Anti-human Ferritin Research Articles

Development of a high-performance immunolatex based on “soft landing” antibody immobilization mechanism

Rabbit anti-human ferritin (anti-hFT) polyclonal immunoglobulin G (IgG) and poly(ethylene glycol) (PEG) were sequentially co-immobilized onto polystyrene submicroparticles (sMPs) to construct sMP/anti-hFT/PEG (SAP) immunolatex. Chemical immobilization of anti-hFT was performed at different pH levels to evaluate variations in antigen recognition. Basic pH disfavored conjugation of anti-hFT to sMPs, but remarkably increased its antigen recognition in comparison to that at neutral pH. We investigated this intriguing phenomenon further by assessing the kinetics of antibody binding, including the time-dependency of immobilization, antigen recognition, and orientation of bound anti-hFT. Therefore, we attributed high antigen recognition to significant electrostatic repulsion between sMPs and anti-hFT at basic pH, which predominately prevented anti-hFT access to sMPs and concurrently promoted anti-hFT orientations suitable for antigen recognition. Subsequent PEG modification maintained such anti-hFT orientation, without which antigen-accessible orientations would have decreased with time. Thus, properly oriented antibody and immediate PEGylation after antibody immobilization contributed to the formation of a high-performance SAP immunolatex.

Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein

A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10-12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called "calorimetric domain", with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single "calorimetric domain", this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains.

Ferritin Contains Less Iron (59Fe) in Cells When the Protein Pores Are Unfolded by Mutation

Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mm) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t1/2) decreased 17-fold), and (iii) decreased alpha-helix (8%). Overexpression (> 4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell 59Fe but increased chelatable iron, 16%, p < 0.01 (59Fe in the deferoxamine-containing medium), and decreased 59Fe in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (59Fe) in ferritin in vivo.

Folding of an antibody variable domain in two functional conformations in vitro: calorimetric and spectroscopic study of the anti-ferritin antibody VL domain

Understanding refolding pathways of recombinant antibody fragments is essential for efficient production of these proteins of high biomedical significance. The recombinant VL domain of mouse anti-human ferritin antibody F11 formed two distinct functional conformations obtained by refolding from bacterial inclusion bodies using two different procedures. Involvement of a dialysis step at pH 2-3 resulted in the VL-1 conformation with fluorescence of the highly conserved Trp-35 residue quenched by the spatially proximal disulfide bond. This conformation was identical to the 'native' VL domain folded in host cells and purified from the cytoplasm. In the absence of the acidic dialysis step, the VL domain adopted a previously unreported conformation, VL-2, that demonstrated prominent fluorescence due to a local structural disorder around Trp-35. Furthermore, VL-2 showed changes in secondary structure and significantly lower stability as determined by differential scanning calorimetry and denaturant-induced unfolding. While more flexible VL-2 binds human ferritin both in solution and after surface adsorption of the antibody domain, the VL-1 conformer needs an adsorption-induced conformational change to allow the access of ferritin to the antigen-binding site. Noteworthy, the two macroscopic conformations constitute kinetically trapped dimers and do not interconvert at elevated temperatures (3 weeks at 37 degrees C or 15 min at 60 degrees C), which indicates a high energetic barrier between them. As a major finding, this paper provides the first description for two stable and functional conformations of an antibody domain.

ENZYME-LINKED IMMUNOSORBENT ASSAY TO QUANTITATE SERUM FERRITIN IN BLACK AND WHITE RUFFED LEMURS (VARECIA VARIEGATA VARIEGATA)

Lemurs in captivity progressively accumulate iron deposits in a variety of organs (hemosiderosis) including duodenum, liver, and spleen throughout their lives. When excessive, the toxic effects of intracellular iron on parenchymal cells, particularly the liver, can result in clinical disease and death. The pathogenesis of excessive iron storage in these species has been attributed to dietary factors related to diets commonly fed in captivity. Tissue iron stores can be directly estimated by tissue biopsy and histologic examination, or quantitated by chemical analysis of biopsy tissue, However, expense and risk associated with anesthesia and surgery prevent routine use of tissue biopsy to assess iron status. A noninvasive means of assessing total body iron stores is needed to monitor iron stores in lemurs to determine whether dietary modification is preventing excessive iron deposition, and to monitor potential therapies such as phlebotomy or chelation. Serum ferritin concentration correlates with tissue iron stores in humans, horses, calves, dogs, cats, and pigs. Serum ferritin is considered the best serum analyte to predict total body iron stores in these species and is more reliable than serum iron or total iron binding capacity, both of which may be affected by disorders unrelated to iron adequacy or excess including hypoproteinemia, chronic infection, hemolytic anemia, hypothyroidism, renal disease, and drug administration. We have developed an enzyme-linked immunosorbent assay to measure serum ferritin in lemurs. The assay uses polyclonal rabbit anti-human ferritin antibodies in a sandwich arrangement. Ferritin isolated from liver and spleen of a black and white ruffed lemur (Varecia variegata variegata) was used as a standard. Ferritin standards were linear from 0 to 50 microg/L. Recovery of purified ferritin from lemur serum varied from 95% to 110%. The within-assay variability was 4.5%, and the assay-to-assay variability for three different samples ranged from 10% to 17%. The assay also measures serum ferritin in several other lemur species.

Folding and Stability of Chimeric Immunofusion VL-Barstar

A chimeric protein VL-barstar that comprises the VL domain of anti-human ferritin monoclonal antibody F11 and barstar, the naturally occurring inhibitor of bacterial RNase barnase, has been constructed for study of structure-function characteristics of chimeric immunoglobulin fused proteins. Such chimeric constructs may be potentially employed for development of bivalent/bispecific antibodies on the basis of the high affinity interaction between barstar and barnase (the association constant is about 10(14) M(-1)). We have developed a protocol for VL-barstar expression in E. coli and purification and refolding from inclusion bodies that yields a homogeneous and soluble form of this protein. Differential scanning calorimetry in combination with fluorescence and CD spectroscopy revealed that the VL-barstar formed well-resolved ordered secondary and compact tertiary structures. However, partial loss of tertiary interactions resulted in low stability of the recombinant protein and the lack of functional activity of the two constituent modules. These conformational features suggest that the protein might be referred to the class of native molten globules, which comprises partially unfolded conformations stabilized under physiological conditions. Since individually expressed VL domain and barstar retain completely folded conformation and stable spatial structure, the incomplete folding of the chimeric protein may be attributed to interaction between heterologous domains, which appears at the folding stage preceding formation of a system of tertiary interactions in both structural modules. The results provide evidence for non-native interactions between heterologous modules that may occur in chimeric proteins composed of taxonomically distinct fusion partners.

Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability.

Chimeric immunotoxins that combine antigen recognition domains of antibodies and cytotoxic RNases have attracted much attention in recent years as potential targeted agents for cancer immunotherapy. In an attempt to obtain a structurally minimized immunofusion for folding/stability studies, we constructed the chimeric protein VL-barnase. The chimera comprises a small cytotoxic enzyme barnase, ribonuclease from Bacillus amyloliquefaciens, fused to the C-terminus of the light chain variable domain (VL) of the anti-human ferritin monoclonal antibody F11. While the individual VL domain was expressed in Escherichia coli as insoluble protein packed into inclusion bodies, its fusion to barnase resulted in a significant ( approximately 70%) fraction of soluble protein, with only a minor insoluble fraction ( approximately 30%) packed into inclusion bodies. The in vivo solubilizing effect of barnase was also observed in vitro and suggests a chaperone-like role that barnase exerted with regard to the N-terminal VL domain. Cytoplasmic VL-barnase was analyzed for structural and functional properties. The dimeric state of the chimeric protein was demonstrated by size-exclusion chromatography, thus indicating that fusion to barnase did not abrogate the intrinsic dimerization propensity of the VL domain. Ferritin-binding affinity and specificity in terms of constants of association with isoferritins were identical for the isolated VL domain and its barnase fusion, and RNase activity remained unchanged after the fusion. Intrinsic fluorescence spectra showed a fully compact tertiary structure of the fusion protein. However, significantly altered pH stability of the fusion protein versus individual VL and barnase was shown by the pH-induced changes in both intrinsic fluorescence and binding of ANS. Together, the results indicate that VL-barnase retained the antigen-binding affinity, specificity and RNase activity pertinent to the two individual constituents, and that their fusion into a single-chain chimeric protein resulted in an altered tertiary fold and pH stability.

Independent folding and conformational changes of the barnase module in the VL-barnase immunofusion: calorimetric evidence

Although stability is critical for in vivo application of immunotoxins, a thermodynamic description of their folding/stability is still lacking. We applied differential scanning calorimetry (DSC) to RNase-based immunofusion comprising barnase, cytotoxic RNase from Bacillus amyloliquefaciens, fused to the light chain variable domain (VL) of anti-human ferritin antibody F11. By analyzing DSC curves recorded with or without preheating and addition of the barnase-stabilizing ligand guanosine 3′-monophosphate, we (i) assigned two well-resolved thermal transitions to the VL and barnase modules of VL-barnase, (ii) demonstrated independent folding of these two modules, and (iii) showed altered stability of the barnase module, which resulted from the dimeric state of VL-barnase.

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance

A direct human ferritin immunosensor was developed using anti-human ferritin monoclonal antibodies (MAbs) immobilized on the gold surface of a self-assembled surface plasmon resonance (SPR) apparatus. A kind of self-assembled monolayer (SAM) prepared by cystamine–glutaraldehyde method was applied to immobilize the MAbs. The reusability of the sensor chip adopting the SAM was found to be better than the other immobilization methods including adsorption, protein A, concanavalin A method. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1 M HCl solution. A linear relationship existed between the angle shifts (millidegrees) and the log values of ferritin concentrations in the range from 0.2 to 200 ng/ml in buffer and human serum. When used for 15 days, the angle shifts were all >95% of those on the response at the first day. A 10 M NaOH solution was used for clearing nonspecific binding in human serum. Correlation coefficient was 0.991 between this SPR method and chemiluminescent immunoassay for determination of ferritin in clinical human serum samples. The SPR sensor offers advantages of simplicity of immobilization, high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.

Liver Ferritin Subunit Ratios in Neonatal Hemochromatosis

Neonatal hemochromatosis is an enigmatic disease. Little is known about iron metabolism in this disease, including the tissue concentration of ferritin or its H and L subunit ratio. The authors report the tissue iron, ferritin, and ferritin subunit content of a child who died at 5 weeks of neonatal hemochromatosis. The child was born at 29 weeks gestation to a mother with lupus, sickle cell trait, and gestational diabetes. The child's severe liver dysfunction led to the clinical diagnosis of neonatal hemochromatosis at 1 week of age. Despite aggressive support, including red cell transfusions and chelation, the child died of an intracranial hemorrhage. Autopsy showed liver fibrosis and iron staining characteristic of neonatal hemochromatosis. Autopsy liver tissue was compared to age-matched control tissue. Soluble protein was analyzed by the Bradford method. Soluble iron (over 90% of total iron) was analyzed by the o -phenanthroline complex. Tissue ferritin and human ferritin controls (Calzyme) were analyzed by Western blotting after SDS-PAGE, identified with sheep anti-human ferritin antibodies (The BindingSite) secondary antibody-fluorescence for detection, and quantified using the Molecular Dynamics Storm 840 phosphorimager and ImageQuant software. Protein, iron, and total ferritin were similar in the normal and neonatal hemochromatosis liver tissues. Ferritin subunits, however, showed an increased H/L-subunit ratio compared to an age-matched control. This first report of a marked increase in the ferritin H/L-subunit ratio may point to an underlying mechanism of disease in this enigmatic disorder.

Liver Ferritin Subunit Ratios in Neonatal Hemochromatosis

Neonatal hemochromatosis is an enigmatic disease. Little is known about iron metabolism in this disease, including the tissue concentration of ferritin or its H and L subunit ratio. The authors report the tissue iron, ferritin, and ferritin subunit content of a child who died at 5 weeks of neonatal hemochromatosis. The child was born at 29 weeks gestation to a mother with lupus, sickle cell trait, and gestational diabetes. The child's severe liver dysfunction led to the clinical diagnosis of neonatal hemochromatosis at 1 week of age. Despite aggressive support, including red cell transfusions and chelation, the child died of an intracranial hemorrhage. Autopsy showed liver fibrosis and iron staining characteristic of neonatal hemochromatosis. Autopsy liver tissue was compared to age-matched control tissue. Soluble protein was analyzed by the Bradford method. Soluble iron (over 90% of total iron) was analyzed by the o -phenanthroline complex. Tissue ferritin and human ferritin controls (Calzyme) were analyzed by Western blotting after SDS-PAGE, identified with sheep anti-human ferritin antibodies (The BindingSite) secondary antibody-fluorescence for detection, and quantified using the Molecular Dynamics Storm 840 phosphorimager and ImageQuant software. Protein, iron, and total ferritin were similar in the normal and neonatal hemochromatosis liver tissues. Ferritin subunits, however, showed an increased H/L-subunit ratio compared to an age-matched control. This first report of a marked increase in the ferritin H/L-subunit ratio may point to an underlying mechanism of disease in this enigmatic disorder.

Partially structured state of the functional VH domain of the mouse anti-ferritin antibody F11

An antibody combining site generally involves the two variable domains, VH from the heavy and VL from the light chain. We expressed the individual VH domain of the mouse anti-human ferritin monoclonal antibody F11. The loss of affinity was not dramatic ( K a=4.0×10 7 M −1 versus 8.6×10 8 M −1 for the parent antibody) and comparable to that previously observed for other VHs. However, the functional VH domain adopted a partially structured state with a significant amount of distorted secondary and compact yet greatly destabilized tertiary structures, as demonstrated by spectroscopic and calorimetric probes. These data provide the first description for a functional antibody domain that meets all the criteria of a partially structured state.

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on a quartz crystal microbalance

A new human ferritin immunosensor was developed using anti-human ferritin antibodies (Abs) immobilized on the gold disc of a quartz crystal microbalance (QCM). Two kinds of self-assembled monolayers (SAMs) prepared by cystamine-glutaraldehyde and cystamine method were applied to immobilize anti-ferritin monoclonal antibodies (MoAbs) and polyclonal antibodies (PoAbs) on the quartz, respectively. The reusabilities of quartz crystal adopting the SAMs were found to be better than those of the other immobilization methods used. The 10 cycles of measurements could be performed on the gold surface of the same crystal regenerated with a solution of glycine·HC1. This sensor system could be continuously performed for 15 days, the relative frequency shifts (the frequency shifts measured are relative to the response at the first day) were all found to be above 95%. A linear relationship existed between the frequency shifts (Hz) and the log values of human ferritin concentrations in the range from 0.1 to 100 ng/ml in buffer and mouse serum. This human ferritin immunosensor had some advantages: high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.

Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: comparison with the completely folded V(L) domain.

Differential scanning calorimetry and spectroscopic probes were applied to study folding and stability of the single-chain Fv fragment (scFv) of the anti-human ferritin antibody F11 and its isolated variable light-chain (V(L)) domain. The scFv fragment followed variable heavy-chain domain (V(H))-linker-V(L) orientation and contained (Gly(4)Ser)(3) linker peptide. The two proteins were produced in Escherichia coli and refolded from denaturant-solubilized inclusion bodies. The isolated V(L) domain demonstrated a typical immunoglobulin fold with well-defined secondary and tertiary structure and was capable of binding human ferritin with K(a) = 1.8 x 10(7) M(-)(1), approximately (1)/(30) of the affinity of the parent F11 antibody. Involvement of this V(L) domain into the two-domain scFv fragment yielded a distorted secondary and significantly destabilized tertiary structure in which neither of the two constituent domains attained complete folding. The thermal unfolding enthalpy of scFv F11 at pH 7.0 was as low as 5. 0 J.g(-)(1) versus 16.3 J.g(-)(1) obtained for the V(L) domain and 24.7 J.g(-)(1) for the parent F11 antibody (mouse IgG2a subclass). Intrinsic fluorescence and near-ultraviolet circular dichroic (CD) spectra, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonate, confirmed partial loss of tertiary interactions in scFv. The spectroscopic and calorimetric properties of scFv F11 under physiological conditions are consistent with a model of a partially structured state with a distorted beta-sheet as a secondary structure and partial loss of tertiary interactions, which closely resembles the alternatively folded A-state adopted by an immunoglobulin at pH 2-3 [Buchner, J., Renner, M., Lilie, H., Hinz, H.-J., Jaenicke, R., Kiefhaber, T., and Rudolph, R. (1991) Biochemistry 30, 6922-6929]. However, scFv F11 demonstrated only an approximately 4-fold decrease in the antigen-binding affinity (K(a) = 1.3 x 10(8) M(-)(1)) versus the parent F11 antibody. The scFv fragment F11 provides the first description of a functional protein trapped under physiological conditions in a partially structured state. This state is either close to the native one in the antigen-binding affinity or, alternatively, initial weak binding of the antigenic epitope induces folding of scFv F11 into a more structured conformation that generates relatively high affinity.

Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra- N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the C H2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1–40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

An immunocytochemical, cytosol, and ultrastructural study of tissue ferritin in breast carcinoma

Increased levels of serum ferritin in breast carcinoma is well known, however, its exact source has been disputed. Weinstein et al found six times the ferritin concentration in malignant tissue as compared to benign tissue. The anaplastic tumors had the highest ferritin concentrations, suggesting that the major site of the increased ferritin was the malignant epithelium. We found in a previous cytosolic and electron microscopic study of tissue ferritin in breast carcinoma that the malignant epithelium is almost certainly the source of the increased ferritin.To better define and expand this study and increase our knowledge of the role of transferrin receptors and iron metabolism in breast cancer, we began an immunocytochemical study of tissue ferritin to compliment our cytosol and ultrastructural observations. This was done with a mouse antihuman ferritin monoclonal antibody. To date 92 specimens have been examined by all three techniques. Immunocytochemical tissue slides showed intense staining of the cytoplasm with immunoperoxidase, and the intensity of the cytoplasmic staining correlated with the cytosolic concentrations and the electron microscopic findings.

Potential for targeting hepatoma with anti-ferritin IgG-daunomycin immunoconjugate

Sheep anti-human ferritin IgG (AHFI) and nonspecific normal sheep IgG (NSIgG) were conjugated with Daunomycin (Dau) separately via a mediator Dextran T40. The conjugates (AHFI-Dex-Dau and NSIgG-Dex-Dau) were labelled with125I and given i. v. into Balb/c nude mice bearingan hepatocellular carcinoma (HCC). The biodistribution of the two conjugates in different organs or tissues 3 days after injection of radiolabels were measured. Selective tumor accumulation of AHFI-Dex-Dau was observed. The specific localization index was at least fourteen times higher in case of the specific AHFI-Dex-Dau when compared to another control conjugate (NSIgG-Dex-Dau) in liver tissues.In vivo treatment experiment, the results demonstrated that conjugation of Dau to IgG could greatly reduce its nonspecific chemical toxicity and significantly increase its ability to inhibit the engrafted tumor growth.

Immunohistochemical demonstration of ferritin in sweat gland and sweat gland neoplasms

Using a rabbit anti-human liver ferritin antibody, we examined the binding patterns of this reagent in normal skin and observed a unique binding pattern limited to the outermost layer of the eccrine duct. Examination of a variety of sweat gland neoplasms revealed 2 distinct patterns. One was the binding of this antibody to the outermost layer of cells in the epithelial cords of syringoma, producing a characteristic ring when seen in cross-section. This pattern of binding did not occur in other neoplasms known to be related to the eccrine duct such as dermal duct tumor and eccrine poroma. Only sparse sporadic binding occurred in other eccrine and apocrine neoplasms. A second characteristic binding pattern, not related to that noted in syringoma and diffuse in pattern, was seen in acrospiroma and in a number of adnexal carcinomas. Diffuse ferritin expression has been described in malignant neoplasms in tissues other than skin. Diffuse ferritin staining of certain sweat gland neoplasms may be an indication of biologic activity and potential aggressivity of these neoplasms.

Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with β- d-galactosidase from Escherichia coli

A highly sensitive sandwich enzyme immunoassay of human ferritin was developed. Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with β- d-galactosidase from Escherichia coli. Using the antiferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube. The range of serum ferritin levels that could be determined using 0.1 μ1 of serum was 0.23–4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 μl of serum. The coefficients of within-assay ( n = 25) and between-assay ( n = 10) variations were 5.9–8.8%. The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92 X(EIA) + 3.0 and 0.99 ( n = 78), respectively. The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of 125I-labelled anti-ferritin IgG used was not sufficiently high.

Methods for establishing a working immunoradiometric assay for serum ferritin

The two-site immunoradiometric assay for measurement of serum ferritin requires purified human ferritin and an avid high-titer antihuman ferritin antibody. Some of the antibody is radioiodinated following purification by immunoadsorption. All methods for preparation of these materials for the assay are described in minute detail. Performance of the assay itself and calculation of results are also described, and attention is drawn to several potential pitfalls. Adherence to these detailed descriptions will help to eliminate difficulties experienced by centers wishing to establish their own working serum ferritin assay.