This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.
We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.
This protocol describes the development and validation of a novel and customized protein microarray assay for the detection and semi-quantification of bacterial strain and isotype-specific antibody responses to C. difficile antigens. We successfully use our C. difficile-specific microarray assay as a promising new tool for the compositional bioanalysis of specific antibody content in patient sera1,2, preparations of IVIg3, and identification of antibody specificities that correlate with poor outcomes in CDI4. We demonstrate how biobanked serum samples and commercial preparations of IVIg can be analyzed on microarray slides, allowing high-quality reproducible profiling of C. difficile pathogen-specific antibody responses in this assay.
Many healthy children and adults have detectable serum IgG and IgA antibodies to C. difficile toxins A and B5,6. These are thought to arise following transient exposure during infancy and following exposure to C. difficile in adulthood. For this reason, polyclonal IVIg has been used off-label to treat both recurrent and fulminant CDI7,8,9. However, its definitive role and mode of action remains unclear. Several studies have shown that the humoral immune response to C. difficile toxins plays a role in disease presentation and outcome. Specifically, asymptomatic patients show an increased serum anti-toxin A IgG concentration compared to patients who develop symptomatic disease10. A demonstrable association has been reported for median anti-toxin A IgG titers and 30-day all-cause mortality11. Several reports have also revealed an association with a protection against recurrence and antibody responses to toxin A, B, and several non-toxin antigens (Cwp66, Cwp84, FliC, FliD, and surface layer proteins (SLPs))12,13,14,15. These observations have spurred the development of the first passive immunotherapy drug targeting C. difficile toxin B (bezlotuxumab), which has recently been approved by the US Food and Drug Administration and the European Medicines Agency for the prevention of recurrent CDI16. Vaccination strategies using inactivated toxins or recombinant toxin fragments are also currently under development17,18,19. These new therapeutic approaches will undoubtedly stimulate the requirement for evaluating humoral immune responses to multiple antigens in large sample sizes.
Today, there is a notable lack of commercially available high-throughput assays capable of simultaneously assessing bacterial strain and isotype-specific antibody responses to C. difficile antigens. There is an unmet need to develop such assays to facilitate future research efforts and clinical applications. Protein microarrays are a method to immobilize large numbers of individually-purified proteins as a spatially organized array of spots onto a microscopic slide-based surface by using a robotic system, which can be either a contact20 or a non-contact printing tool21. The spots may represent complex mixtures such as cell lysates, antibodies, tissue homogenates, endogenous or recombinant proteins or peptides, body fluids or drugs22,23.
Protein microarray technology offers distinct advantages over standard in-house ELISA techniques, which have traditionally been used to assess anti-C. difficile antibody responses. These include an increased capacity for detecting a range of multi-isotype-specific antibodies against a more extensive panel of protein targets, reduced volume requirements for antigens, samples, and reagents, and an enhanced ability to incorporate a larger number of technical replicates, in addition to multiple internal quality control (QC) measures1. Microarrays are therefore more sensitive, accurate, and reproducible and have a greater dynamic range. These factors make microarrays a cheaper and potentially favorable alternative to ELISAs for the large-scale detection of known proteins. However, disadvantages of microarray technology result mainly from the large up-front costs associated with establishing a panel of highly purified antigens and setting up the technological platform.
Protein microarrays have been extensively used over the past two decades as a diagnostic and basic research tool in clinical applications. Specific applications include protein expression profiling, the study of enzyme-substrate relationships, biomarker screening, analysis of host-microbe interactions, and profiling antibody specificity23,24,25,26,27,28. Many new pathogen protein/antigen microarrays have been established, including malaria (Plasmodium)29, HIV-130, influenza31, severe acute respiratory syndrome (SARS)32, viral hemorrhagic fever33, herpesviruses34, and tuberculosis35.
The present protocol relates to the establishment of an easy operating C. difficile reactive antigen microarray assay, which enables accurate, precise, and specific quantification of multi-isotype and strain-specific antibody responses to C. difficile antigens in sera and polyclonal IVIg. Herein, we include representative results pertaining to an acceptable microarray assay performance when compared to ELISA as well as assay precision and reproducibility profiles. This assay could be further developed to profile other clinical samples and sets a new standard for research into the molecular basis of CDI.
1. Preparing a Microarray Plate
2. Printing Arrays with a Contact Robot
3. Storage of the Arrays
4. Array Probing
5. Scanning the Arrays
6. Data Analysis
Figure 1 illustrates a flowchart describing the major steps in the described protocol. Figure 2 shows Spearman correlation tests demonstrating significant agreement between microarray and ELISA for IgG and IgA anti-toxin A and B levels in the patient test sera. Figure 3 shows differential IgG and IgA antibody-class specific antibody responses to toxin A, toxin B, and binary toxin (pCDTb) in patients with CF without diarrhea, CDI patients with diarrhea, and in HC. Figure 4 shows C. difficile antitoxin neutralizing antibody responses in the patient sera. Figure 5 shows the immune reactivity (against C. difficile toxins and SLPs) and neutralizing effect (against C. difficile toxins) of IVIg. Figure 6 shows the immune reactivity (against C. difficile toxins and SLPs) and neutralizing effect (against C. difficile toxins) of the patient sera pre- and post-IVIg administration. Table 1 shows acceptable intra- and inter-assay coefficients of variability of microarray using test sera. Table 2 illustrates a list of immunoglobulins used in this study with relevant concentrations of purified proteins as well as optimized dilutions for sera and secondary antibodies.
Figure 1: General overview of major steps involved in the microarray protocol. The first step is the preparation of the antigens and controls. The subsequent sample dilutions are transferred to a 384-plate in readiness for printing in quadruplicates onto the aminosilane slides. Following drying and blocking of slides, the arrays are incubated with patient sera. After further washing, the biotinylated anti-human immunoglobulin (Ig) of the specified isotype is added. After the final washing and drying steps, the slides are scanned and the resultant images processed with a microarray image analysis software. Please click here to view a larger version of this figure.
Figure 2: Correlation between microarray and ELISA results. The Spearman correlation coefficient was used to assess the level of agreement between the two platforms. When comparing the microarray performance with an in-house enzyme-linked immunosorbent assay (ELISA), A. a good correlation coefficient was observed for toxin A (r = 0.7051; P <0.0001), B. and a moderately good correlation for toxin B (r = 0.5809; P <0.0001). The full ELISA protocol has been detailed elsewhere37. This figure has been reprinted from Negm et al.1 with permission. Please click here to view a larger version of this figure.
Figure 3: Isotype-specific antibody responses to Clostridium difficile toxins. This image sows the serum anti-toxin IgG and IgA responses (toxinotype 0, strain VPI 10463, ribotype 087; toxin A at 200 µg/mL, toxin B at 100 µg/mL), toxin B (C. difficile toxin B-producing strain CCUG 20309; toxin B at 90 µg/mL) and the precursor form of a B fragment of binary toxin, pCDTb (200 µg/mL), in patients with cystic fibrosis (CF) without diarrhea, patients with C. difficile infection (CDI) with diarrhea, and in healthy controls (HC). The serum dilution for IgG and IgA are 1:500 and 1:100, respectively. The differences between the groups were assessed using the Kruskal-Wallis test, followed by Dunn's post hoc test for multiple responses. Compared with the HC (n = 17) and the patients with symptomatic CDI (n = 16), the adult CF patients (n = 16) exhibited significantly higher levels of serum IgA anti-toxin A and B levels; P ≤0.05. The same pattern prevailed for IgG, except that there was no difference in the anti-toxin A IgG levels between the groups. The box and whisker plots represent the median, range, and quartiles. *** P ≤0.0001; ** P ≤0.01; * P ≤0.05. The standardized signals are normalized to the immunoglobulin standard curve. This figure has been reprinted from Monaghan et al.2 with permission. Please click here to view a larger version of this figure.
Figure 4: Neutralizing antibody efficacies to C. difficile toxins A and B in patients' sera. This figure shows the protective neutralizing antibody (NAb) responses to C. difficile toxins A and B [toxinotype 0, strain VPI 10463, ribotype 087, used at a 50% lethal dose (LD50)] in the sera (1:100 dilution; toxin A 2.5 ng/mL, toxin B 0.5 ng/mL) from the HC, the patients with CF without diarrhea, and the patients with CDI with diarrhea. The sera from CF patients exhibited significantly stronger protective anti-toxin NAb responses compared with the sera from the HC (toxins A and B) and from the patients with CDI (toxin A). The differences between the groups were assessed using the Kruskal-Wallis test, followed by Dunn's post hoc test for multiple responses. The box and whisker plots represent the median, range, and quartiles. ** P ≤0.01; * P ≤0.05. This figure has been reprinted from Monaghan et al.2 with permission. Please click here to view a larger version of this figure.
Figure 5: Immune reactivity and neutralizing effect of IVIg to C.difficile antigens. A. This image shows the reactivity of multi-isotype specific antibodies to C. difficile antigens in commercial intravenous immunoglobulin (IVIg) preparations. The heat map illustrates the levels of specific antibody isotypes (IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, and IgM) in three commercially available preparations (see Table of Materials) against seven C. difficile antigens [toxin A (200 µg/mL), toxin B (100 µg/mL), pCDTb (200 µg/mL), toxin B (CCUG 20309; 90 µg/mL), and surface layer proteins (SLPs) 001, 002, and 027 (all 200 µg/mL)] using protein microarray technology. The color code of the heat map is as follows: green (low) to red (high) signal intensity. The signal values represented on the color scale for the heat map are log2-transformed from the arbitrary fluorescence units (AFU). Please note that the AFU has more recently been superseded by the descriptor standardized signals2. The total IgG, IgG1, and IgG2 isotypes gave the highest binding reactivities against toxin A, toxin B, binary toxin (pCDTb), and toxin B (CCUG 20309). B. These plots show the IVIg neutralization efficacy against the native C. difficile whole toxins A and B. They give the percentage of the protective neutralization effect of commercial IVIg products against C. difficile toxins A and B. Each plot represents the median of triplicate experiments at 1:100 dilution. IVIg preparation 1 exhibits the lowest protective effect compared to IVIg preparations 2 and 3, particularly against toxin A. **** P ≤0.0001; * P ≤0.05 (one-way analysis of variance). This figure has been modified from Negm et al.3 with permission. Please click here to view a larger version of this figure.
Figure 6: Immune reactivity and neutralizing effect of patient's sera to C. difficile antigens. A. This figure shows a comparison of antibody reactivities against C. difficile proteins in patients' sera before and after IVIg infusion. The heat map illustrates the expression level of the isotypes (IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, and IgM) in serum samples in seven patients before and after IVIg infusion against seven C. difficile antigens [toxin A (200 µg/mL), toxin B (100 µg/mL), pCDTb (200 µg/mL), toxin B (CCUG 20309; 90 µg/mL), and SLPs 001, 002, and 027 (all 200 µg/mL)] using protein microarray technology. The color code of the heat map is as follows: green (low) to red (high) signal intensity. The signal values represented on the color scale for the heat map are log2-transformed from the AFU. Please note that the AFU has more recently been superseded by standardized signals2. There was a post-infusion enhancement of the total IgG, IgG1, IgG2, and IgG3 reactivities to toxin A, toxin B, and pCDTb. B. This image shows the IgG responses to toxin A, toxin B, and binary toxin (pCDTb), pre- and post-IVIg administration. The total IgG levels against all toxins show a significant increase following the IVIg administration (using the Wilcoxon signed-rank test). Each plot represents the median of triplicate experiments at 1:10 dilution. C. These plots show the neutralization effect against native C. difficile toxins A and B following IVIg administration. A comparison of pre- and post-infusion neutralizing antibody activities shows an enhanced protective effect after the IVIg infusions against the native C. difficile toxins A and B. Each plot represents the median of triplicate experiments at 1:10 dilution. A significant increase in the protective effect against toxins A and B was noted in the patient sera tested post-IVIg infusion (using the Wilcoxon signed-rank test). This figure has been reprinted from Negm et al.3 with permission. Please click here to view a larger version of this figure.
Reproducibility | Toxin A | Toxin B | SLP001 | SLP002 | SLP027 | Toxin B CCUG 20309 | pCDTb |
Intra-assay | 7.70% | 6.40% | 7.40% | 5.10% | 7.60% | 7% | 3.70% |
Inter-assay | 9.10% | 9.10% | 7.40% | 11.20% | 12.8 | 9.70% | 12.50% |
Table 1: Microarray intra-assay and inter-assay precision1. Microarray intra- and inter-assay variabilities were calculated using the sera of 7 patients. Identical samples were assayed on each of two slides at two independent time points. All antigens (n = 7 test and n = 2 controls) were spotted in replicates of five on each array.
Purified protein concentration | Serum dilution | Secondary antibody dilution | |
IgG | 50 μg/ml | 1/500 | 1/20000 |
IgG1 | 200 μg/ml | 1/100 | 1/5000 |
IgG2 | 200 μg/ml | 1/100 | 1/10000 |
IgG3 | 200 μg/ml | 1/100 | 1/5000 |
IgG4 | 200 μg/ml | 1/100 | 1/5000 |
IgA | 50 μg/ml | 1/100 | 1/5000 |
IgA1 | 200 μg/ml | 1/100 | 1/10000 |
IgA2 | 200 μg/ml | 1/100 | 1/5000 |
IgM | 50 μg/ml | 1/500 | 1/20000 |
Table 2: List of immunoglobulins used in this study. The Igs are shown with the relevant purified protein concentrations (µg/mL) and dilutions for serum and secondary antibodies.
In this protocol, we have shown that microarray is a suitable platform for defining humoral immune responses to C. difficile protein antigens in patient sera (Figures 3 and 6) and commercial preparations of IVIg (Figure 5). We have also demonstrated that the microarray technique performs well when compared to conventional ELISA (Figure 2) and shows excellent reproducibility, with intra- and inter-assay variabilities falling within acceptable limits of precision (Table 1).
Critical steps:
A number of critical steps must be followed when building any successful antigen microarray platform. Initially, it is extremely important to run QC experiments. In the QC experiments, different parameters should be evaluated, such as the selection of the appropriate surface chemistry, printing buffers, blocking buffers, dilutions of the serum samples and the secondary antibodies. These experiments must be performed with the aim of delivering a well-validated and reliable technique38,39.
The selection of a suitable protein immobilization process is one of the most important steps in microarray analyses, to ensure a high-quality performance of the tested aminosilane slides, as seen in this study. It is crucial to observe regular and circular spot morphology, high and specific signal intensity, and a clear background. Another factor affecting microarray performance is the printing buffer, which is equally important to achieve the desired surface chemistry to produce uniform and regular spots on the slide.
It is important to select the correct settings for printing as this will allow the optimal size and shape of a spot to be printed. For example, the humidity level should be maintained around 55% during printing, because without humidity the rate of evaporation in the microarray chamber is increased and fewer spots are printed.
Non-specific protein binding is an additional factor affecting the background and spot signals; therefore, appropriate selection of a blocking buffer could reduce any non-specific binding, leading to improved sensitivity and accuracy of the array data39.
Modifications and troubleshooting:
Antigens and serum samples must be aliquoted and stored in smaller storage tubes in the freezer until use, which helps avoid repeated multiple freeze-thaw cycles, which can deteriorate the signal strength of the array. To enhance the chances of a successful experiment, all the reagents must be prepared fresh and be filtered. Cleaning the arrayer before printing and checking the settings are required. Moreover, the slide holder must be kept clean to minimize background noise.
Limitations:
The application of protein microarrays is currently hampered by the stringent demand on surface chemistry in protein microarray fabrication. Here the great variation in the chemical and physical properties of protein molecules necessitates custom-designing unique surface chemistry for different classes of protein and antibody molecules40. Other technical challenges facing the widespread implementation of such technology include the need for expensive specialized equipment and software with allocated bench-space, as well as consideration of maintenance charges, complex data analysis, relative protein quantification, and critically access to purified antigens.
Significance of method with respect to existing/alternative methods:
Microarray technology is similar in principle to ELISA, Meso Scale Discovery, or Luminex immunoassays, but is customizable, can be scaled up to achieve statistical power, relies on only microliter quantities of precious samples, and has the ability to screen sera for multiple protein reactivities. It is therefore particularly suitable for large-scale, historical serum banks and biomarker discovery and screening.
Future applications or directions of method:
Future efforts will be directed towards optimizing the assay for other samples (cell supernatants), using the assay as a prognostic tool for patient stratification, externally validating potential biomarkers using large cohorts in a double-blind fashion, and predicting and optimizing response to immunotherapy (mAb, vaccines). In the future, the microarray technology could be used in a hospital setting as a useful diagnostic tool or to monitor a drug treatment plan over a period of time.
The authors have nothing to disclose.
This research was supported by a Hermes Fellowship to Ola Negm and Tanya Monaghan and through separate funding from the Nottingham Digestive Diseases Centre and the NIHR Nottingham Digestive Diseases Biomedical Research Centre.
BioRobotics MicroGrid II arrayer | Digilab, Malborough, MA, USA | N/A | Contact arrayer used to automated spotting of the antigens onto the slides. |
Scanner InnoScan 710. | Innopsys | N/A | A fluorescent microarray slide scanner with a red (Cy5) laser to read the reaction. |
MAPIX software version 7.2.0 | Innopsys | N/A | Measure signal intensities of the spots. |
Silicon contact pin | Parallel Synthesis Technologies | SMT-P75 | Print the samples onto the slides. |
Thermo Scientific Nalgene Desiccator | Thermo Scientific | 41102426 | To store the new and printed slides. |
384-well plate | Genetix | X7022UN | To prepare the antigens. |
Plate cover | Sigma Aldrich, UK | CLS6570-100EA | To reduce evaporation of the samples. |
Aminosilane slides | Schott, Germany | 1064875 | The slide of choice for printing the antigens. |
Slide holders | GraceBio Labs, USA | 204862 | Divide the slides into identical 16 subarrays. These holders are re-usable, removable, leak-proof wells . |
Candida albicans lysate | NIBSC | PR-BA117-S | Positive control |
Tetanus Toxoid | Athens Research and Technology | 04/150 | Positive control |
Immunoglobulin G (IgG), Normal Human Plasma | Athens research and technology | 16-16-090707 | Purified Native Human Immunoglobulin G IgG, Human Plasma. |
Immunoglobulin G1 (IgG1), Normal Human Plasma | Athens research and technology | 16-16-090707-1 | Purified Native Human Immunoglobulin G1 IgG1, Human Plasma. |
Immunoglobulin G2 (IgG2), Normal Human Plasma | Athens research and technology | 16-16-090707-2 | Purified Native Human Immunoglobulin G2 IgG2, Human Plasma. |
Immunoglobulin G3 (IgG3), Normal Human Plasma | Athens research and technology | 16-16-090707-3 | Purified Native Human Immunoglobulin G3 IgG3, Human Plasma. |
Immunoglobulin G4 (IgG4), Normal Human Plasma | Athens research and technology | 16-16-090707-4 | Purified Native Human Immunoglobulin G4 IgG4, Human Plasma. |
Immunoglobulin A (IgA), Human Plasma | Athens research and technology | 16-16-090701 | Purified Native Human Immunoglobulin A (IgA), Human Plasma. |
Immunoglobulin A1 (IgA1), Human Myeloma Plasma | Athens research and technology | 16-16-090701-1M | Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma. |
Immunoglobulin A2 (IgA2), Human Myeloma Plasma | Athens research and technology | 16-16-090701-2M | Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma. |
Immunoglobulin M (IgM), Human Plasma | Athens research and technology | 16-16-090713 | Purified Native Human Immunoglobulin M (IgM), Human Plasma. |
Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific | Vector Labs | BA-3080 | Goat anti- human IgG (γ-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins. |
Mouse Anti-Human IgG1 Hinge-BIOT | Southern Biotec | 9052-08 | Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins. |
Mouse Anti-Human IgG2 Fc-BIOT | Southern Biotec | 9060-08 | Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins. |
Mouse Anti-Human IgG3 Hinge-BIOT | Southern Biotec | 9210-08 | Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins. |
Mouse Anti-Human IgG4 pFc'-BIOT | Southern Biotec | 9190-08 | Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins. |
Anti-Human IgA, alpha chain specific, made in goat – Biotinylated | Vector Labs | BA-3030 | Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins. |
Mouse Anti-Human IgA1-BIOT | Southern Biotec | 9130-08 | Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins. |
Mouse Anti-Human IgA2-BIOT | Southern Biotec | 9140-08 | Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins. |
Mouse Anti-Human IgM-BIOT | Southern Biotec | 9020-08 | Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins. |
0.2 mm syringe filter | Thermo scientific | 723-2520 | Filter the 5% BSA. |
Bovine Serum Albumin (BSA) | Sigma Aldrich, UK | A7284 | Use 5% BSA for blocking the slides. |
Antibody diluent | Dako, UK | S3022 | To dilute the serum and the secondary antibody. |
Streptavidin Cy5 | eBioscience | SA1011 | Detection of the immune reaction. |
Purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087) | Toxins Group, Public Health England | NA | |
Purified whole C. difficile toxin B (CCUG 20309 toxin B only expressing strain) | Toxins Group, Public Health England | NA | |
Precursor form of B fragment of binary toxin, pCDTb | University of Bath | NA | Produced in E. Coli from wholly synthetic recombinant gene construct. Amino acid sequence based on published sequence from 027 ribotype (http:www.uniprot.org/uniprot/A8DS70) |
Purified native whole ribotype-specific (001, 002, 027) surface layer proteins | Dublin City University | NA | |
Vigam (IVIg preparation 1) | Nottingham University Hospitals NHS Trust | N/A | |
Privigen (IVIg preparation 2) | Nottingham University Hospitals NHS Trust | N/A | |
Intratect (IVIg preparation 3) | Nottingham University Hospitals NHS Trust | N/A |