. The alternative aspects of cα1ourimetric assay and ELISA do not suffer this defect. Reference is made to the attached drawings in which:- -Li££££ il A titration of 25 I- 1 ab e 11 e d anti-HLA-B27 monoclonal antibody on cell lysates of HLA-B27 + and 827 " peripheral blood lymphocytes. Points on each curve represent the mean of triplicate assays + standard error. cpm - counts per minute. Fi H££ 2: A titration of 125 I - 1 ab e 11 ed anti-HLA-827 monoclonal antibody on 1 eu kocyt e /p 1 a t e 1 e t suspensions derived from HLA-B27 + and 827 " subjects. Points on each curve represent the mean of triplicate assays + standard error . ii££ £ Il Direct binding assay of 125 I-labelled anti-HLA- B27 monocoloπal antibody on whole blood derived from a panel of 12 subjects who were either HLA-827" 1 " (subjects 1- 3), HLA-B7 + (subjects 4-8) or who lacked s ecificities (subjects 9-12). Subject 4 was homozygous for HLA-87. Ra ic acti ity bound represents the mean of triplicate assays + standard error. ϋ££ il Inhibition of the binding of 12S I-labelled anti- HLA-827 monoclonal antibody, to the whole blood of HLA-827 heteroz gous, HLA-B7 homozygous, and HLA-B7 heterozygous individuals and to the whole blood of individuals lacking both of these specifici ies following r e i π c ub a io n with various concentrations αf aπti-HLA-B7 mαήocolonal antibody BB7.1. Binding levels with no inhibition by BB7.1 are shown at right. _.!£_!££ §1 Percentage inhibition of the binding of 1 25 I- labelled aπti-HLA-B27 moπαcαlonal antibody to whole blood from HLA-87 homozygous and HLA-B27 heterozygous individuals, fol lowing p r e - i n cu b a t i o π for varying lengths of time with anti-HLA-87 monoclonal antibody (BB7.1 ). lilhere time -= 0, the bl ocking antibody was added immediatel y af ter the r adio 1 abe 1 led antiserum. Points on each curve represent the mean of triplicate assays + standard error.

Claims: CLAIMS: 1. A method αf testing for the HLA-B27 antigen which comprises mixing with blood cel ls or blood cel l lysates a first antibody capable of binding and blocking the HLA-B7 antigen and a second antibody capable of binding to the HLA- B27 antigen and wherein said second antibody is labelled so as to be detectabl e and thereafter detecting for the labelled said second antibody. 2. A method of testing for the HLA-B27 antigen which comprises forming a mixture containing blood cells or blood cel l lysates, a first antibody capable of binding and blocking the HLA-B7 antigen and a second antibody capable of binding to the HLA-B27 antigen, wherein the second antibody is labelled to be detectable and thereafter separating unbound said second antibody from the mixture and detecting for said second antibody bound to HLA-B27 antigen. 3. A method as claimed in claim 1 or claim 2, wherein said second antibody is radio- 1 abe 11 ed. 4. A method as claimed in claim 1 or claim 2, wherein said second antibody is labelled with an enzyme adapted to react with a substrate to produce a detectable change. ¥ 5. A compαsition for testing for the HLA-B27 antigen comprising a mixture containing a first antibody capable of binding and blocking the HLA-B7 antigen and a second antibody capable of binding to the HLA-B27 antigen and wherein said second antibody is labelled so as to be detectable. 6. A composition as claimed in claim 5, wherein said second antibody is r adio - 1 abe 11 ed. 7. A composition as claimed in claim 5 , wherein said second antibody is label led with an enzyme adapted to react with a substrate to produce a detectable change. 8. A method of testin g f or th e HLA-827 anti g en substantial ly as hereinbefore described with reference to any one αf the Examples. 9. A comp ositio n f or testing th e HL A-327 a nt i ge n substantial ly as hereiπbefαrε dεscribed with reference to any one of the Examples. 10. The articles, things, parts, elements, steps, features, methods, processes, compounds and compositions referred to or indicated in the specification and/or claims of the application individually or collectively, and any and all combinations αf any two or more of such.

Inventors: MCKENZIE IAN FARQUHAR CAMPBELL, TRAPANI JOE, MCKENZIE, IAN, FARQUHAR, CAMPBELL, TRAPANI, JOE
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1987
Main classification: G01N33
IPC codes: G01N33