Evaluating in-house anti-serum against B cells with flow cytometry
Suet-Feung CHIN, BSc. (Hons), Soon-Keng CHEONG, FRCP, FRCPA, Yaw-Chyn LIM, MSc, Kok-Lang MOK, BSc. (Hons) and Hussin Noor HAMIDAH, MBChB. DCP.
The applications of antibodies, be it monoclonal or polyclonal, in the diagnostic and research fields are well established. The disadvantage is the high cos1 of comrncrcjally available antibodies. In a diagnostic establishn~ent like ours which also funclions as a training ground for laboratory related personnel, it i s beneficial to be able to produce in-house reagenls. Therefore, we have undertaken this project to produce a rabbit polyclonal antibody against B lymphocytes. We found that the rabbit was a good choice hecaur;e the titre of antibody produced was high and positive reactiotls were still detected at a dilutir>n of 1:38400. The antibody showed significant positive reaction only with the lymphocyte subpop~~lation. A positive reaction was observed between the immunized rabbit serum and B lyinphocy Les but 1101 T lymphocytes. This shows that the antibody was B lyinphocytc specific.
There wa.s a positive correlation bctwcc~l thc pcrccntage of t3 lyrnphocyres labelled using Lhe commercial anti-CD L9 m o ~ ~ o c l o n a l anti body and Ihc in-housc pol yclonal antibody (n= 13, r=0.7, p=0.02). However. the percentage of cells labelled by ihe in-house polyclonal anti-B ivas lower than that by the commercial n~onoclonal anti-CDl9. The fluorescence intensity of the polyclonal antibody was lower than that of the monoclonal. In general, the performance of the in-house polyclonnl antibody can be considered as satisfactory. The rabbi1 serum was stored at - 2 W and no significarit loss of activity was detected tor over a period of 19 months.
K c j ~ ~ u r d s : Pnlyclonal antibody, B lymphocytes, phenotyping, flow cytometry.
INTRODUCTION and hone inarrow aspirates in i ~ o r n ~ a l individu- a l ~ , ~ leukaemic p;ltien~s'-~ and discascs that in- The extraordinary discrimatory power oi' anh-
vulve alterations in the lymphocyte suhpopula- bodies especially monoclonal antibodies, has
tions?-In made it an indispensable tool for both diagnostic
purposes and basic research. The main disad-
vantage is that commercial no no clonal anribod- MATERIALS AND METHODS ies are expensive. In a laboratory that also Prcpara~ion (fimrnunogrtis assumes a role in training per~onnel, dle high
cost may reducc thc hands-on oppo~.tunity of the t r a i n c c s . T h u s . an alternate s o u r c e o f inexpcnsible anti serum would be beneficial. 1n this paper. we describe our experience in pro- ducing polyclonal antibodies from rabbits for iinmunochernical study, determining its Litres and tesling its specificity with flow cylomelry.
Flow cylornetric irnrnunopheno~yping catego- rizes individual cells labelled with fluorochrome conjugated antibodies according to their size (degree of forward light scattering), internal complexity (degree of side light scattering), fluorochro~nc and flur)rcsccnce intensity.'-5 I h e availability of thc flow cyiometer has allowed rhe d e t e r m i n a t i o n o r lhe d i s ~ r i b u t i o n of immunoregulatc~r cells in the peripheral blood
Polyclonal antibodies were raised against hu- man B lymphocytes In rabbits. The cells were isolated frorn a patient with B-chronic lympho- cyric leukaemia (B-CLL) using the P'icoll- h y p a q u e g r a d i e n t dcnuity c c n t r i f u g a t i u n method." l' The isolated cells wcre washed in RPM1 containing 10% foetiil calf serum (PCS).
A cell counr was obtained with ~ h c Coulter Jr.
The cells were resuspended in freezing incdiu~n cutitaining 20% FCS and 10% Two millilitre of the above suspension (cell density of about 10- cclls) were aliyuoted into vials.
The vials are then kept in liquid nitrogen (vapour phase) ovenlight and the11 transferred to the liquid phase for l o n g term s t o r a g e , For immunisation. one vial of stored cells was used.
Addressfarcarrespondenceandrepr~ntrequests Professor Dr Chmng SoonKeng Department of Pathology. Facutyot Medrc~ne, U n ~ v e ~ l t l Kebangsaan Malaysia. Jalan Raja Muda Abdul A z ~ z , 50300 Kuala Lumpur
1 25
The cells were b r o u g h ~ rapidly lo 4OC by warming the vial in a 37°C water bath until the ice had just melted. The cell susperisior~ was washed repeatedly in RPM1 arid phosphate bufl'ered saline (PBS) by cenuifugation at 250g for l'ive minutes t o remove (he DMSO.l"he cells were finally suspended in one niill~litre of injection saline. Ihe cells were tested for vi- ability w i n g Tryphun Blue staining. To obtain a purc uuspcnsiou of B lymphocy~ch. Dyuabeads Pan-T (CD2) was used to remove residual T cclls t h a ~ mn? have been collected during the i sola~ion procedure.
'4nimul rind Irnn~ulaisatinn
The aniinal of choice was a nialc rabbit. The rabbit was bled from the marginul car vein prior
tr) immunisation to obtain nonnal rabbit serum to estnblisli a baseline value. The baseline senirn were aliquoted (0.5 ml/vial) and stored at -20uC. I~nmimizatjons intravenously with pori- fied H cell5 without adjuvant at an intcrval nl' two weeks wcrc carried out. A t e s ~ hlecd rro~rl the marginal car vein was takcn hcfore each subseqt~cnt injcc~ion to asscsh the rabbil's re- .;pon.;c. A boosier was given 1 0 the rabbit when thc titre oi'anribodies produced was high and the rabbi1 was sacrificed 7- 10 days later."
'l'hc scrum collected from Lhe rabbil (hnseliriel iminunized) was diluted in serial doubling dilu- tions from 1:150 to 1:38400. The diluted serum was thcn used to label leucocytes from a normal individual.
An appropria~e atnount of whole blood was lysed w , i ~ h arnnlonium chloride (NH,Cl) (one volume of whole blood to 20 volutne of N HCI'). I
The above suspension was incuhatcd f o r 10 minutes at ri)oni temperature (KT). The cclls were then washcd three times by centrifuging in PRS for fivc niinules at 250g. Thc pcllctccl leucocytes lvcrc resuspended in 0.5 - I .O ml oi' PRS.
A thrcc slep inununolabelling method was clnploycd i .e.:
Firs[ layer: Testfiaseline rabbit scrum (30 nlins / RT)
Second layer: Mouse anti-rabbit immuno- globulin ( 3 0 ~ n i n s / HI') (Dako, lapan)
Thirdlaycr: Goat anti-mouse ilninuno- glcih~rlin conjugated ~ i t h FITC (20 nlins / KT / dark)
(Becton Dickinson, USA)
The cells have to be washed afwr each step.".'b After the final wnsb, the cells are fixed i t ) 1 VC paraformatdehyde. A total of 15000-20000 ta- belled cells were acquired with the FACScan Keserch software in the FACScan (Becton Ilickinson, USA) within 24 hours of preparation and thc data were analysed w ~ t h the LYSYS software. The presence of the antibody w a s delected by the presence of positivc fluorescence reaction as comparccl to thc hascline serum. The cursors for the quadran( were set using the baseline control so that less lhan 2% of cells were positive. The antibody tire can be obtained by determining the highest dilution a t which a posi t ~ v e result remains demonstrable.
V i e treated cells (leucocyles from a normal individual incubated with rabbit serum) that were acquired can he grouped into four different regions according t o thcir light scauer charac- leristics (forward and sidc scuLLer) as shown in Table l . A pictorial cxample of Lhe ~ o n i n g performed is as shown in Fig. I.
Leococytes from a norinal individual was ex- tracted %ith thc ahovc procedure. T lympho- cytes were exlractcd wing CD2 coated Dyna- beads (Dynatcch, LJSA). The purity of the extracted T l y m p h r ~ y ies was wskd by incubating the cells with fluorochrome conjugated anti- C03 and anti-CD19. The stained cells were acquired and analysed. Cross-reactivity between the antiserurn and T lymphocytes was tested by tncubaung both the cells and tbe antiserum (haselineiimmunized rabbit serum) together.
Only the lympliocyte population was analysed.
TABLE l: The light scatter characteristics of leucocyles
Region Leucocy~e hubsels FSC SSC -- Region l (RI) Lymphocytes 1 L Rcginn 2 (R2) Granular 1ylnphocyte.s
& monucyter I! I
R e g m 3 (K3) Granulocytcs I3 H Region 4 (R4) Rcd blooct cells/debrrs L L FSC = forward scatter: SCC
--
side scatter.H = high amount of scattering l = intennediate anloullt of scattering L, = low amount of scattering
EVALUATING ANTI-SERUM WITH FLOW CYTOMETRY
FIG. 1: Zoning of leucocytes based on their light FIG. 2: Immunolabelling of T cells with the in-
scatter characteristics. house polyclonal antibody.
Cross-reactivity with various types of leukaemic cells
Leucocytes were extracted from thirteen indi- viduals diagnosed to have B Acute Lympho- blastic Leukaemia, Acute Myeloid Leukaemia, T-Suppressor Acute Lymphoblastic Leukaemia, Chronic Myeloid Leukaemia with blast trans- formation and B Chronic Lymphocytic Leukaemia. These cells were treated with the three step immunolabelling procedure as above.
The antiserum was also tested with cells that were used as the immunogen. Only the lym- phocyte population was studied. A comparison between the reaction with the in-house antibody
.
and the commercially available CD19 immu- noglobulin was studied.
RESULTS
The rabbit was first bled ten days after the first immunization. Both the baseline and immu- nized rabbit serum at 1 : 150 dilution showed high background when reacted with leucocytes extracted from whole blood of a normal indi- vidual. However, the immunized rabbit serum stained 12% of the cells in region 2 as compared to only 4% stained by the normal rabbit serum.
This shows the presence of an antibody that is staining the lymphocyte population. The rabbit was bled and the titre was tested again 43 days after the first immunization. Each dilution of the immunized rabbit serum is paired with a
similar dilution of the baseline sample which acts as a control. Positive reaction was repre- sented by the cells with fluorescence intensity above the cut-off point set using the baseline serum. The percentage of cells in each region which was positive when reacted with the im- munized rabbit serum at different dilutions was illustrated in Table 2. At the 1:150 - 1:600 dilution of the immunized and baseline serum, a high background was observed i.e. the cells in all four regions reacted strongly with the immu- nized rabbit serum showing fluorescence inten- sity between 10' to 103. This was higher than the fluorescence intensity (less than 102) of the cells reacted with the baseline serum. From dilutions above 1 :4800, only cells in region one and two showed definitive reaction. The rest of the cells in region three and four had low fluo- rescence intensity i.e. unlabelled cells. The immunized rabbit serum had a very high titre i.e.
more than 1:38400. The rabbit was given a booster and was sacrificed 52 days after the first immunization. We decided to use the 52 days serum at a dilution of 1:lOOOO to measure the percentage of B lymphocytes in 13 leukaemic patients diagnosed as different types of leukaemia by immunophenotyping the cell surface markers using commercial monoclonal antibody (Becton Dickinson, USA).
The immunized rabbit serum was found not to be reactive with T lymphocytes as shown in Fig. 2 but with cells extracted from a B-Chronic
Malaysian J Pathol Decwnher 1993
TABLE 2: Percentage of cells in each region labelled by the immunized rabbit serum
Percentage of positive reacting cells Dilution
Region l Region 2 Region 3 Region 4
Lymphocytic Leukaemia (Fig. 3). We tested the performance of the immunized rabbit serum by comparing the percentage of B lymphocytes labelled by the commercial monoclonal anti- CD19 and the in-house polyclonal antibody (Table 3). There was a significant positive correlation between the proportion of B cells labelled with anti-CD19 and the immunized rabbit serum (n= 13, r=0.7, ~ ~ 0 . 0 2 ) . The intensity of labelling between the anti-CD19 and the immunized rabbit serum as compared with their control are represented in Figs. 4 and 5 respec- tively.
We noted that the baseline serum had a higher percentage of non-specific staining (fluorescence
FIG. 3: Immunolabelling of cells extracted from a B-CLL patient with the in-house polyclonal antibody.
intensity range: 1.5- 10 units, mean fluorescence intensity peak: 3.1 units) when compared to the commercial monoclonal control (IgGJ (range:
1.0-2.0 units, mean: 1.5 units). We find that the intensity range for the commercial anti-CD19
TABLE 3: Comparison between the percentage of lymphoid cells from 13 leukaemic patients reacting positively with commercial anti-CD19 and with in-house antibody diluted at 1:10000
Cases CD 19# IRS* Cases CD19 IRS
Case 1-7 : Acute Lymphoblastic Leukaemia of B lineage Case 8-9 : B-Chronic Lymphocytic Leukaemia
Case 10 : Chronic Myeloid Leukaemia with Acute Lymphoblastic Leukaemia Transformation (CD 19 & CD l 0 positive)
Case l I : Chronic Myeloid Leukaemia with Acute Lymphoblastic Leukaemia Transformation (CD 10 positive)
Case 12 : T-suppressor (CD8) Acute Lymphoblastic Leukaemia Case 13 : Acute Myeloid Leukaemia
CD19# = Commercial monoclonal antibody against CD19 antigentpan B (Becton Dickinson, USA) IRS* = Immunized rabbit serum (immunogen: purified B lymphocytes)
EVALUATING AN'!'!-SERCTM WITH FLOW CYT(lMETRY
FIG. 4: Fluorescence intensity of the colllrncrcirl CD1 9 and its control (IgG,).
was between 2-250 fluorescence units (mean fluorescence intensily peak = X8.X unils) and for the in-house polyclonal was between 6-30 fluo- rescence units (mean fluoresence intensity peak
= 12.4 units). Fluorescence units are relative although with proper calibration, the fluorescence units can he directly related to the number of fluorochromes on each cell which is the amount of antibody labelling each ccll."
The outcome of any immu~iization rnay be as- cribed to variations in individual anitnal re- sponse, im~nunogen, the choice of animal. the route of injection, the adjuvant and the dosage
~ c h e d u l e . ~ ' ~ ' ' Rabbit was chosen because it was easily available, cheap, easy to care, robust in the face of quite intensive immunizations and easy to bleed.'? Particulatc or cellular antigens are usually intensely irnmunogenic with the ability to invoke a rapid response. Intravenous immunization is bclicved to producc thc most efficient immune response.15 Our sludy showed the presence of an antibody ten days alter the first immunization that labelled 12% of the cells in region 2 as compared to only 4% labelled by non-immunized rabbit serum. A working anti- w o r n was obtained in about two moulhs.
An antibody needs to be characterized by determining its specificity and titre. The speci- ficity need? to be establ islied in order to evaluate the usefulness of the a n t i h ~ d y . ' ~ In our study, the specificity of the antibody was tested by studying the anlount of positive reactir)n in different rypes of leucocytes from a normal individual and with purified T and B lymphoid cells. It was shown t h a ~ at htgher dilutions the majority of cells showing significant reaclion
were l ymphocytes. The antibody did not react with T lyrnphocytes, T Acute Lyn~phoblastic Leukaemia cells and rnyeloblasls bul with B Chronic Lymphocytic Leukaemizi cells and B Acute Lyrnphoblastic Leukaemia cells. This shows that the antibody was speciric [or B lymphclcytes. It is interesting to note two cases of' Chronic Myelnid Leukaemia with lymphoid blast transformation: onc case showed CD10 and CD19 markers and the other showed only CD10 markers. The cells from the latter did not react with our antiserum. This is consistent ~ 4 t h the immunogen we had used which wcrc puri- fied H lymphocytes from n B Chronic Lympho- cytic Leukaemia patient that' lack the CD10 antigens on then surfaces.
Thc titrc provides information on how much inatcrial WC haye available and the degree of immune response of the immunized anrrnal. The inlmunized rabbit had responded well tu yield an antibody with high titre because positive reaction was still detected at dilutions or l :38400.
Suitable dilution for d ~ e working anlistrum was found to be between 1:48OU and I:3X400. The rabbit serum was stored at -20°C and w c did not experience any significant loss activity over a p e r i d of 19 months.
Thc significant correlation between the per- ccnt;igc of B lymphocytes lahelled by the com- mcrcial anli-CD 19 monoclonal antibody and the in-house polyclonal anti-B showed that the perlbrmance of the in-house polyclonal was satisfactory. However, wc fou~ld that the per- centaye of B cells lahellcd by the in-house anti- B polyclonal antibody was less than the percent- age labelled by the commcrcial anti-CD 19. The mean fluorescencc intcnsity of the commercial an~ibody was highcr than h a t of the in-house polyclorlal antibody. This observation is con- tradictory to che principle of immunostaining
2
W-
W Total= 2564
4 r . ~ ~ T o t * 2564
% T ~ t i l l = 1BR.86 rgnPeak= 2%
Pkchan= 1.00
' bascline Mean= 17.51
L C+ 8i.78
3
FIG. 5 : Pluoresccncc intensity of the in-house polyclonal antibody and its control (bascline)
where indirect lahcllii~g allows the a t t a c h m e n t of scvcral times more fluorochromes tu each antigcn r)n ~ h c cell surface. This causeq the amplification or the fluorescerlce inte11sity.l' Conventional antiscrum will n o t only have anti- hodics t o scvcral determinants but also a fanlily of antihodies of d i r r e r e i ~ t structure and a v i d i t y which cornpetefor e a c h individual d c t c n n i ~ ~ a n l . ~ [ ' Therefore the l o w e r specificily and avidity of t h e antiseruin may contribute to the lower fluorescencu intensity although the t n e t h o d chosen was a m o r c sensitive one as cnrnpared t o t h e direct lahclling inethod of the cornlnercral no no clonal antibody. S o m e subsets uf l y mphu- cy tes (notably natural killer cells) h a v e r e c e p - tors capable of binding iminunoglohulins v i a thcir Fc receptors. This is cspccially a prohlcin when using rabbit polyclonal anti bodics; Ichs wirh goat iinmunoglobulins and even less with m o n c l o n a l antibodies. l h This niay attribute t o t h e higher non-specific binding as seen in baselinc and thc palyoloi~al sera.
ACKNOWLEDGEMENTS
This researcl1 project was fully supported by Ihe IRPA grant N o . 3-07-03-059 from the Ministry of Science, .17echnology and Environ~nent. Ma- laysia. WC wish to Express our gratirude t o the staff of thc Aniinal I-Iouse. llKM for their help i n this projec~.
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