Gave Precipitin Lines Research Articles

Exocellular components of Paracoccidioides brasiliensis: identification of a specific antigen

Yeast forms of Paracoccidioides brasiliensis grown in liquid medium produced exocellular components. Immunodiffusion reactions and immunoprecipitations of 131I-radiolabeled antigenic components with sera from patients having paracoccidioidomycosis (PCM) were used to monitor the isolation of specific constituents. Components having the main antigenic activity (fCon A) were isolated by exclusion from a Bio-Gel P30 column, followed by successive binding of eluted material to a Sepharose-concanavalin A column, and elution. The product contained, from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, a minor 43,000-molecular-weight (MW) component (gp43), a polydisperse high-MW glycoconjugate, and a diffusely migrating 55,000-MW glycoprotein (gp55). Other components, including a 72,000-MW glycoprotein, were irregularly expressed. The high-MW glycoconjugate complex contained, on the basis of methylation and 13C nuclear magnetic resonance data, a branched structure of mainly mannopyranosyl units. These were nonreducing ends, 6-O-, 2-O-, and 2,6-di-O-substituted, and the specific rotation of +16 degrees indicated that the glycosidic configurations of the units were alpha and beta in a ratio of ca. 1:1 (concanavalin A binding indicated that nonreducing ends or 2-O-substituted units or both of alpha-D-mannopyranose were present). A small proportion of nonreducing end units of D-galactopyranose were also present in this polysaccharide. gp55 is a glycoprotein containing a complex carbohydrate moiety with fucose, mannose, galactose, and glucose, either as terminal nonreducing units or substituted in positions indicated by methylation data. Both PCM and normal human sera precipitated the high-MW glycoconjugate from 131I-labeled fCon A preparations, whereas gp55 was unreactive with human sera. gp43 was a specific antigenic component of P. brasiliensis culture filtrates which could be isolated in a pure form by gel filtration column chromatography (Sephadex G150) or by Sepharose-patient immunoglobulin G affinity chromatography. 131I-labeled gp43 reacted equally well with 10 PCM sera and hyperimmune rabbit serum against the band E antigen of Yarzabal at a 10(-3) dilution. At the same dilution, no reaction was detected with sera from normal individuals and from patients with other mycoses. Similarly, only PCM sera and the hyperimmune anti-E serum gave precipitin lines with gp43 in the less sensitive immunodiffusion tests. gp43 consisted of three components, with pI 6.7, 6.4 and 6.2, all of which reacted with PCM serum.

Evidence for a bovine origin of the polyomavirus detected in foetal rhesus monkey kidney cells, FRhK-4 and -6.

Rabbit antisera to the stump-tailed macaque polyomavirus (STMV) which had been shown by immunoelectron microscopy and indirect immunofluorescence to react with the polyomavirus found in FRhK-4 cells (FRKV), also gave precipitin lines in counter-immunoelectrophoresis (CIE) and double diffusion in gel (GD) when reacted with FRKV. The reactions in GD showed identity with that of a rabbit antiserum to FRKV. Naturally occurring antibody to FRKV (anti-FRKV) was found by CIE in 48 per cent of 353 cattle, 1/106 pigs and 1/20 goats but not in any of 13 other species including 45 rhesus monkeys and 97 humans. Each of 9 anti-FRKV positive samples from cattle, the goat serum, but not the pig serum gave a line of identity with the rabbit antiserum to FRKV in GD against FRKV. Detection of anti-FRKV in colostrum deprived newborn calves and in commercial foetal calf sera (FBS) indicates that intra-uterine infection of cattle with FRKV may occur. FRKV adapted readily to growth in secondary calf kidney cultures and grew more rapidly and to higher titres than in the FRhK-4 cultures. We conclude that FRKV is probably another strain of STMV and that the natural hosts of these viruses are cattle and not primates. Evidence of intra-uterine infection of cattle implies that infectious FRKV may be present in some FBS and may thus have gained entry into various susceptible cell lines, particularly primate kidney.

Identification of a human neutrophil angiotension II-generating protease as cathepsin G.

A human neutrophil protease, initially termed neutral peptide-generating protease, has been shown to cleave angiotensin II directly from angiotensinogen and has been identified as leukocyte cathepsin G. When purified neutrophils were disrupted by nitrogen cavitation and fractionated by differential centrifugation, 44 and 24% of the angiotensin II-generating activity was in the lysosomal and undisrupted cell fractions, respectively. Cytochalasin B-treated human neutrophils stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine released beta-glucuronidase, lysozyme, and angiotensin II-generating protease in a dose-dependent fashion, consistent with localization of this protease to the neutrophil granule. Individually purified angiotensin II-generating protease and cathepsin G had similar proteolytic and esterolytic activity for angiotensinogen and N-benzoyl-L-tyrosine ethyl ester on a weight basis, exhibited identical mobilities by SDS-gradient polyacrylamide gel electrophoresis and pH 4.3 disc-gel electrophoresis, and gave precipitin lines of antigenic identity on Ouchterlony analysis with goat antibody to the angiotensin II-generating protease. Thus, the angiotensin II-generating protease of human neutrophils has been identified as cathepsin G on the basis of subcellular localization, substrate specificity, physicochemical characteristics, and antigenic identity.

Purification and immunological studies of glutathione reductase from rat liver evidence for an antigenic determinant at the nucleotide-binding domain of the enzyme

Glutathione reductase has been purified to homogeneity by a method which is an improvement of an earlier procedure (Carlberg, I. and Mannervik, B. (1975) J. Biol. Chem. 250, 5475–5480). The new steps in the purification scheme include affinity chromatography on 2′,5′ ADP-Sepharose 4B. Antibodies to glutathione reductase from rat liver were raised in rabbits and used for analysis of the enzyme by quantitative ‘rocket’ immunoelectrophoresis. Glutathione reductase from human erythrocytes, porcine erythrocytes, and calf-liver gave precipitin lines showing partial identity with the rat liver enzyme in Ouchterlony double diffusion experiments. Enzyme from spinach, yeast ( Saccharomyces cerevisiae), and the photosynthetic bacterium Rhodospirillum rubrum did not give precipitates with the antibodies to the enzyme from rat liver. Titration of glutathione reductase from the different sources with antibodies confirmed the cross-reactivity of the mammalian enzymes; the human enzyme giving the strongest heterologous reaction. No reaction was observed with the enzyme from spinach, yeast, and Rhodospirillum rubrum. NADPH, NADP +, and 2′,5′ ADP were found to inhibit the interaction between antibodies and glutathione reductase from rat liver and human erythrocytes. NADH, glutathione, or glutathione disulfide did not protect the enzyme from reacting with the antibodies. It is concluded that glutathione reductase has an antigenic binding site for the antibodies at the pyridine nucleotide-binding site of the enzyme molecule.

Apoproteins of high density lipoproteins in the urine of normal subjects.

Apoproteins of high density lipoproteins were detected in the urine of normal subjects after the urinary proteins were highly concentrated. By immunoelectrophoresis, all of the urinary apoproteins gave precipitin lines with similar electrophoretic mobility. This suggests that the various apoproteins are present in the same particle. The apoproteins were present only in the ultracentrifugal fraction of density greater than 1.24 g/ml. Neither apoprotein B nor apoprotein E were detected in the urine, suggesting that very low density and low density lipoproteins are not excreted in the urine of normal subjects.

Estrogen induction of plasma vitellogenin in the cockerel: studies with a phosvitin antibody.

The effects of estrogen on plasma vitellogenin have been studied in the cockerel by immunoprecipitation techniques using an antiserum prepared against the egg yolk phosphoprotein, phosvitin. The antiserum gave precipitin lines of complete identity to phosvitin and to vitellogenin which was isolated from hen plasma by DEAE-cellulose chromatography and by affinity chromatography using anti-phosvitin coupled to Sepharose 4B. The cross-reactivity of vitellogenin and phosvitin adds support to the concept that plasma vitellogenin is the precursor phosphoprotein of egg yolk phosvitin. In the three-week old cockerel, anti-phosvitin produced no detectable immunoprecipitate in the plasma. However, after a single sc injection of diethylstilbestrol (2.5 mg), plasma vitellogenin levels began to increase at 4 h and reached a maximum 20-30 h after hormone administration. The increase in plasma levels of triglyceride paralleled those of vitellogenin. These studies suggest that there is no significant time lag in the estrogenic induction of plasma vitellogenesis in the cockerel, the longer lag periods observed by other investigators may be a function of the sensitivity of the assays used for detecting vitellogenin.

Synthesis of very low density lipoproteins in the cockerel. Effects of estrogen.

The effect of estrogen on the synthesis of plasma very low density lipoproteins (VLDL) in the cockerel was studied both in vivo and in vitro. Synthesis was studied by immunoprecipitation techniques with antisera prepared against VLDL and a major VLDL protein. VLDL were isolated from the plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels by ultracentrifugal flotation at d 1.006 g/ml. After delipidation, the lipid-free proteins (apoproteins) were fractionated on Sephadex G-150 and DEAE-cellulose. Both the hen and the estrogen-treated cockerel VLDL were shown to contain an identical apoprotein with a mol wt of approximately 12,000; the apoprotein is designated fraction B. Reduction and S-carboxy-methylation of fraction B resulted in a reduction of the molecular weight by approximately one-half, indicating a dimer-monomer relationship. Antiserum prepared to the hen VLDL dimer protein gave precipitin lines of complete identity to both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density lipoproteins, and plasma; no precipitin line was formed with either hen or cockerel high density lipoproteins. After a single subcutaneous injection of diethylstilbestrol into the cockerel, plasma VLDL protein, cholesterol, and triglyceride increased, reaching a maximum 24--48 h after hormone administration. Liver slices from similarly treated animals were incubated in vitro in culture medium in the presence of [3H]lysine for 2 h. Immunoprecipitable radioactivity in VLDL increased within 2 h of diethylstilbestrol treatment and reached a maximum at 24 h; VLDL radioactivity returned to base-line levels by 72 h. At the peak of induction, newly synthesized VLDL represented 11% of the total soluble protein synthesized. When actinomycin-D (5 mg/kg) was administered simultaneously with estrogen, the induction of VLDL synthesis was totally inhibited. To determine whether the effect of estrogen on VLDL synthesis was mediated at the level of transcription, partially-purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA activity for fraction B was quantitated in a wheat germ translation system. Fraction B mRNA was found to increase from a low base-line value to a maximum 16-24 h after estrogen treatment, returning towards baseline values at 30 h. At the peak of induction, fraction B constituted 12% of the total protein synthesized. The kinetics of induction of fraction B mRNA activity in the cell-free translation system is very similar to that observed in liver slice experiments. This finding suggests that estrogen stimulates VLDL synthesis, at least partially, by enhancing the accumulation of the mRNA for one of their major apoproteins.

Effect of specific antibodies and a 2-macroglobulin on enzymatic activity of trypsin and chymotrypsin

1. (1)Both, antitrypsin antibodies and a 2-macroglobulin abolished the proteolytic activity of trypsin towards the high-molecular, denatured collagen substrate RBB-hide†, but inhibited activity towards the low-molecular synthetic BAPNA by only about 10 per cent. 2. (2) Antichymotrypsin antibodies or a 2-macroglobulin abolished chymotrypsin activity towards RBB-hide. Antichymotrypsin antibodies almost completely inhibited chymotrypsin activity towards the low-molecular synthetic substrate ATEE, but a 2-macroglobulin enhanced activity by about 55 per cent. 3. (3) A low degree of cross-reactivity between antitrypsin (antichymotrypsin) antibodies and chymotrypsin (trypsin), but not elastase was readily demonstrated by enzymatic methods, but not by immunodiffusion techniques. Cross-reactivity between antienzyme antibodies and corresponding zymogens was most pronounced, but affinity for the zymogen, determined enzymatically, was only a small percentage of that for the enzyme. 4. (4) Enzymatic activity of these complexes towards low-molecular substrates could never be completely inhibited, even when a large excess of antibodies or a 2-macroglobulin was present, whereas proteolytic activity was abolished. These observations support the view that the active center of the enzymes is not involved in complex formation with antibodies or macroglobulin, and that diminished enzymatic activity is largely due to steric hindrance by the macromolecule. 5. (5) No equilibration of enzymes between antibodies and a 2-macroglobulin was detectable by enzymatic methods when enzyme-a 2-macroglobulin complexes were incubated with antienzyme antibodies, or when enzyme-antienzyme antibody complexes were incubated with a 2-macroglobulin. However, in immunodiffusion experiments such mixtures gave precipitin lines suggesting interaction of the complexes with the second species of macromolecule.

Antisera produced to purified extracellular pectolytic enzymes from Sclerotinia fructigena.

SUMMARY: Purified isoenzymes of α-l-arabinofuranosidase (AF I, pI value 3·0) and polygalacturonase (PG III, pI 5·5; PGIV, pI 9·7) separated from culture filtrates of Sclerotinia fructigena by isoelectric focusing and gel chromatography were used to produce antisera in sheep. The serum produced to AF I was specific as judged by immunodiffusion, immunoadsorbent column chromatography and binding of fluorescein-conjugated serum to the antigen separated by polyacrylamide gel electrophoresis. Fluorescein-labelled anti-AF I serum was bound to mycelium producing this isoenzyme in liquid culture, and binding was decreased when AF production was repressed with glucose; no labelling occurred on mycelium infecting apple fruit tissue. Data for the anti-PG sera were less conclusive. The antigens gave precipitin lines only with homologous sera, but an anti-PG III immunoadsorbent column bound PG III as well as a smaller proportion of PG IV, while an anti-PG IV column bound neither antigen. Fluorescein isothiocyanate-labelled anti-PG III serum was bound to mycelium of S. fructigena grown in liquid culture and to mycelium-infected apple fruit tissue, but labelled anti-PG IV serum was not bound.

Human High Density Lipoprotein, Apolipoprotein Glutamine II: THE IMMUNOCHEMICAL AND LIPID-BINDING PROPERTIES OF APOLIPOPROTEIN GLUTAMINE II DERIVATIVES

Abstract Apolipoprotein glutamine II (apoLP-Gln-II) is one of the major protein constituents of human plasma high density lipoproteins. The protein is of known amino acid sequence and contains two identical polypeptide chains of 77 amino acids, linked by a single disulfide bridge at residue 6. We have investigated the effects of chemical modification of this disulfide linkage on the physical, immunological, and lipid-binding properties of the protein. ApoLP-Gln-II was reduced and alkylated with three reagents which are selective for the sulfhydryl linkage: (a) iodoacetic acid; (b) the spin label, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)bromoacetamide (TBA); and (c) a fluorescent label, 1-dansyl-2-maleimido ethylenediamine (DME). The three alkylated derivatives were isolated and chemically characterized. It was determined that 0.9 mole of carboxymethylcysteine, 0.8 mole of TBA, and 0.8 mole of DME were present per mole of protein monomer. ApoLP-Gln-II, Cm-apoLP-Gln-II, and TBA-apoLP-Gln-II were reconstituted with phosphatidylcholine. The protein-phospholipid complexes were isolated by ultracentrifugation in sucrose density gradients. The complexes contained, respectively, 1.23, 1.18, and 0.95 mg of phosphatidylcholine per mg of protein, representing approximately 13 moles of phosphatidylcholine per mole of protein. When ribonuclease was reduced and alkylated with iodoacetic acid, incubated with phosphatidylcholine, and subjected to density gradient centrifugation in a way identical with apoLP-Gln-II, the isolated ribonuclease contained less than 0.05 mg of phosphatidylcholine per mg of protein. The helicity of apoLP-Gln-II was reduced from approximately 46 to 37% by reduction and alkylation. This reduction could be reversed by reconstitution with phosphatidylcholine. The helicity of apoLP-Gln-II was increased from 46 to 61% and Cm-apoLP-Gln-II from 37 to 56% by reconstitution with phosphatidylcholine. Similar results were obtained with TBA-apoLP-Gln-II. By contrast, circular dichroism measurements indicated no increase in the α helical content of Cm-RNase when the reconstitution with phosphatidylcholine was performed as with apoLP-Gln-II. The electron paramagnetic resonance spectrum of TBA-apoLP-Gln-II indicated a rapidly tumbling spin label with a high degree of rotational freedom. The mobility of the spin label was not significantly altered by reconstitution with either phosphatidylcholine or with phosphatidylcholine plus cholesteryl oleate. A characteristic fluorescent spectrum of the dansyl chromophore was obtained with DME-apoLP-Gln-II. Reconstitution with phosphatidylcholine did not produce any significant change in the position or intensity of the dansyl fluorescence. ApoLP-Gln-II and each of the alkylated derivatives gave precipitin lines of complete identity with antisera prepared against apoLP-Gln-II. No immunological changes were detected after reconstitution of the apoprotein or its alkylated derivatives with phosphatidylcholine. Our findings show that, while reduction of the disulfide linkage of apoLP-Gln-II may affect the secondary structure of the protein, this effect is seen only in the absence of lipid. The integrity of the disulfide bond does not appear to be critical for binding of phosphatidylcholine. As assessed by EPR and by fluorescent labels selective for the sulfhydryl group, the environment of apoLP-Gln-II at or near residue 6 does not appear to be significantly altered by the interaction with and binding of phosphatidylcholine by the protein.

Biological techniques for studying the allergenic components of nematodes. II. The characterisation of the allergen released by Ascaris suum maintained in saline

The allergen composition of the supernatant after maintenance of adult Ascaris suum in saline, has been examined by sequential allergen challenge techniques. An allergen released into the medium resembled one allergen fraction from a whole worm extract of A. suum which had been separated previously. The maintenance fluid allergen and the whole worm extract allergen, designated allergen A, had molecular weights of the same order (13,000–15,000) and similar electrophoretic characteristics. Further evidence for the identity of the maintenance fluid allergen with allergen A is provided by means of a precipitating antiserum raised in rabbits against purified allergen A. This antiserum gave precipitin lines of complete identity between the antigenic determinants on the maintenance fluid allergen and allergen A.

Fecal SH-antigen in acute hepatitis.

The presence of fecal SH-antigen has not been reported previously. 1,2 Thus, we describe our recent findings. The techniques for the preparation of feces (2.0-3.0 gm instead of 20-30 gm of feces) and for the detection of SH-antigen by the Ouchterlony method were described elsewhere. 3 The sera of a technician (subject 1) and a nurse (subject 2) were used as anti-SH reagents. These antisera were able to detect SH-antigen in 38 and 33 sera, respectively, of a panel of 40 SH-antigen positive reagents. Fecal preparations gave precipitin lines of identity when assayed against these anti-SH antisera, as well as against various reference anti-SH antisera (Fig 1, left ). Lines of identity were also observed when the precipitin pattern of fecal material of an individual was compared with that of serum from the same individual, as well as with other SH-antigen positive sera (Fig 1, right ). Fecal samples from 55 patients

Sporulation in Bacillus subtilis 168. Comparison of alkaline phosphatase from sporulating and vegetative cells.

1. The purification of the ;vegetative' alkaline phosphatase of Bacillus subtilis 168 was simplified by ionic elution of the enzyme from intact cells. 2. The enzyme has a molecular weight of about 70000 and treatment of the enzyme with 10mm-hydrochloric acid or 6.0m-guanidine hydrochloride, beta-mercaptoethanol (0.1m) gives rise to enzymically inactive subunits. 3. The amino acid composition of the enzyme was determined. The N-terminal residue determined by the DNS chloride method is glycine. 4. The properties of this enzyme were compared with the ;sporulation' alkaline phosphatase of the same strain. 5. Although the ;sporulation' enzyme differs from the ;vegetative' enzyme in its physiology of appearance and apparent mRNA stability, an examination of properties of the enzymes revealed no differences. 6. The enzyme from both cell forms is bound to the particulate fraction of cell extracts, but can be solubilized by high concentrations of magnesium chloride; removal of the magnesium chloride, by dialysis, results in precipitation of both enzymes. Both enzymes can be removed from intact cells by ionic elution. 7. The ;vegetative' and ;sporulation' enzymes have identical pH optima, K(m) and K(i) values and electrophoretic mobilities in cellulose acetate. 8. Their half-life is 28min at 65 degrees C and their Q(10) is 1.25. 9. The molecular size determined by gel filtration on Sephadex G-100 is about 69000. 10. ;Vegetative' and ;sporulation' forms gave precipitin lines that were continuous and non-spurred when tested against antiserum prepared against the ;vegetative' enzyme. 11. The ;sporulation' alkaline phosphatase appears to be associated with stage II of sporulation and appears to be induced by something specifically concerned in sporulation and not by phosphate starvation.

Studies on inherited antigenic variation of human serum -lipoprotein by passive hemagglutination. 3. Screening for antibodies in sera from multiply transfused patients.

Sera from 199 multiply transfused patients were tested for antibodies to inherited ^‐lipoprotein antigens by the passive hemagglutination method. Hemagglutinating antibodies were found in 17 sera, whereas 6 gave precipitin lines in double diffusion tests in agar gel. In addition to well established specificities, some specificities not earlier encountered in this laboratory were observed.

Proteins Specified by Influenza Virus in Infected Cells: Analysis by Polyacrylamide Gel Electrophoresis of Antigens Not Present in the Virus Particle

SUMMARY Five polypeptides were found when purified influenza virus was analysed by electrophoresis on polyacrylamide gel. Previous work has shown that hyperimmune convalescent antibody prepared in mice and adsorbed with purified influenza virus which had been disrupted with ether still gave precipitin lines in immunodiffusion tests with extracts of cells infected with influenza virus. These presumed non-structural antigens, labelled with radioactive amino acids, were precipitated with the virus-adsorbed antibody preparation. A comparison of the precipitate and purified virus by polyacrylamide gel electrophoresis revealed three polypeptides which differed from the polypeptides of the virion. These were probably virus specific non-structural proteins.

Comparative studies of antigens from Mycoplasma mycoides and Escherichia coli.

Extracts of sonically disrupted Mycoplasma mycoides and Escherichia coli were fractionated by sucrose density gradient centrifugation. The presence of antigen in each of the fractions was determined by complement-fixation and agar-gel diffusion precipitin tests, in which cow, pig, and rabbit anti-M. mycoides sera and rabbit anti-E. coli serum were used. Fractions of M. mycoides, with a buoyant density of 1.225 or lower, fixed complement with cow and pig anti-M. mycoides sera. These fractions also formed precipitin lines with pig antiserum. Fractions in the buoyant density range of 1.10 to 1.20 fixed complement with rabbit anti-E. coli serum, but precipitin lines were not formed. All E. coli fractions fixed complement and gave precipitin lines with homologous serum. But fractions in the buoyant density range of 1.10 to 1.20 had minimal complement fixation with heterologous M. mycoides sera. The cross-reacting antigens in M. mycoides and E. coli had a buoyant density of 1.10 to 1.20; the specific antigens were isolated from M. mycoides at a buoyant density of 1.08 to 1.02.

The α1-acid glycoprotein variants of certain Caucasian patients

α 1-Acid glycoprotein was isolated in homogeneous state from sera of 44 Caucasian patients with various diseases. The resulting glycoprotein preparations were treated with neuraminidase to cleave off their sialyl residues in order to permit starch gel electrophoresis at pH 5 for the identification of the variants. The relative distribution of the three types of α 1-acid glycoprotein variant patterns was found to be very similar to that of normal Caucasian adults. This finding may be considered to be consistent with the concept of the genetically determined transmission of the α 1-acid glycoprotein variants. The α 1-acid glycoprotein preparations were analyzed by starch gel electrophoresis, immunoelectrophoresis and double diffusion techniques. They appeared homogeneous, revealed apparent electrophoretic mobilities identical to that of pooled normal α 1acid glycoprotein and gave precipitin lines which fused completely with each other and with the precipitin line of normal α 1-acid glycoprotein. The sialic acid content of the α 1-acid glycoprotein preparations was essentially identical to that of the acid glycoprotein derived from pooled normal blood. The seromucoid fraction and the α 1-acid glycoprotein levels of these patients' sera, however, showed a wide range and averaged values that were considerably above the normal.

HUMAN PANCREAS-SPECIFIC ISOANTIGENS.

PANCREAS-SPECIFIC isoantigens have been detected in rabbits1 and rhesus monkeys2 following experimental isoimmunization. It has also been reported that isoantibodies and autoantibodies occur in the sera of patients with various types of pancreatic disease3–5. Attempts were made in this laboratory to confirm the findings in man using the same techniques of agar-precipitation and indirect haemagglutination. Difficulty was experienced since normal serum or globulin often gave precipitin lines in agar. Any specific effect from the interaction of serum from patients with pancreatic disease and human pancreas extracts could therefore not be distinguished from nonspecific precipitation.