Parathyroid hormone reference intervals in adults using second- and third-generation assays.

Parathyroid hormone (PTH) is a routine biochemical analysis, and it varies whether a second- or third-generation assay is used. Information on the levels obtained with different assays and evidence to substantiate local assay-specific reference ranges are important to inform clinical practice. Prior to a shift from the second- to the third-generation PTH assay (Cobas 8000, Roche Diagnostics) in the Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark, a total of 59 EDTA-plasma samples were collected for method comparison (Passing-Bablok). Furthermore, 120 EDTA-plasma samples were randomly obtained from adult blood donors and used for the establishment of reference intervals using the third-generation PTH assay (Cobas 8000, Roche Diagnostics) and two second-generation assays (Atellica, Siemens Healthineers; Alinity, Abbott Laboratories). Method comparison (Cobas 8000, Roche Diagnostics) showed lower levels with the third-generation (y) as compared to the second-generation assay (x) depending on the measurement range (PTH < 10 pmol/L: y = 0.8 (95% CI: 0.7; 0.9) x + 0.3 (95% CI: 0.2; 0.5), PTH ≥ 10 pmol/L: y = 0.6 (95% CI: 0.5; 0.6) x + 3.2 (95% CI: 1.1; 5.2)). Method-specific reference intervals (2.5 and 97.5 percentiles) after the exclusion of samples (n = 31) with 25-hydroxy-vitamin D below 50 nmol/L were: 1.8-8.5 pmol/L (second-generation, Atellica, Siemens Healthineers); 2.4-10.9 pmol/L (second-generation, Alinity, Abbott Laboratories), and 1.8-7.0 pmol/L (third-generation, Cobas 8000, Roche Diagnostics). PTH levels with second- and third-generation assays are not interchangeable. Clinicians should be informed when a laboratory assay is changed, and method-specific reference ranges are needed.

Blood sampling frequency as a proxy for comorbidity indices when identifying patient samples for review of reference intervals.

OBJECTIVES: Indirect data mining methods have been proposed for review of published reference intervals (RIs), but methods for identifying patients with a low likelihood of disease are needed. Many indirect methods extract test results on patients with a low frequency blood sampling history to identify putative healthy individuals. Although it is implied there has been no attempt to validate if patients with a low frequency blood sampling history are healthy and if test results from these patients are suitable for RI review. METHODS: Danish nationwide health registers were linked with a blood sample database, recording a population of 316,337 adults over a ten-year period. Comorbidity indexes were defined from registrations of hospital diagnoses and redeemed prescriptions of drugs. Test results from patients identified as having a low disease burden were used for review of RIs from the Nordic Reference Interval Project (NORIP). RESULTS: Blood sampling frequency correlated with comorbidity Indexes and the proportion of patients without disease conditions were enriched among patients with a low number of blood samples. RIs based on test results from patients with only 1-3 blood samples per decade were for many analytes identical compared to NORIP RIs. Some analytes showed expected incongruences and gave conclusive insights into how well RIs from a more than 10 years old multi-center study (NORIP) performed on current pre-analytical and analytical methods. CONCLUSIONS: Blood sampling frequency enhance the selection of healthy individuals for reviewing reference intervals, providing a simple method solely based on laboratory data without the addition of clinical information.

Analyte stability in whole blood using experimental and datamining approaches.

The analytical stability of laboratory tests relies mostly on internal and external quality control procedures. Summarized patient data has in several studies been shown to be a good supplement for monitoring analytical stability. In our present investigation, we evaluate a datamining method for retrospective evaluation and assessment of analyte stability in whole blood. Results from the laboratory information system were used as the basis for the datamining approach. Blood tests were requested by the general practitioners and drawing of the blood sample was either at the general practitioner's or at the hospital outpatient clinics. We were able to split data into groups based on sample collection place and time to analysis. The datamining approach was compared to experiments where samples were incubated at a single temperature as well as an experiment where the temperatures were changed during incubation. To demonstrate the method, we selected three laboratory tests considered representative: potassium, phosphate, and lactate dehydrogenase. The datamining approach showed results similar to the reference experiment. Furthermore, our results show that the analytes phosphate and potassium were not stable after short storage at a lower temperature.

Hypertriglyceridemia is often under recognized as an aetiologic risk factor for acute pancreatitis: A population-based cohort study.

BACKGROUND: Severe hypertriglyceridemia (HTG) is a well-known risk factor for acute pancreatitis, but updated population-based estimates on incidence of HTG-associated pancreatitis are lacking. METHODS: We identified all individuals with severe HTG (triglyceride level >10 mmol/L [886 mg/dL]) in a population-based sample from 2008 to 2019 and linked these with Danish nationwide health-registers to identify patients with acute pancreatitis. Pancreatitis cases were subsequently confirmed by a detailed medical chart review. Crude and standardized incidence rates were estimated and studied in relation to age, gender and time-period. In addition, aetiological classification designated during index hospitalization, severity and follow-up of individuals with HTG-associated pancreatitis were studied. RESULTS: Among 2146 individuals with severe HTG during the observation period, 75 were diagnosed with acute pancreatitis (3.5%). The mean incidence rate of HTG-associated pancreatitis was 1.4 (95% CI, 1.1-1.7) per 100,000 person years for the total population, for women it was 0.7 (95% CI, 0.5-1.1) and for men 2.0 (95% CI, 1.5-2.6) per 100,000 person-years. The mean incidence rate increased from 0.7 to 1.7 per 100,000 person-years from 2008 to 2019 (ptrend = 0.01). The highest incidence rate of HTG-associated pancreatitis was observed for men in the age group 50-59 years. An elevated triglyceride level was recognized as aetiological risk factor in 35% of patients during index hospitalization. CONCLUSIONS: Only a fraction of patients with severe HTG are hospitalized for acute pancreatitis, but the incidence is increasing. In more than half of patients elevated triglycerides is not recognized as a risk factor for acute pancreatitis during index hospitalization.

Serum index rules prevent risk of analysing uncentrifuged tubes on automated biochemistry analysers.

Around 1.5% of the total clinical biochemistry tests performed in laboratories are affected by preanalytical errors. Large, automated chemistry analysers prevent errors and interference by using control systems such as spectrophotometric measurements to evaluate serum indices, i.e. haemolysis (H), icterus (I), and lipemia/turbidity (L). However, still preanalytical errors can remain undetected. Our laboratory experienced an incident caused by laboratory-induced preanalytical errors, where approximately 100 sedimented lithium heparin samples bypassed centrifugation and entered our automated analyser. Based on index results, we investigated the possibility of using turbidimetry measurement, as a mean to prevent analysis on uncentrifuged sedimented whole blood. 14078 L-indices from 8 days in August 2019 were extracted from the middleware and used to develop and evaluate stop rules. Similarly, a one-day validation dataset was identified in December 2020 and used for an independent validation. Three different types of stop rules were evaluated: (1) A single L-index result above a cut-off; (2) A sequence of an L-index results above a cut-off; (3) A simple moving average of n results above a cut-off. A stop rule using 3 consecutive L-indices of 40-60 was found to be superior. However, practical implementation in the instrument middleware on a Roche Cobas 8000 only allowed a simple moving average of 110 (n = 5). This rule was found to be able to identify and stop sedimented whole blood analysis. Additionally, the rule has minimal impact on daily routine production in the laboratory.

Fully automatic d-lactate assay using a modified commercially available method.

Intestinal infarction is the fast-evolving endpoint of impaired blood perfusion to an intestinal segment which may have fatal outcome. Early diagnosis and treatment within 6 h reduce mortality. Currently, d-lactate is a promising biomarker, however, not available in the acute clinical setting. The aim of this study is implementation of d-lactate analysis in a routine clinical setting. We used a spectrophotometric method, based on enzymatic oxidation of d-lactate by d-lactate dehydrogenase (D-LDH) coupled to the reduction of nicotinamide-adenine dinucleotide (NAD+). The amount of NADH formed in this reaction is equivalent to d-lactate. The primary concern in this method is interfering NADH formed by oxidation of l-lactate by l-lactate dehydrogenase (L-LDH). A commercially available kit for d-lactate measurement was implemented on our existing automated routine laboratory equipment including pH-inactivation of L-LDH. Our setup fulfilled clinical quality goals. We were able to measure d-lactate with an acceptable performance of the analysis and a short turn-around time. The method can be used to distinguish between the expected cut-off for intestinal ischemia around 0.3 mM and the upper reference limit of 0.05 mM. With a turnaround time of just 9 min, the analysis has potential as a readily available detection of circulating d-lactate for early diagnosis of intestinal ischemia.

A simple transformation independent method for outlier definition.

BACKGROUND: Definition and elimination of outliers is a key element for medical laboratories establishing or verifying reference intervals (RIs). Especially as inclusion of just a few outlying observations may seriously affect the determination of the reference limits. Many methods have been developed for definition of outliers. Several of these methods are developed for the normal distribution and often data require transformation before outlier elimination. METHODS: We have developed a non-parametric transformation independent outlier definition. The new method relies on drawing reproducible histograms. This is done by using defined bin sizes above and below the median. The method is compared to the method recommended by CLSI/IFCC, which uses Box-Cox transformation (BCT) and Tukey's fences for outlier definition. The comparison is done on eight simulated distributions and an indirect clinical datasets. RESULTS: The comparison on simulated distributions shows that without outliers added the recommended method in general defines fewer outliers. However, when outliers are added on one side the proposed method often produces better results. With outliers on both sides the methods are equally good. Furthermore, it is found that the presence of outliers affects the BCT, and subsequently affects the determined limits of current recommended methods. This is especially seen in skewed distributions. The proposed outlier definition reproduced current RI limits on clinical data containing outliers. CONCLUSIONS: We find our simple transformation independent outlier detection method as good as or better than the currently recommended methods.

Indirect method for validating transference of reference intervals.

BACKGROUND: Transference of reference intervals (RIs) from multicentre studies are often verified by use of a small number of samples from reference individuals or by the use of one serum sample (Serum X for NORIP RI). Despite recommended and appropriate methods, both have inconveniencies and drawbacks. Several attempts have been made to develop an indirect method, which uses historical data from the laboratory. These methods are retrospective relying on older test results. A near prospective method would be preferable for the laboratories introducing new methods or changing analytical platforms. METHODS: We performed a data mining experiment using results from our laboratory information system covering patients from a large geographic area. Request patterns for patients with assumed healthy characteristics were identified and used to extract laboratory results for calculation of new RI by an indirect method. Calculated RI and confidence intervals (CIs) were compared to transferred NORIP RI verified by NFKK Reference Serum X. RESULTS: We found that our indirect method and NFKK Reference Serum X in general produced similar results when verifying transference of RI. The method produces results for all stratifications. Only single stratifications and one analyte showed unexplained incongruences to the NORIP RI. CONCLUSIONS: Our results suggest using request patterns as a surrogate measure for good health status. This allows for a data mining method for validation of RI or validating their transference, which is likely to be applicable in countries with similar healthcare and laboratory information system.

Reference intervals for the P-Albumin bromocresol purple method.

Correct reference intervals are an important part of test results. As establishing own reference intervals is a very expensive task, the NORIP reference intervals are often transferred for use in Nordic laboratories. The NORIP reference interval on P-Albumin was here compared to current results for laboratories using the bromocresol purple (BCP) method for P-Albumin. External quality control reports were used to investigate the change in levels between the BCP and BCG methods on P-Albumin. An algorithm was built for extracting and isolating the laboratory's healthy subject population. The algorithm was used to extract test results from the laboratory information system. Parametric and non-parametric statistical methods were used to evaluate the P-Albumin test result populations. The indirect method used here clearly shows that the NORIP reference intervals for P-Albumin are not fit for the current bromocresol purple methods. The method was also used to suggest new reference interval limits.

Targeting dexamethasone to macrophages in a porcine endotoxemic model.

OBJECTIVES: Macrophages are important cells in immunity and the main producers of pro-inflammatory cytokines. The main objective was to evaluate if specific delivery of glucocorticoid to the macrophage receptor CD163 is superior to systemic glucocorticoid therapy in dampening the cytokine response to lipopolysaccharide infusion in pigs. DESIGN: Two randomized, placebo-controlled trials. SETTING: University hospital laboratory. SUBJECTS: Female farm-bred pigs (26-31 kg). DESIGN: A humanized antibody that binds to pig and human CD163 was produced, characterized, and conjugated with dexamethasone. In the first study (total n = 12), pigs were randomly assigned to four groups: 1) saline; 2) dexamethasone (1.0 mg/kg); 3) dexamethasone (0.02 mg/kg); and 4) anti-CD163-conjugated dexamethasone (0.02 mg/kg). In the second study (total n = 36), two additional groups were included in addition to the four original groups: 5) anti-CD163-conjugated dexamethasone (0.005 mg/kg); 6) unconjugated anti-CD163. Treatments were given 20 hours prior to infusion of lipopolysaccharide (1 µg × kg × h) for 5 hours. Blood samples were analyzed for cytokines, cortisol, and adrenocorticotropic hormone. RESULTS: In the saline group, lipopolysaccharide increased cytokine and plasma cortisol levels. In both studies, dexamethasone (1 mg/kg) and anti-CD163 dexamethasone (0.02 mg/kg) uniformly attenuated tumor necrosis factor-α peak levels (both p < 0.05) compared with low-dose dexamethasone (0.02 mg/kg). However, dexamethasone 1 mg/kg significantly suppressed plasma cortisol and adrenocorticotropic hormone levels compared with anti-CD163 dexamethasone (0.02 mg/kg; p < 0.05). No significant hemodynamic difference existed between groups. The anti-CD163 dexamethasone drug conjugate exhibited a fast plasma clearance, with a half-life of approximately 5-8 minutes. CONCLUSION: Targeted delivery of dexamethasone to macrophages using a humanized CD163 antibody as carrier exhibits anti-inflammatory effects comparable with 50 times higher concentrations of free dexamethasone and does not inhibit endogenous cortisol production. This antibody-drug complex showing similar affinity and specificity for human CD163 is, therefore, a promising drug candidate in this novel type of anti-inflammatory therapy.

Vitamin B12 reference intervals.

INTRODUCTION: P-Vitamin B12 is a commonly used biochemical test. Evaluation of test results and diagnosis of vitamin B12 deficiency are challenging, and the role of different biochemical methods remains unclear. METHODS: The aim of this study was to establish reference intervals for plasma vitamin B12 concentration using different immunoassays (method 1: Alinity, Abbott Laboratories; method 2: Cobas 6000, Roche Diagnostics; method 3: Atellica IM, Siemens Healthineers). Direct reference intervals were established among blood donors (n = 129) and indirect reference intervals among adult patient results of plasma vitamin B12 concentration requested by general practitioners in the North Denmark Region from 15 August to 15 October 2022 (n = 34,181). Finally, the frequency of low vitamin B12 concentration using different uniform cut-offs was evaluated. RESULTS: Direct reference intervals (2.5-97.5 percentiles) were as follows for method 1: 168-553 pmol/l; method 2: 202-641 pmol/l; and method 3: 211-551 pmol/l. Indirect reference intervals were as follows for method 1: 133-541 pmol/l; method 2: 172-619 pmol/l; and method 3: 182-162-206 pmol/l. When different cut-offs were applied to patient results, the frequency of having a vitamin B12 concentration below 250 pmol/l differed by biochemical method: 33% (method 1), 17% (method 2) and 14% (method 3). CONCLUSION: Measurement of plasma vitamin B12 concentration using different immunoassays revealed results and reference intervals that were not interchangeable. Clinical guidelines for the diagnosis of vitamin B12 deficiency should consider the biochemical methods used. FUNDING: None. TRIAL REGISTRATION: None.

Thyrotropin Receptor Antibodies in Early Pregnancy.

CONTEXT: Thyrotropin (TSH) receptor antibodies (TRAb) are important when distinguishing between Graves' and gestational hyperthyroidism, but sparse evidence exists on the recommended cutoff during pregnancy. OBJECTIVE: This work aimed to establish a method- and pregnancy-specific cutoff for TRAb, to describe the frequency of TRAb positivity in early pregnancy, and to follow up the women in the years after pregnancy. METHODS: This cohort study used the North Denmark Region Pregnancy Cohort and Danish nationwide registers of women in the North Denmark Region who had a blood sample drawn in early pregnancy, 2011 to 2015, that was stored in a biobank for assessment of thyroid function and thyroid autoantibodies. A cutoff value for TRAb was established in a reference cohort (n = 524) and used to identify TRAb-positive and TRAb-negative hyperthyroidism in early pregnancy for evaluation of frequency and follow-up. RESULTS: The method- and cohort-specific cutoff for TRAb in early pregnancy was 0.98 IU/L (95% CI, 0.96-0.99 IU/L). Among women with low TSH in early pregnancy and no known thyroid disease (n = 414), 21 women (5.1%) were TRAb positive and 393 (94.9%) were TRAb negative. Follow-up in the years following the pregnancy (median 8.1 years) revealed that 52.4% of women with TRAb-positive hyperthyroidism and 8.4% of the women with TRAb-negative hyperthyroidism were diagnosed with hyperthyroidism. CONCLUSION: This is the first study to measure TRAb in a large group of women in early pregnancy and to establish a pregnancy-specific cutoff. Results reveal that TRAb-negative hyperthyroidism is predominant in early pregnancy and rarely associated with later development of hyperthyroidism.

Cut-offs for thyroid peroxidase and thyroglobulin antibodies in early pregnancy.

Objective: Thyroid disease in women of reproductive age is mainly of autoimmune origin, and thyroid peroxidase antibodies (TPO-Ab) as well as thyroglobulin antibodies (Tg-Ab) are key markers. Adding to this, much focus in pregnancy is on euthyroid women who are thyroid antibody positive. Evidence to substantiate the cut-offs for the definition of thyroid autoantibody positivity in early pregnant women is warranted. Methods: Stored serum samples from 14,030 Danish pregnant women were used for the measurement of TPO-Ab, Tg-Ab, TSH, and free thyroxine (ADVIA Centaur XPT, Siemens Healthineers). Among all women, a reference cohort of 10,905 individuals was identified for the establishment of antibody cut-offs. Percentile cut-offs for TPO-Ab and Tg-Ab were determined using regression on order statistics (the reference cohort). The established cut-offs were then applied (the full cohort), and frequencies of early pregnancy as well as later diagnosis of hypothyroidism were evaluated. Results: The highest established cut-offs (95th, 97.5th, and 99th percentiles) were 59, 68, and 81 U/mL for TPO-Ab and 33, 41, and 52 U/mL for Tg-Ab. When the cut-offs were applied in the full cohort, 11.0, 10.2, and 9.7% were TPO-Ab positive, whereas 13.3, 12.3, and 11.2% were Tg-Ab positive. Antibody-positive women (TPO-Ab and/or Tg-Ab) had higher median TSH and were more likely to have hypothyroidism in early pregnancy and to be diagnosed with hypothyroidism during follow-up. Conclusions: This large study established and evaluated pregnancy-specific cut-offs for TPO-Ab and Tg-Ab. The findings are important regarding the classification of exposure in pregnancy and assessment of thyroid autoimmunity per se.

Classification of Thyroid Dysfunction in Pregnant Women Differs by Analytical Method and Type of Thyroid Function Test.

CONTEXT: Physiological alterations challenge the assessment of maternal thyroid function in pregnancy. It remains uncertain how the reference ranges vary by week of pregnancy, and how the classification of disease varies by analytical method and type of thyroid function test. DESIGN: Serum samples from Danish pregnant women (n = 6282) were used for the measurement of thyrotropin (TSH), total and free thyroxine (T4), total and free 3,5,3'-triiodothyronine (T3), and T-uptake using "Method A" (Cobas 8000, Roche Diagnostics). TSH and free T4 were also measured using "Method B" (ADVIA Centaur XP, Siemens Healthineers). MAIN OUTCOME MEASURES: Pregnancy week- and method-specific reference ranges were established among thyroid antibody-negative women (n = 4612). The reference ranges were used to classify maternal thyroid function, and results were compared by analytical method and type of thyroid function test. RESULTS: The reference ranges for TSH showed a gradual decrease during pregnancy weeks 4 to 14, a gradual increase was observed for total T4, total T3, and T-uptake, whereas free T4 and free T3 showed less variation. When TSH and free T4 were used, Method A classified 935 (14.9%) with abnormal thyroid function, Method B a total of 903 (14.4%), and the methods agreed on 554 individuals. When TSH and total T4 were used, 947 (15.1%) were classified with abnormal thyroid function, and classifications by either total T4 or free T4 agreed on 584 individuals. CONCLUSIONS: Even when pregnancy week- and method-specific reference ranges were established, the classification of maternal thyroid dysfunction varied considerably by analytical method and type of thyroid function test.

Pregnancy Week-Specific Reference Ranges for Thyrotropin and Free Thyroxine in the North Denmark Region Pregnancy Cohort.

BACKGROUND: Physiological changes in maternal thyroid function during pregnancy necessitate the use of pregnancy-specific reference ranges. Dynamic changes in thyrotropin (TSH) within the first trimester of pregnancy have been reported, but more evidence is needed to substantiate the findings. The objective of this study was to estimate pregnancy week-specific reference ranges for maternal TSH and free thyroxine (fT4) in early pregnancy. METHODS: The study consecutively recruited serum residues from blood samples collected as part of the prenatal screening in the North Denmark Region, 2011-2015. TSH, fT4, thyroid peroxidase antibodies (TPOAb), and thyroglobulin antibodies (TgAb) were measured using an ADVIA Centaur XPT immunoassay. The reference cohort included 10,337 pregnant women who had no thyroid disease or other autoimmune diseases and were TPOAb- and TgAb negative. The main outcome measures were lower and upper reference limits (2.5th and 97.5th percentiles) for TSH and fT4 stratified by week of pregnancy. RESULTS: Blood samples were drawn in pregnancy weeks 4-20 (median week 10), and 92% of the pregnancies ended with a live birth. TSH varied considerably in the first trimester of pregnancy, and the levels were highest in early pregnancy (weeks 4-6: 0.6-3.7 mIU/L) followed by a gradual decline to lower levels in weeks 9-11 (0.1-2.8 mIU/L) and 12-14 (0.03-2.8 mIU/L). Maternal fT4 showed less variation (weeks 4-6: 12-20 pmol/L; weeks 9-11: 13-21 pmol/L; weeks 12-14: 13-20 pmol/L). CONCLUSIONS: The results corroborate dynamic week-specific changes in maternal TSH in early pregnancy. The use of uniform lower and upper reference limits for TSH in early pregnancy may be too simple.

Spectroscopic evidence for the existence of an obligate pre-fibrillar oligomer during glucagon fibrillation.

The 29-residue peptide hormone glucagon has been used as a model system for the study of amyloid-like fibrils. Atomic force microscopy (AFM) studies have detected putative oligomeric species during this lag phase, but this has not been confirmed by any spectroscopic technique. Here we use an attached pyrene group to detect association (excimer formation) between individual glucagon molecules. Our data show that excimer formation precedes fibrillation both at different pHs and with sulfate, and support our original proposal that glucagon fibril formation is preceded by oligomer formation. We suggest that pyrene-labelling may be a useful way to monitor oligomer formation during protein fibrillation.

Functionally fused antibodies--a novel adjuvant fusion system.

Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein [Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566-73.]. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein.

Selection of large diversities of antiidiotypic antibody fragments by phage display.

Antiidiotypic antibodies (Ab2) are needed as tools for a better understanding of molecular mimicry and the immunological network, and for many potential applications in the biomedical and pharmaceutical field. Antiidiotypic antibodies mimicking carbohydrate or conformational epitopes (Ab2beta) are of considerable interest as surrogate immunogens for cancer vaccination. However, it has so far been difficult and tedious to produce Ab2s to a given antigen. Here we describe a fast and reliable technique for generating large diversities of antiidiotypic single chain antibody fragments from non-immunized phagemid libraries using phage display. Key elements are a specific elution with the original antigen followed by trypsin treatment of the eluted phages in combination with the protease sensitive helperphage KM13. This novel method was compared with various conventional selection and elution methods, including, specific elution with or without trypsin treatment, elution with glycine at pH 2.2 with or without trypsin treatment, and elution by trypsin treatment only. The results clearly show that specific elution in combination with trypsin treatment of the eluted phages is by far superior to the other conventional methods, enabling for the first time the generation of a large variety of Ab2s after only two to three rounds of selection, thereby maintaining maximum diversity. We obtained 28 to 88 antiidiotypes out of 96 tested clones after two to three rounds of selection with a diversity of 55-90 %. This was achieved for two carbohydrate (di-, and tetrasaccharides) and one conformational protein epitope using two large naïve libraries and their corresponding monoclonal Ab1. The antiidiotypic nature of the selected scFv-phages was verified by ELISA and immunocytochemistry inhibition experiments.

Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen.

The Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generated so far are IgMs of mouse origin with limited therapeutic potential. Phage-displayed scFv repertoires are an established source for recombinant antibodies; however, we were unable to identify scFvs binding to TF when applying libraries in the standard monovalent display format of phagemid systems. Here, we report on the successful selection of TF-specific antibody fragments using a multivalent scFv phagemid library format based on shortened linkers (one amino acid residue). The libraries were constructed from mice immunized with asialoglycophorin and selected using TF displayed on two different carrier molecules in combination with the proteolytically cleavable helper phage KM13. All isolated clones encoded the same framework genes and the same complementarity-determining regions. After affinity maturation only scFv with the founder sequence were selected from secondary repertoires. This indicates a very narrow sequence window for TF-specific antibodies. Investigating other linker-length formats revealed a clear inverse correlation between linker length and binding activity both as soluble proteins and displayed on phages. The highest affinity was obtained with the tetrameric format. The selected scFv was specific for TF on various carrier molecules and tumor cells and performed well in ELISA and immunohistochemistry. We postulate that scFv phagemid library formats with short linkers (i.e. multimeric scFvs) may, in general, be advantageous in selections for the generation of scFvs against carbohydrate epitopes or other epitopes associated with low intrinsic affinity per binding site), and expect that they will be superior in applications for diagnosis or therapy.