Measurement of high affinity antibodies on antigen-immunoblots

Journal of Immunological Methods 310 (2006) 62 – 66 www.elsevier.com/locate/jim

Research paper

Measurement of high affinity antibodies on antigen-immunoblots Miles D. Chapman ⁎, Geoffrey Keir, Axel Petzold, Edward J. Thompson Department of Neuroimmunology, Institute of Neurology, Queen Square, London, WC1N 3BG, UK Received 13 May 2005; received in revised form 21 September 2005; accepted 2 December 2005 Available online 24 January 2006

Abstract We describe a semi-quantitative method for measuring the relative affinity of antigen-specific oligoclonal IgG bands separated by isoelectric focusing followed by blotting onto antigen-coated membrane and incubation with sodium thiocyanate. When the developed blot is digitised in greyscale, densitograms can be made and peak areas calculated using ImageJ freeware. By expressing peak area as a percentage of the total area under the curve we have shown that there is a statistically significant rise in percentage of peak area for a given band which persists with increasing molarities of sodium thiocyanate. © 2006 Elsevier B.V. All rights reserved. Keywords: Relative antibody affinity; IEF; Thiocyanate

1. Introduction The technique of immunoblotting was originally developed (Moyle et al., 1984) to investigate antigenspecific oligoclonal immunoglobulin G (IgG) patterns in subacute sclerosing panencephalitis (Moyle and Thompson, 1985a) and herpes simplex encephalitis (HSE) (Moyle and Thompson, 1985b) and independently by Dorries and Ter Meulen (1984). After isoelectric focusing (IEF) (Walker et al., 1983), nitrocellulose membrane impregnated with antigen is laid over a gel containing samples focused by IEF. The separated sample protein bands transfer to the membrane by passive blotting where antigen-specific antiAbbreviations: CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; HSA, human serum albumin; HSE, herpes simplex encephalitis; HSV, herpes simplex virus; IEF, isoelectric focusing; IgG, immunoglobulin G; MS, multiple sclerosis; PCR, polymerase chain reaction. ⁎ Corresponding author. Tel.: +44 20 7837 3611; fax +44 20 7837 8553. E-mail address: [email protected] (M.D. Chapman). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.12.004

bodies recognise and bind with the antigens on the membrane and can then be detected immunologically. As described, this technique does not differentiate between low and high-affinity antibodies. In 1978, Shimizu et al. used potassium thiocyanate, a small highly charged chaotrope that disrupts antigenantibody binding, to elute anti-dinitrophenyl antibodies. They found that the mean affinity of their eluted antibodies increased with the molarity of the thiocyanate solution used to elute them. Pullen et al. (1986) investigated the affinity of human anti-rubella antibodies in an enzyme-linked immunosorbent assay (ELISA) using increasing concentrations of thiocyanate to generate a relative affinity index for comparing different sera. Thiocyanate elution followed by ELISA was also employed by Luxton and Thompson (1990) to look at the distribution of antibody affinity in paired serum and cerebrospinal fluid (CSF) samples in multiple sclerosis (MS) and viral encephalitis. For thiocyanate concentrations up to 5 M, these investigators showed that CSF IgG against viral antigens, (measles, herpes, varicella, rubella, toxoplasma, mumps and cytomegalovirus), was

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of lower affinity in MS patients than in patients with viral encephalitis. Further work by Luxton et al. (1995) confirmed this observation. Antigen immunoblotting was employed by Mavra et al. (1992) as part of a strategy to look at the binding of IgG paraproteins against a range of neurotropic microorganisms. Using a qualitative ELISA, thirteen of thirty-four patients with paraprotein bands on IEF demonstrated binding against at least one antigen; after immunoblotting, however, only one patient was positive, showing that antigen immunoblotting can be a very discriminating test. Monteyne et al. (1997) conducted a study comparing the detection of intrathecal synthesis of anti-herpes simplex IgG by antigen-mediated immunoblotting and by calculation of antibody index using ELISA techniques. Twenty-seven paired samples from fifteen patients diagnosed by CSF polymerase chain reaction (PCR) were compared to MS patients acting as controls. Both methods detected the same 14/15 HSE patients, the exception being a patient with relatively benign herpes simplex virus (HSV) type 2 encephalitis, which the authors ascribed to either reduced sensitivity of the assays with regard to type 2 HSV, a low viral load or a false negative assignment. Chapman et al. (2005) used affinity-mediated antigen immunoblotting with chemiluminescent detection to investigate earlier reports (Hughes et al., 2001) that Acinetobacter was involved in an autoimmune mechanism causing MS. They drew negative conclusions but did not quantity their results as their chemiluminescent detection was not linear. The objective of this study was to test systematically whether quantitative densitometric analysis is feasible in a standardised model system using commercially available reagents.

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water and allowed to air dry. Membranes could be stored in a sealed plastic bag at – 20 °C for up to a week. 2.2. Isoelectric focusing and blotting

2. Materials and methods

IEF was carried out according to the method of Keir et al. (1990). Goat anti-human albumin antiserum was diluted 1 / 1000 with distilled water and 4 μl applied into each of 24 spaces on the application foil, in four discrete groups of six. After focusing the gel was blotted onto the albumin-coated Ultrabind, for 10 min under a 1 kg weight as described (Keir et al., 1990). The membrane was then divided into four parts, each with six samples, which were incubated with 0.9% saline containing 0, 1.25, 2.5 and 5 M sodium thiocyanate, respectively, for 30 min at room temperature. The membranes were then washed in twenty changes of running tap water to remove any residual sodium thiocyanate and reincubated in blocking solution for 30 min to ensure that any binding sites uncovered by the sodium thiocyanate incubation were blocked. Blocking solution was decanted and the membranes incubated in 25 ml polyclonal rabbit anti-goat immunoglobulins (Dako, UK), diluted 1 / 1000, overnight at 4 °C. Membranes were then washed five times for 10 min each in 0.025% Tween in 0.2% milk/0.9% saline before incubation with 25 ml of swine anti-rabbit horseradish peroxidase conjugate, diluted 1 / 2500 for 2 h. After five more washes, membranes were visualised by development with ethylaminocarbazole (50 mg of 3-amino-9-ethyl carbazole (Sigma) dissolved in 20 ml ethanol (Hayman), 100 ml working-strength acetate buffer added and 100μl 30% v/v hydrogen peroxide added just before use). All antibody dilutions were made in 0.2% dried skimmed milk in 0.9% saline and all incubations were carried out on a slow rocker.

2.1. Coating the membrane

2.3. Image manipulation

Ultrabind 450 (Gelman, UK) membranes, 10 by 20 cm, were placed in a dry polypropylene incubation chamber and thoroughly soaked with 25 ml of 0.9% saline. 400 μl of a 2.5 g/l solution of human serum albumin (HSA) (Sigma, UK) was mixed into a further 25 ml of saline and added to the membrane in the incubation chamber. The incubation chamber was sealed and placed on a slow rocker at 4 °C overnight. Next morning the solution was decanted and the membrane washed twice briefly in tap water. The membrane was then blocked in 50 ml of 0.9% saline/2% skimmed milk/ 1% Tris (pH 9.2) for at least 30 min. The blocking solution was decanted and the membrane washed in tap

Each dried blot was scanned at 100 dpi (dots per inch) on a GS-690 scanning densitometer (BioRad, UK) and saved as a TIFF file before being converted to Greyscale using Irfanview (www.irfanview.com). The image was then opened in ImageJ, Java-based image processing software, (Rasband, 1997–2005) and calibrated with the Rodbard curve-fitting procedure, using a downloaded step tablet (Epson, US). Rodbard is a four parameter general curve fit procedure proposed by David Rodbard at NIH. Within ImageJ, a box was drawn onto the first lane and used as a frame for the other lanes to obtain densitometric data from equal areas. The area of the peak was outlined (Fig. 2). The proportion of the

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M.D. Chapman et al. / Journal of Immunological Methods 310 (2006) 62–66 Table 2 p values for comparisons between NaSCN incubations in model method Molarity of NaSCN (M)

0M

0 1.25 2.5 5

NS NS b0.0001

1.25 M

2.5 M

5M

NS

NS NS

b0.0001 b0.0001 0.0002

NS b0.0001

0.0002

(Significant values are shown in bold italics, non-significant values are indicated by NS (Not Significant)).

Fig. 1. Examples of IEF trace at A. 0, B. 1.25, C. 2.5 and D. 5 M NaSCN. Representative high affinity band is indicated with arrow.

peak area as a percentage of the total area under the curve was used for statistical analysis. For simplicity, this is referred to as “peak area”. 3. Results On each blot, six replicates were incubated with four different molarities of NaSCN (Fig. 1). One band was identified as the dominant band, being of sufficiently high affinity to be the target. The area of this band was then calculated for each trace in each assay and used to generate the statistics. Some traces were not suitable for peak analysis due to problems that occurred during the running of the gel; principally, skewed traces that could not be accommodated by the software and traces with Table 1 Peak area of target band expressed as a percentage of total area under the curve and intensity relative to 0 M NaSCN Run

1 2 3 6 9 10 12 13 14 15 16 17 18 All

NaSCN (concentration)

Increase in intensity relative to 0 M NaSCN

0M

1.25 M

2.5 M

5M

1.25 M

2.5 M

5M

4.8 5.4 2.9 3.3 3.4 2.5 1.7 1.3 2.1 1.4 1.1 3.1 2.2 2.8

6.0 8.9 3.8 3.7 3.5 3.2 2.7 2.2 2.5 2.5 1.7 4.4 3.0 3.7

6.5 10.8 4.7 5.5 4.1 4.4 3.1 1.8 2.5 2.4 1.5 6.5 4.3 4.6

15.8 15.7 14.8 8.2 9.3 7.0 5.6 3.1 5.9 4.5 3.4 10.0 18.5 9.8

1.25 1.65 1.31 1.12 1.03 1.28 1.59 1.69 1.19 1.79 1.55 1.42 1.36 1.40

1.35 2.00 1.62 1.67 1.21 1.76 1.82 1.39 1.19 1.71 1.36 2.10 2.00 1.63

3.29 2.92 5.10 2.49 2.74 2.80 3.29 2.39 2.81 3.21 3.09 3.23 8.41 3.52

discrete areas of inappropriate colour development, both too heavy and too light. All observations were made on at least three measurements. Twenty blots were run, of which two were unsuitable for any analysis due to skewed traces. Of the remaining eighteen, a further five were rejected on the basis that there were not three or more measurable replicates at each molarity. It can be seen (Table 1) that there were modest increases in the peak area as a percentage at 1.25 (between 1.03 and 1.79-fold) and 2.5 M NaSCN (between 1.19 and 2.10fold), compared to 0 M NaSCN, but a larger increase at 5 M NaSCN (2.39 to 8.41-fold). On average, whilst the chosen peak represented 2.7% (range 1.1 to 5.4) of the total specific IgG at 0 M NaSCN, it was 9.4% at 5 M NaSCN (range 3.1 to 18.5). This equated to a 3.5-fold increase in intensity. The results of these thirteen complete blots were used for further analysis. Using an unbalanced 2-sided ANOVA (General Linear Model) it was possible to demonstrate a significant difference in the pixel density between the chosen peak areas, F(3, 48) = 11.81, p b 0.0001. No direct quantitative statistical difference could be demonstrated between the 0, 1.25 and 2.5 M concentrations (Table 2). Post hoc analysis was done at a categorical level by firstly defining the cut-off as the highest value within the 0 M group, then performing a trend analysis showing an increase in pixel density with increasing concentrations of NaSCN. The trend for an increase across molarity was also shown to be significant (Mantel–Haenszel chi-squared = 18.13, p b 0.001). 4. Discussion The aim of this study was to demonstrate that, in a reproducible system and with statistical significance; relative affinity could be studied using IEF to separate the clones of IgG, affinity-mediated immunoblotting used to capture them and sodium thiocyanate to probe their relative affinities. Salts of thiocyanate are a commonly used chaotrope although urea and acid can also be used. We opted to use sodium thiocyanate in

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accordance with previous work in our laboratory (Chapman et al., 2005; Luxton and Thompson, 1990). Hypothetically, increasing salinity would also increase chaotropism due to the increased ionic strength of the solution but, most commonly, thiocyanate and urea are the chaotropes of choice. Following digitisation of the blot, peak areas could be calculated and compared (Figs. 1 and 2). The peak measured was chosen on the basis that it had the strongest signal. It is possible that some or all of the other peaks remaining at 5 M NaSCN represented the same antibody to a single epitope of HSA. This could not be determined as we could not find an antiserum for determining IgG subtypes in goat serum. Were this the case, then those peak areas would need to be excluded from the calculations thus increasing the ratio of the area of the chosen peak to the background and potentially strengthening the case for our hypothesis. As it is, we could not make this assumption. Ultrabind was chosen as the solid phase because of its ability to covalently bind protein. Covalent binding was considered to reduce the possibility of sodium thiocyanate stripping albumin from the membrane as may happen with nitrocellulose, where the antigen binds non-covalently. Comparable blots on nitrocellulose showed areas that had little or no colour suggesting that albumin either failed to bind or was removed. Also, background staining with ethylaminocarbazole was higher on nitrocellulose than Ultrabind, resulting in the dried nitrocellulose being noticeably brown as opposed to the faint pink or white found with Ultrabind.

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The manufacturers of Ultrabind recommend that colorimetric detection be with chloronaphthol rather than ethylaminocarbazole. Although this does result in even lower background, our work with biological systems (unpublished observations) suggests that some sensitivity is lost. We found that Ultrabind required blocking in 2% w/v dried skimmed milk and 1% w/v Tris in 0.9% saline. When focused samples were blotted onto a milk-coated or Tris-coated membrane, faint IEF patterns could be seen at all molarities of thiocyanate (not shown). When milk and Tris were combined, no patterns could be seen suggesting that milk proteins or Tris alone were not capable of saturating the aldehyde groups of the membrane that covalently bind proteins. Although milk is probably capable of blocking the “matrix” of the membrane, as it does in nitrocellulose, Tris, with its primary amine group, is probably more efficient at blocking the aldehyde groups. We diluted anti-human albumin serum at 1 / 1000 to avoid problems with saturation whereby bands on the developed membrane were visible on the reverse of the membrane. This is thought to be lower affinity antibody that passes through the membrane quickly (Luxton and Thompson, 1989). This was not acceptable to us as we were quantifying the bands and so needed linearity. When the antiserum was diluted to 1 / 2000 we found that the traces lacked consistency. In accordance with previous work (Luxton and Thompson, 1990) we used NaSCN no stronger than 5 M; initial experiments with 10 M NaSCN resulted in nothing detectable remaining

Fig. 2. Densitograms of IEF traces from Fig. 1. A. 0, B. 1.25, C. 2.5 and D. 5 M NaSCN. Peak outlined is the peak calculated as a percentage of total area. Plots not shown at relative scale.

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on the membrane. Given that the commercial antiserum was rich in high affinity antibody to human albumin we incubated for 30 min with NaSCN. Observations in our own laboratory suggest that this should be reduced when studying biological systems as the antigen source may not be pure nor may the antibody response be as strong. However, when investigating the IgG response to an antigen, the oligoclonal response should always be of greater affinity than the polyclonal response as it has undergone affinity maturation. Double antibody detection was used for higher sensitivity and, when applied to biological systems, to allow for comparison with IEF of total IgG if required. Digitised images were saved as greyscale TIFFs because of the universal nature and ease-of-use of these formats. The only drawback was the need for access to a scanner capable of scanning high resolution images; the software used (Irfanview and ImageJ) are both available as free downloads from the internet, ImageJ being a Javabased program that is platform-independent. Peak area, rather than height, was measured (see Fig. 2) to reduce variations in the staining and hence, the background, from one trace to another. To confirm that the antihuman albumin antiserum did contain high affinity antibodies we performed a modified version of the affinity distribution (Luxton and Thompson, 1990) using 0 (saline), 1.25, 2.5, 5 and 10 M NaSCN. This showed that high affinity antibodies were present at 5 M NaSCN. The same batches of commercially available antigen and antiserum were used in all blots to remove two potential sources of variation across all experiments. By using a highly purified antigen and an antiserum rich in specific antibody we have represented the optimal form of the method; “inhouse” or commercial preparations of bacterial and viral antigen will not necessarily be as pure. For this reason, we would recommend that in such experiments preliminary studies are conducted to determine an appropriate coating capacity and sample dilutions should also be studied to determine an optimum dilution for the level of coating. Preferably, the antigen will be in excess, allowing the capture of the maximum amount of antibody, whilst maintaining a low polyclonal background. We suggest that this is a potential method for studying both clonality and affinity of sample IgG to a known antigen or source of antigen and is a practical and economical method for the semi-quantification of relative affinity of individual clones of IgG.

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