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Administration of 5 mg of cyclophosphamide per day for 4 days into neonatal chicks resulted in suppression of natural agglutinin titers up to at least 12 weeks of age. When the administration of cyclophosphamide was delayed until 7 days post-hatching suppression was found up to 8 weeks but titers rose to normal by 10 weeks after initial injections. Injections of cyclophosphamide after 21 days of age did not suppress natural agglutinin titers unless the drug was administered daily for 2 or more weeks.

An immunoabsorbent column was made with antibody to trypsinized human C5. This column removed the chemotactic activity from zymosan-activated serum as well as from C5a des-arg-enriched fractions. Anti-trypsinized C5-absorbed human serum was substituted for unabsorbed human serum in the chemotaxis under agarose system. This resulted in significantly reduced random neutrophil migration with a negligible effect on C5a des-arg or FMLP-directed migration. The results indicate that much of the random migration observed in the chemotaxis under agarose system is due to C5-derived peptides present in normal human serum.

Human C5a des arg was isolated from complement-activated serum by immunoadsorption followed by Sephadex G-75 chromatography. C5a des arg obtained by this 2-step procedure was shown to be immunologically identical to C5a des arg purified by a conventional multi-step method, homogeneous on SDS-polyacrylamide gels, and biologically active. Although this technique yields approximately the same amount of C5a des arg/liter of activated serum as that obtained by conventional methods, its simplicity and relative rapidity make it a practical alternative.

Summary A rapid and reproducible assay method for cytolytic antibodies is described. The procedure is founded on the observation that trypsin digests cells after their exposure to antibody and complement. In practice cells are exposed to various dilutions of antiserum plus complement and injured cells digested by trypsin and the surviving cells are counted. For rapid and accurate counting of cells, the Coulter electronic particle counter was utilized. The electronic impulses registered by the products of the tryptic digestion were negligible compared to those registered by intact cells. In a model system, a horse antiserum against mouse Sarcoma 37 ascites cells was assayed. The cytolytic titer of the serum was expressed as the dilution resulting in lysis of 50% of cells. Assay of antiserum by this method was in close agreement with that obtained by the dye exclusion test. In the range from 24 to 78% lysis, the standard deviation was less than ±3% lysis. Even the low levels of natural antibodies against sarcoma cells present in normal human serum were found to be well within the range of measurement of this method.

Serial dilution with Takatsy loops resulted in exaggerated passive hemagglutination titers with most of the anti-bovine serum albumin sera tested. It appears that certain types of agglutinins adhere to the loop surface and are released only gradually. This adherence, or carry-over effect, was prevented by presoaking loops in gelatin or gelatin-rabbit serum-albumin solutions. Hemolysins did not adhere to loops. In general, hemagglutination reactions performed on plastic trays gave higher titers than those performed in glass test tubes. The quality of the hemagglutination pattern was dependent to a great extent on the type of plastic tray used. As much as a 100-fold difference in titers was obtained depending on the composition of the antiserum diluent. The increase in vibration, in terms of linear displacement (approximately twofold), resulted in an eightfold decrease in titers.

1. A preparation consisting of an oyster mantle and its attached shell has been devised for in vitro studies of shell formation. Shell consisting of typical organic matrix and birefringent crystalline material was elaborated by this preparation, though at a slower rate than in the intact oyster.2. All parts of the mantle were shown to be capable of shell formation in vitro.3. The isolated mantle-shell preparation can be maintained in good condition for several days.

Summary The retention of S35-labeled bovine serum albumin in organs of neonatal and older chicks was compared. The injection of equivalent amounts of S35-BSA (56 mg/kg body wt) resulted in a significantly higher uptake by the spleen of neonatal than of older chicks. The injection into neonatal chicks of a tolerance-eliciting dose of the antigen (1008 mg/kg body wt) resulted in a proportionally higher uptake by spleen. Antigen incorporated in the livers of neonatal and older chicks disappeared at similar rates, whereas rates of disappearance from the spleen differed for the two groups indicating that the antigen retained in the spleen may be different qualitatively in neonatal chicks than in older chicks.

The reaction of HL-A antibodies with the HL-A determinants on the surface of lymphocytes can be blocked by preincubation of lymphocytes with diverse substances such as normal human serum, autologous plasma, fetal calf serum, newborn serum, heat-inactivated rabbit serum and guinea pig serum, serum albumins from various species, lactalbumin, dextrans, synthetic polyglucose, and polyvinylpyrrolidone. The inhibitors can be washed off readily from lymphocytes. The inhibition procedure described here differs from the conventional inhibition tests in that the inhibitor is added first to the target cells. This “masking inhibition” procedure produces substantially greater inhibition than the standard inhibition methods in which the inhibitor is added first to antiserum before the addition of the target cells. The nonspecific nature of masking inhibition indicates that numerous substances adhere to the lymphocyte surface at HL-A sites or closely adjacent sites.

Summary Chicken, sheep and rabbit erythrocytes were stabilized with formaldehyde, pyruvic aldehyde or both, for use in passive hemagglutination reactions. Bovine serum albumin, insoluble BSA, ovalbumin, rabbit γ-globulin, chicken γ-globulin, bovine γ-globulin, E. coli endotoxin, acetylated endotoxin, de-esterified endotoxin, de-esterified and bromacetylated endotoxin, succinylated endotoxin and synthetic polyglucose were found to adsorb firmly on the stabilized cells. For optimal coating of stabilized cells with antigens, critical factors included pH, antigen concentration and the duration of contact with antigen. These conditions differed somewhat from one antigen to another. Coated cells could be stored frozen or lyophilized. Approximately 1 × 10-7 to 1 × 10-8 mg of anti-BSA antibody were detected by using rabbit erythrocytes coated with BSA. This level of sensitivity compares favorably with tannic acid or BDB procedure for cell coating. Antiglobulin tests were applicable in all of the cases tested and were found to increase the hemagglutination titers 10- to 30-fold.

Summary Newly hatched chickens were injected intraperitoneally with 20, 60 and 180 mg of S35-labeled BSA, and the retention of the antigen in various tissues as well as their antibody response to BSA and HGG was determined several weeks later. The amount of antigen retained in the whole chick was estimated to be approximately 0.3% of the originally injected dose when determined 63 days after hatching. The smallness of the amount of antigen retention suggests that the suppression of antibody response is not due to “absorbing out” of the antibody produced. The larger the injection dose at hatching, the larger was the amount of retention, and the larger the amount of retention, the greater was the degree of suppression of antibody response. This relationship suggests that excess antigen retention in antibody forming cells results in suppression of antibody formation. Injection of S35-BSA at hatching did not affect the subsequent antibody response to HGG.

SummarySera from inbred SPF White Leghorn chickens at 6 weeks or 19-22 months of age did not have anti-OA agglu-tinins as determined by the passive HA reaction; 2-day-old chicks also lacked the anti-OA activity. Inbred chickens kept in SPF isolators for 6 weeks, then transferred to a conventional environment for 7 weeks, produced anti-OA agglutinins. Inbred White Leghorn adult chickens conventionally reared had a high anti-OA agglutinin titer. It appeared that natural anti-OA agglutinins are elaborated in response to environmental pathogens in certain lines of chickens. The titer in the individual chicken appeared as early as 10 weeks and was maintained until old age. The presence of natural anti-OA agglutinins did not prevent egg-laying but appears to have delayed it.The HA reaction is specific and can be absorbed.We wish to thank Dr. E. L. Hodapp for valuable assistance during the course of this investigation, and Dr. L. R. Draper for critical review of the manuscript.

SummaryThe sensitivity of the passive hemagglutination reaction was increased considerably by repeated freezing and thawing of antigen-coated erythrocytes. Cells were stabilized by pyruvic aldehyde followed by formaldehyde and then coated with antigen at low pH. After the desired HA titer was attained, the cells were stored in liquid nitrogen. Antigen-coated cells stored at 20° gradually deteriorated during the course of 18 months.

SummaryPassive cutaneous anaphylactic-like (PCA-like) reaction could be elicted in the skin of newly hatched and young chickens with chicken antiserum. The reaction occurred within 20 min following antigen injection, reacted specifically with the antiserum, and the reaction could be prevented with antihistamine. The present findings indicate that differential susceptibilities to anaphylactic reaction exists depending on the developmental stage of the chicken.

SummaryTested by the Schultz-Dale Technic, chicken antibody failed to sensitize the chicken or the guinea pig intestine. Rabbit antibody sensitized guinea pig intestine but failed to sensitize chicken intestine. The minimum doses of histamine and acetylcholine required for contraction of the chicken intestine were considerably larger than those required for guinea pig intestine.We wish to thank Dr. W. E. Vannier for critical review of the manuscript and Mr. H. Crilly and Mr. B. Clark for technical assistance.

SummaryPCA reactions are seldom elicited in conventional adult chickens in contrast to the positive reaction that are readily obtained in young and specific pathogen free (SPF) chickens. Adult germfree chickens also gave postive PCA reactions. Blood exchange experiments were performed among conventional adult chickens, young and SPF chickens. Conventional adult chickens became PCA positive after receiving blood from young or SPF chickens. This suggests that a factor exists in the blood of young and SPF chickens that facilitates the PCA reaction.

SummaryThe sensitivity of the passive hemagglutination reaction was increased considerably by subjecting stabilized rabbit erythrocytes to periodate oxidation, repeated freezing and thawing, or to low pH prior to coating with the antigen BSA. Cells were stabilized by pyruvic aldehyde followed by formaldehyde and then coated with antigen at low pH.

Summary As compared with soluble bovine serum albumin, the use of the heat-denatured protein as an immunizing antigen in rabbits resulted both in a larger number of responding animals and in markedly higher serum antibody titers. In measuring these titers, it was found that the lower the serum titer, the higher was the percentage of the nonprecipitating antibody in relation to the precipitating variety. Evidently, the appearance of nonprecipitating antibody preceded that of precipitating antibody.

Pleural and peritoneal fluids from humans with pathological diseases were examined for the presence of carcinoembryonic antigen-like substances (CEA-LS). Among eight samples tested by a solid phase radioimmunoassay, two pleural fluids and one peritoneal fluid showed significantly elevated CEA-activity. The substances responsible for the CEA-activity were isolated by perchloric acid-extraction followed by two successive Sephadex G-200 chromatography into two pools, Pool I (PI) and Pool II (PII). According to their sedimentation properties, PII was slightly smaller than CEA from tumor tissue-extract (CEA-TTE), while PI was larger than CEA-TTE and approximately twice the size of PII. Micro-double diffusion and antibody binding studies showed that CEA-LS possessed identical antigenic determinants as CEA-TTE, which did not cross-react with normal colon antigen (NCA).

An enzyme-linked immunosorbent assay has been developed to detect human C5a antigen. This ELISA methodology has been shown to be a highly sensitive technique capable of detecting C5a antigen concentrations below 10 ng/ml. The microELISA technique used in this study is specific for human C5a and C5a des arg (C5a antigen) but not for human C5. Conditions to establish sensitivity and specificity are outlined in ths report.

Effects of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), the synthetic chemotactic peptide, and its analogues on blood pressure were investigated in the rabbit, rat, mouse, dog, cat and guinea pig. The administration of FMLP (5 micrograms/kg i.v.) induced the depressor effect in the rabbit but no effect was observed in the other animal species. The only peptide which showed a similar effect as FMLP was N-formyl-L-methionyl-L-methionyl-L-phenylalanine (FMMP) among the peptides relating to FMLP. Tachyphylaxis was observed in the hypotensive action induced by the two peptides.

A number of mono- and oligosaccharides have been utilized as potential inhibitors in radioimmunoassay to identify immunodeterminant groups on the carcinoembryonic antigen molecule. Tri-N-acetylchitotriose, a trisaccharide of β 1 → 4 linked N-acetylglucosamine, result in up to 50 per cent inhibition of binding between carcinoembryonic antigen and its antibody in the radioimmunoassay whereas di-N-acetylchitobiose, N-acetylglucosamine and it β-anomer were less efficient inhibitors. Other monosaccharides, glycosides, and oligosaccharides were inactive or only slightly active at concentrations comparable to or much greater than the tri-N-acetylchitotriose used. These studies indicate that at least a portion of the anti-CEA antibodies have a combining site with an affinity for the trisaccharide unit of two N-acetylglucosamine residues in β → 4 linkage as well as another N-acetylglucosamine moiety or possibly the second β-anomeric linkage of tri-N-acetylchitotriose.

SummaryA reverse passive hemagglutination (RPHA) procedure was developed for the detection of hepatitis B virus associated antigen (HB Ag) in human serum. The comparative sensitivity of six different methods was double agar diffusion

SummaryThe adjuvant effect of colchicine was investigated in the hamster primarily because this species is unique in tolerating high dosage of colchicine. Administration of non-toxic doses of colchicine enhanced hemagglutinin response, but the increase in antibody response was no greater than that reported for the rabbit, a species far more susceptible to the toxicity of colchicine. The increased level of hemagglutinin was for complete antibody; levels of incomplete antibody were not elevated. Increase in the dose of colchicine to near-lethal levels resulted in considerable reduction in antibody response.

Antibodies which were cytotoxic to lymphocytes were found in patients with kidney transplants, Graves' disease, Hashimoto's disease, rheumatic heart disease, and in multiparous women. These cytotoxins were 19S, optimally reactive at 15° C, were autocytotoxic to lymphocytes, and were inactive against granulocytes. The specificity of their reactivity to allogeneic lymphocytes was unrelated to HL-A specificities. In fact, though the sera varied from 1% to 99% in reactivity against a panel of random donors, much of the variation seems to be of a quantitative nature with relatively little specificity. Out of 6,105 x2 comparisons between 110 sera of the diseases mentioned above, 83% were positive and none of the negative x2 values were statistically significant. It is concluded that diverse stimuli elicit the formation of these low avidity 198 antibodies which are directed against a non-HL-A antigen richly represented on lymphocytes. Although their biologic significance may be difficult to show, as with cold hemagglutinins, they could possibly act as a “natural” antilymphocytic serum.

Insoluble angitens may be used to adsorbed specifically antibody from serum. Bovine serum albumin (BSA) has been rendered insoluble, without the loss of antigenic determinants, by heating at pH 3.7. The adsorption of anti-BSA antibody on this material has been made the basis of a method for the estimation of the total antibody contained in antiserums. The insoluble BSA has been found to have a high capacity for the adsorption of specific antibody (0.8 mg of antibody per mg of insoluble BSA) and to retain the native antigenic determinants so that all the antibodies reacting the native BSA were adsorbed. The method provides a rapid and precise estimation of the total anti-BSA antibody content of serums.

Allogeneic antiserums against human lymphocytes were specifically inhibited by M protein from beta hemolytic group A Streptococcus pyogenes, type 1. Analogous M proteins from streptococcus types 3, 4, 5, 6, 12, and 14 had little or no inhibitory activity. The specific inhibition by M protein is not a result of anticomplementary activity or of coating of the lymphocyte surface. Streptococcal polysaccharide and 73 other polysaccharides were inactive. Because all seven HL-A specificities tested were inhibited, it is inferred that M1 protein has a structure common to human histocompatibility antigens.

THE usual procedure for obtaining serum from clotted blood is to detach (rim) the clot from the collecting vessel, allow the clot to retract for several hours, and centrifuge at 500–1,000g. The procedure is not only time-consuming, but often results in much haemolysis ; in addition the yields of serum vary greatly from sample to sample. By using stronger centrifugal force and thromboplastin, I have been able to obtain serum in high yields, within 1 h after bleeding, and with a minimum of haemolysis.

FOR passive haemagglutination tests with proteins, it has been necessary to couple these antigens to erythrocytes by agents such as bis-diazotized benzidine1–3, tolylene-2,4-diisocyanate4 and tannic acid5. During the course of investigations with formalin-treated erythrocytes, we observed that bovine serum albumin (BSA) would adsorb on formalin-treated chicken erythocytes (FCE), as well as on formalin-treated sheep erythrocytes (FSE) without recourse to such coupling agents. This finding suggested a means of simplifying the procedure for coating erythrocytes with proteins for passive haemagglutination tests.