1

Human Antibodies 23 (2014/2015) 1–5 DOI 10.3233/HAB-140276 IOS Press

Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA – based method Mohsen Naseria,∗ , Mohammad Moazzenib , Ali Akbar Purfathollahb and Rahbarizadeh Fatemehc a

Department of Immunology, Paramedical School, Birjand University of Medical Sciences, Birjand, Iran Department of Immunology, School of Medicine, Tarbiat Modares University, Birjand, Iran c Department of Medical Biotechnology School of Medical Sciences, Tarbiat Modares University, Birjand, Iran b

Abstract. BACKGROUND: Dissociation constant (Kd ) is of major significance in immunoassay. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the condition of the assay used. OBJECTIVE: To employ a rapid and simple ELISA- based method for measuring dissociation constants of two Alkaline Phosphatase (ALP) specific MAbs (A1 G8 F7 and A1 G9 G3 ) established in our laboratory. METHODS: A simple and rapid method on enzyme- linked immunosorbent assay (ELISA) was developed for measuring the dissociation constant of antibody – antigen reactions. In this method the ability of increasing amounts of antigen to bind to antibody measured in the fluid phase. RESULTS: Based on the data obtained from this study, the dissociation constants of A1 G8 F7 and A1 G9 G3 MAbs were 3.8 × 10−9 and 4.3 × 10−9 M, respectively. CONCLUSIONS: A1 G8 F7 and A1 G9 G3 MAbs with reasonably high affinity are suggested for used in very immunoassay such as ELISA, immunocytochemistry and immunihistochemistry (APAAP method). Keywords: Dissociation constant, antibody affinity, ELISA, monoclonal antibody, alkaline phosphatase

1. Introduction

Monoclonal antibodies specific for Alkaline phosphatase (ALP) have many applications which Alkaline phosphatase Anti-Alkaline phosphatase (APAAP) complex is one of the most ones. This complex is applicable in many immunohistochemical and immunocytochemical techniques such as diagnosis of various kinds of leukemias, lymphomas, skin diseases, kidney dysfunctions, etc. [1–3].

∗ Corresponding author: Mohsen Naseri, Department of Immunology, Paramedical School, Birjand University of Medical Sciences, P.O.Box: 97178 53577-379 Birjand, Iran. Tel.: +98 0561 4433004; Fax: +98 0561 4440556; E-mail: [email protected].

Antibody affinity is defined as the attractive force or strength between an antigenic determinant and the antibody combining site. The strength of the reaction is dependent on numerous factors, which including fitness of the antigen with its combining site, the distance between the two reactants, the concentration of the antibody, the presence of a multivalent antibody for detection or quantification of its antigen, the sensitivity is mainly dependent on the dissociation constant (Kd ) of the antibody to its antigen [4–6]. Also antibody affinity has been shown to be an important determinant of the efficacy of several laboratory tests, such as hemagglutination, complement fixation, ELISA, precipitation, radioimmunoassay, immunocytochemistry and immunohistochemistry [7–9].

c 2014/2015 – IOS Press and the authors. All rights reserved ISSN 1093-2607/14/15/$35.00 

M. Naseri et al. / Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA

Quantification of the amount of antibody ABSORBANCE AT 492nm

Affinity of many MAbs been determined by radioimmunoassay [10] or sequential – saturation immunofluorescent method [11]. These methods require labeling of either antigen or antibody, which is sometimes difficult to control, dose not always lead to the expected signal and often results in immunological modification of the molecules [12]. To circumvent these problems, homogenous liquid phase methods in which complex formation of antigen and antibody takes place in solution have been replaced to a large extent by heterogeneous solid phase methods where one of the reactants is immobilized. ELISA based methods are possibly the most appropriate for most studies. They permit quantitative measurements of antigen or antibody concentration in solution at very low concentration, they do not require extensive purification procedures or labeling of the reactants, and they make use of simple equipment [12– 16].

3 2.5 2 1.5 1 0.5 0

2.1. Antigen The bovine intestinal ALP (type VIII A) (Sigma, USA) solution was used for the antigen [1]. 2.2. Antibody Two mouse anti-ALP MAbs designated A1 G8 F7 and A1 G9 G3 were selected from large panel of mouse Anti-ALP MAbs, developed in our lab [17]. We produced and characterized the [4] MAbs as described in detail previously both of the MAbs be longed to IgG1 subclass. The MAbs were purified by affinity chromatography using streptococcal protein G (SPG) sepharose 4B (pharmacia, Uppsala, Sweden) from ascetic fluids of Balb/c mice inoculated by the corresponding hybridoma cells. The parity of antibody preparation was monitored by SDS-PAGE according to luemmli [18]. 2.3. Quantification of the amount of antibody (19) The amount of specific antibody to use was determinated by ELISA procedure. This ELISA procedure was setup by performing a series of optimization experiments. All reactions were performed in scaled (to prevent evaporation) mincrotitre polystyrene plates (Maxisorp, Nunc, Denmark) with all reaction volumes of

0.4

0.6

0.8

1

RECIPROCAL OF THE DILUTION

Fig. 1. Plot of the absorbance versus the reciprocal of the dilution of A1 G8 F7 MAbs.

Quantification of the amount of antibody

A1 G9 G3

 R2=0.9203  R2=0.9658

3 2.5 2 1.5 1 0.5 0 0.2

2. Materials and methods

A1 G8 F7

 R2=0.9474  R2=0.6834

0.2

ABSORBANCE AT 492nm

2

0.4

0.6

0.8

1

RECIPROCAL OF THE DILUTION

Fig. 2. Plot of the absorbance versus the reciprocal of the dilution of A1 G9 G3 MAbs.

100 µl. Plate washing was performed three times after each incubation with PBS (0.15 M, PH = 7.2) containing 0.05% tween 20 (Sigma) (PBS /T) for 30 minute. The plates were freshly coated with 1 µg/ml of antigen. Antibody at a range of dilution (10−6 –10−10 M) is incubated on the first ELISA plate in triplicate at 100 µl/ well for 30 min at room temperature, the samples for each triplicate are removed from the three wells, pooled, and transferred in duplicate at 100 µl/ well to the second ELISA plate. The first ELISA plate is then after washing, appropriate dilution (or suppliers recommended) of HRP-conjugated goat anti- mouse immunoglobulin (immunoteach, Czech Rep) (diluted 1 /4000 in PBS-T) was added and the plates were incubated for 30 minute in 37◦ C before washing. OPD (or thophenylen diamine) (Sigma, USA) was used as HRP substrate and after incubation at room temperature the reaction was stopped by addition of 20% H2 SO4 . The optical densities (OD) were measured in 492 nm by ELISA reader (Lab. System, Denmark). The second ELISA plate then is processed as the first plate. The enzyme reaction is stopped after the same incubation time for both plates. The absorbance versus the reciprocal of the dilution of antibody for both of the ELSA plates were plotted (Figs 1, 2).

M. Naseri et al. / Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA

A1G8F7 KLOTZPLOT 10

Y=0.0388+1.097 R2=0.9907

8 [Abt]/[X]

The amount of antibody to be used for affinity determination must be within the linear part of the plot. It is essential that the two slopes differ by less than 10%. If they differ by more the titration must be repeated with less antigens coated on the plates or with reduced incubation time of antibody with the antigen on the plate [20].

6 4 2 0 0

50

2.4. Determination of the dissociation constant

2.5. Calculation of dissociation constant This ELISA procedures measured the ability of increasing amounts of antigen to bind to antibody in fluid phase. Unoccupied binding sites on the antibody are then detected by binding to the same antigen on solid phase. The dissociation constant (Kd ) is calculated through Klotz equation: [Abt ]1 /[X] = Kd / [Ag] + 1 In this equation: [Ab] = [Abt ](A/A0 ) [X] = [Abt ](A0 − A)A0 → Klotz equation: A0 /(A0 – A1 ) = Kd /[Agt ] + 1 1 Ab = Ab Total X = Ab – Ag A: Absorbance A : Primary t 0 Absorbance.

100 (1/Agt)

150

200

Fig. 3. Klotz plot of A1 G8 F7 MAbs (Kd = 3.8 × 10−9 M).

A1G9G3 KLOTZPLOT [Abt]/[X] or A0/(A0-A1)

The equilibrium dissociation constant (Kd ) of antigen/antibody complexes in solution were determined by ELISA technique previously described [5]. In this ELISA procedure placed 100 µl/ well of antigen at 1 µg/ ml in PBS on ELISA plate. The kept plate was upright and wrapped with Clingfilm for overnight at 4◦ C At the same time, in microcentrifuge tubes, set up 250 µl of dilutions of the antigen in PBS-T (100 µg/ml to 0.39 µg/ml) fixed with 250 µl of a pre-determined amount of specific antibody in ELISA buffer 2.4 µg/ml) and was left overnight at 4◦ C. After washing the coated ELISA plates, 200 ml/well skimmed milk powder in PBS were added for 1 h at room temperature to block the ELISA plate and were washed three times with PBS-T. Then 100 µl/well (in duplicate for each assay point) of the antibody, incubated overnight with antigen, were added and leaved on the ELISA plate for 30 min at 37◦ C After washing, HRP-labeled anti-Ig was added and the plates were incubated for 30 min at 37◦ C Following the final washing step, the reaction was revealed with O-Phenylendiamine Dihydrochloride (OPD) (Sigma) substrate. Finally, the reaction was stopped with 20% H2 SO4 and the optical density (OD) was measured by a ELISA reader at 492 nm.

3

12 10 8 6 4 2 0

Y=0.0438+1.083 R2=0.9879

0

50

100 (1/Agt)

150

200

Fig. 4. Klotz plot of A1 G9 G3 MAbs (Kd = 4.3 × 10−9 M).

3. Results 3.1. Determination of amount of primary antibody for incubation with dilution series of antigen We used a solid-phase in competitive coating procedure to detect the amount of primary antibody. Antibody at range of dilutions (5 µg/ml to 1.2 µg/ml) is incubated on the first ELISA plate in triplicate. Then the samples for each triplicate were transferred to the second ELISA plate. The wells were washed and the amount of MAb bound to the plate was determined by developing ELISA as described in the materials and methods section. plotting the absorbance (OD) versus the reciprocal of the dilution of antibody for the two ELISA plates, needed amount of antibody was determined from the liner part of the plot (1.2 µg/ml) (Figs 1, 2). 3.2. Determination of the dissociation constant A constant amount of antibody (2.4 µg/ml) was incubated in solution with various amounts of antigen until equilibrium was reached. Then, the proportion of antibody with remained unsaturated at each concentration of antigen was measured by ELISA. Dissociation constant (Kd =1 /Kd ) of the two AntiALP mouse MAbs was calculated by Klotz equation. Based upon the curves in Figs 3 and 4, the Kd for

4

M. Naseri et al. / Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA Table 1 Results for A1G8F7 clone KD Tube number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

250(µL) 250(µL) antibody ALP (mg/ml) 2.4 25 2.4 15 2.4 12.5 2.4 10 2.4 8 2.4 6.25 2.4 4 2.4 3.25 2.4 1.56 2.4 0.78 2.4 0.39 2.4 0.19 2.4 0.09 2.4 0.00 Negative Control (PBS)

Absorbance 0.123 0.133 0.145 0.135 0.156 0.157 0.227 0.215 0.327 1.032 1.231 1.321 1.351 1.353 0.08

Table 2 Results for A1G9G3 clone KD Tube number 1 2 3 4 5 6 7 8 9 10 11 12

250(µL) 250(µL) antibody ALP (mg/ml) 2.4 100 2.4 50 2.4 25 2.4 12.5 2.4 6.25 2.4 3.125 2.4 1.5 2.4 0.7 2.4 0.36 2.4 0.18 2.4 0.00 Negative Control (PBS)

Absorbance 0.312 0.354 0.364 0.450 0.621 1.354 1.504 1.578 1.555 1.585 1.590 0.302

A1 G8 F7 and A1 G9 G3 MAbs were found to be 3.8 × 10−9 M and 4.3 × 10−9 M, respectively (Tables 1 and 2).

4. Discussion Quantitative measurement of the affinity of an antibody for antigen can provide useful information about the antibody. For example, affinity measurements may be used to screen different isolates of an antibody in order to identify those that are most affective at binding antigen. Also, quantitative measurements of the capacity of an antibody to bind other compounds that are structurally related to the original immunizing antigen can help to establish the likelihood of cross-reacting the antibody to, perhaps undesirably, with other molecules that might accompany the antigen [21–24]. Also knowing the Kd of antibody allows judicious selection of antibody for a specific purpose, such as us-

ing low affinity antibody in an affinity chromatography procedure for antigen purification [25] or high- affinity antibody in immunoassay techniques [8]. Employment of low affinity antibody in an immunoaffinity column would allow purification and elution of the bound antigen with a mild elution buffer avoiding protein denaturation. The use of a high affinity MAb, on the other hand, is regarded as a decisive factor for improvement of the sensitivity of an immunoassay such as ELISA and improvement of stability and specificity of complexes such as APAAP. A large number of techniques have been described for determination of antibody affinity. The most of them include precipitation of complexes by various agents, separation of complexes based on size and electrophoretic mobility, changes in fluorescent properties and equilibrium dialysis [26]. But various enzyme-linked immunosorbent assays or radioimmunoassay methods are currently used to quantify the antibody – antigen interaction. Only indirect competition-enzyme linked immunosorbent assay can provide the real thermodynamic affinity of the antibody for its antigen. They can be applied to a varity of experimental situations, some of which are reviewed in Goldberg et al. [13,27]. The method used here is adapted from friguet.

Acknowledgments This investigation is a part of national projects which are supported by Iranian Council of scientific researches. The authors wish to thank then for their valuable supports. We also offer our thanks to Dr Mostafa Haji Molla Hosseini and Dr Mohammad Javad Rasaei because of their helps during this study.

References [1]

[2]

[3]

S.A. Bobrovnik, Determination of antibody affinity by ELISA. Theory, Journal of Biochemical and Biophysical Methods 57(3) (2003), 213–236. H. Brigitte, S. Isabella, H. Christian et al., Affinity determination of purified IgE and IgG antibodies against the major pollen allergens ph1 p5a and Bet v1a: Discrepancy between IgE and IgG binding strength, Immunology Letters 97 (2005), 81–89. K.S. Chu, S. Jin, J. Guo, M. Jue and J.J. Huang, Studies on affinity constants of hapten-specific monoclonal antibodies using an antibody-immobilized ELISA, Biotechnology Progress 11(3) (1995), 352–356.

M. Naseri et al. / Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA [4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

M.M. Eftekharian, S.M. Moazzeni, A.A. Pourfathollah and A.R. Khabiri, Production of monoclonal antibody against alkaline phosphatase, Human Antibodies 14(1–2) (2005), 17– 21. B. Friguet, A.F. Chaffotte, L. Djavadi-Ohaniance and M.E. Goldberg, Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay, Journal of Immunological Methods 77(2) (1985), 305–319. B. Friguet, L. Dyavacli and M.E. Goldberg, Immunochemical analysis of protein conformation. In: Protein Structure, A Practical Approach, Edited by Creighton T.E., Oxford University Press, Oxford, New York, 1997, pp. 323–349. M.E. Goldberg and L. Djavadi-Ohaniance, Methods for measurement of antibody/antigen affinity based on ELISA and RIA, Current Opinion in Immunology 5(2) (1993), 278–281. M.J. Jacobin, X. Santarelli, J. Laroche-Traineau and G. Clofent-Sanchez, Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for membrane anion exchange chromatography one-step purification, Human Antibodies 13(3) (2004), 69–79. C. Jacobsen and J. Steensgaard, Binding properties of monoclonal anti-IgG antibodies: Analysis of binding curves in monoclonal antibody systems, Immunology 51(3) (1984), 423–430. K. Jaskiewicz, R. Rzepko, A. Dubaniewicz, E. Jassem and K. Raczynska, Pregranulomatous phase of sarcoidosis: Immunohistochemical diagnosis, Acta Histochemica 107(6) (2006), 473–477. R.R. Kumar, A. Meenakshi and N. Sivakumar, Enzyme immunoassay of human epidermal growth factor receptor (hEGFR), Human Antibodies 10(3–4) (2001), 143–147. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227(5259) (1970), 680–685. M.P. Larvor, L. Djavadi-Ohaniance, B. Nall and M.E. Goldberg, Measurement of the dissociation rate constant of antigen/antibody complexes in solution by enzyme-linked immunosorbent assay, Journal of Immunological Methods 170(2) (1994), 167–175. E.E. Loomans, A.J. Roelen, H.S. Van Damme, H.P. Bloemers, T.C. Gribnau and W.J. Schielen, Assessment of the functional affinity constant of monoclonal antibodies using an improved enzyme-linked immunosorbent assay, Journal of Immunological Methods 184(2) (1995), 207–217. M. Naseri, S.M. Moazzeni and A.A. Pourfathollah, APAAP complex: production and usage in immunocytochemical and

5

immunohistochemical staining, Human Antibodies 16(3–4) (2007), 107–115. [16] G.R. Nimmo, A.M. Lew, C.M. Stanley and M.W. Steward, Influence of antibody affinity on the performance of different antibody assays, Journal of Immunological Methods 72(1) (1984), 177–187. [17] D.J. Phillips, T.W. Wells and C.B. Reimer, Estimation of association constants of 42 monoclonal antibodies to human IgG epitopes using a fluorescent sequential-saturation assay, Immunology Letters 17(2) (1988), 159–168. [18] M.D. Qian, J.L. Villeval, X. Xiong, M. Jandrot-Perrus, K. Nagashima, J. Tonra et al., Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library, Human Antibodies 11(3) (2002), 97–105. [19] A. Razai, C. Garcia-Rodriguez, J. Lou, I.N. Geren, C.M. Forsyth, Y. Robles et al., Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A, Journal of Molecular Biology 351(1) (2005), 158–169. [20] R.R. Chalquest, Calculation of antibody affinity in homogeneous and heterogeneous systems, Journal of Clinical Microbiology 26(12) (1988), 2561–2563. [21] N.H. Sigal and N.R. Klinman, The B-cell clonotype repertoire, Advances in Immunology 26 (1978), 255–337. [22] M.W. Steward and A.M. Lew, The importance of antibody affinity in the performance of immunoassays for antibody, Journal of Immunological Methods 78(2) (1985), 173–190. [23] M. Udayachander, A. Meenakshi, N. Sivakumar, R.R. Kumar, S.G. Shankar, C.J. Dean et al., Characterization of monoclonal antibody CIBCNSH3 generated to the human EGF receptor, Human Antibodies 9(3) (1999), 149–154. [24] P.A. Underwood, Problems and pitfalls with measurement of antibody affinity using solid phase binding in the ELISA, Journal of Immunological Methods 164(1) (1993 Aug 26), 119–130. [25] V. Van Heyningen, L. Barron, D.J. Brock, D. Crichton and S. Lawrie, Monoclonal antibodies to human alpha-foetoprotein: analysis of the behaviour of three different antibodies, Journal of Immunological Methods 50(2) (1982), 123–131. PubMed PMID: 6177793. Epub 1982/01/01. eng. [26] M.H. Van Regenmortel and A. Azimzadeh, Determination of antibody affinity, Journal of Immunoassay 21(2–3) (2000), 211–234. [27] J. Yan, S. Wang, J.B. Mi, Z.Q. Guo and W.B. Chang, Production and characterization of anti-recombinant human erythropoietin (rhEPO) monoclonal antibody, Journal of Immunoassay & Immunochemistry 25(1) (2004), 91–101.

Copyright of Human Antibodies is the property of IOS Press and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.