Ludwig A. Sternberger

The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal.

tetrahydrochioride (DAB) to yield a brown reaction product. In the second sequence, antiserum to the second antigen was again followed by anti-immunoglobulin and PAP, and then by hydrogen peroxide and 4-chloro-1-napthol (CN) to yield a blue reaction product. Even though the same anti-immunoglobulin and PAP were used in both sequences, and even though hydrogen peroxide was applied as enzyme substrate twice, no color mixing occurred. Therefore, it has been found unnecessary to remove the immunoreagents of the first staining sequence prior to applying the second sequence. Apparently, the DAB reaction product masked antigen and catalytic sites of the first sequence of immunoreagents and thus prevented interaction with reagents of the second sequence. This conclusion had been derived from attempts at staining the same antigen with the same primary antiserum in the first and second sequence. Only brown reaction product was obtained and no color mixing occurred. However, when either primary antiserum or DAB in the otherwise complete first reaction sequence were progressively diluted, colors became mixed until upon omission of primary antiserum or DAB, standard (control) blue was obtained. Similarly, standard brown was obtained when primary antiserum was omitted in the second sequence. In the pars distalis of the pituitary, separate cells stained brown and blue when pairs of antisera to the following hormones were applied: growth hormone, prolactin, and either ACTH’24 or ovine f1-lipotropin. Separate cells were also visualized when anti-ACTH’24 was followed by anti-LH. When anti-growth hormone was followed by anti-LH, a few cells were mixed in color, even though most cells were either brown or blue. When anti-ACTh’24 was followed by anti-ACTh’3, mixed color staining occurred in cells of the pars intermedia and distalis, and fibers in the pars nervosa were blue.

Phosphorylation protects neurofilaments against proteolysis

During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that phosphorylated neurofilaments are protected against proteolysis, and dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.

The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification.

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.

Phosphorylated neurofilament antigens in neurofibrillary tangles in Alzheimer's disease.

Abstract Neurofibrillary tangles (NFT) are a hallmark of Alzheimers disease (AD), and their presence correlates with the presence of dementia. A major constituent of NFT is the insoluble paired helical filament which shares some antigenic relationships with normal cytoskeletal elements, particularly neurofilaments. If neurofilament proteins (200, 145–160, and 68 kilodaltons [kd]) participate in the formation of NFT, the distribution of these constituents might be expected to be abnormal. To examine this issue, we used immunocytochemical methods to localize phosphorylated and nonphosphorylated epitopes of neurofilament proteins in hippocampal neurons of controls and patients with AD. Normally, the 200-kd neurofilament protein is not phosphorylated in the perikarya of neurons. However, in AD, many pyramidal neurons contained immunoreactive phosphorylated neurofilaments. Patterns of immunoreactivity (linear, flame-shaped, or skein-like within perikarya) greatly resembled the appearance of silver-stained NFT. This pattern of immunoreactivity was not present in hippocampal pyramidal neurons in controls, except in one aged patient in whom adjacent silver-stained sections revealed a few NFT. Patterns of immunoreactivity with antibodies for nonphosphorylated neurofilament proteins were similar in control and AD neurons. Our results indicate that some NFT are associated with abnormal distributions of high molecular weight phosphorylated neurofilament proteins. One domain of the 200-kd protein is believed to be a component of the side arms which link neurofilaments and interact with microtubules. Abnormal interactions of perikaryal neurofilaments could play a role in the genesis of NFT, and this abnormality of the cytoskeleton could contribute to the dysfunction of neurons at risk in AD.

Immunocytochemical studies of neurofilament antigens in the neurofibrillary pathology induced by aluminum

Intrathecal administration of aluminum salts induces accumulation of neurofilaments in axons and perikarya of motor neurons and is associated with impaired axonal transport of neurofilament proteins. Because phosphorylation of the 200-kilodalton (kd) neurofilament protein, thought to be a major component of the sidearms, seems to be important in interactions of neurofilaments with other cytoskeletal elements, we have postulated that aluminum may produce neurofibrillary pathology by altering patterns of neurofilament phosphorylation. To test this hypothesis, antibodies against phosphorylated and non-phosphorylated neurofilament epitopes were used for immunocytochemical analysis of spinal cord sections from aluminum-injected rabbits. In control animals, phosphorylated 200-kd neurofilament proteins were not demonstrable in perikarya of motor neurons. In experimental rabbits, perikarya and proximal axons of affected motor neurons showed striking accumulations of immunoreactivity of one phosphorylated epitope. The presence of phosphorylated 200-kd neurofilament proteins in these regions may have important consequences for the organization of the cytoskeleton and for the transport of neurofilaments. A similar, but not identical, pattern of accumulation of phosphorylated neurofilament immunoreactivity has recently been observed in neurofibrillary tangles in Alzheimers disease.

Quantitative immunocytochemistry of pituitary receptors for luteinizing hormone-releasing hormone.

SummaryIn Araldite sections of male rat pituitaries, stained after embedding by the unlabeled antibody enzyme method with antisera to native luteinizing hormone-releasing hormone (LH-RH) or LH-RH azo-conjugated to bovine serum albumin, localization is confined mainly to the interior of the large, and to a lesser extent to that of the small, secretion granules of the gonadotrophic cells. Plasma membranes are not demonstrated. Except for weak staining in the granules of corticotrophs, no other pituitary cell is stained. Pretreatment of sections with LH-RH (to dilutions of 4 pg/μl) increases staining intensity in the gonadotrophic granules. Other cells are unaffected. The lesser the gonadotroph staining intensity without pretreatment, the greater the increase (more than 23-fold reactivity). Augmented staining is measurable (P<0.001) to antiserum dilutions of 1∶240 000. Pretreatment with des-Glu-1-LH-RH, porcine corticotropin or rat prolactin has no effect. LH-RH-Gly-10(desamide) inhibits. Rat glycoprotein hormones enhance staining with anti-azo-conjugated LH-RH. With antinative LH-RH these hormones enhance weak staining, but inhibit strong staining. Thick vibrotome sections of male rat or rabbit pituitaries stained before embedding reveal specific localization on plasma membrane and gonadotrophic secretion granules, provided the sections have been pretreated with LH-RH (250 pg/μl). The data show that LH-RH after reaction with receptor is not sterically hindered from binding specific antibodies. Receptor may be found in secretion granules, both in the free state or combined with LH-RH. Plasma membrane receptor, on the other hand, was free under the conditions of the experiments. Immunization with LH-RH elicits not only heteroimmune antibodies specific for LH-RH, but also a group of still ill defined autoimmune antibodies, some of which may conceivably be reactive with glycoprotein hormone α-chains.

ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY WITH UNLABELED ANTIBODIES AND THE PEROXIDASE-ANTIPEROXIDASE COMPLEX A TECHNIQUE MORE SENSITIVE THAN RADIOIMMUNOASSAY

Titration curves were developed with antisera to 17-39ACTH (adrenocorticotropin) and 1-39ACTH with the techniques of radioimmunoassay and electron microscopic immunocytochemistry. For the latter method, the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was used to stain normal rat pituitary intermediate lobes. By radioimmunoassay standards, the 17-39ACTH antiserum was of poor quality. Its titration curve exhibited a flat slope and it did not bind a significant amount of labeled antigen beyond a 1: 30 dilution. However, immunocytochemical staining was detected with this antiserum at dilutions as high as 1:1,500. The antiserum to 1-39ACTH was of better quality by radioimmunoassay standards. It bound 45% of the labeled antigen at a dilution of 1:5,000. Immunocytochemical staining intensity was nearly maximal at a 1:5,000 dilution and decreased progressively to a limiting value at 1:16,000. However, when incubation times in the antisera were increased from 3 min to match those of the radioimmunoassay (48 hr) maximal staining was achieved at dilutions as great as 1:512,000 where only trace amounts of the labeled antigen were bound in the radioimmunoassay. It was concluded that the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was sensitive enough to detect antibodies in sera of low titer and/or avidity which are not detected by a radioimmunoassay. The technique holds great promise for a sensitive assay system.

Reaction of Lewy bodies with antibodies to phosphorylated and non-phosphorylated neurofilaments

The reaction of Lewy bodies in the substantia nigra and the sympathetic ganglia was studied in 10 cases of Parkinsons disease and other Lewy body-related conditions, using monoclonal antibodies to non-phosphorylated (02-40) and phosphorylated (03-44 and 07-5) neurofilaments. Although many Lewy bodies were unstained, a peripheral ring of positive immunoreactivity was frequently observed. This reaction was more common and more intense with antibodies to phosphorylated neurofilaments, suggesting a posttranslational phosphorylation of elements in the Lewy body. Although Lewy bodies appear to have some antigenic sites in common with neurofilaments, certain differences between neurofilaments and Lewy body filaments are emphasized.

Distribution of neurofilament antigens after axonal injury

Phosphorylated and nonphosphorylated epitopes of neurofilament (NF) proteins are distributed in different regions of individual neurons. Immunocytochemical methods, with monoclonal antibodies directed against phosphorylated and nonphosphorylated NF, demonstrated nonphosphorylated NF in perikarya and proximal axonal segments of neurons in dorsal root ganglia, while phosphorylated NF proteins were present in axons of these cells. The distribution of these epitopes of NF were examined at various times following injury of axons in the rat sciatic nerve. Between one and 21 days after crush of the proximal nerve, phosphorylated NF were present in neuronal perikarya. We have compared patterns of perikaryal immunoreactivity at one time point (three weeks) following a more distal crush or complete transection of the sciatic nerve. At this time period, following transection/ligation, phosphorylated NF immunoreactivity was not present in perikarya, but abnormal staining was observed after nerve crush. These altered distributions of phosphorylated epitopes of NF are of interest because several recent reports have indicated that similar, but not identical, abnormal staining patterns occur in human neurological diseases, including Alzheimers disease and Parkinsons disease. In accord with previous studies, this investigation indicates that one response of neurons to injury, or to disease, is an abnormal distribution of phosphorylated epitopes of NF proteins.

THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOCYTOCHEMISTRY QUANTITATIVE COMPARISON OF SENSITIVITIES WITH AND WITHOUT PEROXIDASE-ANTIPEROXIDASE COMPLEX

Araldite sections of rat pituitary intermediate lobe were used with anti-17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cyclic PAP molecule, thus preventing the losses of PO that occur during washing when anti-PO and PO have been applied in sequence. Identification of the characteristic, cyclic PAP molecules affords confirmation of specific staining at high resolution. In the sequential application of anti-PO and PO, no PAP molecules are formed, thus making distinction of specific from nonspecific deposition of enzyme reaction products ambiguous. With the use of anti-17-39 ACTH and the intermediate lobe, the unlabeled antibody enzyme method was 16,000-100,000 times more sensitive than radioimmunoassay.